Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas: Volume 2 [2] 0128148314, 9780128148310

Table of contents :
Cover
TRANSPLANTATION,
BIOENGINEERING,
AND
REGENERATION OF
THE ENDOCRINE
PANCREAS,
VOLUME 2
Copyright
Contributors
Part A: Islet auto-transplantation
Section I: Chronic pancreatitis
1
Etiopathogenesis and pathophysiology of chronic pancreatitis
Etiopathogenesis
Alcohol-induced chronic pancreatitis
Autoimmune pancreatitis
Genetic pancreatitis
Enzymatic- and ion-related mutations in PRSS1, SPINK1, CTRC, and CaSR
CFTR mutation
Metabolic disorders
Hyperparathyroidism
Hereditary pancreatitis
Recurrent acute pancreatitis
Ductal obstruction
Pancreas divisum
Sphincter of Oddi dysfunction
Pancreatic trauma
Tropical chronic pancreatitis
Drug toxicity-induced pancreatitis
Idiopathic
Gallbladder dysfunction
Smoking and pancreatitis
Pathophysiology
Exocrine insufficiency
Endocrine insufficiency and diabetes mellitus
Pathophysiology of pain
Calcification
Fatty infiltration
Fibrosis
Pseudocysts
Atrophy
Pancreatic cancer
Molecular mechanisms in the development of pancreatitis
Conclusion
References
2
Nonreplacement treatment of chronic pancreatitis: Conservative, endoscopic, and surgical (resection and drainage procedures ...
Introduction
Clinical evaluation
Abdominal pain
Pancreatic insufficiency
Diagnosis of chronic pancreatitis
Medical and conservative management
Lifestyle modifications
Exocrine pancreatic insufficiency and malnutrition
Pain management
Endoscopic and surgical management of chronic pancreatitis
Endoscopic management of chronic pancreatitis
Management of pancreatic duct stricture
Management of pancreatic duct stones
Management of benign biliary strictures
Surgical management of chronic pancreatitis
Large duct disease
Roux-en-Y lateral pancreaticojejunostomy (Puestow procedure)
Dominant head mass
Pancreaticoduodenectomy
Beger procedure and Berne modification
Frey procedure
Results of clinical trials
Conclusion
References
Section II: Islet auto-transplantation for chronic pancreatitis
3
Requirements for clinical islet laboratories
Introduction
Allogeneic vs autologous islet
Registration with the FDA
Registration with other regulatory agencies
General requirements for a clinical auto islet lab
Establishment and maintenance of a quality program
Facilities and environmental control
The vulnerability of islet cells to contamination
Use of a clean room to manufacture islet cells
Clean room entry and exit
Training
Clean room cost
Environment control
Equipment
Specific equipment needs for an auto islet lab
Telemedicine (communications between the operating room and the clinical islet lab)
Personnel
Procedures
Supplies and reagents
Storage
Recovery
Process and process controls
Labeling
Product tracking
Records
Reporting
Deviation in processing
Inspections
Corrective actions and process improvement
Outcome analysis
References
4
Islet isolation for autotransplantation, following total or near total pancreatectomy
Introduction
Islet isolation
Organ procurement and transport
Laboratory preparation and setup
Islet isolation
Pancreas cleaning, trimming, and cannulation
Pancreas perfusion
Collagenase: Selection and dose
Phase 1: Digestion
Phase 2: Dilution and tissue collection
Islet cell purification
Preparation for administration
Conclusions
References
Further reading
5
Strategies to improve islet yield from chronic pancreatitis pancreases intended for islet autotransplantation
Introduction
Islet cell isolation
Pancreatectomy and pancreas transport
Trimming and cannulation of the pancreas
Enzyme selection and perfusion
Tissue digestion
Tissue recombination
Purification process
Transplant preparation
Conclusion
Conflict of interest
Acknowledgments
References
6
Surgical techniques for total pancreatectomy and islet autotransplantation
Introduction
Indications and contraindications
Patient selection
Candidates
Contraindications
Preoperative testing and assessment
Operative technique
Total pancreatectomy
Principles
Preoperative preparation
Operative procedure
Islet isolation
Islet infusion/transplantation
Minimally invasive surgery
Robotic TP and islet autotransplant
Laparoscopic TP and islet autotransplant
Robotic and laparoscopic TP outcomes
Percutaneous infusion/transplantation of islets
Postoperative care
Complications
Portal vein thrombosis
Islet contamination
Delayed gastric emptying
Biliary anastomotic leak
Rare complications
Outcomes
Salvage pancreatectomy
Pediatric population
Remote processing
QOL after TPIAT
Future
Conclusions
Acknowledgments
References
7
Total pancreatectomy with islet autotransplantation in children
Introduction
Pancreatitis in children
Determining when TPIAT is indicated for the management of children with pancreatitis
Surgical procedure and islet isolation and transplant in children
Postoperative management of the child after TPIAT
Diabetes management after TPIAT
Pain management after TPIAT
GI management after TPIAT
Asplenia management after TPIAT
Short-term and long-term outcomes after TPIAT in children
Surgical outcomes
Pain relief and quality of life after TPIAT
Diabetes after TPIAT
Conclusions
References
8
Islet autotransplantation: Indication beyond chronic pancreatitis
Introduction
Incidence of pancreatogenic diabetes after pancreatic resection
Autologous islet transplantation to prevent or minimize pancreatogenic diabetes in patients requiring total pancreatectomy ...
Expanding indications for IAT: Nonneoplastic diseases beyond chronic pancreatitis
Expanding indications for IAT: Neoplastic benign diseases
Expanding indications for IAT: Neoplastic malignant diseases
Expanding indications for IAT: Milan protocol
Conclusion
References
Section III: Outcomes
9
Postoperative care and prevention and treatment of complications following total pancreatectomy with islet cell autotranspl ...
Introduction
Patient selection and preoperative preparation
Postoperative management and complications
Islet yield and glycemic management
Pain control
Nutrition
Islet cell infusion and its complications
Mesenteric thrombosis
Hemorrhage
Hepatic changes on imaging
Infection
Risk of cancer
Surgical complications
Conclusion
References
10
Metabolic outcomes after total pancreatectomy followed by islet autotransplantation (TPIAT): Mixed blessings☆
History and rationale for TPIAT: Points of view
Favorable metabolic outcomes: Normal levels of glycemia, HbA1c, and β -cell function
Unfavorable metabolic outcomes: Hypoglycemia following meals and exercise and deficient α -cell counter-regulatory respons ...
The need to consider nonhepatic transplantation sites
References
11
Long-term results of TPIAT
Introduction
Surgical outcomes
Cholangitis
Bowel obstruction
Internal hernia
Gastrointestinal function outcomes
Dysmotility, delayed gastric emptying, and slow transit
Exocrine dysfunction
Nutritional deficiency
Pain and quality of life outcomes
What is known about long-term pain/QOL outcomes?
Development of new abdominal pain syndrome
Metabolic outcomes
Long-term islet graft function
Hypoglycemia
Long-term complications of diabetes
Survival and cost of care
Conclusions
References
Part B: Bioengineering and regeneration of the endocrine pancreas
Section I: Pancreas development and regeneration
12
Embryonic development of the endocrine pancreas
Introduction to pancreas development
Pancreas development
Foregut endoderm compartmentalization
Pancreatic buds and branching morphogenesis
Exocrine versus endocrine development during secondary transition
Exocrine cell fate allocation
Endocrine cell fate allocation
Endocrine cell subtype specification: α - and β -cells
Endocrine cell subtype specification: δ, PP, and ε cells
Endocrine development during late gestation
Postnatal islet development and function
Maturation of postnatal islets
Communication between endocrine cells in postnatal islets
α - and β -cell interactions
Interactions with other hormone-expressing cells
Conclusions
Acknowledgments
References
13
Human pancreatic progenitors
Introduction
Induction and patterning of pancreatic endoderm
Conservation of endoderm formation across vertebrates
Nodal signaling initiates endoderm development
Endodermal patterning
Foregut-derived organogenesis and generation of the pancreatic buds
Patterning of the dorsal and ventral pancreas
Dorsal pancreas induction
Ventral pancreas induction
Lineage specification, proliferation, and compartmentalization of pancreatic progenitors
Common pancreatic progenitors arising from the pancreatic bud
Size control
Pancreatic tubulogenesis and the ductal plexus
Endocrine and exocrine specification: The secondary transition
Endocrine differentiation
Exocrine differentiation
Ductal differentiation
Acinar differentiation
Adult pancreatic progenitors
Ductal progenitors
Intraislet progenitors
Exocrine progenitors
Transdifferentiation in the pancreas
Exocrine to endocrine transdifferentiation
Endocrine to endocrine transdifferentiation
Extrapancreatic sources of pancreatic precursors
Biliary tree stem/progenitor cells and biliopancreatic stem/progenitor cells
Mesenchymal stem cells as potential sources for pancreatic endocrine cells
Pancreatic islet-derived MSC
Exocrine pancreas-derived MSCs
Therapeutic modulation of pancreatic regeneration and β -cell mass
Concluding remarks
Acknowledgments
References
14
Strategies to promote beta-cell replication and regeneration
Introduction
Beta-cell replication is the major contributor to the postnatal beta-cell growth
Neonatal period
Partial pancreatectomy
Pregnancy
Insulin resistance
Cell-cell communications regulate beta-cell proliferation
Cross-talk between endothelial cells and beta cells
Cross-talk between macrophages and beta cells
Cross-talk between stromal cells and beta cells
Molecular signaling pathways that control beta-cell proliferation
FoxM1
TGF-beta/SMAD
Concluding remarks
References
15
Diet as a therapeutic approach to diabetes management and pancreas regeneration
Introduction
Proper nutrition is key to the maintenance of β -cell homeostasis and function
Dietary intervention for T1D: Feasibility, outcome, and recent progress
Diet in T2D: The two sides of diet and eating habits as causing factor and potential treatment for T2D
Dietary recommendations for women with gestational diabetes
Clinical relevance of fasting or fasting-like regimens as a nutritional therapeutic approach in diabetes
Molecular mechanisms supporting the potential of fasting and FMD to promote pancreas functional restoration
Conclusion
References
16
The benefits of metabolic/bariatric surgery on diabetes mellitus
Introduction
History of metabolic/bariatric surgery
Metabolic/bariatric surgery for diabetes
Nonbariatric metabolic surgery for diabetes
Metabolic/bariatric surgery mechanisms
Metabolic/bariatric surgery and diabetes mechanisms
The present
The future
Conflict of interest
References
Section II: Scaffolds for endocrine pancreas bioengineering
17
ECM-based scaffolds for pancreas bioengineering
Introduction
Extracellular matrix
Extracellular matrix in pancreatic islets
Collagen
Laminin
Fibronectin
Glycosaminoglycans
Fibrin
Tissue engineered islet scaffold incorporating ECM
Whole organ engineering of pancreas
Whole organ decellularization
Biophysical properties of decellularized organs
Microarchitecture
Mechanical properties
Growth factor retention
Whole organ recellularization
Parenchymal reconstruction
Reendothelialization of the acellular scaffold
Hybrid organs using repurposed biological scaffolds: Liver and kidney to pancreas
Conclusion
References
18
Plasma scaffolds for islet transplantation
Introduction
Islet microenvironment
Integrins and mechanotransduction
Concept of anoïkis
Organoid organization protects islet from anoïkis
Strategies to counteract anoïkis
Molecular approach
Integrin activation
3D culture systems: Scaffolds for islets
Overview
Mechanotransduction
Decellularized gels
Hydrogels
Challenges in reproducing the natural islet environment
Using plasma as a scaffold
Ubiquitous tunable scaffold
Biodegradable scaffold
GFs of interest for islets
Fibronectin
Plasma scaffolds for islets: Drawbacks
Plasma scaffold for islet culture
Plasma scaffold composition
Oxygen diffusion in plasma scaffold
Uses of plasma for other applications
Conclusion
Acknowledgements
Conflict of interest statement
References
19
A biologic resorbable scaffold for tissue engineering of the endocrine pancreas: Clinical experience of islet transplantati ...
Introduction: The intrahepatic site for islet transplantation
Extrahepatic sites for islet transplantation
The greater omentum: A novel site for islet transplantation
Preclinical experience
Clinical experience
Conclusions
References
20
Endothelialized collagen modules for islet tissue engineering
Introduction
Transplantation into the omental pouch of immune competent rats
Subcutaneous transplantation into immune-compromised SCID/bg mice
Future directions
References
Section III: Islet encapsulation
21
Conformal coating
Introduction
Conformal coating technology
Composition of coating hydrogel
The physical phenomena permitting conformal coating—Flow focusing and the Plateau-Rayleigh instability
The conformal coating device, system, and process
In vitro performance of conformally coated islets
In vivo performance of conformally coated islets
Transplantation in syngeneic mouse models
Transplantation in allogeneic mouse models
Conclusion and future directions
Disclosure
References
22
Co-encapsulation of ECM proteins to enhance pancreatic islet cell function
Introduction
Overview of the islet extracellular matrix
Effects of ECM co-encapsulation on β -cell survival
Effects of ECM co-encapsulation on β -cell function
Effects of ECM co-encapsulation on β -cell proliferation
Future directions
References
23
Co-encapsulation of mesenchymal stromal cells to enhance islet function
Type 1 diabetes and islet transplantation
Current issues with encapsulated islet transplantation
Mesenchymal stromal cells
MSC and islet transplantation
Islets and MSC co-encapsulation
Conclusion
References
24
Silk-based encapsulation materials to enhance pancreatic cell functions
Introduction
Silk as a biomaterial
Structure of silk
Silkworm silk
Silkworm silk fibroin
Silkworm silk sericin
Spider silk
Biocompatibility of silk matrices
Biodegradability and bioresorbability of silk matrices
Silk matrices used for islet culture and encapsulation
Hydrogels
Scaffolds
Silkworm silk scaffolds
Spider silk foams
Conclusions and future perspectives
Acknowledgments
References
25
Cell pouch devices
Introduction
Cell encapsulation technology
Macroencapsulation devices
β Air
ViaCyte
Sernova cell pouch
MailPan
Concluding remarks
References
Section IV: Stem cells to generate insulin producing cells
26
Pancreas progenitors
Introduction
Regeneration of pancreatic islets
The role of residing islets
The role of pancreatic duct epithelial cells
The role of pancreatic acinar cells
Biliary tree stem/progenitor cells and the network of hepatic, biliary, and pancreatic stem/progenitor cell niches ( Fig. 1 ...
Pancreatic progenitor cells ( Fig. 1)
Pancreatic duct gland as the niche of pancreatic progenitors ( Fig. 1)
Conclusions
References
27
Human embryonic stem cells (hESC) as a source of insulin-producing cells
Introduction
Current therapeutic approaches and challenges
Pancreatic and islet transplantation: Scarcity, graft loss, and immunosuppression
Diabetes targets of future IPC therapies
Pancreas development
Stem cell-derived β cells
Cell sources, characterization, and requirements for use in β cell differentiation
Dead ends: Pathways to polyhormonal cells which never mature
In vitro vs in vivo: Paracrine signals, cell-cell contact, understanding the signals
Hurdles still preventing a final functional product
Maturation
Evaluating other signals: Endoplasmic reticulum stress and mitochondrial function
Cross talk: Matrix, blood vessels, cell-cell interactions, and paracrine signals
Genome editing for therapeutic advantage
Immunogenicity
Autologous PSCs
HLA-typed haplobanks
Genetic modification to prevent immune rejection
Immune tolerance
Humanized mice
Current clinical trials of stem cells for diabetes
Interspecies organogenesis and stem cells
Conclusion
References
28
Human-induced pluripotent stem cells (iPSC) as a source of insulin-producing cells
Pluripotent stem cells: Embryonic stem cells and somatic cell nuclear transfer
Embryonic stem cells
Somatic cell nuclear transfer (SCNT)
Every cell can be pluripotent: The discovery of induced pluripotent stem cells
iPSC are in the clinic: active protocols in humans
iPSC for β -cell replacement: In vitro differentiation into β -cells
Immunogenicity of iPSC-derived cells
Graft protection
Immunosuppression
Haplobank
Micro/macro-encapsulation
Gene editing: The invisible cell
Safety of iPSC-derived cells
Generation of safer iPSC lines
Removal of pluripotent cells from differentiated cell product
Conclusions
References
29
Ductal cell reprograming to insulin-producing cells as a potential beta cell replacement source for islet auto-transplant r ...
Introduction
Ductal cells as a potential source for beta cell regeneration
In vitro reprogramming of pancreatic ductal cells
In vivo studies on the pancreatic ductal progenitor cells
Controversies regarding endocrine differentiation from ductal lineages
Conclusions and future perspectives
Conflict of interest
Acknowledgments
References
30
Synthetic biology technologies for beta cell generation
Introduction
Synthetic biology
Type I diabetes
Stem cell-based therapy for type I diabetes
Human pancreatic development
Gene-regulatory networks involved in human pancreatic development
Synthetic biology approaches for generating beta cells
Lineage-control networks
Clustered regularly interspaced short palindromic repeats-Cas9
Synthetic messenger RNAs
Synthetic receptors
Synthetic biomaterials
Artificial designer cells
Synthetic biology—Moving toward clinical applications
Roadblocks in generating functionally mature beta cells from PSCs
Roadblocks in engineering artificial beta-mimetic cells
References
Section V: Animal-based platforms for pancreas bioengineering
31
Xenotransplantation of the endocrine pancreas
Introduction
A novel approach to discrepancies in supply and demand
Defining success
A brief history of xenotransplantation
Earliest attempts in xenotransplantation and xenotransfusion
Origins of endocrine transplantation
Advancing to the modern era of xenotransplantation
Optimizing the pig-to-NHP model
The pig-to-NHP as the preferred preclinical model
Laying the foundation: preclinical studies in islet xenotransplantation
Hurdles to free and encapsulated islet xenotransplantation
Encapsulation
Overcoming immediate host responses: pharmacotherapies to prevent the instant blood-mediated inflammatory reaction
Ideal placement of free islets in xenotransplantation
Composite islet-kidney grafts
Cotransplantation of islet xenografts and “regulatory” cells
Genetic modifications to combat IBMIR
Control of the T cell response
Will sensitization to human leukocyte antigens be detrimental to islet xenotransplantation?
Will sensitization to pig antigens preclude subsequent islet allotransplantation?
The induction of immune tolerance: The “Holy Grail” of transplantation
Improving function of porcine islets
Justification for translation to clinical trials
Lessons from early clinical trials
Establishing safety
Determining efficacy
Patient selection
Future directions
Research priorities
Conclusions
Acknowledgment
Conflict of interest
References
32
Interspecies blastocyst complementation
Introduction
Basic principles of IBC
Generation of pancreas with IBC
Transplantability of IBC pancreas
Advantages of IBC
Pretransplant immunogenicity
Posttransplant immunogenicity
Potential for IBC vasculature
Autograft tolerance
Allograft considerations
Efficiency of IBC
Breeding schemes
Gene editing with IBC
Suitability of large animal hosts
Ethical concerns
Outlook
References
Section VI: Tissue engineering technologies applied to ß-cell replacement
33
Bioengineering, biomaterials, and β -cell replacement therapy
Introduction
Immunoprotective barrier strategy
Revascularization strategy
Biomaterials
Hydrogels
Natural hydrogels
Agarose
Alginate
Chitosan
Collagen
Fibrin
Hyaluronic acid
Gelatin
Decellularized ECM-based biomaterials
Synthetic hydrogels
Pluronic
Poly(ethylene glycol)
Methacrylated compounds
Solid biomaterials
Nondegradable biomaterials
Pdms
PEOT-PBT block copolymers
Degradable biomaterials
PGA, PLA, and PLG
Ethisorb
Pva
Islet delivery device fabrication techniques
Scaffold fabrication techniques with random islet distribution
Particulate leaching
Phase separation
Electrospinning
Microencapsulation by droplet generators
Microfabrication techniques (controlled islet distribution)
Pillared wafer
Microthermoforming
Microfluidics
Nanoencapsulation by layer-by-layer techniques
3D Printing in regenerative medicine
3D printing for β -cell replacement therapy
Future outlook
References
Further reading
34
Subcutaneous islet transplantation using tissue-engineered sheets
Introduction
Subcutaneous islet transplantation
Cell sheet engineering in cell transplantation
Cornea
Heart
Esophagus
Liver
Others
Subcutaneous islet transplantation using cell sheet engineering
Our concept of subcutaneous islet transplantation using cell sheet engineering
Cell sources
Cytokines
Previous study
Conclusions
Conflict of interest
References
Section VII: Regulation and funding
35
Regulation for regenerative medicine-based therapies
Regulatory approach to regenerative medicine in the EU
General considerations
A brief historical perspective of the use of cells
Advent of cell and gene therapies
Differences between United States and European Union regulatory framework
Classification procedure
Therapeutic use of pancreatic cells
Pancreas transplant
Pancreatic islet transplant—Encapsulated cells
RM—In vitro creation of pancreatic cells
Conclusion
References
36
Catalyzing beta-cell replacement research to achieve insulin independence in type 1 diabetes: Goals and priorities
JDRF mission
Beta-cell replacement
Islet transplantation
BCR strategies
Clinical trials in beta-cell replacement
Beta-cell replacement: Key considerations
Bridging the gap: Priorities and opportunities
Additional considerations
Conclusion
Acknowledgments
References
Further reading
37
Regenerative medicine technologies applied to beta cell replacement: The industry perspective
Can a “replenishable” (stem cell-derived) beta cell therapy mimic islet transplant therapy?
Immune suppression
Beta cells within devices
Device size
Access to blood supply
Stimulation of fibroses
Can the therapy be removed and replaced?
Can the therapy be mass-produced?
Regulatory approval
Cost and reimbursement
Patient acceptance
Conclusion
References
Further reading
38
Pancreas whole organ engineering
Introduction
Fundamental concepts of tissue development
Whole organ engineering
Three-dimensional bioscaffolds for whole organ pancreas engineering
Whole organ decellularization
Pancreatic whole organ decellularization
Effects of pancreatic organ decellularization on ECM composition, structure, and mechanics
Bioreactors vs in vivo pancreatic organ engineering
Bioprinting of pancreas
Pancreatic tissue engineering
Challenges to current approaches
References
Index
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
Back Cover

Citation preview

TRANSPLANTATION, BIOENGINEERING, AND REGENERATION OF THE ENDOCRINE PANCREAS

TRANSPLANTATION, BIOENGINEERING, AND REGENERATION OF THE ENDOCRINE PANCREAS VOLUME 2 Edited by

Giuseppe Orlando Lorenzo Piemonti Camillo Ricordi Robert J. Stratta Rainer W.G. Gruessner

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-12-814831-0 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

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Contributors

Tomohiko Adachi  Department of Surgery, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan

Melena D. Bellin  Department of Pediatrics, University of Minnesota Masonic Children’s Hospital; Department of Surgery, University of Minnesota Medical Center, Minneapolis, MN, United States

Toshiyuki Adachi  Department of Surgery, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan

Dora M. Berman  Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, United States; Department of Systems Medicine, University of Rome “Tor Vergata”, Rome, Italy

Rodolfo Alejandro  Diabetes Research Institute; Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, United States

Louise Berry  Department of Surgery, University of Minnesota Medical Center, Minneapolis, MN, United States

Ana M. Alvarez  Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, United States

Daniel Bojar  Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland

Domenico Alvaro  Department of Precision and Translational Medicine, Sapienza University of Rome, Rome, Italy

Rita Bottino  Institute of Cellular Therapeutics; AlleghenySinger Research Institute; Allegheny Health Network; Carnegie Mellon University, Pittsburgh, PA, USA

Spencer R. Andrei  Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, United States Matthew Armfield  Department of Pediatrics, University of Minnesota Masonic Children’s Hospital; Pediatric Pain & Advanced/Complex Care Team (PACCT), Minneapolis, MN, United States

Nathaniel W. Brigle  Virginia Commonwealth University, Department of Surgery, Transplant Division, Richmond, VA, United States

Amish Asthana  Wake Forest University School of Medicine, Winston-Salem, NC, United States

Henry Buchwald  Department of Surgery; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, United States

Stephen F. Badylak  McGowan Institute for Regenerative Medicine; Department of Surgery; Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Jane N. Buchwald  Division of Scientific Research Writing, Medwrite Medical Communications, Maiden Rock, WI, United States Vincenzo Cardinale  Department of Medico Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy

David A. Baidal  Diabetes Research Institute; Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, United States

Guido Carpino  Department of Movement, Human and Health Sciences, Division of Health Sciences, University of Rome “Foro Italico”, Rome, Italy

Appakalai N. Balamurugan  Clinical Islet Cell Laboratory, Center for Cellular Transplantation, Cardiovascular Innovation Institute, Department of Surgery, University of Louisville, Louisville, KY, United States

Renee Cercone  Department of Surgery, Northwell Health, New Hyde Park, NY, United States Deborah Chaimov  Wake Forest University School of Medicine, Winston-Salem, NC, United States

Gianpaolo Balzano  Pancreas Translational & Clinical Research Center, IRCCS San Raffaele Scientific Institute, Milano, Italy

Christopher G. Chapman  Center for Endoscopic Research and Therapeutics, Department of Medicine, University of Chicago, Chicago, IL, United States

Ipsita Banerjee  Chemical and Petroleum Engineering; Bioengineering, University of Pittsburgh; McGowan Institute for Regenerative Medicine, Pittsburgh, PA, United States

Amanda Child  University of Maryland School of Medicine, Baltimore, MD, United States

Kaylene Barrera  Department of Surgery, State University of New York, (SUNY) Downstate Medical Center, Brooklyn, NY, United States

Srinath Chinnakotla  Department of Pediatrics, University of Minnesota Masonic Children’s Hospital; Department of Surgery, University of Minnesota Medical Center, Minneapolis, MN, United States

Will Bataller  Virginia Commonwealth University, Department of Surgery, Transplant Division, Richmond, VA, United States

Gaetano Ciancio  Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, United States

Greg J. Beilman  Department of Surgery, University of Minnesota Medical Center, Minneapolis, MN, United States

xi

xii Contributors Kristin P. Colling  University of Minnesota School of Medicine, St Marys Medical Center, Duluth, MN, United States

Rainer W.G. Gruessner  Department of Surgery, State University of New York, (SUNY) Downstate Medical Center, Brooklyn, NY, United States

Marie Cook  Department of Surgery, University of Minnesota Medical Center, Minneapolis, MN, United States

Carolin Hermanns  MERLN Institute for TechnologyInspired Regenerative Medicine, Complex Tissue Regeneration, Maastricht University, Maastricht, The Netherlands

David K.C. Cooper  Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA Alexandria Coughlan  Department of Surgery, University of Minnesota Medical Center, Minneapolis, MN, United States Rick de Vries  MERLN Institute for Technology-Inspired Regenerative Medicine, Complex Tissue Regeneration, Maastricht University, Maastricht, The Netherlands Juan Domínguez-Bendala  Diabetes Research Institute; Dept. of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL, United States Ty B. Dunn  University of Pennsylvania, Philadephia, PA, USA Susumu Eguchi  Department of Surgery, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan Massimo Falconi  Vita-Salute San Raffaele University; Pancreas Translational & Clinical Research Center, IRCCS San Raffaele Scientific Institute, Milano, Italy Austin K. Feeney  Division of Transplantation, Department of Surgery, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, United States Magali J. Fontaine  University of Maryland School of Medicine, Baltimore, MD, United States; Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, United States Benjamin S. Freedman  Kidney Research Institute; Institute for Stem Cell and Regenerative Medicine, University of Washington; Division of Nephrology, Department of Medicine, University of Washington School of Medicine; Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA Virginia Fuenmayor  Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, United States Martin Fussenegger  Department of Biosystems Science and Engineering, ETH Zurich; University of Basel, Basel, Switzerland Maureen Gannon  Department of Medicine, Vanderbilt University Medical Center; Department of Veterans Affairs, Tennessee Valley Health Authority; Department of Molecular Physiology and Biophysics; Program in Developmental Biology; Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, United States Eugenio Gauido  Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza University of Rome, Rome, Italy Michael A. Goedde  Clinical Islet Cell Laboratory, Center for Cellular Transplantation, Cardiovascular Innovation Institute, Department of Surgery, University of Louisville, Louisville, KY, United States

Masaaki Hidaka  Department of Surgery, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan Masataka Hirabaru  Department of Surgery, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan Michael G. Hughes  Clinical Islet Cell Laboratory, Center for Cellular Transplantation, Cardiovascular Innovation Institute, Department of Surgery, University of Louisville, Louisville, KY, United States Abid Hussain  Clinical Islet Cell Laboratory, Center for Cellular Transplantation, Cardiovascular Innovation Institute, Department of Surgery, University of Louisville, Louisville, KY, United States Hajime Imamura  Department of Surgery, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan Marco Infante  Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, United States; Department of Systems Medicine, University of Rome “Tor Vergata”, Rome, Italy G. Janani  Biomaterial and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering, IIT Guwahati, Guwahati, India Marlon Jetten  MERLN Institute for Technology-Inspired Regenerative Medicine, Complex Tissue Regeneration, Maastricht University, Maastricht, The Netherlands Christopher M. Jones  Clinical Islet Cell Laboratory, Center for Cellular Transplantation, Cardiovascular Innovation Institute, Department of Surgery, University of Louisville, Louisville, KY, United States Jagan Kalivarathan  Virginia Commonwealth University, Department of Surgery, Transplant Division, Richmond, VA, United States Mazhar A. Kanak  Virginia Commonwealth University, Department of Surgery, Transplant Division, Richmond, VA, United States David L. Kaplan  Department of Biomedical Engineering, Tufts University, Medford, MA, United States Tatsuya Kin  Clinical Islet Laboratory and Clinical Islet Transplant Program, University of Alberta, Edmonton, AB, Canada Manishekhar Kumar  Biomaterial and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering, IIT Guwahati, Guwahati, India Jonathan R.T. Lakey  Department of Surgery; Department of Biomedical Engineering, University of California Irvine, Irvine, CA, United States

Contributors xiii

Giacomo Lanzoni  Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, United States Emily J. Larkin  University of Maryland School of Medicine, Baltimore, MD, United States Esther Latres  JDRF International, New York, NY, United States Yoojin C. Lee  McGowan Institute for Regenerative Medicine; Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States Elina Linetsky  cGMP Cell Processing Facility, Cell Transplant Center, Diabetes Research Institute; Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, United States Gopalakrishnan Loganathan  Clinical Islet Cell Laboratory, Center for Cellular Transplantation, Cardiovascular Innovation Institute, Department of Surgery, University of Louisville, Louisville, KY, United States Elisa Maillard  Strasbourg University, DIATHEC 7294, Strasbourg, France Biman B. Mandal  Biomaterial and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering; Centre for Nanotechnology, IIT Guwahati, Guwahati, India Adela Helvia Martinez  MERLN Institute for TechnologyInspired Regenerative Medicine, Complex Tissue Regeneration, Maastricht University, Maastricht, The Netherlands Hajime Matsushima  Department of Surgery, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan Jeffrey B. Matthews  Department of Surgery, The University of Chicago, Chicago, IL, United States Kendall McEachron  Department of Surgery, University of Minnesota Medical Center, Minneapolis, MN, United States Raffaella Melzi  Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milano, Italy Alessia Mercalli  Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milano, Italy Giovanni Migliaccio  European Advanced Translational Research Infrastructure in Medicine—EATRIS, Amsterdam, The Netherlands; Center for Biological and Pharmacological Evaluation CVBF, Bari, Italy Samantha A. Mitchell  Division of Transplantation, Department of Surgery, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, United States Sami Mohammed  MERLN Institute for TechnologyInspired Regenerative Medicine, Complex Tissue Regeneration, Maastricht University, Maastricht, The Netherlands

Siddharth Narayanan  Clinical Islet Cell Laboratory, Center for Cellular Transplantation, Cardiovascular Innovation Institute, Department of Surgery, University of Louisville, Louisville, KY, United States Koji Natsuda  Department of Surgery, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan Jon S. Odorico  Division of Transplantation, Department of Surgery, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, United States Shinichiro Ono  Department of Surgery, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan Giuseppe Orlando  Wake Forest University School of Medicine, Winston-Salem, NC, United States Nathalia Padilla  Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, United States Silvia Pellegrini  Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy Laura Perin  GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics, Children’s Hospital Los Angeles, Division of Urology, The Saban Research Institute, University of Southern California, Los Angeles, CA, United States Lorenzo Piemonti  Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milano, Italy; Vita-Salute San Raffaele University, Milano, Italy Antonello Pileggi  Division of Physiology and Pathological Sciences, Center for Scientific Review, National Institutes of Health, Bethesda, MD, United States Catalina Pineda Molina  McGowan Institute for Regenerative Medicine; Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, United States Elizabeth C. Poli  Department of Surgery, The University of Chicago, Chicago, IL, United States Mirza Muhammad Fahd Qadir  Diabetes Research Institute; Dept. of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL, United States Camillo Ricordi  cGMP Facility, Cell Transplant Center, Division of Cellular Transplant, Diabetes Research Institute; Department of Surgery; Department of Biomedical Engineering, University of Miami Miller School of Medicine, Miami, FL, United States L. Rodriguez Rilo  Department of Surgery, Zucker School of Medicine at Hofstra/Northwell, Hempstead; Department of Surgery, Northwell Health, New Hyde Park, NY, United States

Sean Muir  Wake Forest University College of Arts and Science, Winston-Salem, NC, United States

R. Paul Robertson  Divisions of Endocrinology and Metabolism, Departments of Medicine, University of Washington, Seattle, WA, United States; University of Minnesota, Minneapolis, MN, United States

Rita Nano  Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milano, Italy

William Rust  Seraxis, Inc., Germantown, MD, United States

xiv Contributors Sara Dutton Sackett  Division of Transplantation, Department of Surgery, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, United States

Daniel M. Tremmel  Division of Transplantation, Department of Surgery, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, United States

Pratik Saxena  Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland

Bernard E. Tuch  Australian Foundation for Diabetes Research, Maroubra; School of Biomedical Science, Discipline Physiology, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia

Alexander Schwartzman  Department of Surgery, State University of New York, (SUNY) Downstate Medical Center, Brooklyn, NY, United States Sarah J. Schwarzenberg  Department of Pediatrics, University of Minnesota Masonic Children’s Hospital, Minneapolis, MN, United States Michael V. Sefton  Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada Sidharth Sharma  Department of Surgery, State University of New York, (SUNY) Downstate Medical Center, Brooklyn, NY, United States Benjamin Smood  University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA Davide Socci  Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milano, Italy Valeria Sordi  Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy Adam Stell  MERLN Institute for Technology-Inspired Regenerative Medicine, Complex Tissue Regeneration, Maastricht University, Maastricht, The Netherlands Aaron A. Stock  Diabetes Research Institute, University of Miami Miller School of Medicine; Department of Biomedical Engineering, University of Miami, Miami, FL, United States Mitsuhisa Takatsuki  Department of Surgery, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan Alice A. Tomei  Diabetes Research Institute, University of Miami Miller School of Medicine; Department of Biomedical Engineering, University of Miami; Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, United States

Vijayaganapathy Vaithilingam  Australian Foundation for Diabetes Research, Maroubra, NSW, Australia; MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht, The Netherlands Aart van Apeldoorn  MERLN Institute for TechnologyInspired Regenerative Medicine, Complex Tissue Regeneration, Maastricht University, Maastricht, The Netherlands Valentina Villani  GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics, Children’s Hospital Los Angeles, Division of Urology, The Saban Research Institute, University of Southern California, Los Angeles, CA, United States Alexander E. Vlahos  Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada Stuart K. Williams  Department of Physiology, University of Louisville, Louisville, KY, United States Xiangwei Xiao  Division of Pediatric Surgery, Department of Surgery, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States Kunal Yadav  Virginia Commonwealth University, Department of Surgery, Transplant Division, Richmond, VA, United States Manpei Yamashita  Department of Surgery, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan Henryk Zulewski  Department of Biosystems Science and Engineering, ETH Zurich, Basel; Division of Endocrinology and Diabetes, Stadtspital Triemli, Zurich; University of Basel, Basel, Switzerland

C H A P T E R

1 Etiopathogenesis and pathophysiology of chronic pancreatitis Jagan Kalivarathan, Kunal Yadav, Will Bataller, Nathaniel W. Brigle, Mazhar A. Kanak Virginia Commonwealth University, Department of Surgery, Transplant Division, Richmond, VA, United States O U T L I N E Etiopathogenesis

7

Gallbladder dysfunction

17

Alcohol-induced chronic pancreatitis

7

Smoking and pancreatitis

18

Autoimmune pancreatitis

9

Pathophysiology

18

Exocrine insufficiency

19

Endocrine insufficiency and diabetes mellitus

19

Pathophysiology of pain

20

Calcification

21

Fatty infiltration

22

Fibrosis

22

Pseudocysts

22

Atrophy

23

Pancreatic cancer

23

Molecular mechanisms in the development of pancreatitis

24

Conclusion

24

References

25

Genetic pancreatitis Enzymatic- and ion-related mutations in PRSS1, SPINK1, CTRC, and CaSR CFTR mutation Metabolic disorders Hyperparathyroidism

10

Hereditary pancreatitis

12

Recurrent acute pancreatitis

13

Ductal obstruction

13

Pancreas divisum

14

Sphincter of Oddi dysfunction

14

Pancreatic trauma

15

Tropical chronic pancreatitis

16

Drug toxicity-induced pancreatitis

16

Idiopathic

17

Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 2 https://doi.org/10.1016/B978-0-12-814831-0.00001-4

10 11 11 12

5

© 2020 Elsevier Inc. All rights reserved.

6

1.  Etiopathogenesis and pathophysiology of chronic pancreatitis

Chronic pancreatitis is commonly defined as a continuous, chronic, fibro-inflammatory process of the pancreas, characterized by irreversible morphologic changes.1 The pancreas is structurally and functionally divided into two compartments: an exocrine and an endocrine compartment. The exocrine tissue is composed of acini, which are involved in the production and secretion of digestive enzymes and bicarbonate (HCO 3 −). The endocrine compartment contains the islet cells (islets of Langerhans) that synthesize and release hormones including insulin, glucagon, somatostatin, and pancreatic polypeptide into the bloodstream to maintain glucose homeostasis in the body. The exocrine components comprise >95% and the islets makeup 1%–2% of pancreatic mass. Pancreatitis can be either acute or chronic: the two conditions are characterized by significantly different clinical, morphological, and histological changes. Acute pancreatitis (AP) has an abrupt onset and occurs predominantly in response to cell injury which can cause mild or severe complications. Chronic pancreatitis is an inflammatory disease primarily affecting the exocrine compartment, but in later stages, it can affect the endocrine function as well. It is characterized by progressive inflammation and fibrosis of pancreas leading to irreversible damage.2 Fibrosis during chronic pancreatitis ultimately leads to progressive loss of the lobular morphology and structure of the pancreas, deformation of the large ducts and severe changes in the disposition and composition of the islets.3 The major difference between acute and chronic pancreatitis is that the former leads to clinical, histological, and functional resolution of the disease, whereas the latter is a progressive inflammatory process leading to permanent histological irregularity and functional impairment.4 Despite the heterogeneity in the pathogenesis of chronic pancreatitis and involved risk factors, processes

such as necrosis/apoptosis, inflammation, and duct obstruction are invariably present. This leads to irreversible morphological and structural changes resulting in impairment of exocrine and endocrine functions. The prevalence of the disease depends to a large extent on culture and geography. The incidence, prevalence, morbidity, and mortality of chronic pancreatitis vary between countries due to differences in study design, diagnostic criteria, and geography. In the United States, the annual incidence of chronic pancreatitis is estimated to be approximately 5–12/100,000 persons and the prevalence is about 50/100,000 persons.5 Studies have reported higher incidence rates of chronic pancreatitis in Europe compared to the United States. A recent survey conducted in Asia reveals much greater incidence rates of chronic pancreatitis than the United States.6 The etiological risk-factors associated with chronic pancreatitis are multiple and involve both genetic and environmental factors.7 Numerous etiological factors have been identified: alcohol, smoking, genetic/hereditary, efferent duct/obstructive, systemic disease, autoimmune, and idiopathic. Classification of chronic pancreatitis was initially based on the severity of the morphological change of the main pancreatic duct as visualized in ERCP, ultrasound, and Computed tomography (CT) images. Later the disease was categorized into the M-ANNHEIM classification which incorporated the etiology, clinical stage, and severity of the disease.8 More recently another type of characterization evolved based on the etiologic risk factors associated with chronic pancreatitis categorized as Toxic-metabolic factors, Idiopathic, Genetic, Autoimmune, Recurrent acute pancreatitis, and Obstructive chronic pancreatitis (TIGAR-O) classification4 (Fig. 1).

Idiopathic (e.g., Early onset) Obstructive (e.g., Pancreas divisum)

Toxic-metabolic (e.g., Alcoholism) Chronic Pancreatitis Irreversible morphological changes Inflammation and fibrosis

Autoimmune (e.g., Sjogren syndrome)

Genetic/hereditary (e.g., CFTR)

Recurrent acute pancreatitis

FIG. 1  Risk factors of chronic pancreatitis.

A. Islet auto-transplantation



Alcohol-induced chronic pancreatitis

Although diverse risk factors can cause chronic pancreatitis, alcohol abuse is the major cause in the western population constituting up to 90% of the cases.9 Other than alcohol abuse, other toxic insults such as nicotine use and certain drugs can be associated with chronic pancreatitis. Moreover, certain genetic mutation/hereditary conditions predispose individuals to acute/ chronic pancreatic inflammation which may be exacerbated by environmental factors. Acinar cell damage by gain/loss of function by mutation of key proteins involved in activation and inhibition of digestive enzymes may lead to chronic pancreatitis. Genetic/hereditary pancreatitis and cystic fibrosis leading to chronic pancreatitis have an early onset and are characterized by prominent pancreatic calcification. In some cases, obstruction of the duct by stones causes an increase in pancreas ductal pressure and eventual autodigestion of the acinar cells which can progress to develop into chronic pancreatitis. Duct obstruction or blockage by protein plugs or gall stones have been associated with the disease. Chronic obstructive pancreatitis can result from congenital ductal anomalies such as pancreas divisum. The pathogenesis of chronic pancreatitis due to pancreas divisum and sphincter of Oddi disorders remains somewhat uncertain. Clinically, recurrent episodes of AP injury may result in chronic pancreatitis, however, the transition from acute to chronic pancreatitis needs further understanding.10 Autoimmune pancreatitis develops as a result of autoimmunity, and may be either confined to the pancreas alone or may have other organ system involvement as well. Tropical pancreatitis is another type of chronic pancreatitis seen mainly in tropical countries. It occurs usually in young people, involves the main pancreatic duct and causes ductal calculi.11 Additionally, distinctive histologic highlights are represented in several forms of chronic pancreatitis like extensive pancreatic calcification in tropical pancreatitis and a prominent lymphocytic and plasma cell infiltrate in autoimmune pancreatitis.12 Regardless of the etiology, the main histopathological features of chronic pancreatitis are pancreatic fibrosis, acinar atrophy, chronic inflammation, and deformed and obstructed ducts.13 In this chapter, we will discuss the various etiologies, and pathophysiological mechanism surrounding chronic pancreatitis.

Etiopathogenesis Pancreatitis is caused by an initial insult (alcohol consumption, autodigestion, autoimmune reaction) that causes an acute inflammatory event. Pancreatic inflammation causes intense abdominal pain, and, over time, chronic inflammation can cause irreversible morphological changes and fibrosis.14 Pancreatic exocrine and

7

e­ ndocrine function declines because of the replacement of acinar and islet cells with nonfunctional fibrous tissue (Fig. 2). Fortunately, the pancreas has a good functional reserve, and nearly 90% of function can be lost before any clinical manifestation.15

Alcohol-induced chronic pancreatitis The mechanisms by which alcoholism causes pancreatitis are still not well-defined, but it is clear that overconsumption of alcoholic products predisposes an individual vastly to this morbid pathology.16 Although the risk increases with consumption, interestingly, only about 5%–15% of heavy alcohol consumers develop pancreatitis.7, 17 This lends credence to the fact that these mechanisms have yet to be understood: perhaps alcoholism exacerbates the contribution of several other factors rather than being a sole cause of pancreatitis.18 If alcohol consumption is continued at the same level after the first attack of pancreatitis, the chances of subsequent attacks are higher but can be reduced by reduction or complete abstinence of alcohol.19 The small percentage of alcoholics developing chronic pancreatitis indicates that other factors such as smoking, diet or genetic predisposition may increase the susceptibility of disease progression. Certain mutations, such as SPINK1 and cystic fibrosis transmembrane conductance regulator (CFTR) were observed in patients diagnosed with alcoholic chronic pancreatitis.20 The role of alcohol in predisposing to pancreatitis was controversial until the discovery of the CLDN2 gene mutation. Mutations in CLDN2 locus predicts which drinker was at a higher risk of developing chronic pancreatitis, predominantly in the male gender.21 CLDN2 mutation and association with alcohol increase the susceptibility of chronic pancreatitis through a nontrypsin-dependent process by facilitating endotoxinaemia.22 Additionally, PRSS1 mutation may be involved in altering the expression of the trypsinogen gene thereby increasing the susceptibility of alcoholic patients to recurrent AP or chronic pancreatitis.21 Thus, genetic mutations in tandem with alcoholism may be a cause of alcohol-induced chronic pancreatitis. The parenchymal architecture of the pancreas is completely lost, with major changes in the duct ranging from obstruction to dilatation and/or distortion.23 Calcification of various sizes begins to manifest in the ducts and pseudocysts are present in 25%–50% of cases filled with necrotic debris and exocrine enzymes.24 Histological examination revealed accumulation of neutrophils and histiocytes in the necrotic area including sparse distribution of lymphocytes and mast cells in the perineural and perivascular structure.25 Symptoms for alcoholic chronic pancreatitis begins to manifest

A. Islet auto-transplantation

8

1.  Etiopathogenesis and pathophysiology of chronic pancreatitis

Acinar cells

Tail

Duct

Pancreas

ct

tic

du

ea

r nc

(A)

Red blood cell

y

Bod

δ cell Pancreatic acini

Pa Head Duo den um

α cell β cell Islet of Langerhans Macrophage

PSCs (quiescent)

Hypoxia

PSCs (activated)

Insults

Fibrosis

Insults .. ..

.. ..

Recurrent acute pancreatitis

Mononuclear cell infiltrate Oxidative stress

Polymorphonuclear cell Healthy pancreatic lobule

Chronic pancreatitis

Sentinel acute pancreatitis event

Fibrosis Calcifications/stones Dilated pancreatic duct Abnormal duct Duodenal obstruction

(B)

FIG. 2  Pathophysiology of chronic pancreatitis. (A) Anatomy of the pancreas. Anatomically, the pancreas is divided into the head, neck, and body. The exocrine pancreatic function includes the acinar cells, which secrete digestive enzymes that are transported through the pancreatic duct into the small intestine. The endocrine function regulated by the islets of Langerhans cells involves the secretion of pancreatic hormones glucagon, insulin, and somatostatin by alpha, beta, and delta cells respectively. (B) Toxic insults to the acinar cells initiate the first episodes of acute pancreatitis which is characterized by the recruitment of inflammatory cells. Continuous insults lead to recurrent episodes of acute pancreatitis, which activates pancreatic stellate cells and initiate pancreatic fibrosis. PSC—Pancreatic stellate cells.

in the fourth decade of life.26 These symptoms can include endocrine and exocrine dysfunction, pain, and sphincter-related pathologies. The pain begins to reduce in frequency and intensity over time, but gland destruction is underlying which leads to exocrine and endocrine insufficiency. The pancreas becomes severely fibrotic and atrophic in the later stages of the disease. Alcoholic chronic pancreatitis patients have a higher risk of developing pancreatic ductal adenocarcinoma.27 Several mechanisms have been postulated with regards to the pathogenesis of alcoholic pancreatitis. Protein precipitation resulting from alcohol consumption may lead to ductal blockage causing acinar atrophy and fibrosis.28–31 The metabolism of alcohol via the oxidative pathway involving alcohol dehydrogenases and the nonoxidative pathway involving Fatty acid ethyl ester synthases within the acinar cells may result in autodigestive injury leading to pancreatitis (Fig. 3).32–34 Direct damage to acinar cells by alcohol exposure may be due to Endoplasmic reticulum (ER) stress or unfolded protein response and autophagy.35, 36 The effects of ethanol on the pancreas and its related digestive organs also play a role in injury and ­dysfunction.

Ethanol has a pro-secretory effect on gastric acid at lower concentrations and inhibitory effect on secretion at higher concentrations.37 In addition, it has a toxic effect on duodenal epithelium38 and colonic mucosa,39 while it has an inhibitory effect on pancreatic protein secretion.40 These various effects on the pancreas and digestive tract are all contributing factors to pancreatic damage that leads to permanent dysfunction. Although the exact mechanisms of these pathways to cause damage have yet to be elucidated, hypotheses that range from hypoxia 41 due to decreased blood flow and pancreatic fibrosis due to stellate cell activation 42 all provide a greater perspective on how these pathways are integrated. Preclinical models of pancreatitis have reported a shift toward necrotic from apoptotic cell death pathway upon ethanol uptake. This shift has been facilitated by activation of inositol trisphosphate receptors in the ER causing mitochondrial depolarization and less ATP production required for apoptosis.43 Ethanol consumption also inhibits the JAK2/STAT1 pathway resulting in a lack of production of caspases required for apoptosis. Finally, an increase in expression of cathepsin B

A. Islet auto-transplantation



9

Autoimmune pancreatitis

Cytokines

Enzyme activation

Necrosis

Acinar cell Zymogen granules

Stellate cell activation

Ox id stre ant ss

Lysosomes

Mitochondrial depolarisation

Ca2+

Oxidant stress Stellate cell

mRNA CE and FAEE RER

Ac

Ethanol

FIG. 3  Effects of ethanol on acinar and pancreatic stellate cells. Ethanol and its toxic metabolites destabilize lysosomes and zymogen granules [mediated by cholesteryl esters (CE), fatty acid ethyl esters (FAEE)] and trigger increased activation of pancreatic enzymes. Persistent increase in calcium concentration leads to mitochondrial depolarization. Pancreatic stellate cells are activated by ethanol via its metabolite acetaldehyde (Ac) and the generation of oxidant stress. Persistent activation of pancreatic stellate cells leads to the development of pancreatic fibrosis. [The effects of ethanol on acinar cells are shown in red arrows and on stellate cells shown on green arrows]. RER—Rough endoplasmic reticulum.

by ethanol converts trypsinogen to trypsin leading to cell death by autodigestion.44 Mechanistic pathways such as oxidative stress,45 impaired autophagy,36 mitochondrial dysfunction,46 ER stress/unfolded protein response,35 promotion of pro-inflammatory cytokines,47 and increased necrosis44 have all been reported in the pathogenesis of alcohol-induced chronic pancreatitis.16

Autoimmune pancreatitis Autoimmune pancreatitis has been diagnosed in many countries suggesting that it is a worldwide entity.48 It can be identified as a primary pancreatic disorder or secondary to other autoimmune diseases such as IgG4 cholangitis, inflammatory bowel disease, salivary gland disorder, retroperitoneal fibrosis, and mediastinal

f­ ibrosis.49 It was first reported as pancreatitis associated with increased immunoglobulin levels which demonstrated a therapeutic response to steroids.50 It was previously described as a variant of the primary sclerosing cholangitis but presents with IgG4 positive plasma cells identified in multiple organ systems. Thus, this systemic form of autoimmune pancreatitis was called lymphoplasmacytic sclerosing pancreatitis (LPSP) or type 1 autoimmune pancreatitis due to elevated IgG4 levels and presence of LPSP histopathology.51 Another group of patients presenting with similar features as LPSP but distinguished from type 1 autoimmune pancreatitis by the presence of duct-centric granulocytic infiltrate along with duct destruction.52 This type of inflammatory condition was described as idiopathic duct-centric pancreatitis (IDCP) which was mostly seen in young patients.53 Later this type

A. Islet auto-transplantation

10

1.  Etiopathogenesis and pathophysiology of chronic pancreatitis

of disease phenotype was labeled as Type 2 autoimmune pancreatitis, which was characterized by early onset, absence of extrapancreatic involvement, and no association with inflammatory bowel disease or IgG4 elevation.54 Type 1 autoimmune pancreatitis patients often exhibit jaundice, with lack of pain and typically seen in older adults, the male being more common. Pancreas imaging of type 1 autoimmune pancreatitis also shows enlarged pancreatic mass, ductal strictures and infrequently shows AP.54, 55 The diagnosis of autoimmune pancreatitis (type 1 and type 2) has to be initiated after ruling out the possibility of pancreatic cancer which is more common than autoimmune pancreatitis. Steroid treatment to diagnose autoimmune pancreatitis is usually initiated after workup for pancreatic cancer is negative.56–58 Histological examinations for LPSP often showed lymphoplasmacytic infiltration with storiform fibrosis and obliterative phlebitis. Another characteristic histological feature is the positive immunostaining for IgG4 within the infiltrated plasma cells. Autoimmune pancreatitis patient pancreas does not present with fat necrosis, pseudocyst formation, or calcifications, however, lymphoid follicles may be present at the periphery of pancreatic ducts.51, 59 Type 2 autoimmune pancreatitis or IDCP is seen in ­relatively younger patients with a mean age of 40–50 years and no preference in gender. Similar to type 1 AIP, painless jaundice and pancreatic ductal strictures are present. Unlike LPSP, there is no involvement of other organ systems, it is confined only to the pancreas but it is commonly associated concurrently with ulcerative colitis. The presence of inflammatory bowel disease is a supportive finding to diagnose IDCP.60, 61 Diagnosis of IDCP is often challenging even with histopathology, moreover, IgG4 levels are not elevated in 75% of patients with IDCP.62 The histopathological features of IDCP are lymphoplasmacytic infiltration in the periductal regions.53, 59 An important hallmark of IDCP is the infiltration of neutrophils that forms a granulocytic ep­ithelial lesion “GEL.” Additionally, IDCP has reduced the intensity of IgG4+ plasma cell infiltration, unlike LPSP.63 A major finding of autoimmune pancreatitis is a narrow, strictured, or stenosed pancreatic duct.64 In addition, IgG4 sclerosing cholangitis, a common comorbidity with autoimmune pancreatitis, can cause strictures in the biliary tree.64 Pancreatic stone formation can be seen in conditions that involve pancreatic head swelling and narrowing of pancreatic ducts, which are more prevalent in patients with autoimmune pancreatitis.65, 66 The proposed mechanism of action is pancreatic juice stasis in the main pancreatic duct that is found with increased ductal pressure, followed by calcification.67 The difference in the characteristic features of two types of autoimmune pancreatitis has been listed (Table 1).

TABLE 1  Characteristic features of Type 1 and Type 2 autoimmune pancreatitis Characteristic features

LPSP (Type 1 AIP) IDCP (Type 2 AIP)

Age range (years)

50–70

30–60

Gender M:F

3:1

1:1

Percentage of patients with elevated serum IgG

60%

25%

Involvement of other organs

Yes

No

Lymphoplasmacytic infiltration

Yes

Yes

Periductal inflammation

Yes

Yes

Granulocyte epithelial lesion

No

Yes

Inflammatory cell infiltrated

Lymphocytes and Neutrophils, plasma cells lymphocytes, and plasma cells

IgG4 infiltration

Abundant

Low

Steroid treatment relapse

50%

90%.217, 218 In alcoholic chronic pancreatitis, this usually takes 10–20 years. Fat malabsorption leading to steatorrhea usually occurs earlier and is more severe than malabsorption of other nutrients. This is due to an earlier decrease in lipase secretion compared with amylase and proteases,219 higher susceptibility of lipase to acidic pH caused by concomitant impairment of bicarbonate secretion, higher susceptibility of lipase to proteolytic destruction during small intestinal transit, denaturation of bile acids from increased acid and marked inhibition of bile acid secretion.220 Even though the gastric lipase is usually elevated in chronic pancreatitis patients, it is not sufficient to compensate for pancreatic lipase deficiency.221 In contrast, >80% of carbohydrates can still be digested and absorbed in the absence of pancreatic amylase activity 222 and the colonic flora can further metabolize malabsorbed carbohydrates. Pancreatic exocrine function is usually preserved longer and consequently, exocrine insufficiency is milder in “early onset” idiopathic chronic pancreatitis in comparison to alcoholic and “late onset” idiopathic chronic pancreatitis.223

19

Regardless of etiology and type of onset, patients with chronic pancreatitis have a 50%–80% reduction of pancreatic exocrine function compared with healthy controls and 80%–90% show some degree of pancreatic exocrine insufficiency.220 In about 65%–75% of patients, morphologic alterations and functional impairment develop in parallel. Pancreatic exocrine insufficiency without morphologic alterations is rare (16 h of cold storage. In addition to UW, alternative cold storage preservation solutions that include Kyoto solution, Celsior, and histidine-tryptophan-ketoglutarate (HTK) are utilized by many transplant centers. However, UW remains the most commonly used preservation solution for the pancreas, with stellar clinical outcome data.25,34,37,38 In the operating room (OR), once excised, the pancreas is deposited into a sterile 1 L Nalgene jar, prefilled with 500–750 mL of sterile UW. The jar is tightly closed to maintain sterility, wrapped in a securely closed sterile plastic bag commonly utilized in hospital OR. The Nalgene jar is then deposited into a standard cooler commonly utilized for transport of organs designated for transplant, packed with plenty of ice according to the HRSA Organ Procurement and Transplantation Network (OPTN) Policy 16, “Organ and Extra Vessels Packaging, Labeling, Shipping, and Storage.”

Laboratory preparation and setup Given the data discussed earlier and the fact that resected pancreata have added challenges due to the morphological consequences of CP, it is imperative that the resected pancreas is transported to the processing laboratory as soon as possible following surgery, for immediate processing. Hence, it is critical that the processing laboratory is prepared for an impending isolation in advance. Because a pancreas resection surgery is normally scheduled ahead of time, the time of approximate arrival of the pancreas in the laboratory can be easily estimated. Despite the fact that the isolation, purification, and culture of islet cells can be performed in a conventional laboratory outfitted with the necessary equipment, the following discussion should be considered prior to setting up a laboratory space suitable to accommodate the islet isolation process. Publication of the promising results of the Edmonton protocol10 sparked a renewed interest in the field of allogeneic islet transplantation. Within several years following this publication, a number of centers were reporting significantly improved clinical outcomes at 1 and 3 years following transplant. Encouraged by such positive developments Food and Drug Administration (FDA) took another look at clinical uses of allogeneic islet cells, considering it as a

A. Islet auto-transplantation

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4.  Islet isolation for autotransplantation

­ otentially marketable therapy for the treatment of pap tients with severe forms of type 1 diabetes (T1D). Due to the fact that at the present time, in the United States, allogeneic islet cells are regulated as a biologic and a drug and islet cell transplantation is considered an experimental procedure, all relevant clinical studies must be performed under an Investigational New Drug (IND) application. This means that the process utilized to manufacture allogeneic islet cells must meet applicable federal regulations.39 The FDA recommends that islet cell processing should be done in a good manufacturing laboratory (GMP) that meets all applicable FDA regulations.39 Although these recommendations have been made for allogeneic islet products transplanted under the auspices of cGMP (21 CFR Part  210 and 211), IND (21 CFR Part 312), and regulations for biologics (21 CFR Part 600, 601, and 610), it is good practice to follow these recommendations for autologous islet cell processing as well. Isolation, culture, and preparation for transplant of human islet cells in an environmentally controlled and monitored positive pressure laboratory can facilitate a significant reduction in the risk for cross contaminating an islet product. A controlled laboratory environment, designed, tested, and certified to meet ISO-Class 7 standards (previously Class 10,000), can also prevent notable changes in ambient temperature which might affect the isolation and purification process. The islet isolation and purification procedure can be performed in a laboratory outfitted, at minimum, with the following equipment: at least two interconnected biological safety cabinets (BSCs) are necessary, although three BSCs are preferable to spatially separate pancreas cleaning and cannulation, organ distention and digestion/dilution, and tissue recombination steps in separate sterile environments. Additionally, several (ideally twofour) large capacity refrigerated centrifuges are preferable during the tissue recombination step, to collected large volumes of pancreatic tissue digest in the shortest possible time to minimize islet cell damage. At least one COBE 2991 Cell Processor (Terumo, Lakewood, CO) is necessary, but two are preferable, to complete islet purification step when the islet cells (endocrine fraction) are purified from the acinar (exocrine) tissue. Ideally, purification step should be performed in the cold as quickly as possible to assure that islet cells remain metabolically inactive during their exposure to potentially damaging purification solutions. Placing 2 COBE 2991 in a cold room or refrigerated space is of significant advantage. Other highly recommended noncritical equipment (i.e., equipment which does not come into direct contact with the islet product) includes a balance (analytical and top loading), a pH meter, at least two peristaltic pumps, several timers, several portable pipette aids, —three to four stir plates, temperature probes and a digital temperature reader (Biorep Technologies, Miami, FL) to monitor

the temperature throughout the procedure, and a water bath necessary for media preparation and warming up the circuit during the islet isolation process. Having a refrigerator that can accommodate all the reagents and media utilized during the islet isolation process is quite beneficial, although not necessary. If the space cannot accommodate a refrigerator, a portable cooler can be utilized. In addition to general laboratory equipment listed above, Biorep Technologies (Miami, FL) manufactures specialized equipment designed specifically to optimize the islet isolation process. Biorep Technologies has been manufacturing and distributing medical devices for islet cell isolation for the last 20 years, and is well known to islet cell transplant centers in the United States, and around the world. The company is the original manufacturer of islet isolation-specific equipment that includes Ricordi isolation chamber (Fig.  1) and chamber accessories (silicone marbles, thermo-probe, screen, O-ring, stand, silicone tubing, and temperature monitor), Ricordi islet isolator and accessories (Ricordi isolator tubing set, Ricordi chamber clamp, heating coil, sampling port), gradient mixer, automated islet cell counter, and corresponding counting dishes. Ricordi isolation chamber, chamber accessories, and perfusion sets can be obtained in a reusable form or individually packaged and sterilized, suitable for a single application, which is preferable when setting up a cGMP compliant facility. The new molded Ricordi chambers are now offered in two different sizes: 500 and 600 mL (Fig.  1). The chamber consists of the lower cylindrical portion and the top conical part, separated by a stainless steel mesh or a perforated stainless steel plate of a predetermined pore size. The reusable or autoclavable chamber material is Ultem polyetherimide (Fig.  1), a tough, rigid biocompatible plastic of superior thermostatic strength; it can withstand prolonged exposure to high temperatures and repeated cleaning and sterilization. Chambers made of this material have a characteristic translucent quality and are light amber in color. Each chamber comes with a defined size screen and an O-ring to facilitate sealing of the chamber once it is closed. While the 600 mL chamber uses a nondisposable clamp to hold the bottom and the top part together, the 500 mL uses three screws to seal the chamber. Stainless steel or plastic are both acceptable materials to use for the digestion chamber. However, nontoxic, durable, and biocompatible plastic is preferable as it affords the operator a direct observation of the pancreatic tissue in the chamber, during the digestion process. In addition, plastic chamber is much lighter compared to the stainless steel one, and is, therefore, much easier to handle. The lower portion of the isolation chamber has two single molded inlet openings connected to a size 16 silicone tubing (Biorep Technologies, Miami, FL). This tubing is

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FIG. 1  The molded Ricordi isolation chamber, made of Ultem polyetherimide, available in two different sizes: 500 and 600 mL. Available in disposable and autoclavable versions. The chamber has two entry ports in the base. The female luer connector for the thermoprobe is located in the side of the base of the chamber. Courtesy: Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW. Automated method for isolation of human pancreatic islets. Diabetes 1988;37(4):413–420.

connected to stainless steel coil heated by the Ricordi islet isolator, which controls the temperature and pumps the necessary solution into the chamber. The metal outlet on the top part of the chamber is connected to a larger size silicone tubing (size 17, Biorep Technologies, Miami, FL) used to alleviate increased pressure exerted on the islets during the high-speed collection phase of the islet isolation procedure. The use of a stainless steel screen placed between the upper and lower parts of the digestion chamber is essential as it maintains the pancreas in the lower half of the digestion chamber. IT allows for filtration and release of the digested tissue through the screen and upper part of the chamber. In other words, while the stainless steel screen allows for the continuous release and dilution of islet cells, larger nondigested pieces of tissue remain in the chamber, in close contact with the enzyme solution during the digestion step. We recommend using 530 μm screen for most of the donor organs. However, smaller pore size screen of 380 μm may be required for younger donors. In addition, seven to nine silicon nitride or stainless steel marbles are placed in the digestion chamber before it is closed. These are used to prevent the pancreas from moving in unison with the chamber and enhance the digestion process. In the past, standard glass marbles, most often found in toy stores, were used during the digestion. However, silicon nitride marbles have the same bounce as glass marbles, and have an added benefit of not chipping over time with glass chips finding their way into the islet product. The silicon nitride marbles are approximately 5/8″ (15.875 mm) in diameter and can be easily sterilized.

It is critical that digestion and, to some extent, dilution phases of the islet isolation procedure are closely monitored. Ricordi isolation chamber is equipped with a side port to accommodate the temperature monitoring transducer (temperature probe). Biorep Technologies offers two different options of temperature probes: (i) a Biorep Thermoprobe with a female Luer fitting and (ii) a BNC (Bayonet Neill– Concelman connector) thermocouple with a Thermowell connector. To monitor the temperature, the probe is connected to the Mon-a-Therm temperature monitor. Ricordi islet isolator, a perfusion apparatus and perfusion trays can be obtained from Biorep Technologies as well. The Ricordi isolator was developed to assist in the dissociation process by automating and standardizing pancreas digestion. The instrument accurately controls chamber agitation, temperature, and flow. Advanced software uses a variety of sensors to monitor and ensure a controlled and safe isolation process. The software has the ability to store a time-lapse record of every parameter used during the procedure. It is important to remember that the digestion step is dependent on the enzymatic digestion process as well as mechanical action that facilitate the breakdown of the donor pancreas into exocrine and endocrine components. Although the Ricordi isolator is a highly recommended piece of equipment, it is not required as the chamber can be agitated manually, by an experienced operator, and the digestion circuit can be heated by placing the heating coil in the water batch heated to 42–43°C. The perfusion apparatus, however, is useful for a controlled perfusion of the pancreas, once it is cannulated through the pancreatic duct. Perfusion tray is usually used in concert with

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the perfusion apparatus, with the organ connected to the tray using the network of perfusion tubing provided in the prepackaged, presterilized perfusion set (Biorep Technologies, Miami, FL). The islet isolation laboratory should be well stocked with all the necessary supplies and reagents utilized during the islet isolation procedure. Prepackaged and presterilized disposable and nondisposable supplies should be organized in packs and placed in the laboratory prior to the start of the isolation. The laboratory should be set up and prepared before starting the procedure. This applies not only to laboratory setup but media preparation as well. Islet isolation is a complex procedure that consists of several distinct steps; it requires highly trained and knowledgeable personnel for a proper, problem-free performance. Three-person team is necessary for the successful and timely execution of the procedure. As mentioned above, the laboratory should be set up in advance, with the all reusable equipment and supplies, disposables, and media arranged before the organ arrives in the laboratory. The laboratory should be set up and arranged so that the islet isolation process can begin without any delay, once the pancreas arrives in the laboratory. Once process begins, it should flow without interruptions from the arrival of the pancreas in the lab, its cleaning and decontamination, distention with the enzyme solution, digestion, and dilution phase, recombination of the resulting digest, purification of the islet cells from the acinar tissue, if necessary, and preparation of the final product for infusion, in order to achieve optimal results. The role of each member

of the isolation team should be considered so that team members’ responsibilities do not overlap. While two members of the team are setting up BSCs for pancreas decontamination and cleaning, distention, digestion, and tissue recombination, the third person should be preparing sufficient volume of media required throughout the isolation process. The team should also assure that all the necessary equipment is functional, calibrated, and is set at the required temperature, where required.

Islet isolation It was Paul Lacy, who was the first to demonstrate that rat islets could be successfully isolated and transplanted.40 Several years later, Mirkovitch and Campiche were the first to successfully transplant free islet grafts by injecting the dispersed graft into the spleen of pancreatomized dogs.41 David Sutherland, working at the UMN, was first to attempt IAT in human subjects, to prevent PD following near TP and TP.42,43 Although first series of transplants were technically successful, the results were largely inconsistent. A loss of function was reported in several patients several months following IAT.42,43 Early challenges were corrected by significant improvements in surgical care and islet isolation technique. Ricordi automated method for islet isolation (Fig.  2), first published in 1988, pioneered a significant improvement in the islet isolation process. It allowed for the continuous release of the islets liberated from the exocrine tissue during the digestion phase, thereby protecting them from any further enzymatic action and preventing

Sampling port Temperature monitor Digestion chamber Shaking device

Collecting flask Dilution solution Recirculating cylinder

Heating circuit

Pump

FIG. 2  Automated method for the isolation of human pancreatic islets. Courtesy: Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW. Automated method for isolation of human pancreatic islets. Diabetes 1988;37(4):413–420.

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overdigestion of the endocrine tissue. By significantly reducing the islet cell loss during the digestion process, this innovation ultimately resulted in significant improvement to both quality and quantity of the islet tissue.44 The digestion process is judged complete when only ductal tissue was left in the chamber, with no or small amount of pancreatic tissue remaining. The fact that the Ricordi method resulted in the complete dissociation of pancreatic tissue, with a significant improvement to the quantity and quality of the isolated islet cells, set this method apart from what was done and published previously.44 New and improved enzyme blends, effective use of large-scale purification methods, and routine application of a number of additives during the islet isolation process all contributed to further improvements in the islet isolation yield and quality of the isolated cells, as well as the utilization of the isolated tissue for transplant.20,45–49 Of substantial benefit is the fact that isle preparations for IAT are transplanted fresh, within a few hours of the completion of the islet isolation process.

Pancreas cleaning, trimming, and cannulation When the pancreas is received in the laboratory, the temperature of the preservation solution is measured using a thermocouple and recorded in the batch record. A 3 mL sample is collected for the assessment of microbial contamination, that is, aerobic, anaerobic, and fungal organisms. Sterility testing can be performed by any appropriately accredited and licensed laboratory, according to the USP Sterility Testing Standards. It is imperative to keep the organ cold from the time of pancreatic resection to the beginning of enzymatic digestion; low-temperature environment slows down endogenous enzyme activity and subsequent tissue degradation.25,37 Following sampling, the pancreas is transferred to a stainless steel tray filled with cold 1 L of trimming solution (Corning, # 99-676-CM) (Fig.  3).

FIG. 3  Upon receipt in the laboratory, the pancreas is cleaned of extra fat and connective tissue prior to cannulation. During cleaning, the organ is maintained at 4–8°C.

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In order to maintain low temperature of the trimming solution, the cleaning tray is placed inside a larger stainless steel tray filled with frozen sterile normal saline (B Braun, CA): 2:1 ratio of frozen sterile saline to cold sterile saline is utilized in our laboratory. The same can be accomplished working in the cold room, in which case neither the second tray, nor frozen saline is necessary. Normal saline is packaged in 1 L double bags and is maintained at −20°C; hence it’s frozen well in advance. Saline ice cubes can be prepared by braking frozen saline using a hammer or mallet. To protect the integrity of the outer plastic bag, saline bag should be wrapped in a sterile half sheet. To maintain the sterility of the inner bag, the outer bag is removed in the BSC, and saline cubes are poured into a larger stainless steel tray. Once the cleaning tray assembly is prepared, 1 L Nalgene jar containing the pancreas in UW is aseptically opened in the BSC, and the pancreas moved to the cleaning tray filled with 1 L of cold trimming solution containing 1 g/L Cefazolin Sodium USP. The presence of the antibiotic in the trimming solution allows for pancreas surface decontamination. Initially, morphologic observations are recorded based on visual observation of the pancreas evaluated for degree of fat, quality of the flash, degree of blood, edema, texture, general condition (intact, capsular damage parenchymal damage), and gross pathology. The quality of the pancreatic tissue retrieved from CP patients undergoing pancreatic resection is much worse compared to that obtained from deceased, heart-beating donors. Pancreata excised from these patients are often fibrotic, with large degree of calcification, and otherwise damaged by a prolonged inflammatory process, which makes the isolation of sufficient number of islets from these organs quite challenging.25,50 All visual observations are recorded in the batch record. A trained member of the isolation team cleans the pancreas of access fat and connective tissue, making sure that the capsule stays intact to preserve the quality of intraductal perfusion. The organ is then rinsed in three consecutive sterile 1 L beakers, each containing 500 mL of phenol-free Hank’s balanced salt solution (HBSS, Corning, NY), deposited in a new sterile 1 L Nalgene jar and weighed on a pretared scale to calculate the amount of enzyme required. This calculation is based on the weight of the pancreas, that is, enzyme activity/g of pancreas weight. However, the degree of fibrosis and calcification, as well as the age of the patient, play an important role in this calculation; a higher enzyme concentration is required to digest organs with extensive gross pathology, from older donors. At our center to achieve best distention results, the pancreas is cannulated once it is split at the neck, into body and tail, and head portions. However, depending on the severity of pancreatitis and the degree of prior surgical intervention, the pancreas might arrive in the

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lab as a whole organ, a portion of it, or in multiple pieces. In the worst-case scenario, when the pancreas is in several pieces, with varying degree of calcification and/ or fibrosis, the tissue must be repeatedly injected with the enzyme solution, using a syringe-needle assembly. In case of a whole pancreas, the best result is achieved when the pancreas is subdivided at the neck into bodytail and head portions. Each portion is cannulated using a 14G–24G catheter, although the size of the catheter should be chosen based on the size of the duct, and the degree of fibrosis and calcification. In the worst-case scenario, stainless steel catheter should be used. The catheter must fit firmly into the duct, which can be accomplished by suturing it in place, to avoid back flow during perfusion. With the organ trimmed of access fat and tissue and both lobes of the pancreas cannulated, the organ is deposited into a sterile 1 L Nalgene jar, and moved to the perfusion tray (Biorep, Miami, FL) connected to the perfusion apparatus (Biorep, Miami, FL) prefilled with the enzyme solution, prepared while the pancreas is cleaned.

Pancreas perfusion Collagenase aids in rapid dissociation of the stromal component of the gland, while preserving the anatomical integrity of the endocrine tissue. It’s been the preparation of choice ever since the technique for collagenase digestion for dissociation of pancreatic tissue was first described by Moskalevski in 1965.51 He used a crude preparation of Clostridium histolyticum to isolate islets from guinea pig and rat pancreata. In the most quoted article that describes a method for islet cell isolation, Lacy reported a modification of the Moskalewski’s technique. He disrupted the exocrine tissue of a rat pancreas by retrograde perfusion fluid delivered into the pancreatic duct, under pressure. The tissue was then minced, the exocrine and endocrine tissue separated by collagenase digestion, and the islets handpicked using a dissecting microscope.52 Since then it became clear that when collagenase is infused into the main pancreatic duct, it is broadly distributed within the human exocrine and endocrine tissue.53,54 This is not surprising, as approximately half of the human islets are “peri-ductal,” that is, lying adjacent to the ductal tissue.53,55,56 Because CP results in variable progression of tissue damage and fibrosis of diseased pancreas, islet yields from such organs are variable. UMN reported that islet yields in CP patients receiving IAT at UMN have ranged from nearly 0 to >500,000 IEQ.16 This is quite understandable; as often happens in organs with varying degree of CP-related tissue damage, even distribution of the enzyme during perfusion via the pancreatic duct in CP pancreata is particularly difficult. In those cases when automated perfusion is not

possible, the organ should be injected with the enzyme solution in order to achieve some degree of pancreatic tissue dissociation. Effective intraductal delivery of the collagenase solution into the main pancreatic duct via a pressurized injection at a constant rate is crucial to the subsequent ability to isolate viable islet cells.25,57 This is a critical step in the isolation process. A consistent delivery of the enzyme solution to the entire interstitial compartment of the pancreas ensures a complete digestion of the organ and maximizes the yield and quality of the islet cells that are ultimately infused back into the patient. Many clinical islet transplant centers load the enzyme solution into the pancreas by retrograde injection into the body and tail of the pancreas, and anterograde injection to the head of the organ. This is accomplished using a 30 mL sterile disposable syringe following cannulation of the pancreatic duct. This approach, although widely used, does not allow for continuous monitoring of the pressure during the distention, or the temperature of the enzyme solution. It was Lakey, who introduced the idea of an alternative approach to pancreas perfusion; this method involves perfusing the organ via the pancreatic duct using a recirculating perfusion device system25,57 that includes a recirculating pump. This approach provides continuous control over perfusion pressure and collagenase temperature during pancreas distention. Lakey et  al.57 showed that controlled perfusion via the pancreatic duct allowed an effective delivery of the enzyme solution throughout the pancreatic parenchyma and achieving maximal distension throughout the organ. This led to an increased recovery of the islets with no detrimental effect on subsequent in  vitro islet function. A modern semiautomated perfusion system (Biorep Technologies, Mimi, FL) controls temperature, pressure, and speed, and is equipped with a peristaltic pump, pressure and temperature sensors, a heater, a touch screen, and an advanced software with data storage capability. The instrument combines the convenience of an automated system with manual control made possible through the use of the touch-screen interface. This semiautomated system is utilized by several centers that participated in multicenter registration clinical trials of islet transplantation completed in 2016. We demonstrated this perfusion method and a highly purified collagenase Liberase HI resulted in a significantly improved islet cell yield and quality.20 During the perfusion process, the temperature should be kept between 4°C and 14°C, while the injection pressure is maintained between 60 and 80 mmHg for the first 4 min of the distention, and between 160 and 180 mmHg for the remainder of the time. In our center, the distention process usually takes between 10 and 12 min. Perfusion pressure can vary depending on the condition of the organ (degree of calcification and fibrosis) and damage to

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the pancreatic capsule. While the pressure can be low for damaged and leaking organs, it can increase dramatically for an organ with abnormal ductal anatomy, severe fibrosis, or a misplaced catheter. Another important parameter to keep a note of during the distention process is the flow rate of the collagenase. In our hands, the flow rate ranges between 40 and 60 mL/min during the first 4 min of the distention, and between 90 and 115 mL/min for the remainder of the time. A flow rate below 30 mL/ min is indicative of either a system leak or a misplaced cannula. When this occurs, the situation should be corrected immediately by clamping or suturing the leak, as the latter usually results in a suboptimal perfusion and distention of the pancreas, eventually resulting in partial digestion of the pancreatic tissue.58 Depending on the degree of organ damage (parenchymal fibrosis and calcification), the pancreas can fail to distend and has to be distended manually, by repeatedly injecting cold enzyme into the pancreatic parenchyma, to achieve some degree of distention. This is usually accomplished using a sterile 30 mL luer-lock syringe connected to a sterile 18–20 G needle. In some cases, the distal portion of the body and tail portion of the pancreas fail to distend as a result of alterations in the pancreatic duct due to inflammation, fibrosis, or calcified deposits. In such cases, it is useful to make a transverse cut close to the distal nondistended portion of the organ and cannulate the duct in an attempt to better distend this portion of the gland. At the end of the distention process, the pancreas is cleaned of the remaining fat, connective tissue, capsule, sutures, and cannulae. Following additional trimming, the organ is cut into several smaller pieces. This step should be performed as quickly as possible, to limit the exposure of the pancreatic parenchyma to the collagenase. In case of a normal organ from a healthy deceased donor and depending on the size of the pancreas, the organ is normally cut into 9–11 pieces, each 1–2.5″ in length. When dealing with pancreatomized organs, the tissue can be cut into smaller pieces to achieve the best possible digestion results.

Collagenase: Selection and dose Translating islet isolation technique that worked well in small animal models to large animal models and human organs proved to be difficult. A major obstacle to successful human and canine pancreas dissociation was the low enzymatic activity of the bacterial collagenase. Additionally, a number of reports indicated that a combination of collagenase and protease enzymes is necessary for best tissue dissociation and successful recovery of islets from large animal and human organs.20,59–61 The development of Liberase HI (Roche, Indianapolis, IN) in 1994, a highly purified enzyme blend that consisted

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of two collagenase isoforms from C. histolyticum (class 1 and class 2 collagenases) and a NP with very low endotoxin activity, has helped eliminate lot-to-lot variability in enzymatic activity. The use of Liberase HI resulted in consistently superior islet yield and quality compared to that obtained when unpurified collagenase products were utilized.20,59,60 Liberase HI also provided a convenient formulation in terms of its packaging, based on which a single vial was dissolved in a desired enzyme solution volume. The formulation of Liberase HI was later changed in such a way that two highly purified collagenase isoforms, class 1 and class 2 collagenases were packaged separately from NP. Using this enzyme combination, most centers utilized collagenase: NP in 1:1 ratio.21,62 FDA’s request for better characterization of tissue dissociation enzymes utilized during the manufacture of human allogeneic islet cells in clinical trials resulted in withdrawal of Liberase HI from the market. A difficult transition from Roche Liberase HI to a different cGMP-grade enzyme combination, NB-1 collagenase (NB-1) supplemented with NB NP manufactured by SERVA Electrophoresis GMBH (Heidelberg, Germany) ensued. The latter was utilized during the National Institutes of Health (NIH)-funded Clinical Islet Transplantation (CIT) consortium trials, starting in 2007. Several transplant centers found that Serva’s NB-1 collagenase (NB-1) and NB NP (NB) mix failed to provide islet yields comparable to those obtained using Roche Liberase.62–65 This led to modifications to islet isolation protocols to increase islet yields, including selecting specific lots of Serva enzymes or modifying the sequence of enzyme addition used during the islet isolation procedure.62,63,65 At the present time, several purified recombinant collagenase preparations are available from Roche (Indianapolis, IN), Serva (Heidelberg, Germany), and VitaCyte (Indianapolis, IN). These are utilized with the original NP or in combination with NP manufactured by a different company.21,66 Szot et al. demonstrated that using a mixture of cGMP-grade collagenase NB-1 and NB NP (SERVA Electrophoresis GMBH, Heidelberg, Germany) and a modified islet isolation process to account for the activity of Serva collagenase, consistently resulted in high-quality islet cell preparations suitable for clinical transplantation.63 Balamurugan reported superior islet cell yield, and improved islet cell structural integrity and quality, using VitaCyte collagenase with Serva NP.66 This enzyme combination was reported to produce total islet yields of >200,000 IEQ in 90% of attempted autologous isolations.66 A dose of collagenase utilized for digestion varies between centers. While some centers utilize collagenase at a constant concentration63,66,67 disregarding the weight of the organ, others tailor collagenase dose according to

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the pancreas morphology and weight, which was shown to result in more consistent islet cell yields. For example, UMN varies the collagenase dose from 22 to 30 Wunch units/g and NP dose from 1.5 to 3.0 DMC units/g of pancreas weight, depending on the organ characteristics and weight.68 For islet isolation from resected pancreata from CP patients, we have utilized Liberase MTF (recombinant collagenase, Indianapolis, IN) and Serva Collagenase-AF-1 (recombinant collagenase, SERVA Electrophoresis GMBH, Heidelberg, Germany) collagenase, both supplied as cGMP grade. We have found that a combination of either Liberase MTF (Indianapolis, IN) and Serva-AF-1 (SERVA Electrophoresis GMBH, Heidelberg, Germany) with NP-AF (SERVA Electrophoresis GMBH, Heidelberg, Germany) results in superior islet cell quantity and quality. We normally utilize 20–22 U/g of collagenase with 1.8–2.0 U/g of NP-AF, increasing the dose of NP-AF for severely fibrotic and damaged organs. For larger organs, we recommend that the volume of the enzymatic solution and the amount of both collagenase and NP be adjusted accordingly. We normally prepare the enzyme in 400 mL HBSS with 10 U/mL heparin, although the volume of the enzyme solution can be adjusted up for larger organs.66

Phase 1: Digestion Once the collagenase is injected, it begins to bind to the tissue almost immediately. Hence, any unnecessary delay between the end of the distention and the beginning of digestion should be avoided. At the present time, most islet isolation centers around the world utilize a semiautomated method for islet isolation, first described by Ricordi in 198844 (Fig.  2). The method incorporates several key elements: (i) minimal traumatic action on the islets cells, (ii) continuous digestion of the pancreatic tissue, while free islets are progressively released from the digestion chamber, and (iii) minimal operator intervention in the digestion process to limit handling and damage to the pancreatic tissue. At the conclusion of the digestion process which incorporates both enzymatic and mechanical components, the only part of the pancreatic tissue left is the fibrous network of ducts and vessels that remain in the Ricordi chamber. This method has proven to be even more superior to manual methods used previously, as these utilized a significant traumatic component to disrupt the pancreatic tissue and liberate islet cells. Along with the pancreas, seven marbles made of silicon nitride (Biorep Technologies, Miami, FL) and sufficient volume of the digestion solution to fill the bottom part of the digestion chamber, are added to the Ricordi chamber to aid in the enzymatic dissociation of the pancreatic tissue. Two parts of the Ricordi chamber are

separated by the 533 μm stainless steel screen and held together by four screws that should be tightly in place, and sealed by the rubber O-ring, placed in position to prevent leaks during the tissue dissociation process. The chamber has a port in its upper part and two smaller ones in the lower part. Size 16 Masterflex tubing (Cole Parmer, Vernon Hills, IL) fits the larger port in the upper part of the chamber, while size 17 Masterflex tubing connects to two smaller ports in the bottom part of the digestion chamber. The smaller size tubing is secured in the Masterflex pump (Cole Parmer, Vernon Hills, IL) and is connected to the heating coil placed in the Ricordi isolator (Biorep Technologies, Miami, FL) or a water bath preheated to 43 °C. The ends of the smaller and larger tubing are placed inside the recirculation reservoir filled with extra collagenase solution. The pancreas is cut into 9–11 similar sized pieces approximately 1″ × 2.5″ in length and aseptically transferred to the Ricordi digestion chamber, along with the silicone marbles. Once the 533 μm stainless steel screen is in place, the digestion chamber is tightly closed and sealed to prevent leakage. When the chamber is closed, the digestion circuit is complete. The tubing that connects the heating coil to the recirculation container is placed in the Masterflex pump (Cole Parmer, Vernon Hills, IL) to promote recirculation within the digestion circuit. The chamber has an outlet for a temperature probe that allows for temperature monitoring during the digestion process. The tubing has a port that connects to a sterile disposable syringe utilized to collect samples of the digest during the digestion process. At the start of the digestion process, the pump speed is set to 300 mL/min in order to fill the digestion circuit as quickly as possible. Care must be taken to eliminate bubbles from the tubing system and the digestion chamber. Once the circuit is full, the pump speed is adjusted down to 150 mL/min, while the digestion chamber is gently rocked to mix its contents. Over the next 5–7 min, the circuit temperature gradually increases to 35–37°C. Once the target temperature is reached, the heating coil is partially/completely removed from the water bath and the chamber is shaken more forcefully, to provide a mechanical component to the enzymatic dissociation process. Close monitoring of tissue dissociation during the digestion process is critical to prevent overdigestion of the pancreatic tissue resulting in islets of poor quality. At our center, we collect a 2–3 mL sample from the syringe connected to the outlet port in the tubing circuit. We typically start collecting samples at 7–8 min into the digestion process. At this point, the solution in the recirculation reservoir is cloudy as the pancreatic tissue begins to dissociate. Samples should be collected every 2 min, which allows for a detailed observation of tissue dissociation. Once collected, the sample is

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stained with a few drops of freshly prepared dithizone [diphenylthiocarbazone (DTZ)] solution inside a small, 35 mm Petri dish, and quickly evaluated, under a light microscope, for quantity and cluster size of acinar tissue, number of islets, quantity (percent) of free islets, and quantity (percent) of fragmented (overdigested).69 Islet score is calculated based on each of the above observations. Several successive samples that contain increased quantities of free islets and acinar tissue that occupies most of the visual microscope objective observation field indicate a completion of the digestion, or recirculation phase (digestion/phase 1), and the beginning of the dilution phase (phase 2). This is a critical point which profoundly impacts the outcome of the isolation process. Generally, it is imperative to keep digestion/phase I as short as possible, to prevent inferior islet cell yield and quality at the end of the isolation process. Some centers reported adjusting digestion settings in order to compensate for the off-target enzyme dose (calculated by subtracting the trimmed weight of the pancreas from the original tissue weight). They report raising or lowering the digestion temperature to compensate for insufficient enzyme dose, and/or slow progress of digestion.21 When the digestion phase progresses too slowly, addition of extra collagenase and/or NP directly into the digestion circuit that lead to improved islet cell yields and quality has been reported.21,50,62 The digestion phase of islet cell isolation process is a critical step that impacts the outcome of the whole process. It is essential that digestion parameters such as temperature set point and rate of increase, enzyme dose, and digestion switch point are determined by previously trained and qualified personnel. Pancreata resected from CP patients are commonly severely fibrotic and hard. Additional complications in the architecture of the pancreatic parenchyma can be a result of previous surgical procedures in an attempt to reduce inflammation and pain. Fibrotic tissue is even more resistant to collagenase action, and often results in reduced islet cell yield. As mentioned above, maintaining the digestion temperature in the higher end of a normal temperature range can increase enzyme activity. Balamurugan et  al. described a modification to the Ricordi method, when unusually low amount of pancreatic tissue was observed in samples collected during the digestion pace. In order to achieve a more complete digestion of the pancreatic tissue, free islets already released into the digestion circuit were collected along with the digestion solution right after they were released.21 The digest was centrifuged, free islets were pelleted by centrifugation and transferred to a fresh, cold collection solution, while the supernatant containing the active enzyme was collected and recycled into the digestion circuit.21 This resulted in a higher dose of enzyme

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reintroduced into the digestion chamber, leading to a near complete or complete digestion of tissue remaining in the digestion chamber.21 In addition to fibrosis, other donor characteristics critically impact the outcome of the digestion phase. Donor age, for example, is significantly associated with the isolation outcome.70,71 Younger donors are generally healthier, exposed to fewer pathogens, and have fewer medical complications compared to older donors. Islets from younger donors have been reported of higher quality and exhibit better function.70,72 However, consistent recovery of sufficiently high islet yields for clinical transplantation from younger donors70 remains a challenge. Islets from such donors are often mantled or embedded in surrounding acinar tissue following digestion. This changes the density of the islet cells, making it difficult to purify them from the surrounding nonendocrine tissue during COBE processing using currently utilized density gradient centrifugation methods.70 Methods that include an extended enzyme recirculation (distention) step followed by a static incubation devoid of any manipulation, removal of free islets released early in the digestion step and rescue purification were demonstrated to result in improved yields of functional islets from younger donors.21,70,72 Additional process modifications add to the success of isolating greater numbers of highly functioning islets cells from younger donors. These include the use of a smaller size Ricordi digestion chamber (250 mL) effective in concentrating the enzyme activity and shortening the length of the digestion phase, and use of a smaller pore size mesh in the digestion chamber to ensure better filtration of the circulating cell clusters and removal of the larger size tissue clusters. In general, temperature of the digestion circuit, length of the distention phase, circulation speed, enzyme dose, force of mechanical shaking, and duration of the digestion step are all factors that can be modified to accommodate for inherent variations in the condition of the pancreatic parenchyma caused by different CP pathology. A detailed understanding of how these parameters influence the outcome of the isolation process is essential to understand how to modify these in order to affect the rate of tissue dissociation, minimize the amount of the undigested tissue left in the Ricordi digestion chamber, and maximize quantity and quality of the resulting islet cells.

Phase 2: Dilution and tissue collection This phase of the islet isolation process is closely associated with the digestion phase, and can be considered an extension of the latter. All the islet cells collected during this step have to be protected from further action of collagenase. Failure to inhibit collagenase activity

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invariably leads to cell degradation. Dilution begins by adjusting several parameters: • flow rate to 230 ± 20 mL/min, • temperature of the digestion chamber to ≤30°C, by removing the heating coil from the water bath, and • opening the closed digestion circuit by removing inlet tubing (Masterflex, size 17) from the recirculation container and placing it in the 4 L intake container filled with room temperature RPMI-1640 media (Corning, NY). If a large number of mantled or embedded islets are observed in the sample at the time of the switch from a digestion to a dilution phase, the chamber temperature should remain at 30–38°C during the dilution phase. The first four digest fractions are collected in four 1 L sterile Erlenmeyer flasks filled with cold 400 mL RPMI1640 (Corning NY), supplemented with 200 mL 25% human serum albumin (HSA), 0.2 U/mL insulin, 10 U/mL heparin. Samples should be collected throughout the dilution/collection process to evaluate the number and condition of the islet cells, and condition of the acinar tissue. If needed, additional collection containers with 25% HSA to provide a final concentration of 1.5%. When islet cells are no longer observed in the sample stained with DTZ, dilution/collection phase is judged complete. At this point, the digest remaining in the system should be collected, addition of the fresh collection media to the system should be discontinued, and the recirculation pump should be stopped. As tissue is collected, it is aseptically transferred to as many sterile 250 mL conical tubes as necessary, and centrifuged at 170×g, at 2–8°C for 3–5 min. The digest is collected by decanting the supernatant from each 250 mL conical and transferring the pelleted tissue to a sterile 1 L collection container filled with cold wash solution that consists of Cold Storage/Purification Stock Solution (Corning, NY) supplemented with, 0.625% HSA 25%, 10 U/mL heparin, 0.2 U/mL insulin, and 2% Pentastarch Solution (Corning, NY). The latter prevents exocrine cells from swelling and becoming less dense, which can negatively affect the efficiency of the downstream purification process. Balamurugan et  al. discussed the issue of small, ~3 mm in diameter, calcified deposits found in the digest.68 If present, such deposits should be removed from the tissue pellet prior to the purification step and washed to free islet cells trapped in the crevices of these structures, before the calcified particles are removed and discarded. When the dilution is judged complete and all the tissue is combined in the same 1 L flask containing wash solution, the tissue is mixed gently by swirling the flask several times to assure that the tissue is completely resuspended and no clumps are present. The tissue is equally distributed into as many 250 mL conical tubes as necessary and centrifuged. This is to eliminate the s­ upernatant

and recombine tissue digest pellet from several 250 mL conical tubes into a single one. Once the issue is combined in a single tube, the tissue is resuspended in a total volume of 200 mL of wash solution. To obtain an accurate count, the tissue digest must be uniformly resuspended by gently rocking the 250 mL conical, to disrupt any remaining issue aggregates. Duplicate 100 μL samples are quickly collected from the middle of the 250 mL conical, and transferred to a 35 mm counting dish containing 1 mL of wash solution/media/­phosphate-buffered saline (PBS) and several drops of freshly prepared DTZ solution. Care must be taken not to collect 100 μL counting samples in succession. A 250 mL conical containing the digest must be gently but thoroughly mixed between samples. The packed pellet volume is estimated by filling a 250 mL conical containing the tissue digest with 200 mL of wash solution or purification solution (depending on whether the purification step is necessary), centrifuging it at 170×g, 2–8°C for 3–4 min, and estimating the tissue volume by reading gradations on the 250 mL conical tube. Packed tissue pellet volume and counting samples are important determinants in whether the purification step is necessary. Islet isolations from deceased allogeneic donors yield digest volumes of well over 20 mL, necessitating the purification step using isopycnic gradient purification to separate islet cells from the bilk of acinar tissue. Autograft pellet volumes of 300,000 IEQ or >3000 IEQ/kg is desirable, however, patients have been shown to achieve insulin dependence even with lower yields. The isolation procedure as described by Ricordi remains to be the preferred method, with the use of Liberase which results in higher yields compared to the previously used collagenase P. A detailed description of the islet cell isolation is provided in this book in section “Islet Isolation.” In brief, the pancreas is loaded into an isolator which consists of two stainless steel chambers with a mesh in between. Glass marbles are added to the chambers along with a solution of Liberase or collagenase. The chamber is connected to a shaker, and the digested pancreas passes through the mesh. This is then cooled to inactivate the enzymatic digestion. The solution is further filtered through a smaller mesh, and islets are purified using a series of centrifugation and gradients. The resultant pellets are then resuspended.4, 23

Islet infusion/transplantation If islet isolation is performed at the same facility, this procedure is started before or at the time of gastrointestinal reconstruction. If performed at an outside facility, the patient’s abdomen is closed and the patient is extubated. The patient will return to the operating room after arrival of the islet isolation product. Alternatively, the infusion may be performed by interventional radiology. Prior to islet infusion, Etanercept 50  mg IV, and Anakinra 100 mg subcutaneously, and a heparin bolus of 35 units/kg (islet drip also contains 35 μ/kg heparin) are administered. Etanercept, a tumor necrosis factor (TNF)

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inhibitor and Anakinra, an interleukin (IL)-1 antagonist serve as antiinflammatory agents to improve islet grafting and are often used in islet allotransplantation. It is assumed that these drugs have similar effects in autotransplantation.24 The use of Anakinra potentiates the effect of Etanercept, leading to improved insulin content and reduction in apoptosis as shown in animal studies.25 Heparin is added to prevent thrombosis of the portal vein and its branches. If after completed isolation the islets are infused in the operating room, the liver via portal vein infusion serves as the engraftment site. In preparation, a stay suture using 6-0 polypropylene is placed on the splenic vein (or any other chosen portal vein tributary) stump. A 14F angiocath is then introduced and can be secured with the stay suture or held in place (Fig. 5). Using the previously described assembly (Fig. 6), the islets are infused and the portal pressure is measured. Heparin is administered intravenously at 20–40 U/kg. The portal vein pressure should be measured at baseline and then intermittently assessed before infusion and about every 15 min during infusion. If the portal vein pressure is >25–30 mmHg, islet infusion should be halted for a while until the portal

Minimally invasive surgery TPIAT using minimally invasive techniques has also been described. Giulianotti et al. described the first robotic distal pancreatectomy with IAT in 2009, in a patient with CP. The islets were transfused via a mesenteric vein; the robot was not used for the infusion.27 The first robotic TP for a patient with CP was described in 2010 by Marquez et  al. The islets were obtained from the distal pancreas specimen and transfused through a Pfannenstiel incision via a mesenteric vein of a small bowel loop.28 The first fully robotic TPIAT, using the Da Vinci system for both pancreatic resection and islet infusion, was performed by Galvani et al. at the University of Arizona. The first series of robotic TPIAT using the whole pancreas was also first reported in Galvani et al. in 2013.29 Laparoscopic TP was described later, with the first two fully laparoscopic pylorus and spleen preserving total pancreatectomies reported in 2013 by Dallemagne et al. for IPMN and neuroendocrine lesions.30 Fan et al. described the first series of laparoscopic TPIAT in 2017. Their technique involves resection of the pancreatic head and duodenum followed by a distal pancreatectomy. Conversion to open was required in two out of their 20 patients due to difficult anatomy and prior surgery.31 The islets were transfused via a 14–18 g laparoscopic needle using a 12-mm trocar into the splenic vein stump.

(A)

(B) FIG.  5  (Top) 14 g angiocath in the splenic vein stump. (Bottom) Visible islet cell clumps.

vein pressure drops. If it does not drop, the duodenal wall, the omentum, and the cul-de-sac have been used as additional engraftment sites. Core liver biopsies of the right and left lobes are obtained after infusion of the islets. These biopsies frequently demonstrate engraftment of the islets within the portal venous system (Fig. 7). After islet infusion, a gastric or jejunal feeding tube may be placed, especially in patients with a poor nutritional status. Before the abdomen is closed in standard fashion, the viability of the spleen is reassessed and if tears, bleeding or swelling of the spleen are noted then a splenectomy should be performed. Vaccinations include polyvalent pneumococcal vaccine (Pneumovax 23), Haemophilus influenzae b vaccine (HibTITER), and meningococcal polysaccharide vaccine prior to discharge when medically stable.26 Drains are usually placed as per the surgeon’s discretion, but it is not uncommon to place at least 1 JacksonPratt (JP)-drain in the pancreatic bed.

Robotic TP and islet autotransplant Except for minor changes, the principles for robotic TPIAT are the same as for open TPIAT.29, 32, 33 The patient is placed in the supine position with both arms tucked. After induction of general anesthesia,

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Operative technique

(A)

109

(B)

FIG. 6  (Left) Assembly for transfusion of islet cells and portal vein measurement. (Right) Processed islet cells.

(A)

(B)

FIG. 7  (Left) H&E stained auto-islet in native liver after transplantation via splenic vein. (Right) IHC stain for insulin on auto-islet transplantation.

a­ rterial and central vein lines are placed. The operating table is rotated in such a way that it allows for docking of the robot from the patient’s head. Ports are placed as depicted in Fig. 8. Prior to docking of the robot, the gastrocolic ligament is taken down laparoscopically. A Nathanson retractor is placed to retract the stomach anteriorly. Next, the robot is docked from the head of the patient. In the robotic procedure, the tail of the pancreas is mobilized first, along with dissection of the splenic artery and vein. The space between pancreatic tail and spleen is divided using an endovascular stapler. The blood supply to the tail of the pancreas via the splenic vessels is preserved until final en-bloc removal of the pancreas in order to minimize warm ischemia of the islets. All retroperitoneal attachments of the distal pancreas are taken down. The splenic artery is dissected at its take-off from the celiac artery and the splenic vein at the confluence with the SMV.

Next, the right colon and duodenum are mobilized. An extended Kocher maneuver is performed providing exposure to the SMV. The uncinate process is separated from the superior mesenteric vessels by dividing all venous tributaries with the harmonic scalpel or by suture ligation. The first portion of the duodenum is divided proximally with a linear cutting stapler roughly 3–5 cm distal to the pylorus, followed by division of the common bile duct. The pancreas is then completely mobilized, attached only by its vascular pedicle. After administration of intravenous heparin (50– 70 IU/kg), the splenic artery, GDA, and splenic vein are divided using an endovascular stapler. The splenic vein is divided approximately 3 cm proximal to the confluence to leave a relatively long stump for the islet infusion. Protamine is administered intravenously to neutralize the heparin effect. The pancreas is then removed via a 6–7 cm Pfannenstiel incision. The spleen is only removed

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(A)

(B)

(C)

(D) FIG. 8  (A and B) Port placement for robotic TPIAT. (C and D) Docking of DaVinci Si and positioning of intraoperative personnel. Adapted from Galvani CA, Rodriguez Rilo H, Samame J, Porubsky M, Rana A, Gruessner RW. Fully robotic-assisted technique for total pancreatectomy with an autologous islet transplant in chronic pancreatitis patients: results of a first series. J Am Coll Surg 2014; 218(3):e73–78.

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Postoperative care

if does not appear viable, as the right gastroepiploic and short gastric vessels usually remain intact. While the gastrointestinal reconstruction takes place, the islets are isolated in the laboratory. A hepaticojejunostomy using running 4-0 polydiaxanone sutures is performed. The duodenojejunostomy is performed with 3-0 V-Loc sutures in two layers, 50 cm from the hepaticojejunostomy. A modified jejunojejunostomy Braun anastomosis (to reduce bile reflux gastritis) can also be performed using a GIA stapler. The mesenteric defects are closed with 3-0 V-Loc sutures. Laparoscopic TP and islet autotransplant A 12-mm port is placed in the umbilicus. An additional 12-mm port is placed on the right and a 5-mm port on the left side of the abdomen. The gastrocolic ligament is divided. The GDA is identified, ligated, and divided. A tunnel is created posterior to the neck of the pancreas, with dissection of the portal and SMVs. The first portion of the duodenum 3–5 cm distal to the pylorus or a distal gastrectomy is performed using an EndoGIA stapler. A cholecystectomy is performed. The neck of the pancreas is divided, followed by a pancreaticoduodenectomy. As described by Fan et al., the head of the pancreas is then given to the extraction team and processed. Thus, the pancreas is not removed en-bloc. The distal pancreas is removed separately, after division of the splenic artery followed by the splenic vein. The specimen is also sent for islet isolation processing. A hepaticojejunostomy is performed using 4-0 barbed sutures and suture clips. A standard jejunojejunostomy is performed. The islets are infused via the portal vein using a 16 g needle via a 12-mm port site.31 Robotic and laparoscopic TP outcomes Outcomes in minimally invasive TP with or without IAT are limited to small volume (n 3000 IE/ kg in open series). Robotic-assisted procedures although noted to have increased operative times (600 vs 469 min, P = .014), had less blood loss (220 vs 705 cc, P = .004).31, 33, 34 In a direct comparison of robot-assisted TPIAT compared to open, there were no significant differences in outcomes.33 Further long-term and greater power studies are needed to further characterize the role of robotic TP.

Percutaneous infusion/transplantation of islets Interventional radiology can also assist with infusion of islets. This method is useful in patients undergoing delayed infusion of islets, for example, in facilities

­ ithout on-site isolation capabilities. The procedure was w first performed in 1999 by Weimar et  al., who utilized computed tomography and fluoroscopic techniques to cannulate the portal vein.35 Since then sonographic techniques have also been described and reviewed, demonstrating low complication rates and adequate long-term results.36 Difficulties and complications associated with this method include repeated attempts at cannulation, bleeding, hemoperitoneum, hemothorax, and portal vein thrombosis. The major advantage for patients undergoing percutaneous infusion is that they do not require general anesthesia. Sedation with versed and fentanyl usually suffices. Using fluoroscopy or ultrasound guidance, an appropriate puncture site is identified, and local anesthesia is administered. A branch of the right portal vein is identified and accessed with a 22-g Chiba needle as described by Owen et  al. An 18 g guidewire is introduced and advanced to the portal vein. A sheath is then advanced over the wire to the portal confluence. Under fluoroscopy, a venogram is performed to confirm placement. Islet infusion is then conducted as previously described with frequent assessment of portal venous pressure measurements. Embolization of the tract with a gelatin sponge has been described, however, with smaller catheters has been performed less often. Postoperative care consists of bed rest with follow up ultrasound of the portal vein.37

Postoperative care Postoperatively, patients are admitted to the intensive care unit. The nasogastric tube is left in place. Patients are maintained on an intravenous insulin drip postoperatively “to rest the islets,” with a goal of titrating blood sugar levels between 80 and 120 mg/dL. Pain control can be achieved via narcotics, parenteral acetaminophen, nonsteroidal antiinflammatory drugs (NSAIDs), lidocaine infusion, and/or fentanyl patch with consultation from the pain management and anesthesia teams. Specifically, the pain management team needs to follow the patient closely throughout the hospitalization. If gastrostomy and jejunostomy tubes are present, they are placed to gravity. Antimicrobial prophylaxis with meropenem, vancomycin, and fluconazole is administered for several days and discontinued if cultures are negative. A vascular ultrasound of the abdomen is obtained in the ICU to assess portal vein patency. If the estimated portal pressure on the duplex ultrasound increases >15 cm H2O, enoxaparin 0.5 mg/kg twice per day is administered. Postoperatively, the patient receives Anakinra 100 mg SC on postoperative days 1–7. Etanercept is given on

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postoperative days 3, 7, and 10 (if still in hospital). As previously noted, Anakinra and Etanercept (section “Islet cell transfusion/transplantation”) work in concert to reduce inflammation and improve islet graft survival and function. Patients can be started on total parenteral nutrition (TPN) postoperatively per institutional guidelines. With guidance from endocrinology, patients are started on long-acting and premeal doses of insulin, a regimen that is continued for several weeks, in order to rest the implanted islets. Close outpatient follow up by the endocrinologists is essential for titration of the insulin regimen. Nasogastric tube and enteral nutrition can be gradually started. Patients are usually continued on insulin for several weeks to rest the islets cells. For patients who become independent from insulin, C-peptide levels are obtained monthly during postoperative follow-up to assess function of the transplanted islets. Pancrelipase (Creon) is also used liberally after TP. The manufacturer’s recommendation postpancreatectomy per meal dose is 72,000 units, however, we titrate the dosage based on the improvement in patient steatorrhea.

Complications The rate of postoperative complications after TPIAT ranges widely, but has been reported to be as high as 30%–40%.38 Complications of TPIAT include portal vein thrombosis, islet contamination, delayed gastric emptying, intra-abdominal abscess, intra-­ abdominal hemorrhage, sepsis, hypoglycemia, and wound complications.

Portal vein thrombosis High portal pressures after completed infusion have been associated with portal vein thrombosis. Large tissue volume (TV) is also a risk factor in the development of portal vein thrombosis. Purification of islets is dependent on a series of gradients, therefore, the yield and purity of islets can be variable. Wilhelm et al. recommended that a TV 6-month duration and at least one of the following: • Pancreatic calcifications on computerized tomography scan. • At least two of the following: ≥4/9 criteria on EUS, compatible ductal or parenchymal abnormalities on secretin MCRP; abnormal endoscopic pancreatic function tests (peak HCO2 ≤ 80 mmol/L) • Histopathology confirmed diagnosis of chronic pancreatitis • Compatible clinical history and documented hereditary pancreatitis (PRSS1 gene mutation) • History of recurrent acute pancreatitis (more than one episode of characteristic pain associated with imaging diagnostic of acute pancreatitis and/or elevated serum amylase or lipase greater than three times upper limit of normal) 2. At least one of the following: • Daily narcotic dependence • Pain resulting in impaired quality of life, which may include: inability to attend school, recurrent hospitalizations, or inability to participate in usual, age-appropriate activities 3. Complete evaluation with no reversible cause of pancreatitis present or untreated 4. Failure to respond to maximal medical and endoscopic therapy 5. Adequate islet cell function (nondiabetic or C-peptide positive) a Criteria were formally implemented in 2008.

TP-IAT. Table 1 lists the current criteria used to diagnose RAP and CP at our institution. Contraindications to offering the procedure include active alcohol or drug abuse, poorly controlled psychiatric issues, inability to comply with postoperative cares, and significant cardiac or respiratory disease that would lead to inordinately a high operative risk.16 In addition, preexisting liver disease, including cirrhosis, portal hypertension or portal vein thrombosis also contraindications for major pancreatic resection or intraportal islet transplant.12, 16 Currently, pancreatic cancer or pancreatic neoplasms, and morbid obesity are contraindications to the procedure at our institution, but some centers have performed TP-IAT in these patients. Lastly, patients with C-peptide negative diabetes will not benefit from the IAT portion of the procedure so TP alone is offered without the IAT.17

Postoperative management and complications Islet yield and glycemic management The goal of the auto-islet portion of the TP-IAT is to prevent the complete loss of the endocrine function of the pancreas (via loss of insulin producing β-cells and the counter-regulatory hormone of glucagon) and

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Postoperative management and complications

­ revent development of brittle diabetes. Although many p patients do require insulin after surgery, almost all remain on a stable dose with well-controlled glucose levels and minimal to no hypoglycemia. Insulin independence rates range from 10% to 47% in large series, with an average of 1/3 of patients remaining insulin independent following TP-IAT.10, 12, 13, 18 It is important to remember that TP-IAT is performed not to prevent diabetes, but to prevent brittle type 3c diabetes and its devastating and life-threatening hypoglycemia. In this light, almost all patients benefit from TP-IAT; as 90%–100% of patients remain c-peptide positive (a marker of continued islet cell function) in a long-term follow-up.10, 12 Patients with an islet yield of >5000 islet equivalents per kilogram (IEq/kg) are 100% c-peptide positive and 94% are able to maintain a HgbA1c of 5000 islets/kg body weight can be transplanted (see Chapter 9 and Ref. 12). The surprising thing is that total pancreatectomy followed by islet autotransplantation (TPIAT) is a procedure that remains unknown to the majority of practicing physicians. This is evidenced by the absence of this topic in the vast majority of CME courses and in textbooks of Internal Medicine, Gastroenterology, and Endocrinology. Even in textbooks of General Surgery and General Transplantation this topic is given only fleeting attention, if any at all. Why this should be the case is a puzzlement. Hopefully, the remainder of this chapter helps convince the reader that the inherent risks of TPIAT are outweighed by the excellent metabolic outcomes in pancreatic islet function and quality of life in general (see following chapter by McEachron et al.).

Favorable metabolic outcomes: Normal levels of glycemia, HbA1c, and β-cell function The number of islets of Langerhans in the adult human pancreas is usually estimated as approximately one million. They comprise 2%–3% of the total pancreatic mass, with the remainder of the pancreas being exocrine tissue, including the pancreatic ducts. The islets themselves are comprised primarily of alpha and beta cells. Beta cells synthesize and secrete insulin, proinsulin, and C-peptide into the hepatic venous portal blood in the basal state. This is amplified by rapid secretory responses when the islet is stimulated with glucose, amino acids, GLP-1, and secretin. Insulin delivery from beta cells into the islet periportal system also has the important function of inhibiting the alpha cells from secreting their primary product, glucagon. The glucagon response is triggered quickly by the development of hypoglycemia and is the earliest hormonal response to counter-regulate low blood glucose levels by activating liver glycogenolysis that liberates free glucose, which in turn enters the hepatic venous return into the inferior vena cava and the general circulation. This sequence rapidly increases systemic blood glucose levels and corrects hypoglycemia. Second responses that quickly follow the glucagon response is the epinephrine response from the adrenal medulla, as well as the norepinephrine response from

the sympathetic nervous system inside the pancreas and from the brain. If these normal interconnections ­between beta cells, alpha cells, and adrenergic cells are ­interrupted, serious consequences are prolonged periods of hypoglycemia and attendant desensitization to hypoglycemia with loss of the normal symptoms, such as warmth, hunger, tachycardia, and decreased mentation. These important components of the hormonal cross-talk that regulates islet function are seriously compromised by removal of the pancreas and introduction of isolated islets into hepatic sites (see later). Successful prevention of postpancreatectomy diabetes caused by autoislet transplantation requires approximately 350,000 islets, which is >5000 islets/kg body weight.12 Greater than 2500 islets/kg are required to maintain a recipient insulin-free albeit with mild-­ moderate hyperglycemia (Fig.  1, Ref. 12). In this large series from the University of Minnesota, use of fewer than 2500 IE/kg was associated with only 70% of recipients achieving HbA1c levels 90% when recipients received >5000 IE/kg (Fig.  2). Similarly, with use of 5000 IE/kg provided C-peptide positivity for >95% of recipients. The functional β-cell mass of islet engraftment can be most accurately determined in vivo by measuring the functional βcell secretory capacity. The test is termed glucose potentiation of arginine-induced insulin secretion (GPAIS). Alternatively, C-peptide rather than insulin responses may be measured. This metabolic test is a simple one involving only an intravenous injection of arginine before a glucose infusion and once again 60 minutes after the glucose infusion in an outpatient

FIG.  1  The percent of TPIAT recipients who were free of insulin treatment at 6, 12, 24, and 36 months posttransplant as a function of the number of islets transplanted. Vertical stripes = 5000 islets/kg. Reproduced from Sutherland DER, Radosevich DM, Bellin MD, et  al. Total pancreatectomy and islet autotransplantation for chronic pancreatitis. J Am Coll Surg. 2012;214:409–424.

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Unfavorable metabolic outcomes: Hypoglycemia following meals and exercise and deficient α-cell counter-regulatory response

FIG. 2  The percent of TPIAT recipients who were C-peptide positive

(boxes with horizontal stripes) or maintained HbA1c levels  36 weeks

Reduced insulin requirement by 16%

84

Adult/nondiabetic CM

High-M alginate

Kidney capsule

> 180 days

Urine porcine C-peptide-positive

21

Dufrane et al.

Adult/diabetic CM

Alginate MCD

Subcutaneous

> 6 months

Diabetes correction > 6 months

22

Adult + pig MSCs/ diabetic CM

Alginate MCD

Subcutaneous

> 32 weeks

Diabetes correction > 32 weeks

Dufrane et al.

Veriter et al.

CM, cynomolgus monkey; MCD, monolayer cellular device; MSC, mesenchymal stromal cells; NICCs, neonatal islet-like clusters; PLL, poly-l-lysine; PLO, poly-l-ornithine. Modified from Zhu HT, Lu L, Liu XY, et al. Treatment of diabetes with encapsulated pig islets: an update on current developments. J Zhejiang Univ Sci B. 2015;16(5):329-343.

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Laying the foundation: preclinical studies in islet xenotransplantation

Islet infusion into the portal vein

IBMIR

Islet Thrombus formation (Thrombin)

Lysis (C3a, C5a, MAC...)

Platelet Fibrinogen Leukocytes TF FVIIa

Coagulation activation & platelet activation and aggregation

Complement activation & infiltration of leukocytes

iC3b Red blood cell

FIG. 2  Overview of IBMIR. The contact between blood and islets triggers the activation of coagulation that is mediated through tissue factor (TF). As a result, thrombin is generated, leading to fibrinogen deposition. Attachment of platelets to islets further increases the procoagulant effect. Complement (iC3b) is deposited on the islet surface, C3a and C5a are activated, attract leukocytes, and promote formation of membrane attack complex (MAC) which mediates the lysis of islets. (FVIIa = activated coagulation factor VII). Reproduced with permission from Liu Z, Hu W, He T, Dai Y, Hara H, Bottino R, Cooper DKC, Cai Z, Mou L. Pig-to-primate islet xenotransplantation: past, present, and future. Cell Transplant. 2017;26(6):925–947.

Oxygen tension in the portal vein is low, which may trigger islet apoptosis. Direct exposure of islets to the blood results in a substantial loss of islets from IBMIR. It is estimated that IBMIR reduces the number of successfully transplanted islets in the portal system by 60% within the first few hours or days.6,93,98,116–119 Furthermore, the islet graft cannot be retrieved, and liver biopsies usually do not yield sufficient islets for analysis. While better control of IBMIR and graft rejection could improve outcomes of portal transplantation (described above), alternative sites for free islet transplantation must continue to be explored to improve the clinical outcome.6,55,113 Although the pancreas may be considered the natural site for islet transplants, it is not easily accessible, and the risk of pancreatitis would be significant. Therefore,

alternative sites (other than the portal vein) have been explored (Table 4).55,113 These studies have been directed to sites where the islets are not immediately exposed to blood, and thus protected from IBMIR/early graft loss. Potential transplant sites include the gastrointestinal submucosal space, omental pouch, striated muscle, and bone marrow.55,120–127 Although a current trial is examining the omentum as an alternative site for islet allotransplantation, the other locations have been largely studied in small mammals or pigs.128,129 Islet transplants into the renal subcapsular space in animals under optimized protocols have demonstrated some success as an alternative to intraportal islet infusion, but have shown limited success in humans.130–139 Failures are likely due to islet ischemia after transplantation.55,130,131

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31.  Xenotransplantation of the endocrine pancreas

FIG. 3  Binding of human IgM and IgG antibody to pig islets (xenogeneic) (A–B) and to human islets (allogeneic) (C–D). IgM (green, A, C), IgG (green, B, D), insulin (red), nucleus (DAPI/blue). Yellow indicates colocalization of insulin and IgM/IgG. The greatly increased binding of human IgM and IgG to pig islets (compared to human islets) is obvious. Reproduced with permission from van der Windt DJ, Marigliano M, He J, Votyakova TV, Echeverri GJ, Ekser B, Ayares D, Lakkis FG, Cooper DK, Trucco M, Bottino R. Early islet damage after direct exposure of pig islets to blood: has humoral immunity been underestimated? Cell Transplant 2012;21(8):1791–1802.

Composite islet-kidney grafts One mechanism to increase the speed of revascularization and engraftment lies in composite islet-kidney transplants. Pig islets have been transplanted under the kidney capsule in autologous or syngeneic settings of pig littermates, allowing revascularization and proliferation of islets in the absence of immune and inflammatory responses.132–135 Some weeks later, the composite islet-kidney graft was transplanted into the ultimate pig recipient which, if MHC mismatched with the composite graft donor tissue, received immunosuppressive therapy. Successful engraftment and immediate function of both transplanted tissue/organ proved the validity and clinical potential of the approach. There has also been success in the immunosuppressed NHP model.136,137 A xenogeneic model would be necessary to make these studies truly clinically relevant. However, a xenogeneic model was originally troubling because of the difficulty in maintaining viability and function of a porcine kidney graft in an NHP. With improved genetic engineering of the organ-source pig (and effective immunosuppressive therapy of the NHP host), this hurdle has now been overcome.140,141 The ultimate goal would

be for the composite islet-kidney to be implanted in patients with end-stage renal disease and diabetes, with the aim of curing the renal failure and controlling glucose metabolism.

Cotransplantation of islet xenografts and “regulatory” cells Cotransplantation has also been studied by combining porcine islets with mesenchymal stem cells (MSCs) or Sertoli cells (SCs). MSCs and SCs have been shown to function across species lines, and possess anti-­ inflammatory, regenerative, and immunomodulatory properties that promote revascularization of islets after xenotransplantation.22,142–150 SCs help create tight junctions to isolate germ cells from the blood in the testis, but there is inconclusive evidence suggesting that SC cotransplantation may improve islet survival in humans.55,85,151–153 Nevertheless, SCs, and especially MSCs, have considerable potential in islet xenotransplantation to diminish acute (and probably chronic) islet graft loss.55,154 The ability to obtain large quantities of porcine MSCs and SCs from the identical, genetically engineered pig islet donor may be a significant advantage.55,142,143

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Gastric submucosal space Pancreas

Liver

Renal capsule Spleen

Skin

Omentum

Muscle

Efficacy of clinical trial

Good

Poor

Not reported

Poor

Limited Experience

Limited experience Not reported

Limited experience

Patient safety

Safe

Safe

Safe

Safe

Safe

Safe

Possible pancreatitis

Safe

Oxygen tension

Low

Not reported

High

Low

Not reported

High

Not reported

Not reported

Vasculature

Rich

Poor

Not reported, but probably rich

Poor

Rich

Rich

Not reported

Rich

Site of insulin released by the graft

Liver

Not reported

Portal vein

Systemic circulation

Portal vein

Portal vein

Not reported

Systemic circulation

Surgery

Invasive, some complications

Invasive

Invasive

Non-invasive

Easy

Easy (endoscopy)

Difficult

Easy

IBMIR

Yes

Not reported

Yes

Not reported

Not reported

Not reported

Not reported

Not reported

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TABLE 4  Comparison of different sites for free islet xenotransplantation

Modified from van der Windt DJ, Echeverri GJ, Ijzermans JN, et al. The choice of anatomical site for islet transplantation. Cell Transplant 2008;17(9):1005–1014.

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Genetic modifications to combat IBMIR Genetic knockin, knockout, and knockdown models in the pig have been developed to combat IBMIR/ hyperacute rejection.10,20,65,73,155–160 Varying degrees of success have been demonstrated in a number of genetically modified pig strains to prevent immediate islet destruction in the pig-to-NHP model and reduce the need for pharmacologic immunosuppressive therapy to the host.10,15,20,24,55,66,161–163 The identification of Gal on pig cells that is bound by human anti-pig antibodies was a major milestone in combating hyperacute rejection of solid organ xenografts.65,164–168 In 2003, the first GTKO pigs were introduced, lacking the enzyme responsible for adding Gal to the oligosaccharides of pig endothelium and islets that cross-react with host antibodies.167 Since then, two other carbohydrate epitopes have also been targeted, namely Nglycolylneuraminic acid (Neu5Gc) and Sda (the product of β-1,4-N-acetylgalactosaminyltransferase).55,155,156,158,167,169 Since the first successful use of GTKO pigs in islet xenotransplantation, improved methods of genetic manipulation, including the CRISPR technology (Table  5), have facilitated attempts to reduce injury by protecting the graft from the primate immune response (Table 6).61,65,112,167,168,170–180 A recent model developed by Kirk and colleagues demonstrated that islets transplanted from GTKO and WT donor pigs into NHPs developed relatively similar IBMIR responses as measured by insulin, complement, antibodies, neutrophils, and macrophages.181 This model quantitatively highlights the specific difficulties in islet xenotransplantation compared to allotransplantation, demonstrating increased IgM, cellular infiltration, and apoptosis even in GTKO xenogeneic islet donors.182 As an alternative or additional approach, human complement-regulatory proteins, for example, CD46, CD55, and CD59, have been expressed on pig islets TABLE 5  Timeline for application of evolving techniques for genetic engineering of pigs employed in xenotransplantation

TABLE 6  Selected genetically modified pigs currently available for xenotransplantation research Complement regulation by human complement-regulatory gene expression CD46 (membrane cofactor protein) CD55 (decay-accelerating factor) CD59 (protectin or membrane inhibitor of reactive lysis) Antigen deletion α1,3-Galactosyltransferase gene-knockout (GTKO) Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene-knockout (CMAH-KO or Neu5Gc-KO) β4GalNT2 (β-1,4-N-acetylgalactosaminyltransferase) gene-knockout (β4GalNT2-KO) Suppression of cellular immune response by gene expression or downregulation CIITA-DN (MHC class II transactivator knockdown, resulting in swine leukocyte antigen class II knockdown) Class I MHC-knockout (MHC-IKO) HLA-E/human β2-microglobulin (inhibits human natural killer cell cytotoxicity) Human FAS ligand (CD95L) Human GnT-III (N-acetylglucosaminyltransferase III) gene Porcine CTLA4-Ig (cytotoxic T-lymphocyte antigen 4 or CD152) Human TRAIL (tumor necrosis factor-alpha-related apoptosisinducing ligand) Anticoagulation and anti-inflammatory gene expression or deletion von Willebrand factor (vWF)-deficient (natural mutant) Human tissue factor pathway inhibitor (TFPI) Human thrombomodulin Human endothelial protein C receptor (EPCR) Human CD39 (ectonucleoside triphosphate diphosphohydrolase-1) Anti-inflammatory, anti-apoptotic (and anticoagulant) gene expression Human A20 (tumor necrosis factor-alpha-induced protein 3)

Year

Technique

Human heme oxygenase-1 (HO-1)

1992

Microinjection of randomly integrating transgenes

2000

Somatic cell nuclear transfer (SCNT)

Human CD47 (species-specific interaction with SIRP-α inhibits phagocytosis)

2002

Homologous recombination

2011

Zinc finger nucleases (ZFNs)

2013

Transcription activator-like effector nucleases (TALENs)

2014

CRISPR/Cas9a

a

Porcine asialoglycoprotein receptor 1 gene-knockout (ASGR1-KO) (decreases platelet phagocytosis) Human signal regulatory protein α (SIRPα) (decreases platelet phagocytosis by ‘self’ recognition) Prevention of porcine endogenous retrovirus (PERV) activation PERV siRNA

CRISPR/Cas9, clustered randomly interspaced short palindromic repeats and the associated protein 9

PERV KO

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(and organs) and have been demonstrated to protect against the effects of human complement activation.12,55,161,163,183–189 Similarly, transgenic expression of human coagulation-regulatory proteins (e.g., tissue factor pathway inhibitor, thrombomodulin) have reduced the development of thrombotic microangiopathy and consumptive coagulopathy in pig organ grafts in NHP recipients, and may play a role in protecting pig islets from IBMIR.20,55,190–192 A promising milestone for islet xenotransplantation was achieved by van der Windt et al. in 2009, who used islets from pigs expressing hCD46, successfully achieving insulin independence for > 1 year in diabetic monkeys (Fig. 4).12

433

Neonatal islets expressing human complement-­ regulatory proteins, CD55 and CD59, on a GTKO background have been shown to attenuate IBMIR-associated pathological events in immunosuppressed baboons, with reduced complement activation and thrombin generation.193 Using multiple human transgenes, including complement and coagulation inhibitors, Bottino et  al. further demonstrated modulation of IBMIRmediated early islet loss, even though this did not consistently translate into better long-term outcomes.20 Simultaneous transgenic modifications in islet donors for GTKO/hCD46/CMAH (Neu5Gc)-KO demonstrated a near-complete reduction in IgM and IgG responses.

FIG.  4  (A) Blood glucose and pig C-peptide levels in a streptozotocin-induced diabetic cynomolgus monkey before and after intraportal transplantation of islets from a pig expressing the human complement-regulatory protein, CD46. No exogenous insulin was administered after the transplant. The normoglycemic monkey was electively euthanized after 12 months. Day 0 = day of islet transplantation. (B) Insulin immunostaining (in red) of a liver section in a monkey recipient of islets from a pig transgenic for human CD46, showing a healthy pig islet 12 months after transplantation. (Magnification × 200). Reproduced with permission from van der Windt DJ, Bottino R, Casu A, Campanile N, Smetanka C, He J, Murase N, Hara H, Ball S, Loveland BE, Ayares D, Lakkis FG, Cooper DK, Trucco M. Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets. Am J Transplant. 2009;9(12):2716–2726.

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Combined ­pharmacologic therapy with multiple transgenic modifications may prove to be the best method to prevent IBMIR in islet xenotransplantation. Importantly, genetic modifications that delete pig antigens or use promotors to target expression of transgenes in β-cells do not appear to reduce islet function.55,73,160,193,194 The differentiation between IBMIR and rapid ­antibody-mediated rejection of the pig islet graft is unclear and, indeed, they have many similar features. Even if not identical, many of the genetic modifications to reduce IBMIR also reduce antibody-mediated rejection.55,128,195–207 the reduction in antigen expression, and expression of human complement- and ­coagulation-regulatory proteins have all been shown to be protective to pig organs and islets.12,15,20,161–163 If IBMIR and the innate immune response can be controlled by genetic engineering of the islet-source pig, then efforts can be focused on the suppression of the adaptive (cellular) immune response.54,61

Control of the T cell response The genetic manipulations and pharmacologic therapy outlined above attempt to protect against the innate immune system and the early loss of grafts from humoral immunity. However, they do not adequately target prevention of the adaptive immune response (cellular rejection), including the T cell response that causes lymphocytes to infiltrate the graft and induce an elicited antibody response.11,208–210 Moreover, the innate and adaptive immune systems are not mutually exclusive. An organ from a pig genetically modified to combat the innate immune response may also allow reduced immunosuppressive therapy to control the adaptive immune response.73,211–213 For example, there is evidence that the absence of expression of Gal on the graft and the expression of a human complement-regulatory protein both reduce the T cell response.214,215 Nevertheless, it is very likely that some exogenous immunosuppressive therapy will be required to prevent the T cell response to transplanted pig islets and organs for the foreseeable future. T cells require activation through their T cell receptor and peptide-MHC complexes (signal 1) with additional costimulation (signal 2) in order to induce a cellular proliferative response and the secretion of cytokines. Conventional immunosuppressive therapy (directed to blocking the activation and proliferation of resting T cells) has to date proved unsuccessful in fully protecting a pig xenograft.209 In contrast, therapy directed to block T cell costimulation (signal 2), first introduced into pig-to-NHP xenotransplantation by Buhler et al. proved much more

effective.209 Chief among the costimulation-activating pathways is the CD154 (CD40 ligand)-CD40 p ­ athway.216 This provides an attractive target to prevent the adaptive immune response.11,217 Indeed, an anti-CD154 monoclonal antibody (anti-CD154mAb) was the first costimulation blockade agent used in xenotransplantation, and has been employed widely since then in preclinical islet xenotransplantation studies.168,209 Its efficacy is illustrated by the studies of Park and his colleagues who have achieved insulin independence in diabetic monkeys for approximately 2  years by transplanting adult wild-type (WT, i.e., genetically-­ unmodified) pig islets under the cover of anti-CD154mAb therapy (Table  7).24 These remarkable results could not be duplicated when this group replaced anti-CD154mAb therapy with other ­costimulation-blockade agents, including anti-CD40mAb.14,218 In contrast, when genetically engineered whole organ pig grafts are transplanted, anti-CD40mAb-based regimens have proved entirely successful.140,141,219,220 Despite the success of Park’s group using WT pig donors with costimulation blockade-based immunosuppressive therapy, a combination of islet transplantation from multi-transgenic pigs and novel exogenous immunosuppressive therapy holds the most promising future for islet xenotransplantation (Table 7).10,20,24,55,66,193 Together, these can reduce the need for intensive conventional immunosuppressive therapy, ultimately reducing the likelihood of adverse complications, thus reducing the barriers to successful clinical practice.6,80 Although anti-CD154mAb has helped establish normoglycemia from several months to over 2 years in pigto-NHP models, prior Phase I and II clinical trials of organ allotransplantation demonstrated a risk of thromboembolic events, underpinning the balance between efficacy and safety.8,11,12,14,20,112,168,211,213,218,221,222a A recent study, however, demonstrated this agent’s safety in pig-to-NHP islet xenotransplantation, potentially making it a candidate for use in clinical islet transplantation.109 Whether the lack of thromboembolic events after islet xenotransplantation in the pig-to-NHP model was related to the low antigen load of islets (compared with a solid organ) or to other factors remains to be ascertained. Modifications of the molecular structure of this compound aimed at reducing the potential prothrombotic effects are underway and may prove clinically relevant. Anti-CD40mAb is a promising alternative that continues to be explored, particularly when the islets are derived from genetically engineered pigs.11,14,16,168,223–226 Other costimulationblockade agents, for example, CTLA4-Ig, have been found to be less successful when used alone.13,14,227–230 In addition, there are specific genetic modifications that can be made to the pig donor tissues to reduce the

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TABLE 7  Experience with the xenotransplantation of free islets from wild-type pigs in immunosuppressed NHPs genetically-engineered pigs in NHPs (± immunosuppressive therapy) Reference

Donor/recipient

Immunosuppressive therapy

Maximal graft survival (days)

(A) WILD-TYPE PIGS IN IMMUNOSUPPRESSED NHPS Hering et al.8

Adult/CM

FTY720 + rapamycin + anti-IL-2R + anti-CD154

> 187

Cardona et al.11

Neonatal/rhesus monkey

CTLA4-Ig + rapamycin + anti-IL-2R + anti-CD154

> 260

Thompson et al.14

Neonatal/rhesus monkey

CTLA4-Ig + rapamycin + anti-IL-2R + anti-CD40

> 203

16

Neonatal/rhesus monkey

MMF + CTLA4-Ig + LFA-3-Ig + anti-IL-2R + anti-LFA-1

114

Shin et al.

Adult/rhesus monkey

ATG + CVF + rapamycin + anti-TNF + antiCD154mAb(+ Treg)

> 603

Shin et al.218

Adult/rhesus monkey

ATG + CVF + rapamycin + adalimumab + antiCD40mAB(+ tacrolimus or belatacept)

60

Thompson et al. 24

(B) GENETICALLY-ENGINEERED PIGS IN NHPS (± IMMUNOSUPPRESSIVE THERAPY) Mandel et al.163

hCD55 fetal/CM

Cyclosporine + steroids + cyclophosphamide or brequinar > 40

GnT-III adult/CM

None

5

hCD46 adult/CM

MMF + ATG + anti-CD154mAb

> 396

GTKO neonatal/rhesus monkey

MMF + anti-CD154mAb + anti-LFA-1mAb + CTLA4-Ig

249

Chen et al.

GTKO/hCD55/hCD59/hHT neonatal/baboon

MMF + ATG + tacrolimus

28

Bottino et al.20

Multi-transgenic adult/CM

MMF + ATG + anti-CD154mAb

> 365

162

Komoda et al.

12

van der Windt et al. 15

Thompson et al. 161

anti-LFA-1, anti-lymphocyte function-associated antigen-1 monoclonal antibody; ATG, antithymocyte globulin; CM, cynomolgus monkey; GnT-III, Nacetylglucosaminyltransferase-III; hHT, human α(1,2)fucosyltransferase; MMF, mycophenolate mofetil; Treg, autologous regulatory T cell infusion. Modified from (a) Park CG, Bottino R, Hawthorne WJ. Current status of islet xenotransplantation. Int J Surg. 2015;23(Pt B):261–266 and (b) Zhu HT, Yu L, Lyu Y, Wang B. Optimal pig donor selection in islet xenotransplantation: current status and future perspectives. J Zhejiang Univ Sci B. 2014;15(8):681–691.

cellular response, for example, (i) insertion of a mutant (human) MHC class II transactivator gene, resulting in downregulation of swine leukocyte antigen (SLA) class II expression, (ii) deletion of expression of SLA class I (SLA class I-KO), or (iii) insertion of an immunosuppressive gene, for example, CTLA4-Ig55,198,202,230,231 (Table 6).

Will sensitization to human leukocyte antigens be detrimental to islet xenotransplantation? It is well known that patients who have received blood transfusions or organ transplants from human donors, or have been pregnant, can develop antibodies directed toward human leukocyte antigens (HLAs). If the patient then requires an organ or cell transplant, this condition may make it difficult to identify a human donor against which the patient has no preexisting antibodies. There is some evidence that allosensitization to HLAs would not preclude successful xenotransplantation, although there is considerable conflicting evidence in this respect.232,233

Will sensitization to pig antigens preclude subsequent islet allotransplantation? If sensitization develops to a pig xenograft, the limited data available to us at present suggest that the recipient would be at no immunological disadvantage to subsequently undergo allotransplantation when a donor becomes available.232,234,235

The induction of immune tolerance: The “Holy Grail” of transplantation The ultimate goal of islet xenotransplantation is to induce a state in which the host immune system recognizes the transplanted pig islets as “self” and makes no effort to reject them.6 If such immunologic “tolerance” could be achieved, immunosuppressive therapy, which may have detrimental effects on the host (and the graft), could eventually be discontinued.10,61 Xenotransplantation has the advantage that the timing of the transplant is known well in advance, allowing

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31.  Xenotransplantation of the endocrine pancreas

manipulation of the host’s immune system (without the constraints of timing that occur in allotransplantation using deceased donors). Nevertheless, we suggest that tolerance induction can only be seriously considered when the early hurdles to pig islet xenotransplantation, for example, IBMIR and the innate immune response, have been fully overcome.80 Most efforts to induce tolerance to both allografts and xenografts have revolved around either (i) donor-specific hematopoietic progenitor cell transplantation in an effort to induce a state of chimerism or (ii) concomitant donor-specific thymus transplantation.236

Improving function of porcine islets Although the pig is considered a good xenogeneic donor, issues remain in establishing sufficient insulin production and glucose control after islet xenotransplantation. The relatively poor secretory function of porcine islets and their relative inability to respond to metabolic stimuli compared to healthy islets from NHPs or humans is often overlooked.73 Pigs utilize less insulin, require lower levels of C-peptide, and maintain higher blood glucose levels compared to NHPs (Table  8).54,160,194,222a Isolated porcine islets secrete three to six times less insulin than human islets when stimulated with glucose in vitro.73,237–240 Unfortunately, this cannot be explained by the lower insulin content of porcine islets, and underscores the field’s poor understanding of porcine β-cell physiology, which has predominately focused on smaller animal models that are more easily obtained.73 Identification of the optimal age and strain to improve proliferation and increase the quantity of islets is only part of the solution.20,73,241 Just as genetic

TABLE 8  Fasting blood glucose, C-peptide, insulin, and glucagon levels in monkeys (Macaca fascicularis), pigs, and humans Cynomolgus monkeys222a

Pigs222a

Humans

Blood glucose (mmol ·L− 1)

2.2–4.1 (3.2)

4.0–5.2 (4.8)

3.9–5.6222b

C-peptide (nmol ·L− 1)

0.47–3.14 (1.39)

0.11–0.32 (0.16)

0.17–0.66222c

Insulin (pmol ·L− 1)

15–201 (109)

7–12 (9)

34–138222c

Glucagon (pmol ·L− 1)

18.7–179.4 (54.3) 11.3–13.8 (12.5)

5.7–28.7222c

Data are presented as ranges (mean). C-peptide (P  9 yr

Insulin requirement reduced by 30%

Valdes-Gonzalez et al.88

NICCs + SCs (encapsulated)

Subcutaneous

None

> 3 yr

Insulin requirement reduced from 19-28 IU/d to 6IU/d

Valdes-Gonzalez et al.87

NICCs (encapsulated) Subcutaneous

None

> 7.7 yr

Insulin requirement reduced by 33% (in > 50% of patients)

Wang et al.9

NICCs

CsA + MMF + prednisolone

> 1 yr

Insulin requirement reduced by 33%–62%

Hepatic artery

> 1 yr OKT-3 + tacrolimus  + sirolimus + prednisolone

Insulin requirement reduced 33%–62%

CsA + MMF

Not available

Not available

NICCs (encapsulated) Peritoneal cavity

None

> 52 week

1/14 showed full graft function for a period of time

Matsumoto et al.250 NICCs (encapsulated) Peritoneal cavity

None

> 600 day

HbA1c < 7.0%; reduced insulin and severe episodes of hypoglycemic unawareness

23

Matsumoto et al.

ATG, anti-thymocyte globulin; CsA, cyclosporine; FICCs, fetal islet-like cell clusters; MMF, mycophenolate mofetil; NICCs, neonatal islet-like clusters; SCs, Sertoli cells. MMF = mycophenolate mofetil; NICCs = neonatal islet-like clusters; SCs = Sertoli cells. Modified from Rood PP, Cooper DK. Islet xenotransplantation: are we really ready for clinical trials? Am J Transplant. 2006;6(6):1269–1274.

­ ccurred in these trials, highlighting the safety of clinical o application, but underpinning the need for further studies to improve efficacy.54,251–253

Establishing safety In 2016, the IXA published an executive summary and a seven-chapter consensus statement regarding the prospect of taking porcine islet xenotransplantation into the clinic.54,61,254–259 This was its first update since its original statement in 2009, and highlights new guidelines aimed at accelerating the use of genetically modified pigs. In doing so, their hope was to introduce preclinical guidelines that are less demanding, but maintain patient safety.259 Key to establishing effective clinical trials—indeed a maxim of medicine—is to first ensure safety of the patient and minimize undue risk. Porcine xenotransplants could be associated with zoonotic transmission of microorganisms to the recipient, and possibly even to close contacts and the community. The 2003 US Food and Drug Administration (FDA, updated in 2016) and 2008 First World Health Organization Consultation on Regulatory Requirements for Xenotransplantation

Clinical Trials acknowledged that the possibility of a zoonotic “epidemic” was of sufficient concern when defining the regulatory framework and principal guidelines for xenotransplantation.54,260,261 All pig cell nuclei contain viruses or virus remnants known as porcine endogenous retroviruses (PERV) (just as human cell nuclei contain human endogenous retroviruses [HERV]). These are dormant in their host and not associated with any specific diseases. Despite initial concerns that PERV could become activated in humans, there has never been a report of in vivo infection between pigs and humans.89,262,263 Furthermore, although recent studies have shown successful inactivation of PERV in pig DNA, most of those working in xenotransplantation do not believe this will be necessary.255,264 Today, most experts agree that, using the appropriate precautions, there is minimal risk that porcine xenotransplantation would spread a communicable disease.61,265 Pigs used in clinical trials must be housed in biosecured “designated pathogen-free” facilities that eliminate most potentially pathogenic microorganisms. With good manufacturing practices and established standard operating procedures, the risk is considered minimal.61

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31.  Xenotransplantation of the endocrine pancreas

The IXA executive summary outlines recommendations on screening the patient for donor-derived pathogens and pathogens that may be introduced during islet preparation that would ensure safety in well-planned pilot clinical studies. Transplant recipients would not require monitoring for pathogens initially absent from the islet-source pig.61,258,259 The risk of infection secondary to administered immunosuppressive therapy and other risks typical of any transplant or novel therapeutic intervention would require monitoring. These risk-benefit determinations must be acknowledged when obtaining informed consent.56,61,266 In light of their own recommendations, the US FDA will review proposals for clinical trials of xenotransplantation if they contain a plan for patient follow-up.261

Determining efficacy The 2016 IXA guidelines have been slightly revised since 2009 regarding the preclinical justification for transitioning to clinical trials after success in preclinical pigto-NHP models. In 2016, it was suggested that successful treatment without graft rejection be present for at least 6  months, and preferably 12  months, in four of six (or five of eight) consecutive NHPs to warrant clinical trials with potential success in free islet xenotransplantation requiring immunosuppressive therapy.54,61 However, the IXA was hesitant to provide definitive guidelines that might inhibit discussion with regulatory authorities or restrict clinical trials unduly. A minority of experts suggested that a reduced period of graft function (3 months) in NHPs could be sufficient, if the primary goal was improved glycemic control (demonstrated by the absence of recorded hypoglycemic events), rather than a complete insulin independence. However, due to the delayed production of insulin when transplanting embryonic/fetal/ neonatal islets, several months of follow-up might be required to demonstrate reduced insulin requirements or insulin independence.11,13,54 A significant minority of those working in this field also recommended that the requirement for NHP experiments should not be generalized, but rather developed by investigators and regulatory agencies in light of their experience and objectives. Importantly, these shorter durations differ from the US FDA recommendations to conduct animal experiments for 12–24 months.261 Regardless, the IXA acknowledges that preclinical studies using NHPs are at times essential, and always recommended. Similar and perhaps more lenient guidelines were suggested for encapsulated islet xenotransplant studies. Given the difficulty of carrying out studies in NHPs in Europe, the variability in experience and objectives, and the important theoretic ability to transplant encapsulated islets (or islets protected solely by SCs or MSCs) without immunosuppression, a minority of experts

recommended that no work in NHPs may be necessary.54,61 Nevertheless, in either the case of free or encapsulated islet xenotransplantation, preclinical studies should be sufficiently rigorous to establish safety and provide optimism for success in clinical trials, but need not be so demanding as to require prolonged experimentation to ensure success, as this might adversely affect patients who could truly benefit from islet xenotransplantation.54,61,259 It is possible that the lack of preclinical success in NHPs limited the efficacy of past clinical trials.25,53,85,89,267,268 However, it is important to note that the earliest published clinical trials in islet xenotransplantation were performed before the original IXA consensus statement for undertaking clinical trials in 2009.56,61,249,263,269,270 The current guidelines for initiating clinical trials have evolved to keep up with advances in technology, and it is important to recognize that the current era of islet xenotransplantation necessitates established preclinical safety and efficacy in the pig-to-NHP model before proceeding to human trials.61,255,264

Patient selection Defining a clinical study population after preclinical success requires a population with a favorable ­benefit-risk ratio. The US FDA regulations insist that patient selection should focus on, among other criteria, those who “(i) have serious or life-threatening diseases for whom adequately safe and effective alternative therapies are not available except when very high assurance of safety can be demonstrated, and (ii) have potential for a clinically significant improvement with increased quality of life following the procedure.” 61,261,271,272 In light of these guidelines, the IXA recognizes a narrow population of eligible patients that comprises diabetics experiencing recurrent and severe hypoglycemia unawareness despite optimized medical management.61,259 Other potential candidates who have been identified include those with diabetes (with poor glycemic control), end-stage renal disease (who require kidney allotransplantation and might benefit from islet xenotransplantation), and “brittle” diabetics who lack timely access to islet allotransplantation.61,259

Future directions Research priorities The IXA has proposed establishing an IXA Clinical Trial Advisory Committee, whose role would be to advise and serve as an informative body—but not to regulate— for research programs considering initiating clinical trials (and possibly also to advise regulatory agencies on

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References

the scientific aspects of such trials).54 In 2016, IPITA-TTS released a joint executive summary on the future of βcell replacement therapy.6 With regard to islet xenotransplantation, they determined that the first priority is preventing IBMIR with a multifaceted approach that specifically utilizes genetically modified pigs. Second, they encouraged developing an effective and clinically relevant anti-rejection regimen that might be based on disrupting CD40/CD154 signaling.6

Conclusions Roughly 1.25 million Americans have T1Ds, with an additional 40,000 diagnosed each year.273 Progress toward making islet xenotransplantation a clinical reality has grown exponentially. Certainly, hurdles remain, and even success in pilot clinical trials would not guarantee broad applicability. Nevertheless, hope for transplanting tissues across species is no longer quixotic or science fantasy. It is a profound reality that, within the reader’s lifetime, islet xenotransplantation will transform the management of diabetes. In all of this success, we are reminded of Sir Frederick Grant Banting, who shared the Nobel Prize in Medicine and Physiology with John James Rickard Macleod in 1923 for the discovery of insulin. “It is not within the power of the properly constructed human mind to be satisfied. Progress would cease if this were the case.” 274

Acknowledgment Work on xenotransplantation at the University of Alabama at Birmingham is supported in part by NIH NIAID U19 grant AI090959.

Conflict of interest No author declares a conflict of interest.

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sharing independence of beta-cell microfilaments. Endocrinology. 2012;153(10):4644–4654. Mourad  NI, Nenquin  M, Henquin  JC. Amplification of insulin secretion by acetylcholine or phorbol ester is independent of ­beta-cell microfilaments and distinct from metabolic amplification. Mol Cell Endocrinol. 2013;367(1-2):11–20. Yang  Y, Gillis  KD. A highly Ca2 +-sensitive pool of granules is regulated by glucose and protein kinases in insulin-secreting INS-1 cells. J Gen Physiol. 2004;124(6):641–651. Bottino  R, Balamurugan  AN, Smetanka  C, et  al. Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies. Xenotransplantation. 2007;14(1):74–82. Ekser  B, Cooper  DK. Overcoming the barriers to xenotransplantation: prospects for the future. Expert Rev Clin Immunol. 2010;6(2):219–230. Graham  ML, Bellin  MD, Papas  KK, Hering  BJ, Schuurman  HJ. Species incompatibilities in the pig-to-macaque islet xenotransplant model affect transplant outcome: a comparison with allotransplantation. Xenotransplantation. 2011;18(6):328–342. Rood  PP, Cooper  DK. Islet xenotransplantation: are we really ready for clinical trials? Am J Transplant. 2006;6(6):1269–1274. Matsumoto S, Abalovich A, Wechsler C, Wynyard S, Elliott RB. Clinical benefit of islet xenotransplantation for the treatment of type 1 diabetes. EBioMedicine. 2016;12:255–262. Elliott RB, Living CT. Towards xenotransplantation of pig islets in the clinic. Curr Opin Organ Transplant. 2011;16(2):195–200. Sgroi  A, Buhler  LH, Morel  P, Sykes  M, Noel  L. International human xenotransplantation inventory. Transplantation. 2010; 90(6):597–603. Wynyard  S, Nathu  D, Garkavenko  O, Denner  J, Elliott  R. Microbiological safety of the first clinical pig islet xenotransplantation trial in New Zealand. Xenotransplantation. 2014;21(4):309–323. Cozzi  E, Tonjes  RR, Gianello  P, et  al. First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 1: update on national regulatory frameworks pertinent to clinical islet xenotransplantation. Xenotransplantation. 2016;23(1):14–24. Spizzo T, Denner J, Gazda L, et al. First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 2a: source pigs—preventing xenozoonoses. Xenotransplantation. 2016;23(1):25–31. Cowan  PJ, Ayares  D, Wolf  E, Cooper  DK. First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter  2b: genetically modified source pigs. Xenotransplantation. 2016;23(1):32–37. Rayat  GR, Gazda  LS, Hawthorne  WJ, et  al. First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 3: Porcine islet product manufacturing and release testing criteria. Xenotransplantation. 2016;23(1):38–45. Denner J, Tonjes RR, Takeuchi Y, Fishman J, Scobie L. First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter  5: recipient monitoring and response plan for preventing disease transmission. Xenotransplantation. 2016;23(1):53–59. Hering  BJ, O’Connell  PJ. First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 6: patient selection for pilot clinical trials of islet xenotransplantation. Xenotransplantation. 2016;23(1):60–76.

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260. World Health Organization. First WHO Global consultation on regulatory requirements for xenotransplantation clinical trials. In: Paper presented at The Changsha CommuniqueChangsha, China, November 19–21, 2008; 2008. 261. US Department of Health and Human Services Food and Drug Administration Center for Biologics Evaluation and Research (CBER). Guidance for Industry: Source Animal, Product, Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans; 2016. 262. Paradis  K, Langford  G, Long  Z, et  al. Search for cross-­species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. The XEN 111 Study Group. Science. 1999;285(5431):1236–1241. 263. Elliott RB, Escobar L, Garkavenko O, et al. No evidence of infection with porcine endogenous retrovirus in recipients of encapsulated porcine islet xenografts. Cell Transplant. 2000;9(6):895–901. 264. Niu  D, Wei  HJ, Lin  L, et  al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science. 2017;357(6357):1303–1307. 265. Cooper DKC, Pierson 3rd RN, Hering BJ, et al. Regulation of clinical xenotransplantation-time for a reappraisal. Transplantation. 2017;101(8):1766–1769. 266. Vanderpool  HY. The International Xenotransplantation Asso­ ciation consensus statement on conditions for u ­ndertaking clinical trials of porcine islet products in type 1 diabetes— Chapter  7: Informed consent and xenotransplantation clinical trials. Xenotransplantation. 2009;16(4):255–262.

267. Check E. Diabetes trial stirs debate on safety of xenotransplants. Nature. 2002;419(6902):5. 268. McKenzie IF, d’Apice AJ, Cooper DK. Xenotransplantation trials. Lancet. 2002;359(9325):2280–2281. 269. Sykes M, d’Apice A, Sandrin M, Committee IXAE. Position paper of the Ethics Committee of the International Xenotransplantation Association. Transplantation. 2004;78(8):1101–1107. 270. Sykes  M, Sandrin  M, D’Apice  A. Ethics Committee of the International Xenotransplantation A. Guidelines for xenotransplantation. N Engl J Med. 2003;349(13):1294–1295. 271. European Medicines Agency (EMEA) Committee for Medicinal Produce for Human Use (CHMP). Guideline on Xenogeneic CellBased Medicinal Products; 2009. 272. Health Research Council of New Zealand Gene Technology Advisory Committee. Guidelines for Preparation of Applications Involving Clinical Trials of Xenotransplantation in New Zealand; 2007. 273. American Diabetes Association. Type 1 research highlights. http://www.diabetes.org/research-and-practice/we-are-research-leaders/type-1-research-highlights/; 2018. (Accessed January 20, 2018). 274. Collip  J. Frederick Grant Banting, discoverer of insulin. Sci Monthly. 1941;52(5):472–474.

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32 Interspecies blastocyst complementation Benjamin S. Freedman Kidney Research Institute, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Division of Nephrology, Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA O U T L I N E Introduction

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Introduction Both the supply of pancreas transplants, as well as their quality, is insufficient for the number of patients who need them. For transplantation purposes, it is important that the graft be human, to avoid provoking a severe rejection response that occurs in response to organs from other species.1,2 Ideally, it would be possible to find a way to produce human organs such as the pancreas on-demand in a fully immunocompatible way, from stem cells. As for other organs, the complexity of the pancreas poses a challenge for stem cell-based bioengineering as a therapeutic strategy in humans. Human pluripotent stem cells (hPSC) can be differentiated into pancreatic islet cells, but the resulting structures (or organoids) are immature, variable in composition, and are furthermore contaminated with non-pancreas cells that could pose Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 2 https://doi.org/10.1016/B978-0-12-814831-0.00032-4

a risk of tumorigenesis.3–5 In addition, these structures produced in vitro are tiny, avascular, and lack the architectural complexity of the pancreas. Although a more sophisticated tissue could conceivably be bioengineered, bioprinted, or produced as a scaffold, the techniques for accomplishing this remain in their infancy.6–9 The bioengineering and stem cell differentiation fields therefore remain distant from producing a true therapeutic alternative to allograft pancreas transplant. An alternative strategy is to grow a human pancreas in a host species, such as a pig. In theory, such a methodology would enable farming of human organs, which could be harvested as needed for transplantation. An emerging technique to accomplish this feat is interspecies blastocyst complementation (IBC). In IBC, pluripotent stem cells from a donor species are implanted within the embryo of a different host species to fill an organ niche, which consists of a deficiency in the host’s

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ability to develop specific cell types. Here, the basic features of IBC and its successes to date will be reviewed, and both technical limitations as well as potential ethical issues arising from this technology will be discussed.

Basic principles of IBC The starting point for blastocyst complementation is an early embryo “host” (typically blastocyst-stage) that is incapable of forming a particular type of embryonic structure. For instance, the host embryo may carry lossof-function mutations in a gene essential for the development of a particular organ, such as Pdx1 in the mouse pancreas. As the host develops, this deficiency would produce a niche (vacuum) that can only be filled by cells from another source.10,11 The supplementation of the host blastocyst embryo with healthy “donor” stem cells (lacking the deficiency) creates a situation in which the donor and host cells combine to make a functional embryo. Blastocyst complementation was first tested between cells and embryos of a single species, Mus musculus. Originally, it was demonstrated that immune cells of the blood lineage (B and T lymphocytes) could be complemented by transferring wild-type mouse pluripotent stem cells into blastocysts deficient in Rag2.11 Decades later, it was determined that a similar strategy could work for the pancreas.10,12 This was followed by successful mouse-to-mouse blastocyst complementation in other solid organs, such as kidney, heart, and eye, as well as vascular endothelium.12–14 In addition, single-species blastocyst complementation has been shown to be possible in larger animal species, such as pigs.15 IBC, introduced in 2010, is a cross-species variation on the classic blastocyst complementation technique, in which the donor stem cells originate from a species different than the host embryo.10 This produces a chimera, containing cells from two different species (Fig.  1). In the absence of an organ niche, chimerism between two species would be very difficult to achieve.16–20 In contrast, in IBC, the incorporation of an organ niche into the experiment produces a developmental pressure that enables interspecies chimerism to succeed, albeit at low rates.

Generation of pancreas with IBC The pancreas was the first solid organ to be generated using IBC and remains the best characterized.10 In rodents, expression of Pdx1 is required to generate pancreas. Mice or rats lacking a functional copy of Pdx1 are unable to form pancreas and do not survive long after birth. Pdx1−/− embryos therefore have a developmental pancreas niche that needs to be filled.

FIG. 1  Schematic representation of human pancreas generation with IBC. A theoretical example of pancreas generation in a pig is shown. Note that a small proportion of pancreas tissue is of pig origin.

Taking advantage of this property, IBC was used to generate a mostly rat pancreas in a Pdx1−/− mouse.10 The pancreatic epithelium in these chimeras appeared to be entirely of rat origin, as assessed by a genetically encoded green fluorescent protein (GFP) tracing label in the donor rat iPS cells (Fig.  2). The pancreas itself was of normal morphology and size for a mouse, contained both exocrine and endocrine tissues by marker analysis, and responded to glucose challenge.10 Although most IBC animals failed to reach adulthood, two did survive, according to the original report.10 The rat-specific GFP label persisted into adulthood in the pancreatic epithelial cells of these animals, and was estimated to be present in ~80% of the cells within the organ. The remaining 20% of cells were of mouse origin, and suggested to be of non-epithelial lineages, although their specific fates were not described in detail. The achievement of IBC pancreas provides general insight into the cell-intrinsic nature of solid organ formation. Although it is known that a Pdx1−/− knockout mouse cannot form pancreas, it is less clear whether Pdx1−/− cells can contribute to a pancreas when mixed with wild-type cells (i.e., benefit from a “neighbor”

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FIG. 2  Generation of IBC rat pancreas in a mouse. Rat iPS cells expressing GFP were implanted inside a mouse blastocyst deficient in Pdx1, and the resultant mouse was sacrificed and imaged for GFP expression. Abdominal organs are labeled with abbreviations.

e­ ffect). The IBC results suggest that the intrinsic Pdx1−/− cells of the embryo remain incapable of contributing to a pancreas when mixed with wild-type neighbors. Rather, the vast majority of cells within the pancreas derive from the implanted IBC donor stem cells. This is an interesting finding that could be further investigated by carefully quantifying the appearance of mouse cells within IBC pancreas tissue, to rule out any contribution to the epithelial lineages independent of Pdx1 for specification.

Transplantability of IBC pancreas The clinical vision for IBC is to utilize it as a method to produce transplantable grafts. To test this as a therapeutic strategy, it is necessary to transplant an IBC graft out of the host into a diseased member of the donor species. Although this could conceivably done by transplanting rat-in-a-mouse pancreas tissue into diabetic rats, it was difficult to generate a sufficient mass of rat IBC pancreas tissue in mice for transplantation, because the rat pancreases grown in mice are mouse-sized and much smaller than rats.10,21 The converse experiment—transplantation of mousein-a-rat pancreatic graft into a diabetic mouse—has been successfully achieved.21 In this study, each single rat, harboring biallelic null alleles of Pdx1, was capable of producing a rat-sized IBC pancreas comprising primarily of mouse cells. Both exocrine and endocrine cells of the pancreas in these rats were primarily of mouse origin. About 200 islets could be harvested from each rat pancreas, which was sufficient to implant two diabetic (streptozotocin-induced) mice beneath the kidney capsule. Transplantation of the IBC islets rapidly and dramatically reduced serum glucose in these animals. Although the sample size was low, this rescue of glucose levels persisted for over 300 days after the transplant, and was rapidly reversible when the kidney containing the islets was removed from the animal by nephrectomy (Fig. 3). Thus, IBC islets were both necessary and sufficient to

sustain the low serum glucose levels in the diabetic mice.21 This work suggests that IBC may be a viable therapeutic strategy, at least for the pancreas. Although the transplantation of IBC islets is undoubtedly a success, it should be noted that no transplantation of whole solid organs has not yet been accomplished using IBC, including pancreas. Thus, the transplantability of IBC pancreas is not yet fully explored.

Advantages of IBC The major advantage of IBC over other methods is its ability to generate fully formed and functional organs of macroscopic size. Other methodologies, such as cadaver islet transplantation, differentiation of pluripotent stem cells in vitro, or bioprinting technologies based on spatially ordering cells in prearranged geometries, cannot produce complete organs. In contrast, the organs generated using IBC are very similar to the natural organs present in the host’s body, because they use the host as an incubator to generate the organ in a way that perfectly matches the organogenesis process. It is somewhat vexing, therefore, that IBC transplantation experiments have to date been limited to islet engraftment, rather than successful transplantation of entire organs. Undoubtedly, whole organ transplant is one of the most attractive potential applications of IBC, but it remains speculative. In addition to the pancreas, many other organs could be theoretically generated with IBC for the purposes of transplantation. IBC could therefore emerge as a competitor for existing allograft solid organ transplantation, which remains the gold standard of treatment in many organ systems. Despite substantial interest in IBC as a source of human pancreas and other organs, its adoption is limited by questions of practicality, technical feasibility, and ethics (Table  1). In the ensuing sections, we will consider some of these issues in detail, in an attempt to define a path forward for IBC in the pancreatic lineage.

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FIG. 3  Functional transplant of IBC islets. Serum glucose levels in two mice (red and blue “X” lines) transplanted beneath the kidney capsule with IBC mouse islets grown in biallelic Pdx1 mutant (Pdx1mu/mu) rat hosts. As a negative control, rat islets were transplanted, or islets were from monoallelic mutant rat hosts (Pdx1+/mu).

TABLE 1  Challenges in the application of IBC in humans Challenge

Possible solution(s)

Low efficiency of chimerism

Carefully match stem cells with embryo host

Failure of human IBC in rodents

Attempt in large domesticated species

Inefficient host breeding

SCNT host; CRISPR IBC

Late-stage rejection in host

Harvest graft early; immunosuppress host

Acute rejection in recipient

Acute immuosuppression with transplant

Hyperacute rejection in recipient

IBC vasculature; xenotransplantoptimized host

HLA mismatch

HLA-edited universal donor cells

Brain or gonadal chimerism ethics Restrict differentiation to target organ

Pretransplant immunogenicity One of the unanswered questions regarding the viability of IBC organs is their vulnerability to immune attack. In general, interspecies grafts provoke severe immune reactions. Thus, immune responses must be considered both pretransplant (during development of the IBC chimera) and posttransplant (after harvest and implantation of the graft).

It is remarkable that interspecies chimeras can exist at all, given the extreme nature of xenotransplant immune responses.2,22 The existence of IBC animals would suggest that immune tolerance is induced by the co-­ development of the chimeric donor cells within the host. However, we do not yet know the extent to which this tolerance induction might factor into the success rate of IBC, which is generally low. Performing IBC in immunodeficient hosts could conceivably improve the efficiency of IBC, if tolerance induction is a major issue in the success of the procedure. Although tolerance does appear to be induced in IBC embryos to some degree, there are concerns that rejection could occur during long-term development. A study in which IBC mouse pancreas was generated in rats noted that the host rats suffered from a form of immunologically mediated juvenile diabetes accompanied by lymphocyte invasion of acinars and islets, and pancreatic deterioration both structurally and functionally.21 This is fascinating as it suggests that the IBC organ may be attacked by the host’s immune system, even though it developed together with the rest of the embryo and clearly enjoys some measure of immune tolerance. It is also noteworthy that most of the IBC animals born to date have not remained viable into adulthood.10,21 Whether this is due to a rejection event, inadequate functionality of the IBC organ, or a consequence of off-target chimerism in other tissues is not yet clear.

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Posttransplant immunogenicity After transplantation, concern shifts to possible rejection of the organ in the donor/recipient species due to the contaminated cells of the IBC host species. We do not yet fully understand how organs like the pancreas form and how many different cell populations may be present. Even a small population of cells, if derived from the non-recipient species, could potentially initiate a rejection event in the recipient. The ensuing destruction of this population, or the rejection event itself, may compromise the function of the IBC organ, or cause systemic problems. For this reason, it is critical to test the safety and longevity of IBC grafts in animal models with functional immune systems, as was done in the IBC mouse-in-rat islet transplantation experiments.21 In those experiments, when IBC islets were transplanted into mice, the graft recipients were dosed with tacrolimus and a set of anti-inflammatory monoclonal antibodies at implantation and for the ensuing 5 days. This appears to have been sufficient to offset any severe immune rejection events that might have endangered the graft. Immunosuppressive therapy was discontinued after 5 days, and the mice survived for many months afterwards with normal glycemic control. As the islets were probably not 100% donor-derived, it does appear that at least some foreign-species cells can be tolerated by the transplant recipient, when immunosuppressed in an acute way following the operation. Given the attractive potential of IBC for whole organ transplantation, it is worth considering why islets were transplanted instead of whole pancreas in the rodent model. While it is true that whole pancreas transplant in the mouse requires substantial technical skill to accomplish the surgery, it can be successfully performed, and would have made a more dramatic demonstration of transformative potential of IBC than islet transplantation. Size mismatch between organs of rat and mouse, although considerable, may also have been a surmountable challenge, just as children can be transplanted with organs taken from adults. Why, then, was whole IBC pancreas transplant not performed? A possible answer to this question may lie in the observation that IBC organs are only partial chimeras. Although 80% of cells within an IBC pancreas may be derived from the donor species, that still leaves 20% of cells of host species.10,21 These host species cells may present difficulties for transplantation of the whole IBC organ. In particular, the vasculature derives from a distinct germ layer from the pancreatic epithelium, and therefore is host-derived in IBC pancreas. Transplantation of the pancreas into the donor species would necessarily involve this host-derived blood supply. Interactions

between circulating blood cells from the donor species and the endothelial wall of the host species could provoke a “hyperacute” rejection event, such as the rapid clotting of blood that occurs in pig kidneys when transplanted into primates.1,2,23 Pig endothelia provoke this response because they express galactosyl-α1,3,-galactose and other carbohydrate antigens against which humans and other primates have specific, circulating humoral antibodies. Acute immunosuppression might not be sufficient to prevent such a hyperacute rejection event. Thus, there is a certain advantage to purifying islets and transplanting them in the absence of the entire pancreas, in that it avoids direct interaction between the recipient immune system and the interspecies vasculature. Although the graft of islets beneath the kidney capsule is likely to be less functional than an intact pancreas, it may be a less risky approach, and suitable for proof of principle of IBC graft function after transplant.

Potential for IBC vasculature Admittedly, limiting IBC to avascular grafts such as pancreatic islets partially defeats the greatest advantage of doing IBC in the first place, which is to generate a fully functional, intact organ. To generate an IBC organ including a vasculature that is wholly derived from the donor species would therefore be a more ideal therapeutic strategy. To begin to address this possibility, same-species blastocyst complementation experiments have been performed to generate mice with exogenic vasculature.14 In these experiments, a loss-of-function Flk1 mutant was used as the host embryo. Implantation of mouse pluripotent stem cells produced chimeric mice (about 10% of all live births) in which both the vasculature and the hematopoietic lineage appeared to be ~100% donor-­ derived. Other components of the blood vessels, such as the smooth muscle, were a mixture of donor and host cells. In the same study, the authors tested the ability of rat cells to generate IBC endothelium within a mouse. Unfortunately, this resulted in embryonic lethality.14 Thus, it may not be possible to generate a viable IBC chimera with wholly donor vasculature (e.g., a live rat with mouse blood vessels), given the pleiotropic and central role of the vasculature in organizing and nourishing the body. It is notable, however, that IBC vasculature embryos in this experiment did survive until gestational day 9.5. This may be sufficient time for certain embryonic rudiments to form. Thus, one strategy may be to remove embryonic rudiments from IBC embryos at an early time point, while the embryo is still viable, and use these for transplantation. Such a strategy would depend on

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whether the rudiments could mature sufficiently in the context of the new recipient. Alternatively, it is conceivable that there may be subtypes of vascular endothelium that are specific to the pancreas, as has been suggested for other solid organs.24 If so, the pancreas-specific vasculature may have specific genetic vulnerabilities that can be exploited to create a pancreatic vasculature niche. This could be combined in trans with Pdx1 or an equivalent gene to create a donor pancreas with a donor vascular tree within a host species. Such technologies will require substantive advances in our understanding of vascular developmental biology at the organ-specific level and our ability to manipulate those boundaries. Besides the vasculature, there may yet be other types of systemic cells that cannot be made wholly human. For instance, resident blood cells (e.g., lymphocytes or dendritic cells) in a human IBC pancreas would still derive from the nonhuman host. These would need to be depleted from the graft, or else accepted as a contaminant that could introduce safety concerns. Notably, the same genetic mutations that are permissive for the creation of IBC vasculature also generate IBC blood cell populations,14 providing a possible avenue of attack for this lineage.

Autograft tolerance A final, but not insignificant, immune consideration is the allogeneic immunogenicity of the eventual IBC graft. Ideally, an IBC transplant would be an autograft. It is furthermore possible to conceive of an autograft strategy, using induced pluripotent stem (iPS) cells for IBC. iPS cells can be derived from practically any patient’s somatic cells, by reprogramming these cells with genes expressed in embryonic stem (ES) cells and growth ­factors.25,26 An iPS IBC organ would therefore be a true “perfect match” for its recipient. Although in theory we would not expect any adverse reaction to the human cells within an iPS IBC organ, in practice we cannot know for certain whether such cells might provoke some form of immune response. The immune system is highly complex and sensitive, and we do not yet fully understand its intricacies. As the iPS IBC organ has developed outside of the body into which it is being transplanted, there is a chance that its antigen presentation may in some way differ from the recipient. There is some suggestion that such immune responses are a general outcome of iPS cell-derived transplants (e.g., those generated purely in vitro), although most of the data suggests that these are likely to be safe, at least in animal models.27–29 The possibility of autologous rejection is compounded by the consideration that the iPS IBC organ has ­developed

in the presence of external cues from the host species (e.g., the mouse) cells for its entire life. During this time, the organ may have incorporated host antigens, and could present them, even though the organ cells themselves are autologous with the recipient. Such foreign antigen presentation could conceivably provoke an autoimmune response. Thus, even autologous transplantation strategies are not necessarily foolproof in IBC, and need to be tested first in animal models. In this regard, it is encouraging that IBC mouse pancreatic islets were well tolerated in the C57BL/6 inbred background, which was syngeneic with the stem cells used to derive them, and is analogous to an autograft.21 Nevertheless, it is important to demonstrate that the same holds true for other species, which may be more distant evolutionarily than mouse and rat.

Allograft considerations While autograft IBC is attractive as an ideal strategy, in practice it may be very difficult to generate “personalized” IBC grafts tailored for each individual patient. Generating iPS cells can be a challenging and an expensive proposition. Furthermore, every iPS cell line is different with regard to its ability to differentiate, and it is possible that certain ones will work better for IBC than others. Significant quality control effort must accompany the process of generating any individual cell line to ensure that it does not become tumorigenic or otherwise carry mutations that could damage the host.29 Given the low efficiency of IBC to begin with, adding a requirement to optimize the process for each individual cell line may well place the technology out of reach. Even if these considerations can be overcome, if autologously derived, the IBC graft will carry the same mutations that caused disease in the recipient’s pancreas, which may result in the production of an IBC pancreas with disease (or require a costly genetic correction step to avoid this, if the genes involved can be identified). Thus, at least in the short term, it seems unlikely that iPS IBC will be developed at the level of personalized therapy. One possibility is to instead utilize allograft iPS cells, which could be produced from a subset of human leukocyte antigen (HLA)-matched founder lines.30,31 The resultant IBC allografts could be HLA-matched to the patient recipient, to reduce the likelihood of acute or chronic rejection events. Although such an approach might be convenient, and could conceivably improve the supply chain for these organs, it is worth considering whether an IBC allograft strategy could realistically compete with deceased donor pancreas as a source of transplant. After all, such an IBC allograft pancreas would still require HLA matching and immunosuppression, much like a deceased donor pancreas, and would come with all of the added risks associated with IBC, in addition to

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its technical difficulty and expense. Pancreatic islets are also immunologically complex, for instance, expressing HLA-G, and the effect this complexity might have on IBC transplantation is not yet clear.32 Thus, allograft IBC may not be a viable approach in today’s market and in lieu of further research. Alternatively, multiple groups are engaged in developing “universal donor” cell lines that are engineered to express a limited set of HLA molecules that enable them to evade the immune system and cannot be readily recognized or rejected by a host.33,34 These could make a useful source of donor pluripotent stem cell IBC. Such organs could have clear superiority over deceased donor organs, in that they would not require HLA matching or immunosuppression. In addition, only one founder pluripotent stem cell line could suffice for thousands of recipients. This would be a true “off the shelf” pancreas for transplant, and a worthy successor to deceased donor pancreases. Attractive as this vision may be, there are also certain risks associated with HLA-engineered cells. For instance, should any cell within the organ become tumorigenic, it is likely that the tumor could evade the immune system of the host, due to its natural invisibility to the immune system. We do not fully understand how the immune system will interact with universal cells over long periods of time, particularly in humans. There is also a substantial risk that the universal cells could become contagious and spread from person to person, like certain cellular cancers found in canines and Tasmanian devils. These risk factors need to be assessed in animal models and mitigated to avoid adverse outcomes of universal cell IBC transplantation, compared to conventional transplant modalities.

Efficiency of IBC The efficiency of IBC is currently very low. In the original report of pancreas IBC, 10 mice with rat pancreas were born, out of a total of 139 implanted embryos.10 In addition, only 2 of the 10 that were born survived to adulthood. While this level of success may be tolerable for certain mouse experiments, where a large litter is born every 3 weeks, it is unlikely to be useful in larger, more clinically relevant species with much longer gestational times. Fundamentally, why the success rate of IBC is so low is not yet understood at the mechanistic level. It is clear that the IBC success rate is substantially lower than same-species blastocyst complementation. Even in the absence of IBC, interspecies chimerism is lower than intraspecies chimerism, and this is true even in rodents, which are relatively tolerant of chimerism between different species.35 This may be attributable to mismatches

between the pluripotent stem cells of the donor species with the host embryo at the stage of the blastocyst, or later stages such as the induction of pancreas. As we have already discussed, the role of cross-species immune rejection is currently poorly understood in blastocyst complementation, and may also contribute to the generally low success rate of IBC. Attempts have been made to produce better matching between host embryo and donor IBC cells, in the hopes of increasing the efficiency of IBC. One issue has been that pluripotent stem cells from different species have strikingly different properties. Mouse ES cells, for instance, tend to stabilize in vitro in a “naïve” pluripotent state, which is highly compact and do not depend on fibroblast growth factor signaling. In contrast, human ES cells grow in a “primed” state that is closer to the epiblast stage of the embryo, depend on fibroblast growth factor, and forms disc-like colonies in culture. As might be predicted, these two types of cells do not mix particularly well in the setting of a blastocyst.16–20 Recent work has attempted to identify culture conditions for hPSC (including both embryonic and iPS cells) that enhance chimerism during blastocyst complementation in rodents, possibly by mimicking the naïve state.18,36–38 Although most of this work has been performed on mice, finding appropriate match is particularly important in non-rodent species. In one recent study, the potential for chimerism between human stem cells and two large domestic species, cattle and pigs, was explored.13 These studies suggested that the pig could in some cases exhibit a very low degree of chimerism from human cells, although this likely represented less than one-tenth of 1% of all cells in the embryo. Such experiments have only been performed in the absence of IBC. A critical experiment, which has not yet been performed, is to evaluate human chimerism in these species in the context of IBC. Whether IBC could be successfully performed using human cells in a pig embryo is not yet clear, from a technical standpoint.

Breeding schemes The genetics of producing blastocyst complementation offspring are rather complicated. Due to the nature of the complementation-associated mutations, which prevent the formation of essential organs, the host animals can rarely be bred as homozygotes. The simplest approach is to breed heterozygotes (e.g., Pdx1+/−), but due to the Mendelian rules only ~25% of all resultant embryos are homozygous nulls and appropriate hosts for IBC. To identify appropriate host embryos, one strategy is to screen for homozygous null embryos using b ­ lastocyst

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polymerase chain reaction (single cell biopsy of the blastocyst, followed by PCR (polymerase chain reaction) of the mutant locus), which could then be selected for IBC. Although this approach can reduce the number of embryos needed for implantation, it would need to be timed carefully to coordinate it with transferring of the embryos into the pseudopregnant mother. It should also be noted that heterozygous breeders may also manifest disease phenotypes, due to haploinsufficiency. For instance, heterozygous Pdx1 rats exhibit a diabetic phenotype.21 The impact of such illnesses on breeding efficiencies must therefore also be taken into consideration when heterozygotes are utilized. An alternative breeding scheme to increase efficiency of IBC incorporates blastocyst complementation within the host species to generate suitable parents. For instance, to generate rat pancreas in mouse, the male parent was a chimeric Pdx1−/− mouse that had received complementing wild-type cells as a blastocyst. This mouse could be mated with multiple Pdx1+/− female mice to obtain litters, each of which was 50% Pdx1−/−. If a female chimera could be obtained, the ratio could be increased to 100%. This illustrates how creative application of blastocyst complementation can be used to circumvent some of the limitations associated with the technique. In a similar vein, tetraploid complementation can be used as an alternative to conventional Mendelian breeding schemes to generate a host soma of the desired genotype. This begins by intentionally fusing together the cells of a two-stage wild-type blastocyst into a single cell, using electrical current. Without complementation, the resultant tetraploid cell will proceed through the early stages of embryonic development, and will be capable of generating extra-embryonic tissues of the embryo, but will not be able to generate soma in the longer term. To perform tetraploid complementation, the tetraploid blastocyst or morula can be injected with ES cells of the desired genotype. The resulting soma will derive entirely from the injected ES cells. To generate chimeras (e.g., for IBC), ES cells carrying the host knockout mutation can be injected together with the complementing cells.39 In an IBC setting, the resultant embryo would be guaranteed to be a knockout, circumventing the inefficiency issue that plagues Mendelian breeding schemes. Although this is an interesting approach, there are certain limitations. Tetraploid complementation is a specialized technique that may not be readily available to many laboratories. It is typically performed only in mice, and may not work in other species with more clinical relevance for human IBC. Blastocyst complementation via tetraploid complementation has only been demonstrated within the mouse species, using mouse donor and mouse host cells in a tetraploid embryo—IBC has not yet been demonstrated.39

Another possible methodology that could potentially be used to increase the efficiency of IBC breeding is somatic cell nuclear transfer (SCNT), in which a somatic nucleus is implanted into an enucleated egg to produce a zygote. SCNT, which is also known as reproductive cloning, offers complete control over the donor genome, and is used relatively frequently in pigs to generate mutants.40,41 It is compatible with blastocyst complementation techniques, as a means of generating a knockout host, as was demonstrated for same-species blastocyst complementation of pancreas in pigs.15 The drawback of this approach, however, is that SCNT is itself less efficient than straightforward breeding schemes, and comes with a substantial risk of embryo loss or disease. It is not difficult to imagine, however, that the process of using SCNT to generate IBC hosts might be optimized in a choice species, by selecting the most compatible cell lines for this process as nuclear donors, which would result in a streamlined approach for the large-scale “farming” of organs.

Gene editing with IBC A relatively new approach to circumvent classical breeding schemes is to utilize genome editing in host embryos to target a gene or genes of interest. The CRISPR (clustered regularly interspaced short palindromic repeats) gene editing system has been used to accomplish this in the context of IBC in the mouse.13 The CRISPR system uses a nuclease (Cas9) that can be directed to specific loci in the genome by a short RNA sequence, called a guide RNA.42,43 Mismatch repair of the resultant double-stranded break frequently introduces short insertions or deletions (indels) at the targeted locus. This is a powerful methodology for editing the genome, and particularly for disrupting genes. To test the potential of CRISPR for IBC, a guide RNA targeting a gene required for organogenesis of the pancreas, heart, or eye was injected into mouse zygote-stage embryos, together with the Cas9 enzyme.13 This produced knockout host blastocysts, which were then injected with rat iPS cells that had been transiently exposed to a fluorescent label, into elicit IBC. The resultant chimeras contained high contribution of rat cells in the organ of interest, with substantially lower contribution elsewhere in the body. Although the CRISPR approach has substantial advantages in terms of its ease of use, there are some concerns regarding its reproducibility and efficiency.44 In particular, the likelihood of mosaicism in such embryos is a concern that needs to be more fully addressed. Because the mutation is introduced at the zygote stage, there is a substantial risk that knockout will occur only in a proportion of the embryo’s cells. This could lead to a false positive rate

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Ethical concerns

of apparent chimerism, which would be enhanced by the presence of host species cells. Indeed, transplantability experiments designed to show clinical relevance have eschewed using the first generation of gene edited rat hosts, in favor of using “F1” second-generation animals with confirmed germline mutations.21

Suitability of large animal hosts To date, IBC has only been demonstrated in rodent species. For the approach to work for humans, however, it will likely to be necessary to successfully execute IBC with human donor cells in a large animal species. This is for two reasons: first, hPSC have consistently been unsuccessful in engrafting in rodent embryos; and second, a rodent host is unlikely to be of sufficient size to produce functional organ for a human being. To date, however, no one has been successful in using human cells for IBC in any animal species. Practically speaking, it is important to find a species in which human IBC might actually work, that is, the embryos should be sufficiently compatible to successfully produce a human IBC organ in the host. Nonhuman primates would seem to be a logical choice from an evolutionary point of view, but the ethical and financial challenges of performing experiments in such species, outside of their natural habitat, makes them less than suitable for these types of applications. Thus, a domesticated species typically farmed and consumed by our society would be a far better choice, if IBC could be made to work. From a xenogenicity standpoint, the same considerations that make a host more suitable for xenograft also apply for IBC. Pigs, for instance, have certain advantages for xenotransplantation, including their wide availability, domesticated nature, and organ structure similar to humans.1,2 However, pigs also have specific drawbacks including endogenous viruses that might spread to humans,40,45 and antigens that provoke a hyperacute rejection response in primates.1,2 To some degree these concerns can be ameliorated using genetic strains designed to improve cross-species xenotolerance or safety.40,45–47 It might be possible to perform IBC in such strains, as well, to reduce the risk of graft rejection or recipient infection.

Ethical concerns There are significant ethical concerns surrounding the creation of animals containing human cells. Although IBC enriches for chimerism in a particular organ, a low degree of chimerism may also arise in non-IBC organs.13 IBC donor hPSC can potentially differentiate into any of

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the body’s somatic cell types. In particular, there is a concern that human brain or germ cells would be present in an IBC chimera that was implanted with hPSC.44,48 The presence of human brain cells being generated inside animals would raise questions about the consciousness of the organism and whether such a chimera would have human rights. The brain is a particularly sensitive organ for pancreas IBC, because Pdx1 is expressed not only in the pancreas but also in certain types of cells in the brain.49 Thus there is a risk that generating Pdx1 chimeras would create a niche in the brain, as well as in the pancreas. In addition to the brain, there is also concern over the potential of human cells to enter the germline of another species. This could occur, for instance, if a mutation were present that enabled the generation of human eggs or sperm within an animal. This could create a situation where human reproduction becomes uncoupled from speciation, and perhaps even lead to hybrids between humans and other species. Human germ cells, even very rare ones, would potentially enable chimeras to mate with one another and conceive wholly human embryos inside their reproductive organs, which would raise contentious questions regarding the rights of those embryos. Due to concerns such as these, it is currently illegal to create human-animal chimeras in Japan. The Japanese group that invented IBC was therefore compelled to transfer its studies to the United States to perform experiments in pigs with human cells. Even in the United States, the National Institutes of Health has instituted a funding moratorium on these types of experiments, due to the ethical uncertainties surrounding this research strategy. Thus, while not illegal in the United States, financial support for IBC studies is limited to private funds.44,48 One proactive approach to reduce the ethical risk is to utilize genetic tools to direct differentiation of the donor cells into the lineage of choice, rather than off-target lineages. This has been tested in mouse same-species blastocyst complementation, using donor stem cells that were genetically engineered to express Mixl1, a transcription factor that biases early embryonic cells toward an endodermal fate. When Mixl1 was forcibly expressed during the first several days of embryonic development, either using a doxycycline controllable promoter or expression from the OCT4 promoter, the resultant embryos showed reduced contribution of the donor stem cells to non-endodermal germ layer derivatives, such as the fur coat.39 However, the extent to which this skewed the differentiation away from off-target lineages was not carefully quantified, and it is possible that some cells escaped selection. Other strategies may also be possible. For instance, rather than implant hPSC, it might be possible to instead implant a more differentiated stem cell specific to

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the organ of interest (for instance, pancreatic progenitor cells). These would be restricted to a specific cell fate and would unlikely contribute to other organs such as brain or gonads. It is also worth noting that the levels of chimerism between humans and domestic large animals in the absence of IBC is extremely low to date, and therefore may ultimately be negligible at the functional level.13 When embryos from different species have been combined into one in the absence of IBC, chimerism has only been successfully achieved between certain closely related species such as mouse and rat, or goat and sheep.10,50,51 Although there are examples of hPSC contributing to rodent embryo formation, chimerism levels are very low and do not produce viable animals.16–20 As a result, in IBC, donor cells contribute mainly to niche cell types but not in a significant way to the rest of the embryo. The formation of gallbladder in chimeric animals also suggests a strong need for a survival-promoting niche to produce mature structures. Mice naturally possess gallbladders while rats do not. Although rat blastocysts can tolerate a significant degree of chimerism from mouse cells (>20% of cells in certain lineages), gall bladders were never observed in such interspecies chimeras.10 Thus, an empty organ niche is necessary, but may not be sufficient, to drive IBC in the absence of a biological need for the organ in question. There is also a strong ethical argument to be made in favor of the potential benefits of IBC for patients in need of transplantation, many of whom may never receive an allograft transplant due to the restrictions of current waiting lists and the global organ shortage. If IBC can be made fully immunocompatible, without the need for chronic immunosuppression, for instance via an autograft strategy as described above, this would also represent a major advantage over existing organ replacement therapies, which are associated with strong side effects of the immunosuppressive medications. Thus, the ethical concerns surrounding IBC may be surmountable, either from a technical or a philosophical perspective, and development of IBC in a safe and responsible manner through preclinical research should continue. Nevertheless, as this work proceeds toward more clinically relevant experiments with human cells, it is important that it do so carefully, to avoid outcomes that traverse ethical lines. Such experiments could be widely perceived as “going too far,” and could usher in a backlash against scientific research.

Outlook Demonstration that IBC pancreas can be generated and used for islet transplantation in the mouse is an important advance and proof of principle experiment.

Such experiments have not yet been performed to generate an IBC human pancreas. At this time, there remain more challenges in this field than solutions. To achieve the grand vision of IBC in a therapeutic context, critical issues, both technical and ethical, need to be addressed in preclinical models. Creative solutions using gene editing and other disruptive technologies, combined with trial-and-error approaches, are likely to be required to advance IBC into a viable option for the clinic. As this work develops, presumably there will come a point at which the benefits of having a fully formed, functional, and mostly human IBC pancreas available for transplant will outweigh the risks of not receiving such a transplant, at least for certain patients. At that point, IBC pancreas will be ready for clinical trials. Such trials must be performed very slowly and carefully, initially with islets and subsequently with whole pancreases, with abundant monitoring of the patients for both acute and chronic complications, including rejection and infection. As with allograft transplantation of solid organs, this is likely to be a long process, which will require patience and persistence to achieve successfully. The reward may be a far more functional system for pancreas transplant, with substantial increases in availability, safety, and efficacy, than is currently possible.

References 1. Chen G, Qian H, Starzl T, et al. Acute rejection is associated with antibodies to non-gal antigens in baboons using gal-knockout pig kidneys. Nat Med. 2005;11(12):1295–1298. 2. Cooper  DK, Ezzelarab  MB, Hara  H, et  al. The pathobiology of pig-to-primate xenotransplantation: a historical review. Xenotransplantation. 2016;23(2):83–105. 3. Pagliuca FW, Millman JR, Gurtler M, et al. Generation of functional human pancreatic beta cells in vitro. Cell. 2014;159(2):428–439. 4. Russ HA, Parent AV, Ringler JJ, et al. Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro. EMBO J. 2015;34(13):1759–1772. 5. Saber N, Bruin JE, O’Dwyer S, Schuster H, Rezania A, Kieffer TJ. Sex differences in maturation of human embryonic stem cell-­ derived beta cells in mice. Endocrinology. 2018;159(4):1827–1841. 6. Baidal  DA, Ricordi  C, Berman  DM, et  al. Bioengineering of an intraabdominal endocrine pancreas. N Engl J Med. 2017;376(19):1887–1889. 7. Kang HW, Lee SJ, Ko IK, Kengla C, Yoo JJ, Atala A. A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat Biotechnol. 2016;34(3):312–319. 8. Norotte  C, Marga  FS, Niklason  LE, Forgacs  G. Scaffold-free vascular tissue engineering using bioprinting. Biomaterials. 2009;30(30):5910–5917. 9. Miller JS, Stevens KR, Yang MT, et al. Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nat Mater. 2012;11(9):768–774. 10. Kobayashi T, Yamaguchi T, Hamanaka S, et al. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell. 2010;142(5):787–799. 11. Chen J, Lansford R, Stewart V, Young F, Alt FW. RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development. Proc Natl Acad Sci U S A. 1993;90(10):4528–4532.

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12. Usui J, Kobayashi T, Yamaguchi T, Knisely AS, Nishinakamura R, Nakauchi H. Generation of kidney from pluripotent stem cells via blastocyst complementation. Am J Pathol. 2012;180(6):2417–2426. 13. Wu J, Platero-Luengo A, Sakurai M, et al. Interspecies chimerism with mammalian pluripotent stem cells. Cell. 2017;168(3):473–486 e415. 14. Hamanaka S, Umino A, Sato H, et al. Generation of vascular endothelial cells and hematopoietic cells by blastocyst complementation. Stem Cell Reports. 2018;11(4):988–997. 15. Matsunari  H, Nagashima  H, Watanabe  M, et  al. Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs. Proc Natl Acad Sci U S A. 2013;110(12):4557–4562. 16. James  D, Noggle  SA, Swigut  T, Brivanlou  AH. Contribution of human embryonic stem cells to mouse blastocysts. Dev Biol. 2006;295(1):90–102. 17. Mascetti  VL, Pedersen  RA. Human-mouse chimerism validates human stem cell pluripotency. Cell Stem Cell. 2016;18(1):67–72. 18. Gafni  O, Weinberger  L, Mansour  AA, et  al. Derivation of novel human ground state naive pluripotent stem cells. Nature. 2013;504(7479):282–286. 19. Theunissen TW, Friedli M, He Y, et al. Molecular criteria for defining the naive human pluripotent state. Cell Stem Cell. 2016;19(4):502–515. 20. Wu  J, Okamura  D, Li  M, et  al. An alternative pluripotent state confers interspecies chimaeric competency. Nature. 2015;521(7552):316–321. 21. Yamaguchi  T, Sato  H, Kato-Itoh  M, et  al. Interspecies organogenesis generates autologous functional islets. Nature. 2017;542(7640):191–196. 22. Cowan PJ, Tector AJ. The resurgence of xenotransplantation. Am J Transplant. 2017. 23. Hawthorne  WJ, Griffin  AD, Lau  H, et  al. Experimental hyperacute rejection in pancreas allotransplants. Transplantation. 1996;62(3):324–329. 24. Marcu R, Choi YJ, Xue J, et al. Human organ-specific endothelial cell heterogeneity. iScience. 2018;4:20–35. 25. Takahashi  K, Yamanaka  S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–676. 26. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861–872. 27. Guha P, Morgan JW, Mostoslavsky G, Rodrigues NP, Boyd AS. Lack of immune response to differentiated cells derived from syngeneic induced pluripotent stem cells. Cell Stem Cell. 2013;12(4):407–412. 28. Araki R, Uda M, Hoki Y, et al. Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells. Nature. 2013;494(7435):100–104. 29. Mandai  M, Watanabe  A, Kurimoto  Y, et  al. Autologous induced stem-cell-derived retinal cells for macular degeneration. N Engl J Med. 2017;376(11):1038–1046. 30. Turner  M, Leslie  S, Martin  NG, et  al. Toward the development of a global induced pluripotent stem cell library. Cell Stem Cell. 2013;13(4):382–384. 31. Baghbaderani BA, Tian X, Neo BH, et al. cGMP-manufactured human induced pluripotent stem cells are available for pre-clinical and clinical applications. Stem Cell Reports. 2015;5(4):647–659. 32. Cirulli V, Zalatan J, McMaster M, et al. The class I HLA repertoire of pancreatic islets comprises the nonclassical class Ib antigen HLA-G. Diabetes. 2006;55(5):1214–1222.

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33. Rong  Z, Wang  M, Hu  Z, et  al. An effective approach to prevent immune rejection of human ESC-derived allografts. Cell Stem Cell. 2014;14(1):121–130. 34. Gornalusse GG, Hirata RK, Funk SE, et al. HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nat Biotechnol. 2017;35(8):765–772. 35. Yamaguchi T, Sato H, Kobayashi T, et al. An interspecies barrier to tetraploid complementation and chimera formation. Sci Rep. 2018;8(1):15289. 36. Ware  CB, Nelson  AM, Mecham  B, et  al. Derivation of naive human embryonic stem cells. Proc Natl Acad Sci U S A. 2014;111(12):4484–4489. 37. Theunissen TW, Powell BE, Wang H, et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell. 2014;15(4):471–487. 38. Yang  Y, Liu  B, Xu  J, et  al. Derivation of pluripotent stem cells with in  vivo embryonic and extraembryonic potency. Cell. 2017;169(2):243–257 e225. 39. Kobayashi  T, Kato-Itoh  M, Nakauchi  H. Targeted organ generation using Mixl1-inducible mouse pluripotent stem cells in blastocyst complementation. Stem Cells Dev. 2015;24(2):182–189. 40. Yang  L, Guell  M, Niu  D, et  al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science. 2015;350(6264):1101–1104. 41. Lai L, Kolber-Simonds D, Park KW, et al. Production of alpha-1,3-­ galactosyltransferase knockout pigs by nuclear transfer cloning. Science. 2002;295(5557):1089–1092. 42. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012;109(39): E2579–E2586. 43. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816–821. 44. Freedman BS. Hopes and difficulties for blastocyst complementation. Nephron. 2018;138(1):42–47. 45. Niu  D, Wei  HJ, Lin  L, et  al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science. 2017;357(6357):1303–1307. 46. Higginbotham  L, Mathews  D, Breeden  CA, et  al. Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-to-primate kidney transplant model. Xenotransplantation. 2015;22(3):221–230. 47. Iwase H, Liu H, Wijkstrom M, et al. Pig kidney graft survival in a baboon for 136 days: longest life-supporting organ graft survival to date. Xenotransplantation. 2015;22(4):302–309. 48. Bourret  R, Martinez  E, Vialla  F, Giquel  C, Thonnat-Marin  A, De Vos  J. Human-animal chimeras: ethical issues about farming chimeric animals bearing human organs. Stem Cell Res Ther. 2016;7(1):87. 49. Song J, Xu Y, Hu X, Choi B, Tong Q. Brain expression of Cre recombinase driven by pancreas-specific promoters. Genesis. 2010;48(11):628–634. 50. Rossant J, Frels WI. Interspecific chimeras in mammals: successful production of live chimeras between Mus musculus and Mus caroli. Science. 1980;208(4442):419–421. 51. Fehilly  CB, Willadsen  SM, Tucker  EM. Interspecific chimaerism between sheep and goat. Nature. 1984;307(5952):634–636.

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C H A P T E R

33 Bioengineering, biomaterials, and β-cell replacement therapy Rick de Vries, Adam Stell, Sami Mohammed, Carolin Hermanns, Adela Helvia Martinez, Marlon Jetten, Aart van Apeldoorn MERLN Institute for Technology-Inspired Regenerative Medicine, Complex Tissue Regeneration, Maastricht University, Maastricht, The Netherlands O U T L I N E Introduction

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Biomaterials Hydrogels Natural hydrogels Decellularized ECM-based biomaterials Synthetic hydrogels Solid biomaterials Nondegradable biomaterials Degradable biomaterials

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Introduction Clinical islet transplantation (CIT) is a promising therapy for type 1 diabetes. However, the success of CIT is limited by a decrease of islet mass caused by mechanical stress, a lack of oxygen flow toward the islets due to lack of vascularization, an immediate blood-mediated immune response, a relatively high concentration of immunosuppressive drugs as well as exhaustion of islets due to glucotoxicity in the intrahepatic environment.1–4 Extrahepatic islet transplantation with the help of a ­tailor-made implant could provide an improvement on the issues discussed above. Such a β-cell replacement device should lead to improved islet viability, and therefore a reduction in the amount of donor organs needed for transplantation. Moreover, islet or β-cell delivery devices Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 2 https://doi.org/10.1016/B978-0-12-814831-0.00033-6

Islet delivery device fabrication techniques Scaffold fabrication techniques with random islet distribution Microfabrication techniques (controlled islet distribution)

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can potentially offer enhanced maintenance of glycemia, reduction, or even absence of the need to use exogenous insulin, resulting in a lower risk of long-term complications linked to type 1 diabetes such as nephropathy, retinopathy, and neuropathy.5 There are several requirements for devices used in extrahepatic transplantation sites including the ability to house a sufficient number of islets, or β cells, the presence of a dense vascular network in close proximity, or the capacity to initiate the formation of one, allowing for swift revascularization ensuring an efficient exchange of nutrients, metabolites, and hormones, and maintenance of adequate oxygen tension. Other preferred characteristics should be a minimal or no inflammatory response against the materials used in the device, which could potentially lead to islet loss, easy surgical accessibility

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with minimal operative risks, the ability for graft recovery and noninvasive monitoring. Several extrahepatic transplantation sites have been suggested including underneath the kidney capsule, within the bone marrow, spleen, the greater omentum, gastric submucosal space, intramuscular, endovascular, peritoneal cavity, and epididymal fat pad, each with their own (dis)advantages.6–12 Different strategies can be taken to realize extrahepatic transplantation like the use of an immunoprotective encapsulation device, or a macroporous delivery device aimed to restore revascularization upon implantation, see Fig. 1.

Immunoprotective barrier strategy Both CIT and macroporous delivery devices rely on systemic administration of immunosuppressive drugs to prevent implant rejection by the host. Nevertheless, patients undergoing chronic immunosuppressive therapy are exposed to considerable side effects including a

significant risk for higher rates of malignancies and opportunistic infections, as well as loss of islet mass.13 Cell encapsulation technology offers an alternative strategy, where a passive barrier (either a dense hydrogel network or a polymer membrane) separates implanted cells from the hostile immune system (see Figs. 1 and 2). This barrier has a certain cut-off, allowing diffusion of low-molecular-weight molecules (nutrients, glucose, insulin, and oxygen) through the barrier, while larger components of the immune system are blocked. The pore size, or, in case of hydrogels, mesh size, determines the effectivity of such barriers. Pores in the range of several micrometers ensure cellular immunity against cytotoxic T-lymphocytes, while humoral immunity can only be achieved by pore radii in the range of 10 nm.13 Graft rejection can be further aggravated by inflammation stemming from tissue irritation during surgery, and the implantation of a foreign (bio)material within the human body. The innate immune system will be activated, and neutrophils, macrophages, and basophils start releasing cytokines and aid in wound repair. Some cytokines are

FIG. 1  Schematic overview of immunoprotective and macroporous revascularization device strategy principles. (A) Immunoprotective strategy in which islets are seeded in a device consisting of two microporous membranes that allow free diffusion of glucose, oxygen, and insulin, while blocking immune cells. Over time, vasculature will grow over the device, but islets will still depend on diffusion through a barrier to obtain nutrients. (B) Revascularization approach in which islets are seeded within a device consisting of macroporous membranes that allow free diffusion of glucose, oxygen insulin, and immune cells, as well as blood vessel ingrowth over time.

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Revascularization strategy

FIG. 2  The immunoprotective and revascularization approaches for hydrogel encapsulation. (A) Immunoprotective strategy in which are embedded within a nano/microporous hydrogel that allows free diffusion of glucose, oxygen, and insulin while blocking immune cells. Over time, vasculature will grow over the gel keeping islets depending on diffusion. (B) Vascularization approach in which islets are encapsulated within a degradable hydrogel that allows free diffusion of glucose, oxygen, and oxygen, as well as immune cells and vasculature over time.

considered harmful for the implanted islets, and can lead toward a significant decrease in islet mass within a week posttransplantation.14 The three major islet-destructive cytokines include interleukin (IL)-1β, interferon (INF)-γ, and tumor necrosis factor (TNF)-α.15,16 However, their hydrodynamic radii of 2.2, 3.1, and 3 nm are comparable to that of insulin (3.0 nm), suggesting that engineering pore sizes in the submicrometer range is not enough to realize complete immunoprotective devices.17–19 The success of the encapsulating strategy does not solely rely on barrier architecture. Permeability is another key feature of the encapsulation strategy. Encapsulated islets will develop a necrotic core if it takes too long before nutrients and oxygen can diffuse toward them. Permeability of a construct depends on multiple parameters, including diffusion coefficient, hydrodynamic drag on the moving solute and both polar and hydrophobic interactions between the barrier material and the macromolecule passing through, indicating the complexity of creating a suitable barrier.13 However, the limiting factor in the immunoprotective barrier strategy is the prevention of direct blood access toward the implanted islets, making islets dependent of diffusion to obtain oxygen and nutrients and the appropriate secretion of insulin upon glu-

cose levels. Protection against the host immune system is therefore traded for low oxygen levels leading to low islet survival due to a suboptimal environment.

Revascularization strategy Revascularization strategies are based on promoting reconnecting islets to the host vasculature to restore a proper oxygen tension to maintain high islet viability and restore the endocrine function quickly after transplantation. This is crucial since glucose responsiveness of β cells (and therefore glucose homeostasis) is highly dependent on oxygen.20,21 Islets are exposed to a pO2 of 40–60 mmHg within the pancreas, which is quickly lost upon islet isolation due to lack of blood supply. Directly after transplantation islets, or β cells, solely depend on diffusion until the vasculature is restored. It has been found that pO2 levels of transplanted islets are only around 5 mmHg after implantation under the kidney capsule, in the liver and the spleen.22,23 In these hypoxic conditions, insulin secretion is expected to be significantly reduced by several orders of magnitude compared to islets within their native pancreatic e­nvironment.

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Appropriate “islet revascularization” might be achieved by the use of novel implantation techniques, realized by the using so called “macroporous islet-delivery devices.” These devices should have large enough pore sizes that allow for the host microvasculature to connect with the islets embedded in the implant, providing direct access to nutrients and oxygen. Previous studies by Sharkawy et al. and Madden et al., have indicated that pore size is an important factor influencing the amount of neovascularization which can occur, ideally a pore diameter of 30–40 μm is recommended.24,25 Another crucial factor is that these bioengineered constructs should provide sufficient mechanical protection for islets, to retain their native rounded morphology and allow for surgical handling and device retrieval if needed. Several biomaterials can be chosen in order to fulfill these criteria. One could use either natural (e.g., gelatin, fibrin, gelatin, collagen, and decellularized ECM) or synthetic materials (e.g., pluronics, methacrylated hydrogels like GelMA, PDMS, PLG, PLA, Ethisorb, PEOTPBT, and PVA) to form implants, which are all discussed in more detail in the following sections. Depending on the chosen biomaterial, several implant fabrication techniques can be followed with varying control over islet distribution throughout the constructs. Scaffold fabrication techniques with random cell distribution include particulate leaching, phase separation-based techniques and electrospinning, while high control of islet distribution can be achieved by formation of individual, porous pockets that constrain one or multiple islets. These pockets can be realized by different techniques, including thermoforming, the use of pillared wafers or microfluidics. The highest control of islet distribution can be attained by direct three-dimensional (3D) printing of islets. Recent developments in both direct 3D i­ slet-printing and printing of rigid islet-supporting structures are discussed. Lastly, future directions of the revascularization strategy are addressed.

Biomaterials The European Society for Biomaterial (ESB)’s current definition of a biomaterial is “a material intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ, or function of the body.”26 Biomaterials can be derived either from natural or synthetic origins. With regard to natural biomaterials, these are processed biopolymers originating from living organisms. In contrast to synthetic materials, which are generated in a controlled laboratory environment, where one can use a variety of chemical synthesis approaches utilizing metallic components, polymers, ceramics, or composite materials. Both biomaterial types can be used and adapted for medical applications.

Hydrogels In contrast to polymeric scaffolds where cells are seeded on the surface of biomaterials, hydrogels provide a congenial environment to embed cells within the biomaterial itself. Depending on the hydrogel it could potentially enable cells to migrate and proliferate in any direction in a 3D environment thereby mimicking the native properties of tissues. Hydrogels are a class of cross-linked polymeric materials capable of absorbing and retaining large quantities of water.27 The gel can be described as a solid jelly-like material that can have properties ranging from soft, weak almost liquid appearance to a hard, tough almost solid appearance. In general, gels are defined as a diluted cross-linked system, which exhibits no flow when in the steady state, since by weight, gels are mostly liquid, yet they behave like solids due to a 3D cross-linked network within the liquid. In this sense, hydrogels can be employed as a cell extracellular matrix (ECM) and be tailored to mimic, or replace, native tissues. Hydrogels in tissue engineering are classified into two groups: naturally derived hydrogels (biopolymers) such as agarose, gelatin, fibrin, collagen, chitosan, and alginate, or synthetically derived hydrogels such as Pluronic and polyethylene glycol (PEG). Some hydrogels are able to mimic part of native tissue environment as they possess several essential characteristics of the native ECM.28 These ECM-like properties allow cell encapsulation in a highly hydrated, mechanically strong 3D microenvironment. However, both natural and synthetic hydrogels have their limitations. Natural hydrogels generally have weak biomechanical properties, while synthetic counterparts lack major biological components such as molecular motifs for integrin binding to allow for cell adhesion or migration.29 The biocompatibility of hydrogels is influenced by their inherent hydration levels. Hydrogels can absorb up to 1000 times their original weight in water in aqueous media without dissolving,30 making them ideal for cell encapsulation. Because they are highly permeable to oxygen, nutrients, and other water-soluble compounds, hydrogels are attractive materials for fabrication of complex tissue constructs.31 Although a variety of hydrogel matrices have been tried for islet encapsulation and implantation, they all have issues with biocompatibility, inflammatory responses, biodegradability and stability, islet viability, and sustainable insulin production, indicating that the optimal hydrogel configuration is yet to be determined.

Natural hydrogels Although it is impossible to describe all the biological hydrogels and their variations described in the literature, in the following paragraph we focus on some typical examples used today in the field of tissue engineering and islets (Fig. 2).

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Agarose Agarose is a naturally derived polysaccharide molecule which undergoes gradual gelation at low temperatures and liquifies at the temperatures ranging from 20°C to 70°C, depending on the hydroxyl ethylation.32,33 In solid state, agarose is brittle, but maintains its shape for a long period of time at a broad range of temperatures. Low cell adhesion and spreading suggest that agarose is a poor material for adherent cell culture; ­however, it serves well as a mold material for the generation of highly reproducible cell aggregates.34 Its biological properties can be improved by blending with other hydrogels such as collagen. Agarose has been used in ­extrusion-based bioprinting resulting in good cell viability and highly stable and controllable construct geometries. In this particular study mesenchymal stem cells (MSC) retained a rounded as opposed to a flattened morphology, due to the hydrophobic nature of agarose and the lack of cell-binding motifs which prevented cell attachment.35 In regard to islet transplantation, an agarose-based microcapsule design was used to physically isolate islets from immune cells while inhibiting complement activation.36 Islets were microencapsulated using agarose combined with a soluble complement inhibitor, resulting in protection from complement cytotoxicity in both in vitro and in vivo experiments. This agarose based controlled drug delivery strategy lead to prolonged islet graft survival in rat-tomouse xenotransplants providing strong evidence that local inhibition of complement system may be a promising method in xenotransplantation.37 Alginate Alginate is a natural anionic polymer and has been isolated from Azotobacter vinelandii, several Pseudomonas species, and algae.38 Alginate is a linear polysaccharide composed of 1,4′-linked β-d-mannuronic acid (M) and αl-guluronic acid (G) residues in different sequences. The ratio of G and M blocks depends on the source of algae used for alginate extraction. Alginates with a high guluronic acid content are preferred for applications where a more rigid structure is required like 3D printing.14 Alginates with a higher mannuronic acid content are preferred for applications where more pliable gels are desired.14 Alginate’s main advantage is its net negative charge, positively charged biomaterials are commonly described to provoke an inflammatory response, and its ability to electrostatically cross-link using positively charged ions such as Ca2+ and Ba2+, which does not require elaborate complex engineering knowledge or facilities for hydrogel creation. Alginate supports cell growth and exhibits high biocompatibility provided it is extensively purified to remove cytotoxins. Water and other molecules can be trapped by capillary forces in an alginate matrix, whereas these molecules are still able to diffuse. These features make alginate hydrogels interesting

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for bioprinting strategies. Apart from its high biocompatibility, this low-cost marine material normally forms hydrogel under mild cell-friendly conditions. For these reasons, numerous material scientists and bioengineers have employed alginate as a component in the design and fabrication for various types of cell encapsulation. Preparation of islet-alginate microbeads have been shown to be a promising candidate for immune protection in light of their low inflammatory potential39,40 as well as functional performance in mice models.41,42 Several studies using similar microbeads and human donor islets highlighted the beneficial effects of alginate encapsulation on human islet functionality.43 An initial in vitro study revealed subtle functional differences between alginate encapsulated, and free islets but, none of these differences indicate that encapsulation increases susceptibility to negative effects of acute hypoxia. Indeed, the outcome of several parameters indicated better resilience toward hypoxia.43 This was a positive finding in relation to alginate’s potential use in encapsulation for CIT. An examination of gene expression of ­alginate-encapsulated islets was undertaken to report global gene expression analysis. It showed that alginate microencapsulation does not alter mRNA or miRNA expression of human islets isolated at three different centers in the world. These data suggest that microencapsulation with alginate is safe for human islets and other cells, at least based on these in  vitro studies.44 With alginate being the most studied biomaterial for islet encapsulation, it currently has reached the level of clinical trials using allogeneic and xenogeneic sources for islet transplantation. However, capsule stability, biocompatibility, and reproducibility of these systems remain a significant concern. There is a long history of biocompatibility issues surrounding alginate-based islet transplants, which are prone to early graft failure due to fibrosis,45–49 although some progress has been made in this regard through advanced alginate purification, chemical modification, and optimized capsule fabrication protocols.46,50–55 However, the processes for improving alginate biocompatibility are complex and expensive; thus, an alternative synthetic hydrogel with similar properties is appealing. Chitosan Chitosan is a linear polysaccharide molecule obtained from deacetylation of chitin, and has a wide range of applications in tissue engineering such as cartilage regeneration, devices with hemostatic and antibacterial activity, formation of sponge like scaffolds, and fabrication of wound dressings.56 In a study by Byun et al., it was reported that porcine islets were stably encapsulated with chitosan-catechol, which effectively prevented dissociation of porcine islets after isolation, which allowed retention of the morphology of porcine islets in response to mechanical strength.57,58 Therefore, robust porcine islets

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in combination with chitosan-based encapsulation could be developed as an effective treatment of type 1 diabetes. Due to its weak mechanical properties, chitosan can be used for cell encapsulation, but not for the fabrication of large 3D tissue engineered scaffolds unless the material is chemically modified to enable ­cross-linking.59 For instance, chitosan has been used in bioprinting of perfusable vessel-like microfluidic channels, where chitosan was cross-linked by an ionic cross-linker, sodium hydroxide (NaOH) to form a stable hollow construct.60 The polymer and the cross-linker solution were printed simultaneously using a coaxial nozzle unit with the cross-linker solutions in the inner core diffusing out toward the polymer resulting in hollow tubes. Collagen Collagen is a biomaterial with excellent biocompatibility, biodegradability and low antigenicity. There are at least 16 different collagen types in the human body. The most prominent variants are type I, II, and III (or collagen I, II, and III), which make up approximately 80%–90% of all the collagens in the body.61 Collagen gels and solutions are widely used for building bioengineered scaffolds, or 3D cell culture systems, some of them constructed by 3D bioprinting, but usually by more classical techniques such as freeze drying or particle leaching. Collagen matrices facilitate cell attachment and growth due to the presence of abundant ­integrin-binding domains. Although collagen type I has been used in bioprinting, it has limitations as it remains in a liquid state at low temperatures and forms a fibrous structure with increased temperature or at neutral pH. Also control over homogenous distribution of cells in collagen is not trivial, as gravity can pull cells or cell aggregates down before gelation takes place. Low mechanical properties, fast degradability, and instability along with the abovementioned issues necessitate the use of supportive hydrogels or additional cross-linking protocols for collagen to enable the creation of complex scaffold configurations. Collagen can be used to improve outcomes of islet transplantation in three direct ways: (1) islets can be cultured on collagen, or embedded in 3D collagen gels, to temporarily maintain differentiation and endocrine function before transplantation62; (2) after the culture period, cells along with the collagen gels can be transplanted into a recipient host to provide a biological scaffold for revascularization of islets; and (3) islets can be encapsulated with collagen immediately after isolation and directly transplanted into a recipient host.63 In the endocrine pancreas, type I, III, and V collagens are present surrounding islets, and near islet capillaries in various intensities between the pancreas of rat, dog, pig, and human.64 Collagens I and IV can often be observed to colocalize with insulin positive cells, im-

plying that β-cells produce collagen I and IV. Collagen IV is localized around the basement membrane and near capillaries during pancreatic development, while in adult human islets, collagen IV can also be found in between ­insulin-positive cells and islet capillaries. Despite c­ ollagen providing a more natural cell culture substrate then tissue culture polystyrene, islets cultured in collagen gels have a tendency to lead to noninsulin producing cells due to dedifferentiation of β-cells.65 Interestingly, human islets cultured on collagen-coated tissue culture polystyrene did not lead to loss of β-cells due to dedifferentiation as well as maintenance of insulin gene expression.62 Murine islets embedded in a fibroblast-populated collagen matrix can significantly improve graft survival after transplantation.66 In this study diabetes reversal occurred in diabetic C57BL/6 (B6) mice and it required a smaller islet mass compared to the noncollagen supported controls.66 In another study, islets in scaffolds absorbed with collagen IV were able to improved glycemia significantly faster, than scaffolds coated with either fibronectin, laminin, or serum.67 Furthermore, when a suboptimal numbers of human islets are seeded in collagen IV-containing scaffolds, they can show improved graft function compared to noncollagen containing controls.68 Encapsulating islets using combinations of collagen, collagen and VEGF, collagen and FGF, or collagen and angiogenic factors, all resulted in protection against immune destruction, improved function, revascularization, and differentiation.63,69–74 Since collagen has low immunogenicity, absorbability, and shows good biodegradation, while promoting cell survival, function, and phenotype, and its current use in the clinic it is a prime biomaterial for supporting islets or β cells within an engineered bioartificial endocrine pancreas.75 Fibrin Fibrin is a hydrogel formed by the enzymatic reaction between thrombin and fibrinogen, the key proteins involved in blood clotting. It supports extensive cell growth and proliferation,76 plays a significant role in wound healing and has been used in the fabrication of skin grafts.77,78 The fibrin network is comprised of filaments forming as soft complex that allows a high degree of deformation without breakage. This is an essential factor in the vascularization of large tissue constructs and provides an effective in vitro model for analysis of the fundamentals of the angiogenic process.79 The disadvantage of using fibrin for in vivo applications is the possibility of a severe immune reaction or transmission of infectious diseases when heterologous proteins are used. In order to increase the efficacy and safety of fibrin hydrogels, either bacteria and viruses ought to be inactivated, or fibrinogen and thrombin must be produced as recombinant proteins by mammalian cell lines.

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Biomaterials

In addition, the degradation of fibrin is rapid, which is not conducive to long-term tissue culture. Since fibrin also contains the amino-acid motif Arg-Gly-Asp (RGD), a ligand to a large group of integrin receptors,80 it has been shown to significantly reduce isolated islet cell death.81 Studies with the rat insulinoma cell line, INS-1, using RGD peptides, fibronectin, and fibrin gels as well as human islets in 3D fibrin cultures all show enhanced β-cell function and survival, as well as significant improvements in proliferation and angiogenesis.82–93 These improvements of β-cell function are believed to be associated with the ability of fibrin to maintain cellcell contact and enable cellular responses to growth factors both ex and in vivo.83,84 Fibrin provides protection against apoptotic stimuli, demonstrated by the fact that young porcine islets cultures in fibrin were found to be resistant to hydrogen peroxide treatment.94 Fibrin was able to improve islet function and survival in diabetic mice that received fibrin-cultured islets as opposed to mice receiving free-floating islets, whereby human c-peptide levels significantly increased in mice having received the fibrin-treated islets.84 In a larger animal model the use of fibrin reduced the required marginal islet mass up to 90% in a subcutaneous xenogeneic porcine islet transplantation model95 In addition, fibrin reduces islet fragmentation into single cells, cells which have been shown to undergo an integrin-mediated form of apoptosis known as anoikis, often seen in free-­ floating transplantation procedures.96 As such, fibrin is a great candidate to use during islet transplantation to improve transplantation outcomes by decreasing islet fragmentation. Hyaluronic acid Hyaluronic acid is a linear nonsulfated glycosaminoglycan that is ubiquitous in almost all connective tissues and a major ECM component of cartilage.97 It is comprised of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine moieties linked by alternating β-1,4 and β-1,3 glycosidic linkages98 Hyaluronic acid is widely used in tissue engineering due to its excellent biocompatibility and ability to form flexible hydrogels.99 One of the early applications of HA in the medical field is in eye surgery procedures, such as cataract extraction. Nowadays its a very popular ingredient in skin care products and widely used as a (dermal) filler in plastic surgery. Hyaluronic acid is favored due to its role in early embryonic development, role in wound healing, cell friendly nature, and controllable mechanics, architecture and degradation. Allogeneic transplants of islets in HA-collagen-derived (HA-COL) hydrogels in diabetic rats were able to reverse diabetes and showed no evidence of graft rejection, or fibrosis in the recipients for as long as 18 months.100 These results corroborate excellent glycemic control observed in vivo of these implants,

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and it demonstrates the robust long-term biocompatibility and immunoprotective capacity of the HA-COL gel. Given the long history of biocompatibility issues surrounding alginate-based islet transplants, which are prone to early graft failure due to fibrosis,45–49,101 HA could serve as an alternative biomolecule for islet transplantation. Several of these aforementioned studies have demonstrated the potential of an HA-COL-derived hydrogel as an effective alternative to alginate for longterm immunoprotected islet transplantation. Clinical outcomes of alginate-encapsulated islet transplants have been disappointing, and these promising results support further investigation into HA-COL hydrogels for the encapsulation of islets and β cells. Gelatin Gelatin is a biodegradable polypeptide derived from the partial hydrolysis of collagen.102 As such, gelatin retains the RGD sequence from its precursor, is less immunogenic, and promotes cell adhesion, differentiation, migration, and proliferation.103 Still, even though gelatin shows better cytocompatibility and induces vasculature under in  vivo conditions, its weak mechanical strength limits its use in such applications as pancreatic tissue engineering. However, its mechanical strength can be improved by preparing it as an interpenetrating polymer network (IPN), which is defined as a physical mixture of at least two polymers that have been synthesized or cross-linked in the presence of each other with no significant degree of covalent bonds between them. This results in the creation of hybrid ­gelatin-based hydrogels, such as gelatin/alginate, gelatin/hyaluronan, and gelatin/fibrinogen mixtures, which still have unique features of gelatin, excellent biocompatibility, rapid biodegradation and nonimmunogenicity.104 Lee et al. showed that islet spheroids that were embedded in a collagen-alginate microsheet functioned well in diabetic mice leading to normoglycemia during a 4-week study.105 Some of these hybrid gelatin hydrogel combinations have been used in 3D bioprinting technologies due to physical (i.e., structural and morphological), chemical, and biological functional properties which make them suitable for tissue engineered constructs.106–113 The use of these hybrid gelatin hydrogels is not entirely trivial as was shown by Muthyala et  al.,114 who used 3D porous gelatin-polyvinylpyrrolidone (PVP) semiinterpenetrating network scaffolds to culture pancreatic islets. Polymer interpenetrating networks can be defined as a physical mixture of at least two polymers synthesized or cross-linked in the presence of each other with no covalent bonds in between, usually done to create mechanically stronger and stiffer hydrogels. Their findings suggested that gelatin-PVP semi-IPN scaffolds cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)

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carbodiimide hydrochloride (EDC) could support and maintain islet cells for 1  month. However, substitution of the EDC cross-linker by glutaraldehyde (GTA) lead to a loss of morphology and functionality of islet cells after 4 days indicating that the same biomaterial can lead to a completely different outcome if the fabrication process is altered.

Decellularized ECM-based biomaterials Decellularized ECM (dECM)-based materials involve decellularization of a tissue of interest by removing the cells while preserving the ECM. The ECM is then crushed into a powder form and dissolved in a cell friendly buffer solution to formulate the final product. A carrier polymer can be used to increase the solubility, to tune the viscosity, or to induce/enhance postcross-­ linking of the biomaterial. Recently, Pati et al.115 showed that dECMs from three tissues could be solubilized into so called “bioinks” and used for 3D printing. While many “bioinks” are compositionally simple, dECM bioinks contain a complex mixture of ECM components characteristic of the initial tissue source, and as a result, more closely resemble the native tissue. Although the mechanical properties of dECM bioinks do not mirror the original tissue, they represent a promising addition to biomaterials available for bioprinting because ECM proteins are among the most conserved proteins and often play a key role in cell behavior.116 Important is that decellularization removes xenogenic or allogenic cellular contents theoretically producing a minimally immunogenic scaffold with a native intact structure for new tissue regeneration. ECM molecules bind to integrin receptors that are expressed by islets, and these receptors modulate cell-cell and cell-ECM interactions to regulate functional islet survival.117 Examples of fibrous ECM proteins in the pancreas are collagen, laminin, and fibronectin. The importance of ECM for islet function has been shown in several studies demonstrating its role in organizing interactions between and behavior of endocrine cells, vascular endothelial cells, neural cells, and immune cells. These interactions enable the rapid exchange of oxygen, nutrients, metabolites, signaling hormones, and of course islet-related hormones such as insulin and glucagon.118 Hence the increasing interest to modify synthetic polymers with ECM molecules to improve cell-material interactions. Although it goes a bit beyond the scope of this chapter there are numerous strategies to enhance biomaterials with specific ECM molecules, varying from simply adsorbing molecules on the surface, electrostatic binding via and intermediate biomolecule such as heparin, to more sophisticated chemistry using tailor-made linkers to covalently bind proteins to the surface ensuring their correct presentation to cell membrane receptors.

Synthetic hydrogels Pluronic Pluronic F-127 is a trade name for synthetic polymer poloxamer-based polymeric compound. It exhibits a polymeric architecture consisting of two hydrophilic blocks between a hydrophobic block making it an effective surfactant.119 There are 11 types of Pluronic polymers which differ by molar mass, percentage of composites, functionality, and temperature of cross-linking which can vary from 10°C to 40°C.61 Pluronic copolymer structures erode quickly and cannot hold structural integrity for longer than a few hours; however, Pluronic, in combination with other hydrogels such as PEG, is useful for drug delivery and controlled release applications.120 An example of how Pluronic can be applied in the case of pancreatic cells was shown by Fullagar et al.121 In their study bilirubin, an endogenous antioxidant, was nano-encapsulated in a Pluronic-chitosan construct. The uptake of bilirubin by murine pancreatic cells improved dramatically increasing their viability under hypoxic stress. The reversible properties of Pluronic can be useful in fabrication of complex constructs. Small strands of Pluronic in its solid form (at room temperature or higher) can be embedded into a second type of hydrogel and then placed at 4°C to liquefy. This procedure creates perfusable channels within bulky cell-laden constructs after washing away the liquid Pluronic in which cells can be seeded.122 One can imagine that β cells or islets can be encapsulated in the surrounding hydrogel, while the open channels can be seeded with endothelial cells to stimulate vascularization after implantation. Poly(ethylene glycol) Poly(ethylene glycol) is widely used in medical and nonpharmaceutical products.107,123–125 It is a linear polyether hydrophilic compound which can be conjugated with proteins,126,127 enzymes,125 liposomes,128 and other biomolecules. It has been employed as a drug delivery agent,124,126,129 and used in biosensing or signaling,130 and pharmaceutical applications126 PEG is generally considered as a hydrophilic polymer resistant to protein adsorption. Hence, some cells, particularly chondrocytes and osteoblasts, encapsulated in PEG survive well even without the addition of biological constituents.131 Other cell types may require additional specific cell adhesion moieties, such as RGD peptides or fibronectin. PEGylation of biomaterials or medical devices inhibits protein adsorption making PEG a very useful synthetic polymer for fabrication of nonfouling implants in regenerative medicine. In a study by Phelps et al.132, biofunctionalized polyethylene glycol maleimide (PEG-MAL) hydrogels were

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Biomaterials

engineered as a vascular inductive β-cell delivery vehicle by the incorporation of VEGF, a potent stimulator of angiogenesis. After implantation of this delivery device at the small bowel mesentery they observed revascularized islets within 4  weeks, indicating that biofunctionalized PEG-MAL hydrogels are very effective at stimulating neoangiogenesis to support the encapsulated islets. Methacrylated compounds As previously discussed, numerous biopolymers can be used as building blocks for the creation of bioengineered scaffolds in regenerative medicine and islet transplantation specifically. However, their biomechanical instability still limits their use in bioprintable implants. The addition of methacrylate (MA) or diacrylate (DA) groups to the amine-containing side groups of a biopolymer such as collagen leads to generation of mechanically stable hydrogels (GelMA) at 37°C. GelMA, comprises a denatured form of collagen with methacrylate groups conjugated to its amine side groups, and it has been used in several tissue engineering applications due to its favorable biological and tunable mechanical characteristics.133,134 Directly after 3D printing a porous GelMA construct can be exposed to UV light to cross-link the GelMA monomers in order to stabilize and fix the shape of the construct.135 One of the major limitations of PEG is its poor mechanical strength at low molecular weight, addition of diacrylate (DA) of methacrylate (MA) can be used to create cross-links leading to increased mechanical stability. A downside to the use of methacrylated polymers is that DA and MA require photo-cross-linking, usually in the presence of a cytotoxic photo-initiators, and that exposure to UV light can lead to apoptosis when cells are embedded in the device before cross-linking, a technique often used in 3D printing in the literature. Methacrylated biomaterials where also used for the microencapsulation of porcine islets by Hillberg et al.136. Their biocompatible polymeric delivery system, combined alginate and a photo-cross-linkable methacrylated glycol-chitosan (MGC) biomaterial, resulting in the formation of ∼600 μm diameter capsules.136 Good islet viability and insulin release was demonstrated in vitro over the course of a month, and in  vivo transplantation in mice of the capsules demonstrated good biocompatibility and a minimal fibrotic response.

Solid biomaterials In contrast to hydrogel-based cell delivery devices used in tissue engineering, solid polymer-based devices are usually highly porous, allowing for cell infiltration and thus the formation of a dense vascular network inside the implant. The macroporous structure and accompanying vascularization allows for an optimal transport of nutrients, oxygen, and hormones to and from the

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cells. Solid porous scaffolds for extrahepatic islet transplantation can be manufactured from several different polymers, frequently used biomaterials are for example poly(glycolide-l-lactide) (PLG)67,68,137–141 and PDMS.142,143 Usually, nondegradable biomaterials with a minimal foreign body response and a proven track record in other clinical applications are chosen. The main advantage of solid versus hydrogel-based islet delivery devices is their mechanical strength and potential retrievability after implantation. Some of the most used solid polymers used in the field of islets are depicted in Fig. 3.

Nondegradable biomaterials PDMS Polydimethylsiloxane (PDMS) is a nondegradable synthetic polymer that belongs to the polysiloxanes, characterized by the presence of a silicon-oxygen-silicon (Si–O–Si) linkage in the backbone often referred to as silicones. The chemical formula of silicones is generally [R2SiO]n, where R is an organic group such as an alkyl (methyl, ethyl) or phenyl group. By varying the –Si–O– chain lengths, side groups, and cross-linking, it is possible to synthesize a wide range of materials with unique properties. PDMS exhibits no reaction, is stable and resistant to extreme environments. PDMS’s high durability is a result of its intrinsic material properties such as high hydrophobicity, low surface tension, and chemical and thermal stability. PDMS had been shown to be biocompatible and is therefore generally considered suitable for medical applications. Among silicones, PDMS has the longest history as implant material.144 Since mid-1940s, silicon has been used for a range of medical products: initially as coating for glass ware and needles to prevent blood clotting, and it subsequently was used more and more in medical devices, including catheters, blood oxygenators and in, for example, bile duct, urethra, hearth valve replacement. One of its most extensive applications over the last 40  years has been in esthetic and reconstructive surgery (e.g., breast implants). The long-term durability of these implants has been shown up to 32  years after implantation (confirming that no degradation took place).145,146 However, the use of silicones is limited by its low load-bearing capacity due to its poor mechanical tensile, abrasion, and tear strength. Initial studies carried out with PDMS scaffolds for extrahepatic islet transplantation showed problems in seeding and homogenous distribution of islets within the scaffold, due to the hydrophobicity of the material leading to poor cell attachment and low islet retention after implantation. Most of these problems were overcome by and additional processing step by coating the scaffolds with fibronectin, or immersing islets in fibrin gel before seeding, showing that PDMS can still be an interesting biomaterial for future CIT.142

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FIG. 3  Examples of typical hard polymer biomaterials used in the creation of β-cell replacement devices: PLA poly(lactic acid), PGA poly(gly-

colic acid), PLG poly(lactic acid-co-glycolic acid), PDS (poly-dioxanone), PVA (polyvinyl alcohol), PDMS (polydimethylsiloxane), and PEOTPBT (poly(ethylene oxide terephthalate)-poly(butylene terephthalate)).

PEOT-PBT block copolymers PEOT-PBT is a family of thermoplastic polymers, which consist of a block copolymer comprising polyethylene oxide terephthalate (PEOT) and polybutylene terephthalate (PBT). The nature and composition of the polymer blocks can vary, giving rise to materials with different mechanical properties and degradation rates (including nondegradable or very slow degradation) depending on their clinical application.147,148 PEOT is amorphous, soft, and hydrophilic, while PBT is crystalline and hydrophobic.149 In PEOT-PBT block copolymers, PEOT segments are covalently bound to PBT as one chain. This physical linking makes the polymer easy to process. The biomechanical properties of PEOT-PBT copolymers including elasticity, strength, and toughness can all be easily tailored depending on their clinical application by varying the polymer blocks and molecular weight of the PEG used during synthesis.150 PEOT-PBT block polymers have been developed for application in tissue engineering, that is, skin, cartilage, bone and muscle, and have and are being used in the clinic as dermal substitute, bone fillers, cartilage implants, cement stoppers, and in controlled drug delivery.151,152 Recent studies have proven its potential in islet transplantation and use in a macroporous islet delivery device. In these studies, macroporous thin-film microwell array devices

where creating using a technique based on a combination of microthermoforming and high-frequency-pulsed laser drilling leading to micrometer thin sheets in which islets can be seeded and be kept separated from each other. After transplantation of islets with these devices into diabetic mice efficient remission of diabetes in these mice was observed, while the blood vessel distribution and ingrowth seemed to be associated with the location of β cells in the islets indicating that good revascularization is key to β-cell function and their survival.153–156

Degradable biomaterials PGA, PLA, and PLG Poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and their copolymer poly(lactic acid-co-glycolic acid) (PLG) are the most widely used synthetic degradable polymers in medicine. Among them, PGA has the simplest structure and is more hydrophilic than PLA. PGA is highly crystalline, therefore has a high melting point and low solubility in organic solvents, which could difficult its use. Nevertheless, its crystallinity is lost rapidly by copolymerization with PLA. PGA was used in the development of the first totally synthetic absorbable suture.157 PLA and PGA have been already approved for many medical applications, for example, sutures, screws, and

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Islet delivery device fabrication techniques

tacks, membranes for guiding tissue regeneration in dental application, internal bone fixation devices, implantable drug delivery microspheres, and systems for cartilage and meniscus repair. Further manipulation of the polymer 3D architecture, mechanical and structural integrity as well as biodegradability can increase the range of potential applications. Thus, these polymers can be additionally used in the design of vascular and urological stents and skin substitutes and scaffolds for tissue engineering.158,159 Its potential to be used as islet transplantation devices has been investigated subcutaneously, using different fabrication techniques.160–162 The copolymerization of PGA and PLA to form PLG exhibit several advantages for medical application. It has mechanical strength, biocompatibility and is FDA approved.163–165 As mentioned with PLA and PGA, it is also possible to tailor PLG degradation time, ranging from days to years.166 PLG degradation rate can be altered by varying the formulation and initial molecular weight. Thus, a copolymer of 50% glycolide and 50% dl-lactide degrades faster than the homopolymers.167 Additional devices, such as microspheres, microcapsules, nanoparticles, pellets, implants, and films have been fabricated using these polymers. Due to the possibility of tailoring their degradation rate, PLG has also been used as carrier for drug delivery.168 However, it has been shown that PLG degradation gives rise to acidic products that could decrease the pH of the implant surroundings, which is undesirable for its proper integration.169 Additionally, due to its high price and hydrophobicity, the actual medical application is limited,170 including islet extrahepatic transplantation. Nevertheless, most studies showed that PLG scaffolds improved islet viability, enhance maintenance of islet morphology and induced normoglycemia of the implanted animals.68,140,141 Ethisorb Is a synthetic absorbable implant made of polyglactin 910 and poly-p-dioxanone (PDS). Polyglactin 910 is a type of PLG, made from 90% glycolide and 10% l-lactide. Those copolymers have been used commercially as multifilament suture materials (e.g., Vicryl; Ethico, Somerville, JN) since 1970. PDS is a polymer of multiple repeating ether-ester units which degrades by hydrolysis. For medical applications, it is used in the preparation of surgical sutures. Unlike polyglactin 910, PDS is a uni-filament suture material. It is used in orthopedics, plastic surgery, drug delivery, cardiovascular, and tissue engineering applications.171,172 Both, Polyglactin 910 (i.e., Vicryl) and PDS have been combined to fabricate a synthetic absorbable woven mesh called Ethisorb. Ethisorb patches consists of a porous Vicryl structure, which allows cell infiltration and implant integration, and a PDS film coating, that retains cells (or islets). Its use is approved by the FDA for treatment of encephalitis and spinalis defects, for example, in repair of orbital floor defects. Ethisorb patches have

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also been investigated as islet delivery device in dogs. The dogs were treated with islet autotransplantation after pancreatectomy in the omental pouch, normoglycemia in the diabetic animals was restored up to 2 months while in the control group, without a delivery device, no normoglycemia was observed.173 Although the number of animals used in this study was small it showed the potential beneficial use of transplanting islets with the help of a porous mesh-like islet delivery device in the omentum. PVA Polyvinyl alcohol (PVA) is a synthetic polymer derived from polyvinyl acetate through partial or full hydroxylation. It is easily degradable by biological organisms, and is a solubilized crystalline structure in water.174 It is widely used in industry (as paper coating and textile sizing) as well as in commercial, medical, and food sectors.175 PVA has substantial tensile strength, flexibility, hardness, and gas barrier characteristics. Its chemical and physical properties can be modified by varying the percentage of hydrolysis.172 Due to PVA’s properties, low protein adsorption, hydrophilicity, biocompatibility, high water solubility, and chemical resistance, this polymer has been extensively used in medical devices. Some of the most common medical uses of PVA are in soft contact lenses, eye drops, embolization particles, tissue adhesion barriers, and as artificial cartilage and meniscus.176 PVA was used in a sponge like disk-shaped device for subcutaneous islet transplantation in a syngeneic mouse model.161 Diabetic mice treated with islets in a PVA disc showed relatively positive outcomes although no further improvement of normoglycemia over time was observed a phenomenon the authors suggested could have been caused by a loss of islets due to the device configuration rather than the biomaterial itself.

Islet delivery device fabrication techniques After selecting a biomaterial, several different fabrication strategies can be used to shape a biomaterial into a desired configuration. The manufacturing method will depend on the material properties of the biomaterial and the desired device configuration. Several fabrication techniques will be discussed in the following paragraphs, ranging from low-to-high control of islet distribution, as described in Table 1.

Scaffold fabrication techniques with random islet distribution Particulate leaching This technique is one of the most conventional methods to create porous 3D constructs for tissue engineering purposes. The technique consists of dissolving the raw

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TABLE 1  Overview of different manufacturing techniques used for the creation of islet delivery devices based on random to high control over islet distribution within the device Random islet distribution

Controlled islet distribution

Micro

Macro

Nano

Micro

Macro

Droplet generator

Particulate leaching

Microfluidics layer by layer

Microfluidics

Thermoforming

Phase separation Electrospinning

biomaterial and adding porogens to the solution (usually salt crystals). The mixture is then often casted in a discoidal shape and the polymer is allowed it to cross-link, creating a 3D scaffold. The porogen can then be washed away, leaving an empty pocket within the construct. The size of the particles therefore directly corresponds to the final pore size. Specific porosities can be obtained by varying the salt-to-polymer ratios, as demonstrated by Brady et al. 250–425 μm in PDMS, and Kaufman-Francis et al. 300–600 μm in polylactic glycolic acid and polylactic acid (PLGA/PLA).143,177 This is, therefore, a technique that allows obtaining pores within a narrow range of sizes and tailored porosity. However, the options for obtaining specific pore geometry and pore distribution are limited. The technique is further limited by the fact that most resulting pores are closed, while interconnected pores are needed for cells to migrate within the construct upon seeding. Pore leaching is therefore often combined with gas foaming techniques used to create open and interconnected pores.141 Gibly et  al. showed long-term glycemic control in mice using this approach to implant 2000 human islets within a PLG implant with a pore size of 250–425 μm.178 Phase separation Demixing, or phase separation, of solvent-biomaterial solutions is a principle that is frequently used in other fabrication methods for macroporous hydrogels. An example of these methods is freeze drying, or lyophilization, after which hydrogels are often named cryogels. A mixture of solvent and biomaterial is rapidly cooled to induce phase separation, after which the solvent is removed by sublimation under vacuum, creating a void. The technique is suitable to create interconnected, open structures for a wide range of materials, including natural (agarose, chitosan, fibroin) and synthetic hydrophilic polymers.179–182 Nonetheless, tuning the pore size is rather difficult since hydrogel architecture is dictated by the kinetics of the thermal quenching process. In addition, structures often show weak mechanical properties and formation of a surface skin, a thickened area at the liquid-air interface183 Cui et al. have previously used freeze drying to produce chitosan sponges with pore size ranging from 200 to 500 μm, and subsequently seeded rat

Pillared wafer 3D printing

islets to attempt long-term in vitro culture within a 3D structure.182 They were able to show stable insulin release of islets up to 2 weeks. In addition, Borg et al. used freeze-drying to form starPEG-heparin cryogels with a pore size of 228 ± 130 μm to capture islets. Islets showed similar in vitro insulin secretion in these freeze dried porous devices after glucose stimulation compared to free islets used as control.184 Electrospinning Electrospinning is a fabrication technique based on a dry spinning process first developed over 70 years ago.185 It produces fine polymer fibers from a liquid (polymer solution) by means of electrostatic forces. In brief, a polymer solution is released from a syringe connected to a needle and forms a hemisphere at the tip of the needle due to surface tension. In this process a high-voltage source will generate a large electrical potential between the tip of the needle and a collector, which is creates attractive forces high enough to break the surface tension. The polymer solution will form a polymer jet directed toward the collector which solidifies before reaching it. The use of stationary or dynamic collectors will give rise to either randomly oriented or perfectly aligned nanofiber meshes. The diameter of the fibers generated, ranged from several microns down to 100 nm diameter or less. High polymer viscosity and a high DC voltage in the range of several tenths of kVs are both prerequisites for this technique to work properly.186 Electrospinning has been widely used for tissue engineering since it provides nanofiber structures that can simulate the 3D extracellular environment that cells find in the body. The interconnected porosity and high surface area of electrospun scaffolds enhance cell attachment and distribution as well as transport of nutrients and oxygen.187 The scaffolds can be additionally functionalized by coatings, or combined with other molecules that will be gradually derived (drugs or signaling molecules). Electrospinning offers fabrication with an aligned morphology as well high density of small pores.188 The technique has been used with more than 200 polymers, both synthetic and natural.189,190 In the case of biopolymers, they need to be combined with synthetic polymers in order to facilitate the electrospinning process and

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provide physicochemical and mechanical properties that cannot be obtained with only natural polymers. Even though electrospinning is one of the most used and promising techniques for developing constructs that resemble the native tissue, it also has some limitations. Fiber diameter is difficult to control, the resulting scaffolds exhibit inadequate mechanical strength for ­ load-bearing applications, and cellular infiltration and ingrowth is insufficient.188,191 From a technical point of view, solvent evaporation is one of the most critical problems in electrospinning. The reason is that solvents should be completely evaporated before the polymer jet reaches a collector but, controlling the evaporation rate in practice is still challenging. Nevertheless, electrospun 3D scaffolds have been used for a wide range of medical applications such as bone 192 and cartilage repair,193 regeneration of nerves,194 muscle,195 and blood vessels.196 Additionally, several electrospun biomaterials have been explored for the creation of islet macroencapsulation devices.154,197–200 Microencapsulation by droplet generators As previously mentioned, hydrogels are primarily used for cell encapsulation due to their physical properties and because they permit efficient diffusion of gasses, nutrients, metabolites, and therapeutic products. Primarily encapsulation is used to protect allogeneic cells from the host’s immune system. If successful then microencapsulation can offer an interesting solution for donor shortages, since it might allow for the use of islets from nonhuman species or engineered ­insulin-producing cells.70 Cell encapsulation technology can be divided over three categories: macro-, micro-, or nanoscale encapsulation. Macro-encapsulation of cells aims to retain a large amount of cells or islets in one single large device, while microencapsulation involves the encapsulation of individual cells or islets in microbeads or microfibers using a droplet generator, or microfluidic device.201 Nanoencapsulation, involves applying a very thin polymer coating on the outside of cells or islets. In general, the goal is to ensure that encapsulated cells retain their native rounded morphology and secretory function.70,202–207 Wolters et  al. developed an air jet droplet generator that produces small, uniform, and smooth alginate beads.208 This type of micro-droplet generator was used by many other groups afterwards to conduct a number of cell encapsulation studies.209

Microfabrication techniques (controlled islet distribution) Pillared wafer Many researches are now dedicated to manufacturing constructs with a high control of cell distribution. The idea behind controlling the position of cells or cell

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a­ggregates in tissue engineered scaffolds is that one can optimize mass transport to and from the cells to optimize their survival and function. This especially important for β cells since they depend highly on an optimal oxygen supply and preventing islets or β cells from aggregating together by physical separation using specially designed scaffolds might help overcome this problem. Cells are mostly confined to well-defined pores or microwells to retain individual cell aggregates that allow exchange of nutrients and oxygen, while preventing the formation of larger tissue clumps. One technique of realizing these dedicated pockets is the use of a pillared substrate collector (often manufactured from PDMS) during electrospinning,200 or a pillared PDMS or silicon mold during polymer film casting.155 In the latter situation a dissolved polymer can be casted on a pillared template (mold) and after solvent evaporation a thin polymer film remains. After careful demolding an array of small similar indentations in a thin polymer film is left. The molds are usually made in a cleanroom environment by a combination of reactive ion etching with the help of a photomask and photopolymerizable SU8 polymer from silicon wafers. Sometimes and intermediate step is done using the same technology to create by creating a mold from PDMS using a wafer template. The flexibility and elastic properties of PDMS allows for the creation of rubber-like elastic molds, which depending on the biomaterial used for film casting can have some advantages during the demolding process. This microfabrication technique allows for a very controlled and reproducible film design resulting in a very reproducible homogeneous well distribution and uniform microwell geometry.200 The microtextured casted films are by definition dense and nonporous, but phase separation and particulate leaching can be used to introduce small pores. The technique is limited by both the biomaterial that is being used for casting as well as the design of the mold. It is especially vital that the PDMS or silicon mold and polymer can still be separated without fracturing after solvent evaporation, which will be dictated by the film thickness and polymer and mold’s mechanical properties. This technique creates what is described a 2.5D environment, since flat films are a two-dimensional substrate, while microtextures, or microwells can provide limited variation in height in the Z-direction in the order of several 100 μm. Microthermoforming Control over islet, or β-cell distribution can also be achieved by shaping thermoplastic thin films (produced e.g., by film casting or electrospinning) into a microarray of wells using microthermoforming previously demonstrated by Buitinga et al.154. Thin films are placed on top of a metal mold with a desired pattern. The thin films are then covered with a backing material with a lower

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glass transition temperature Tg then the b ­iomaterial. Subsequently, these are placed in a hot press that is heated to allow deformation of biomaterial and backing film. The thin biomaterial film is compressed into the metal mold with the help of the softer backing material, and after cooling a micropattern is then fixed in place. One can design the metal mold in such a way that microwells can be created which serve as a physical barrier between individual islets. The idea behind this concept is that microwells can prevent cell aggregation and preserve the natural rounded morphology of islets or β-cell aggregates. Similar to the aforementioned film-casting technique one can introduce large pores within the thin films that allow for revascularization after implantation using various techniques. Pores with random size and distribution can be obtained by particulate leaching or phase separation, whereas more uniformly distributed and equally sized pores can be obtained by using pillared wafers or high-frequency-pulsed laser drilling techniques.154 Microfluidics Microfluidics can be utilized to produce fiber-shaped immunoprotective implants with highly degree of control over the distribution of islets within these fibers. A PDMS-or glass-based microfluidic device is designed comprising micrometer size converging channels for mixing specific hydrogel components to create solid hydrogel fibers with a uniform diameter smaller than 250 μm. If designed correctly, this microfluidic device can be used to encapsulate cells and islets in the core of the hydrogel microfibers with great accuracy.202 The microfibers can be minimally invasively implanted via a micro-catheter under the kidney capsule. A main advantage of microfibers is that they are easier to retrieve compared to individual microcapsules after implantation.210 Soft lithography is generally used to manufacture several microchannels in which main channel supplies a hydrogel precursor and islet, or β-cell mixture, while coaxial channels can supply a hydrogel cross-linking agent. Upon convergence of the aforementioned channels the hydrogel-precursor containing the cells mixes with the cross-linking agent initiating solidification of the microfiber. A common biomaterial used in a microfluidic device is alginate and its crosslinker CaCl2. In a noncross-linked state and at low concentrations an alginate solution still has a very low viscosity enabling relative nonrestrictive flow causing minimal shear stress on the encapsulated cells or islets.210–212 More biologically relevant microenvironments can be created by incorporating ECM molecules, such as collagen, within the hydrogel precursor solution to mimic ECM.211 Fabrication of PDMS microfluidic devices is, however, time consuming and prone to errors or batch-to-batch variations, and it requires

a­ ccess to an expensive cleanroom facility to enable creation of high-resolution molds. The recent introduction of 3D-printed lithography has shown to bypass some of these problems, significantly reducing manufacturing times and errors in these molds, and providing more design flexibility.212 An alternative approach was studied by Onoe et al. who used small glass capillaries instead of a PDMS based device to form core-shell microcapsules with a hydrogel shell and a core containing cells in an ECM mixture.210 Depending on the choice of hydrogel used in this system, the shell can be digested and removed to obtain capsules that solely consist of islets and ECM proteins. In addition, they have shown that microfibers can be weaved into a larger macroscopic construct by using a bespoke microfluidic weaving machine for easier handling by creation of a mesh containing encapsulated cells. Nanoencapsulation by layer-by-layer techniques Nanoencapsulation offers several advantages over microencapsulation. In particular, ultrathin layers should significantly improve mass transport of essential nutrients to the islets and reduce the total volume of material and cells that is transplanted. In addition, with nanoencapsulation, the small size of the capsules does not only allow for transplantation into a vast number of alternative sites within the body, but it can be used for regular CIT as well. Furthermore, nanoencapsulation could be used to protect many cell types other than primary islets. Taking encapsulation technology research even further, Diabetes Research Institute (DRI) scientists are now developing “bioactive” nanocapsules by tethering certain antiinflammatory agents to the islet surface that will allow it to “defend” itself against an attack from the immune system, thus allowing transplantation with minimal or no immunosuppression. As an alternative to microencapsulation strategies that create rather large capsules around cell clusters of different sizes, Tomei et  al. have designed and optimized a novel approach for conformal coating of islets, resulting in thin, uniform coatings of reproducible thickness on different sizes of islets.213 In vitro these conformally coated islets exhibit no delay in glucose-stimulated insulin secretion, or loss of function during. They showed that in syngeneic diabetic mice, conformally coated islets can restore and maintain euglycemia for over 100 days with no foreign body reaction, and normal vascularization around the encapsulated islets. Similarly, Krol et  al. used a multilayer nanoencapsulation technique in order to reduce the size of the capsule with the ultimate goal to create a multilayer system with different properties in each layer, or a specific region of the capsule.214 They showed positive outcomes for both the endocrine function and immunoprotective properties. These first studies with

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3D Printing in regenerative medicine

multilayer nanocapsules have shown that nanoencapsulation is a possible good alternative to the more space-consuming and random islet-trapping technique of microencapsulation. Conversely, Gattas-Asfura et al. used encapsulation of viable tissues via l­ayer-by-layer polymer assembly for cell surface engineering, with nanoscale control over the capsule properties.215 They reported the development of a hyperbranched ­polymer-based, ultrathin capsule, expressing biorthogonal functionality and tailored physiochemical properties. Hyperbranched polymers provide a flexible platform for the formation of covalently stabilized ultrathin coatings on the outside of viable cells and tissues. In addition, hyperbranched polymers can be used to present a surface with many chemical functional groups suitable for biorthogonal conjugation of growth factors, or immunomodulatory compounds.

3D Printing in regenerative medicine 3D printing, a form of additive manufacturing, is a technique in which materials are deposited in a layer-bylayer fashion to create a 3D object dictated by computeraided design (CAD). It can be used to create complex structured objects with high architectural control and resolution, and has since its invention more than a d ­ ecade ago triggered the imagination of researchers in the biomedical field from the beginning.216 The 3D (bio)printing process consists of three steps: preprocessing, processing/ printing, and postprocessing.217,218 During preprocessing a CAD file is created that functions as the blueprint for the printing process. During the processing/printing step the actual construct is manufactured via bioprinting. The main challenge during this step is to choose the right combination of 3D technique and biomaterials used for printing.218 Postprocessing includes cell seeding, proliferation and differentiation of cells, and tissue formation within the printed construct.217,218 Currently there are three different techniques which are most used for bioprinting, each has their own advantages and disadvantages, which are summarized in Table 2. There have been multiple attempts to directly print living tissues. For example, bioengineers working in bone research are focused on the development of a suitable bioink that enables sufficient diffusion of nutrients to cells in order to create viable large centimeter-sized tissue engineered ­implants.217,220–222 The aim of this research is to develop a graft that can bridge and regenerate large critical sized bone or cartilage defects. Another example is the development of printed skin grafts to replace traditional skin grafts. In the last few years different groups were already able to print viable small models of skin tissues,223–225 but significant improvements are still needed to enable true clinical translation.

TABLE 2  Current 3D printing technologies used for bioprinting217–219 Micro extrusion printing

Laser-assisted bioprinting

Thermal and piezo-electric forces are used to expel droplets of bioink

Mechanical or pneumatic forces are used to dispense bioink in continuous stream

Direct laser pulses are applied onto a metal target on which bioink is deposited, leading to a jet of liquid solution deposited onto substrate

+ High production speed

+ High viscosity bioinks possible

+ High viscosity bioinks possible

+ Ease of accessibility

+ High cell density possible

+ Nozzle free technique

+ Affordable

+ Affordable

+ High precision

Inkjet bioprinting

+ Several biological + Easy to scale-up components can be printed next to each other

+ High resolution

+ Cost convenient technique for 3D porous cell-laden constructs - Frequent nozzle clogging

- Distortion of cellular structure

- Lower cell viability due to heat

- Only for low viscosity bioinks

- Lower cell viability due to pressure and shear stress

- Time consuming

- Often poor mechanical properties

- Only for high viscosity bioinks

- Only for photocross-linkable biomaterials

3D printing for β-cell replacement therapy Several bioprinting techniques, mostly relying on micro extrusion printing, have been explored to improve CIT (see Fig.  4). Farina et  al. used medical-grade polylactic acid (PLA) to create a 3D-printed nanogland device; a discoidal encapsulation device composed of two layers, in which the inner surface was patterned with ­micrometer-sized reservoirs to house individual islets.226 These micro-reservoirs were connected to an array of square microchannels on the outside to allow for vascularization. They used a prevascularization strategy in which their device was first implanted subcutaneously without cells. Once vascularization in and around the device was deemed sufficient, islets were loaded through a loading port into the device. Implantation of the nanogland device in mice lead to comparable insulin secretion levels as kidney capsule transplantations. In a follow-up study, the ­hydrophilicity

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FIG. 4  Different 3D printing strategies for tissue engineering purposes. Direct cell-loaded hydrogel printing encapsulates islets within a porous design to diminish diffusion-related hypoxia. Hard polymer scaffold printing is focused on printing a rigid backbone that is subsequently loaded with an islet-bearing hydrogel. Microfluidic fiber printing consists of hydrogel-encapsulated islets with either a core-shell configuration or a solid fiber. Tissue strand printing is a biomaterial free approach in which tissue strands are formed within a hydrogel shell and subsequently used as “bioink” for 3D printing.

and surface roughness were altered by plasma treatment to increase cell attachment to these PLA devices.160 A further improvement involved coating with ­poly-l-lysine (PLL) and endothelial cell attachment factor to further increase cell material interactions. These proof of concept studies shown the ample possibilities of device design and manufacturing with 3D printing. However, regulatory affairs dictate that all aspect of such a complex device design should be tested for safe use in a clinical setting and that perhaps device design should perhaps focus on rather simple and easy to manufacture solutions to enable quick clinical implementation. PLA has

also been used in a hybrid approach in which fibrin was used to seed β cells into a 3D-printed device in order to retain the cells in the macroporous device. In this study stem cell-derived β cells (SC-βs) were first mixed with a degradable fibrin gel, which was subsequently pipetted onto a 3D-printed macroporous PLA device.227 The authors used a finite element model of o ­ xygen-diffusion and consumption to theoretically optimize the design and architecture of the device while also taking into consideration the SC-β aggregate dimensions used for seeding. Subcutaneous implantation of these constructs in mice lead to a good insulin secretion response to glucose

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Future outlook

stimulation after 12 weeks of implantation, but unfortunately the absence of blood glucose values makes it difficult to exactly interpret the metabolic implant function. This study showed that by combining mathematical modeling with CAD and 3D printing the implant design and fabrication can be tailored to meet a specific in vivo application in a very controlled manner. Daoud et  al. have shown that it is also possible to combine different techniques by utilizing a combination of pore leaching and 3D printing.228 In their study, PLGA was dissolved, mixed with a sodium chloride solution, subsequently bioprinted and immersed in water to dissolve the salt crystals. This technique resulted into scaffolds with ~100-μm-thick strands. By controlling the fiber interspacing, it was possible to create several constructs with different porosities and pore sizes. Isolated human islets were then embedded into an ECM gel and seeded into the polymer scaffold after which they were kept for 10  days in culture. They showed preservation of islet functionality and good survival for this period. A completely different approach which can be considered as another form of 3D printing, using live tissue as “bioink,” includes the use of coaxial extrusion and microinjection to create scaffold-free tissue strands.229 In this technique tubular alginate strands function as semipermeable capsule in which a cell pellet is seeded and cultured for a certain period in which the cells proliferate to form a tissue strand in the center. The alginate is subsequently removed resulting in scaffold-free cylindrical strands made from cells. The authors used mouse insulinoma β TC3 cells and proved that they were able to retain their viability and insulin secretion after printing with this scaffold-free fabrication technique. However, loss of viability of TC3 cells over time was significant, related to diffusion limitations within the ~625 μm diameter fibers. Tissue constructs bigger than 200 μm generally require vascularization for sufficient oxygenation and nutrient supply to ensure long-term viable and functional islets.230 In this particular case the fusion of individual tissue strands over time can potentially further limit nutrient diffusion. Nevertheless, scaffold-free tissue strands have already been used for 3D printing of articular cartilage into large constructs, and might therefore be an interesting strategy for islet bioprinting in the future once the diffusion limitations have been solved.231 To date, there is only one paper in the literature published from Marchioli et  al. (see Fig.  4) describing a method for direct 3D printing of primary pancreatic islets into a delivery device.232 In this study 3D bioprinting was used to create alginate-based porous scaffolds in which islets were directly embedded during the printing process. Different alginate mixtures with gelatin were tested with rat insulinoma β-cells (INS1E), and human and mouse islets were successfully 3D-printed into macroporous 3D rectangular scaffolds. Both INS1E cells and isolated islets

were able to retain their natural rounded morphology and showed good viability. The individual islets were kept separated by the alginate matrix which prevented the formation of large tissue clumps. Most islets could be found within 200 μm distance from the outside, which is comparable to most studies focused on microencapsulation. Although viability studies showed that the islets were able to withstand the printing process, the glucose responsiveness of 3D-printed islets was much lower than free floating controls. Interestingly, the islets were capable restoring full functionality after degradation of the alginate and retrieval from the scaffold. This indicates that the decreased functionality observed by the authors was not a result of the printing process itself but a suboptimal “bioink.” In order to be able to print a 3D stable scaffold using a hydrogel-based “bioink” the material used needs to meet certain demands. In order for a 3D scaffold to retain its shape during the printing process a minimal material viscosity is needed. Unfortunately, the concentration of alginate used in microencapsulation, typically 2%–3%, which still allows for a good glucose response is not viscous enough for 3D printing. Alginate at this concentration collapses on itself resulting in a dense hydrogel mass with no structure. In order, to use alginate for 3D printing one has to either increase its concentration or add a supporting material with a higher viscosity. In the aforementioned study gelatin was added to enable 3D printing, but the authors concluded that this had decreased the mesh size of the “bioink” too much, significantly decreasing mass transport. In order for direct 3D printing of islets or β cells specifically tailored hydrogels are needed to ­enable fully functional 3D-printed islet delivery devices to work properly. New synthetic biomaterials, or modified alginate are needed which combine a high viscosity with a large enough mesh size allowing for efficient diffusion of nutrients and hormones through the hydrogel barrier and creation of complex 3D architectures at the same time (Fig. 5).

Future outlook Advances in the development of biomaterials for tissue engineering and regenerative medicine are providing unprecedented opportunities for current and future clinical therapies. In this chapter, the use of different biomaterials and fabrication techniques is described in the domain of β-cell replacement therapy for type 1 diabetes. While each biomaterial and fabrication technique presented provide specific improvements to a final β-cell delivery device, none thus far encompass all criteria required to overcome the challenges presented by islet biology and current clinical treatment for complete endocrine pancreatic function restoration. Among the ­future outlooks are combining the advantages of

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FIG. 5  Example of 3D-printed islets in a macroporous alginate-based construct (top right panel). Prior to printing islets were transfected with GFP for later visualization after printing (left panel). In this study the 3D constructs were implanted subcutaneously to see if islets were able to survive the 3D printing process and implantation afterwards. Islets can be found throughout the construct in close vicinity to the surrounding tissue (bottom right panel). Images based on a study by Marchioli G, van Gurp L, van Krieken PP, et al. Fabrication of three-dimensional bioplotted hydrogel scaffolds for islets of Langerhans transplantation. Biofabrication 2015;7(2):025009.

­ ifferent types of biomaterials promoting revascularizad tion and thwarting immune and inflammatory response and develop a limitless supply of insulin-producing β cells to replace primary islet transplants. One of the biggest challenges in pancreas bioengineering is the delivery of oxygen and nutrients to β cells inside delivery devices. Ongoing research on this topic has led to the investigation of different microfabrication strategies that can promote revascularization of an implanted device by incorporating angiogenic growth factors or support cells like endothelial or MSC. Another approach to overcome the some of the challenges mentioned above can also be sought by converting bioinert synthetic polymers into “bioactive” immunoprotective scaffolds. ECM molecules such as collagen, gelatin, or fibronectin can be incorporated within delivery devices, either by coating or by impregnating them throughout the biomaterials of which the device is composed. ECM proteins have been known to stimulate the infiltration of endothelial cells into a transplanted device thereby promoting revascularization.233–235 Moreover, a recent study by Llacua et al. demonstrated the significant role of ECM in reducing cytokine-mediated cell death in ­immuno-isolated human islets.236 The advent of 3D bioprinting can aid in improving vascularization since inclusion of ECM proteins and support cells can be done simultaneously when devices are built in a very controlled and precise manner.

3D bioprinting can also aid in the precise deposition of pro-angiogenic factors, such as VEGF or bFGF, which are known to enhance islet viability and function, in a device.237 Besides incorporating ECM proteins into the construct, co-encapsulation of other cell types along with β cells have proven to be beneficial, in terms of inducing revascularization or modulating the immune response. For instance, co-transplantation of islets with MSC showed enhanced islet viability and function, possibly due to the immunomodulatory and antibacterial activities presented by MSCs.238,239 Several factors, ranging from those that augment vascularization to those that hold ­oxygen-generating properties or even inhibit the immune response, can all be used to functionalize biomaterials. The most interesting application of 3D bioprinting is the fabrication of a prevascularized islet delivery device.240 A prevascularized device can promote rapid local revascularization, thus fast delivery of oxygen and nutrients, of islets as was reported by Hiscox et al. who implanted islets with the help of a collagen-based prevascularized implant.241 This particular advantage, direct creation of prevascularized islet encapsulating devices, renders easily accessible implantation sites, such as the subcutaneous space, a not highly vascularized site, potentially more hospitable leading to better β-cell survival and function.242 By offering a fast and highly controlled method of fabrication allow to precisely deposit cells and materials in a predefined shape, 3D bioprinting technology holds a

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Future outlook

huge potential in making prevascularized devices readily available in the future in any shape and size (Fig. 6). In order to increase further device complexity, 3D printing approaches can also be combined with more conventional scaffold fabrication methods like particle leaching, as previously shown by Daoud et al.228 Other approaches include laser drilling to generated capillary-sized channels within transparent hydrogels to guide cell growth and vascularization.243,244 Alternative to 3D printing a scaffold directly, a sacrificial reverse template can be 3D printed and subsequently filled with a biomaterial. In this way interconnected microchannels can be created, by dissolving the 3D-printed template, which later can be used for ingrowth of vasculature within the scaffold.245 Another ongoing challenge in β-cell replacement therapy of diabetes is the scarcity of human donors and the chronic use of immunosuppressive drugs. While several groups, including Melton’s246,247 and Kieffer’s248 groups, are investigating the possibility of directly differentiating embryonic or induced pluripotent stem (iPSC) cells into β cells, it is also important to consider other pancreatic cell types in tissue engineering of the pancreas. Native pancreatic islets maintain physiologic blood glucose levels via complex hormonal interactions between the different pancreatic cell types (α, β, δ, γ,

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and ε cells). To recapitulate these complex hormonal interactions, future devices should ideally encompass different types of cells rather than only β cells. In this regard, 3D bioprinting technology can play a unique role as it offers the flexibility to deposit groups of cells with high precision in any spatial distribution programmed into the CAD file used. In conclusion, an optimal islet or β-cell delivery device should encompass supporting cells or molecules, preferably minimize inflammatory responses against the allogeneic tissue and biomaterial to maximize cell survival from the beginning to obtain a sufficient long-term endocrine function. The use of advanced scaffold fabrication techniques can be used to create a tailor-made delivery device which protects islets, retains the endocrine phenotype and maximized exchange of nutrients, oxygen, and hormones in a highly vascularized microenvironment other than the intra vascular site currently used in CIT. The most optimal situation would be if a device can support islets and β cells in such a way that nutrient and hormones are exchanged in a nonlimited manner. The distribution of islets or β cells should not interfere with their function and survival, endocrine function and phenotypes are preserved by providing an optimal microenvironment, and the biomaterials used should not induce cell stress

FIG. 6  Schematic depiction of a possible optimal islet delivery device in which islets or β cells are positioned in a controlled manner, with space in between them to ensure optimal mass transport. Local immunomodulation can be achieved via controlled drug delivery of immunomodulating agents, while the addition of biomolecules such as extracellular matrix proteins can be used to avoid rejection of allogeneic cells, or decrease a potential foreign body response to the delivery device by mimicking native tissue. The use of support cells, or biomaterials functionalized with growth factors can further aid in improved tissue engraftment, vascularization, and creation of an optimal beta cell microenvironment.

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and an inflammatory or foreign body response. Ideally, allogeneic cells should not be rejected without systemic immunosuppression, while retrieval or renewal of the cells and the device can be done in a relative minimal invasive manner. It is clear now that a multidisciplinary approach is needed, in which bioengineering and biomaterial science should go hand in hand with cell biology, immunology, and transplantation medicine to develop a clinically relevant β cell replacement device which can mimic the endocrine pancreas kinetically and metabolically in the best way possible.

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Further reading 249. Elliott RB, Escobar L, Tan PL, Muzina M, Zwain S, Buchanan C. Live encapsulated porcine islets from a type 1 diabetic patient 9.5 yr after xenotransplantation. Xenotransplantation. 2007;14(2):157–161.

250. Orive  G, Hernandez  RM, Gascon  AR, et  al. Cell encapsulation: promise and progress. Nat Med. 2003;9(1):104–107. 251. Jung KC, Park C-G, Jeon YK, et al. In situ induction of dendritic cell–based T cell tolerance in humanized mice and nonhuman primates. J Exp Med. 2011;208(12):2477. 252. Hering BJ, Wijkstrom M, Graham ML, et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates. Nat Med. 2006;12(3):301–303. 253. Hering Bernhard J, Cooper David KC, Cozzi E, et al. Executive summary. Xenotransplantation. 2009;16(4):196–202. 254. Chen  Y, Stewart  JM, Gunthart  M, et  al. Xenoantibody response to porcine islet cell transplantation using GTKO, CD55, CD59, and fucosyltransferase multiple transgenic donors. Xenotransplantation. 2014;21(3):244–253.

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C H A P T E R

34 Subcutaneous islet transplantation using tissue-engineered sheets Shinichiro Ono⁎, Tomohiko Adachi⁎, Masataka Hirabaru⁎, Hajime Matsushima⁎, Hajime Imamura⁎, Masaaki Hidaka⁎, Koji Natsuda⁎, Toshiyuki Adachi⁎, Manpei Yamashita⁎, Mitsuhisa Takatsuki⁎, Tatsuya Kin†, Susumu Eguchi⁎ ⁎

Department of Surgery, Nagasaki University, Graduate School of Biomedical Sciences, Nagasaki, Japan †Clinical Islet Laboratory and Clinical Islet Transplant Program, University of Alberta, Edmonton, AB, Canada O U T L I N E

Introduction

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Introduction The outcomes of insulin-independent islet transplantation have significantly improved since the introduction of the Edmonton protocol with a steroid-free immunosuppressive regimen,1 but the long-term survival remains unsatisfactory.2 According to the annual report of the Collaborative Islet Transplant Registry (2017), 1086 recipients received islet allotransplantation with 2150 infusions between 1999 and 2015 (http//www.citregistry. org). However, approximately 73% of these recipients required multiple transplantation procedures in order to maintain the islet function. In recent years, good results with an insulin-independent rate of 50% have been reported,3,4 but multiple islet transplantation is still

Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 2 https://doi.org/10.1016/B978-0-12-814831-0.00034-8

Our concept of subcutaneous islet transplantation using cell sheet engineering

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Cell sources

491

Cytokines

491

Previous study

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Conclusions

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Conflict of interest

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References

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r­equired for insulin independence. Several factors are related to islet graft loss in intrahepatic islet transplantation, including instant blood-mediated inflammatory reaction,5 hypoxia, liver immunity,6 the risk of exposure to high concentrations of immunosuppressive drugs,7 and procedure-related complications, such as portal vein thrombosis and hemorrhaging.8,9 To overcome these problems and improve the results of islet transplantation, several studies on changing the transplantation site have been conducted, with alternate sites including the kidney capsule, pancreas, spleen, omental pouch, thymus, and subcutaneous cavity.10–15 Among them, we focused on subcutaneous sites because of their easy accessibility during the transplantation procedure and ease of performing a biopsy if needed.

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34.  Subcutaneous islet transplantation using tissue-engineered sheets

However, while the subcutaneous cavity is an ideal site for islet transplantation, the outcomes of subcutaneous islet transplantation remain unsatisfactory because of poor vascularization. Prevascularization of subcutaneous sites using basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) has been attempted,15–17 and many reports that further develop subcutaneous transplantation have recently been published.18,19 Recently, tissue engineering cell sheet technology has been applied to various ends, such as improving the condition of the cornea, esophagus, heart, and cartilage.20 In addition, these cell sheets can preserve cellular junctions, the endogenous extracellular matrix (ECM), and the integrative adhesive system,21 helping to maintain the cell functions. This technology has been applied for subcutaneous islet transplantation. In this article, we introduce the current status of subcutaneous islet transplantation, and subcutaneous islet transplantation using tissue-engineered sheets.

Subcutaneous islet transplantation The subcutaneous space is an attractive site for islet transplantation due to the technical safety of surgeries performed therein with minimal invasion and the superficial characteristics facilitate graft imaging, biopsy, retrieval, and ability to transplant a large amount of tissue due to its huge area. In the 1970s and 1980s, several researchers attempted to change the accepted ideal transplantation site from the liver via the portal vein to the subcutaneous space; however, they failed to show sufficiently improved outcomes.22–24 The major reasons for the poor engraftment to the subcutaneous space are believed to be the poor oxygen tension and poor blood supply due to the insufficient number of blood vessels with a lack of early neovascularization. Indeed, early angiogenesis is considered necessary for successful subcutaneous islet transplantation.25 To address this issue, previous studied have induced angiogenesis using polyvinyl alcohol and polyglycolic acid (PGA) polymers to create vascularized organoids. However, the vascularized of islets which were contained in the PGA polymers showed the improving effect on glucose tolerance in syngeneic mouse, the transplant efficiency was inferior to the kidney subcapsular transplantation.11 Another strategy proposed to address these limitations involves implanting a device to control the release of acidic fibroblast growth factors, such as bFGF,25–27 VEGF,28 and HGF.16 Studies on that approach have shown good results and many researchers have continued similar studies. Another approach to achieve vascularization of the transplant site involves inserting the artifacts and biological origin materials have

attempted by many researchers. Pillegi et al. attempted to achieve neovascularization using cylindrical stainless mesh,15 while Kriz et al. used polymeric mesh to these ends.29 Both authors showed favorable neovascularization at the site and achieved euglycemia in rat models. Biological-origin materials using collagen-chitosan30 and hydrogel-type fibrin31,32 have also shown good effects and potential utility in subcutaneous transplantation. Pepper et al. described the successful performance of device-less subcutaneous islet transplantation.33–35 They implanted a catheter made from nylon to induce a foreign body reaction, which led to the creation of a highly vascularized engraftment site. Before transplantation, the catheter was withdrawn, and syngeneic murine islets were transplanted. The recipient naïve mice showed insulin independence for more than 1-year posttransplant without immunosuppression. Recently, Komatsu et  al. described that oxygen inhalation after transplantation showed good results in the rats with in prevascularized subcutaneous sites.19 Bertuzzi et al. used an agarose rod containing the cyclic oligopeptide SEK-1005 to induce local vascularization, leading to the formation of granulomatous tissue containing regulatory T cells.36 At this site, a transplanted islet successfully engrafted without immunosuppression. Recent studies have shown evidence that subcutaneous islet transplantation can be a viable alternative in animal models. Although islet transplantation requires a deceased donor, it is necessary to consider that a deceased donor suddenly occurs and there is no grace of prevascularization.

Cell sheet engineering in cell transplantation Cell sheet engineering using temperature-­responsive culture dishes was introduced by Okano et  al.37,38 UpCell (UpCell; CellSeed, Tokyo, Japan) is coated with the ­temperature-responsive polymer poly(N-isopropyl acrylamide), which can change the sheet’s character from hydrophobic to hydrophilic at temperatures under 32°C. Cells cultured on UpCell can be retrieved as a sheet without dispase using trypsin.39 The advantage of this technique is that the ECM and cell-to-cell junctions as well as the cell function itself can be maintained by maintaining the individual components, including adhesion factors and growth factors, without an artificial scaffold. This novel technology has been applied in ­various fields of regenerative medicine (Table 1).

Cornea Corneal transplantation is widely used for patients whose vision has been impaired due to damage involving the eye. However, the limited number of donors

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Cell sheet engineering in cell transplantation

TABLE 1  Cell sheet engineering in cell transplantation No.

Author

Applications

Cell source

Animals

References

1

Yang

Cornea

Oral mucosal epithelial cells

Human

[40]

2

Nishida

Cornea

Oral mucosal epithelial cells

Human

[37]

3

Shimizu

Heart

Chick cardiomyocytes

Rat

[41]

4

Shimizu

Heart

Neonatal cardiomyocytes

Rat

[42]

5

Memon

Heart

Skeletal myoblast

Rat

[43]

6

Kondoh

Heart

Skeletal myoblast

Hamster

[44]

7

Hata

Heart

Skeletal myoblast

Canine

[45]

8

Sawa

Heart

Skeletal myoblast

Human

[46]

9

Ohki

Esophagus

Oral mucosal epithelial cells

Canine

[47]

10

Kanai

Esophagus

Epidermal cell

Porcine

[48]

11

Ohki

Esophagus

Oral mucosal epithelial cells

Human

[49]

12

Kobayashi

Esophagus

Oral mucosal epithelial cells

Porcine

[50]

13

Ohashi

Liver

Hepatocyte (mouse)

Mouse

[51]

14

Sakai

Liver

Hepatocyte (human)

Mouse

[52]

15

Shiroyanagi

Bladder

Urothelial cell

Canine

[53]

16

Kanzaki

Lung

Skin fibroblast (rabbit)

Rat

[54]

17

Akizuki

Periodontal

Periodental ligament cell

Canine

[55]

and complications such as rejection remain issues in this field. Cell sheets using autologous oral mucosa epithelial cells have been used to address these problems because of their histological approximation with the native cornea epithelium.37,40 Nishida et al. described a successful case of cell sheet transplantation fabricated from autologous oral epithelial cells for patients with bilateral total corneal stem cell deficiencies. The cell sheets were directly implanted on the denuded cornea surface without sutures, and the transplanted corneal showed a clear and smooth appearance. The transplanted corneas were completely reepithelialized within in 4 weeks in all treated eyes. During the mean follow-up period of 14 months, the transplanted corneas maintained their transparency.40

Heart Severe heart failure is a serious condition directly linked to death, and new treatments are being investigated. The regeneration of cardiomyocytes using cell sheets was first proposed by Shimizu et al.41 They created an electrically communicative three-dimensional cardiac construct of bilayer cell sheets using cardiomyocytes. These cardiomyocyte cell sheets showed spontaneous and synchronous pulsation.42 In further experiments, the developed a four-layer neonatal rat cardiomyocyte cell sheet that showed spontaneous beating for 1-year

after transplantation with a heart tissue-like structure, neovascularization and growth.56,57 Sekiya et  al. elucidated the mechanism underlying this neovascularization and showed that the transplanted sheet migrated in order to connect to the recipient vascular network.58 Although autologous skeletal myoblast transplantation has also been used for myocardial cell therapy, efficacy was insufficient due to cell loss and mechanical stress of delivery method.59 With the incorporation of tissue engineering, skeletal myoblast cell sheets resulted in the long-term survival without cell loss at the transplanted site and the attenuation of cardiac dysfunction and remodeling.43–45 In the clinical setting, Sawa et  al. transplanted autologous myoblast sheets into patients with a decreased heart function who were using left ventricular assist devices.46 The heart function recovered in three of the four patients treated due to left ventricle reverse remodeling and improvements.

Esophagus Endoscopic submucosal dissection (ESD) is now widely indicated for treating early-stage esophageal cancer. Although ESD shows beneficial effects due to its low invasiveness, extensive ESD, defined as 10-cm and full-circumferential ESD, can cause esophageal stricture. Tissue engineering cell sheets using autologous oral epithelial cells or epidermal cells has helped prevent

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34.  Subcutaneous islet transplantation using tissue-engineered sheets

esophageal stricture after ESD in animal m ­ odels.47,48 Furthermore, tissue engineering cell sheets using autologous oral epithelial cells have shown safety and efficacy in clinical settings.49 Kobayashi et  al. used this technique into the refractory esophageal stricture model.50 However, refractory esophageal stricture is treated by endoscopic balloon dilation, re-stricture occurs frequently due to tear readhesion after dilatation. The tissue engineering cell sheets prevented re-stricture by inhibiting inflammation at ulcer sites and preventing atrophy and fibrosis of the muscular layer. This new technology has a potential to change the indication of ESD and treatment method for refractory esophageal stricture in the clinical setting.

Liver Several cell therapies for patients with liver dysfunction have been developed. Because the liver is composed of various cells and exerts various functions, reconstruction of liver structures and the maintenance of their functions can be difficult.60 To address these issues, Ohashi et  al. introduced hepatocyte cell sheet engineering under the kidney capsule and successfully engineered a functional system in mice.51,61 However, because of the invasiveness of kidney capsule transplantation, they switched the transplantation site to the subcutaneous cavity.62 They successfully created a uniformly continuous hepatocyte sheet using mouse cells with a spatial two- or three-dimensional functional liver system at the subcutaneous cavity. Sakai et  al. recently developed a human hepatocyte sheet and confirmed its functionality in mice.52 Although subcutaneous hepatocyte sheet transplantation represents a new approach to treating liver failure patients, further studies will be needed to confirm its potential efficacy.

Others Other applications using cell sheet engineering include bladder augmentation using urothelial cells,53 lung air leak sealant using skin fibroblasts,54 and periodontal

treatment using periodontal ligament-derived cells.55 Cell sheet engineering is expected to be applied using various cell types in a number of fields going forward.

Subcutaneous islet transplantation using cell sheet engineering Cell sheet engineering also has been applied in islet transplantation (Table  2). Shimizu et  al. first reported the outcomes of subcutaneous islet cell sheet transplantation using a temperature-responsive culture dish.63 Islet cells were obtained from rats, reduced to single cells using trypsin, and cultured on temperature-­responsive culture dishes. This bioengineered uniform cell sheet showed glucose responsiveness and survived with functionality in  vitro. Saito et al. further reported that subcutaneous islet cell sheet transplantation was able to maintain the long-term function in STZ-induced diabetic SCID mice. Ohashi et  al. used cryopreserved islet cells to create cell sheets and showed their functionality in  vitro.65 The same group investigated a suitable human-­ derived ECM on a temperature-­responsive culture dish and concluded that human-laminin-332 was an optimal ECM.70 However, while tissue engineering islet sheet transplantation was able to achieve a euglycemia state at subcutaneous sites, the efficiency was still not high. Fujita et al. changed the transplantation site of the islet sheet from the subcutaneous cavity to the liver surface in order to improve the therapeutic efficiency and achieved favorable results.66

Our concept of subcutaneous islet transplantation using cell sheet engineering Our concept of cell sheet islet transplantation involves using a cell sheet as a scaffold for islets that produces cytoprotective cytokines and ECM, thereby enabling transplantation while maintaining

TABLE 2  Subcutaneous islet transplantation using cell sheet engineering No.

Name

Cell source

Constituent cells

Animal (recipient)

Transplantation site

References

1

Shimizu

Lewis rat

Uniform

Lewis rat

Subcutaneous

[63]

2

Saito

Lewis rat

Uniform

SCID mouse

Subcutaneous

[64]

3

Ohashi

Lewis rat

Uniform (cryopreserved)

In vitro

In vitro

[65]

4

Fujita

Lewis rat

Uniform

SCID mouse

Liver surface

[66]

5

Hirabaru

Wistar rat

Islets with MSCs sheet

SCID mouse

Subcutaneous

[67]

6

Matsushima

Human

Islets with Fibroblast sheet

In vitro

In vitro

[68]

7

Imamura

Human

Islets with several kinds of sheet

In vitro

In vitro

[69]

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Cytokines

FIG. 1  Our concept of islet cell sheet transplantation involves using MSCs and fibroblast sheets as a scaffold for islets that produce cytokines and extracellular matrix, thereby enabling transplantation while maintaining intercellular binding.

­intercellular binding (Fig. 1). Cell sources were seeded onto 35-mm-diameter temperature-responsive culture dishes. Following an additional 48–72 h of culture at 37°C, the cell sheets were detached spontaneously from the dish by reducing the culture temperature from 37°C to 20°C (Fig.  2). Hematoxylin and eosin (H&E) staining showed that the islets were attached to the cell sheet (Fig. 3), and insulin-positive cells were confirmed by immunostaining (Fig.  4). Furthermore, transmission electron microscopy revealed that the scaffold sheets contained multiple cell layers that established cell-to-cell connections via the formation of tight and gap junctions. Under anesthesia using isoflurane, we transplanted the islets + scaffold sheets into the subcutaneous cavity of STZ-induced diabetic SCID mice in order to evaluate the efficacy of our subcutaneous transplantation (Fig. 5).

Cell sources Previous studies have shown that MSCs have the beneficial ability to regenerate myocardial and skin tissue as well as restore bone cartilage damage through their stem cell activity.71–73 Furthermore, MSCs have demonstrated cytoprotective effects, such as modular effects on immune reactions,74,75 regulation of inflammatory reactions,76,77 inhibition of apoptosis,78 and promotion of vascularization,67,68 through the secretion of cytokines. In islet transplantation, co-transplantation of islets and MSCs reduced the number of islets required to reverse diabetes by promoting graft revascularization. The secretion of VEGF and neuro growth factor by MSCs played an important role in improving vascularization. In addition, co-transplantation of MSCs with islets also helped to improve the graft function and exert protective effects on islets69,79 by secreting angiogenic and antiapoptotic cytokines, such as VEGF, interleukin (IL)-6, IL-10, HGF, and transforming growth factor beta 1 (TGF-β1).80–84 Fibroblasts are also well known to promote vascularization and improve islet viability via the secretion of VEGF, FGF, and HIF-1α,85–88 in addition to being easy to harvest. Furthermore, fibroblasts promote tissue formation between connective tissue and the ECM, helping to maintain the cell structure by producing numerous molecules, such as collagen, proteoglycans, and fibronectin.89

Cytokines

FIG. 2  The tissue engineering islets + MSC sheets were harvested by low-temperature treatment after 72 h of coculture.

Several cytokines are known to exert beneficial effects on islets80,90 and to have pro-angiogenic activity.91 IL-6 is a multifunctional cytokine involved in antigen-­specific immune responses and inflammatory reactions.92,93

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34.  Subcutaneous islet transplantation using tissue-engineered sheets

FIG. 3  H&E staining showed that the sheets adhered to the islets.

Insulin staining 100 mm

FIG. 4  Rat insulin immunostaining of islets cocultured with MSC sheets.

Previous study

FIG. 5  The tissue engineering islets + MSC sheets were implanted into the subcutaneous cavity of STZ-induced diabetic SCID mice.

It also promotes insulin secretion via glucagon-like peptide-1 secretion and is regarded as having an antiapoptotic effect on islets.94 IL-6 is considered a revascularization factor involved in the function and survival of islets when cocultured with MSCs.89 TGF-β1 gene therapy was shown to accelerate the production of heat shock protein 32 and X-linked inhibitor of apoptosis protein. Heat shock protein 32 has a protective effect on islets, while X-linked inhibitor of apoptosis protein exerts an antiapoptotic effect on islets.95–97

In a previous study, we successfully performed subcutaneous islet and bone marrow-derived MSC (BM-MSC) sheet transplantation using tissue engineering to reverse diabetes mellitus in a rodent model.98 To explore the clinical applicability of this approach, we performed similar experiments using human fibroblasts sheet and islets. Fibroblast sheets also exert beneficial effects on the in vitro survival and function of human islets by secreting several cytokines and producing ECM.99 We therefore compared human fibroblasts, BM-MSCs, and adipose-derived MSCs (ADSCs), all of which exert protective effects on human islets in vitro. Each tissue engineering cell sheet showed favorable effects on the outcome of transplantation.100 Although our concept of subcutaneous islet transplantation using cell sheet does not require prevascularization, in terms of efficacy, further development is needed. Experiments in large animals are currently in progress.

Conclusions Cell sheet islet transplantation using MSCs and fibroblast sheets which producing cytoprotective cytokines and ECM is a suitable as a scaffold for islets. Although further studies are needed, this novel approach may be a useful strategy for subcutaneous islet transplantation and may become a possible therapeutic option.

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References

Conflict of interest The authors declare no conflicts of interest in association with the present study.

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39. Kobayashi J, Okano T. Fabrication of a thermoresponsive cell culture dish: a key technology for cell sheet tissue engineering. Sci Technol Adv Mater. 2010;11(1):014111. 40. Nishida K, Yamato M, Hayashida Y, et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med. 2004;351:1187–1196. 41. Shimizu  T, Yamato  M, Kikuchi  A, Okano  T. Two-dimensional manipulation of cardiac myocyte sheets utilizing temperature responsive culture dishes augments the pulsatile amplitude. Tissue Eng. 2001;7:141–151. 42. Shimizu T, Yamato M, Akutsu T, et al. Electrically communicating three-dimensional cardiac tissue mimic fabricated by layered cultured cardiomyocyte sheets. J Biomed Mater Res. 2002;60:110–117. 43. Memon IA, Sawa Y, Fukushima N, et al. Repair of impaired myocardium by means of implantation of engineered autologous myoblast sheets. J Thorac Cardiovasc Surg. 2005;130:1333–1341. 44. Kondoh H, Sawa Y, Miyagawa S, et al. Longer preservation of cardiac performance by sheet-shaped myoblast implantation in dilated cardiomyopathic hamsters. Cardiovasc Res. 2006;69:466–475. 45. Hata  H, Matsumiya  G, Miyagawa  S, et  al. Grafted skeletal myoblast sheets attenuate myocardial remodeling in pacing-­ induced canine heart failure model. J Thorac Cardiovasc Surg. 2006;132:918–924. 46. Sawa Y, Yoshikawa Y, Toda K, et al. Safety and efficacy of autologous skeletal myoblast sheets (TCD-51073) for the treatment of severe chronic heart failure due to ischemic heart disease. Circ J. 2015;79:991–999. 47. Ohki  T, Yamato  M, Murakami  D, et  al. Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-­ engineered autologous oral mucosal epithelial cell sheets in a canine model. Gut. 2006;55:1704–1710. 48. Kanai N, Yamato M, Ohki T, Yamamoto M, Okano T. Fabricated autologous epidermal cell sheets for the prevention of esophageal stricture after circumferential ESD in a porcine model. Gastrointest Endosc. 2012;76:873–881. 49. Ohki T, Yamato M, Ota M, et al. Prevention of esophageal stricture after endoscopic submucosal dissection using tissue-­engineered cell sheets. Gastroenterology. 2012;143:582–588. 50. Kobayashi S, Kanai N, Tanaka N, et al. Transplantation of epidermal cell sheets by endoscopic balloon dilatation to avoid esophageal re-strictures: initial experience in a porcine model. Endosc Int Open. 2016;4:E1116–E1123. 51. Ohashi K, Waugh JM, Dake MD, et al. Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases. Hepatology. 2005;41:132–140. 52. Sakai  Y, Yamanouchi  K, Ohashi  K, et  al. Vascularized subcutaneous human liver tissue from engineered hepatocyte/fibroblast sheets in mice. Biomaterials. 2015;65:66–75. 53. Shiroyanagi  Y, Yamato  M, Yamazaki  Y, Toma  H, Okano  T. Urothelium regeneration using viable cultured urothelial cell sheets grafted on demucosalized gastric flaps. BJU Int. 2004;93:1069–1075. 54. Kanzaki  M, Yamato  M, Yang  J, et  al. Dynamic sealing of lung air leaks by the transplantation of tissue engineered cell sheets. Biomaterials. 2007;28:4294–4302. 55. Akizuki  T, Oda  S, Komaki  M, et  al. Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs. J Periodontal Res. 2005;40:245–251. 56. Shimizu T, Yamato M, Isoi Y, et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ Res. 2002;90:e40. 57. Shimizu  T, Sekine  H, Isoi  Y, Yamato  M, Kikuchi  A, Okano  T. Long-term survival and growth of pulsatile myocardial tissue grafts engineered by the layering of cardiomyocyte sheets. Tissue Eng. 2006;12:499–507.

58. Sekiya  S, Shimizu  T, Yamato  M, Kikuchi  A, Okano  T. Bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential. Biochem Biophys Res Commun. 2006;341:573–582. 59. Leobon B, Garcin I, Menasche P, Vilquin JT, Audinat E, Charpak S. Myoblasts transplanted into rat infarcted myocardium are functionally isolated from their host. Proc Natl Acad Sci U S A. 2003;100:7808–7811. 60. Bhatia SN, Balis UJ, Yarmush ML, Toner M. Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. FASEB J. 1999;13: 1883–1900. 61. Ohashi K, Kay MA, Yokoyama T, et al. Stability and repeat regeneration potential of the engineered liver tissues under the kidney capsule in mice. Cell Transplant. 2005;14:621–627. 62. Ohashi K, Yokoyama T, Yamato M, et al. Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets. Nat Med. 2007;13:880–885. 63. Shimizu  H, Ohashi  K, Utoh  R, et  al. Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus. Biomaterials. 2009;30:5943–5949. 64. Saito T, Ohashi K, Utoh R, et al. Reversal of diabetes by the creation of neo-islet tissues into a subcutaneous site using islet cell sheets. Transplantation. 2011;92:1231–1236. 65. Ohashi  K, Mukobata  S, Utoh  R, et  al. Production of islet cell sheets using cryopreserved islet cells. Transplant Proc. 2011;43: 3188–3191. 66. Fujita I, Utoh R, Yamamoto M, Okano T, Yamato M. The liver surface as a favorable site for islet cell sheet transplantation in type 1 diabetes model mice. Regen Ther. 2018;8:65–72. 67. Milovanova  TN, Bhopale  VM, Sorokina  EM, et  al. Hyperbaric oxygen stimulates vasculogenic stem cell growth and differentiation in vivo. J Appl Physiol. 2009;106:711–728. 68. Jiang M, Wang B, Wang C, et al. Angiogenesis by transplantation of HIF-1 alpha modified EPCs into ischemic limbs. J Cell Biochem. 2008;103:321–334. 69. Ito T, Itakura S, Todorov I, et al. Mesenchymal stem cell and islet co-transplantation promotes graft revascularization and function. Transplantation. 2010;27:1438–1445. 70. Yamashita S, Ohashi K, Utoh R, Okano T, Yamamoto M. Human laminin isotype coating for creating islet cell sheets. Cell Med. 2015;21:39–46. 71. Sasaki  M, Abe  R, Fujita  Y, Ando  S, Inokuma  D, Shimizu  H. Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol. 2008;180:2581–2587. 72. Amado  LC, Saliaris  AP, Schuleri  KH, et  al. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci U S A. 2005;102:11474–11479. 73. Nöth  U, Steinert  AF, Tuan  RS. Technology insight: adult mesenchymal stem cells for osteoarthritis therapy. Nat Clin Pract Rheumatol. 2008;4:371–380. 74. Sato K, Ozaki K, Oh I, et al. Nitric oxide plays a critical role in suppression of T-cell proliferation by mesenchymal stem cells. Blood. 2007;109:228–234. 75. Gieseke  F, Bohringer  J, Bussolari  R, Dominici  M, Handgretinger  R, Muller  I. Human multipotent mesenchymal stromal cells use galectin-1 to inhibit immune effector cells. Blood. 2010;116:3770–3779. 76. Ortiz LA, Dutreil M, Fattman C, et al. Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury. Proc Natl Acad Sci U S A. 2007;104:11002–11007. 77. Yagi  H, Soto-Gutierrez  A, Navarro-Alvarez  N, et  al. Reactive bone marrow stromal cells attenuate systemic inflammation via sTNFR1. Mol Ther. 2010;18:1857–1864.

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78. Block GJ, Ohkouchi S, Fung F, et al. Multipotent stromal cells are activated to reduce apoptosis in part by upregulation and secretion of stanniocalcin-1. Stem Cells. 2009;27:670–681. 79. Sakata N, Chan NK, Chrisler J, Obenaus A, Hathout E. Bone marrow cell cotransplantation with islets improves their vascularization and function. Transplantation. 2010;27:686–693. 80. Choi SE, Choi KM, Yoon IH, et al. IL-6 protects pancreatic islet beta cells from pro-inflammatory cytokines-induced cell death and functional impairment in vitro and in vivo. Transpl Immunol. 2004;13:43–53. 81. Rehman J, Traktuev D, Li J, et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation. 2004;109:1292–1298. 82. Aksu AE, Horibe E, Sacks J, et al. Co-infusion of donor bone marrow with host mesenchymal stem cells treats GVHD and promotes vascularized skin allograft survival in rats. Clin Immunol. 2008;127:348–358. 83. Boumaza  I, Srinivasan  S, Witt  WT, et  al. Autologous bone marrow-­derived rat mesenchymal stem cells promote PDX-1 and insulin expression in the islets, alter T cell cytokine pattern and preserve regulatory t cells in the periphery and induce sustained normoglycemia. J Autoimmun. 2009;32:33–42. 84. Yeung TY, Seeberger KL, Kin T, et al. Human mesenchymal stem cells protect human islets from pro-inflammatory cytokines. PLoS One. 2012;7:e38189. 85. Jalili  RB, Moeen Rezakhanlou  A, Hosseini-Tabatabaei  A, Ao  Z, Warnock  GL, Ghahary  A. Fibroblast populated collagen matrix promotes islet survival and reduces the number of islets required for diabetes reversal. J Cell Physiol. 2011;226:1813–1819. 86. Liu D, Xiao H, Du C, Luo S, Li D, Pan L. The effect of fibroblast activation on vascularization in transplanted pancreatic islets. J Surg Res. 2013;183:450–456. 87. Perez-Basterrechea M, Obaya AJ, Meana A, Otero J, Esteban MM. Cooperation by fibroblasts and bone marrow-mesenchymal stem cells to improve pancreatic rat-to-mouse islet xenotransplantation. PLoS One. 2013;8:e73526. 88. Ono M. Molecular links between tumor angiogenesis and inflammation: inflammatory stimuli of macrophages and cancer cells as targets for therapeutic strategy. Cancer Sci. 2008;99:1501–1506. 89. Park KS, Kim YS, Kim JH, et al. Trophic molecules derived from human mesenchymal stem cells enhance survival, f­unction,

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and angiogenesis of isolated islets after transplantation. Transplantation. 2010;89:509–517. 90. Matsuda T, Suematsu S, Kawano M, et al. IL-6/BSF2 in normal and abnormal regulation of immune responses. Ann N Y Acad Sci. 1989;557:466–476. 91. Kamimura D, Ishihara K, Hirano T. IL-6 signal transduction and its physiological roles: the signal orchestration model. Rev Physiol Biochem Pharmacol. 2003;149:1–38. 92. Karaoz  E, Genc  ZS, Demircan  PC, Aksoy  A, Duruksu  G. Protection of rat pancreatic islet function and viability by coculture with rat bone marrow-derived mesenchymal stem cells. Cell Death Dis. 2010;1:e36. 93. Kutty RK, Nagineni CN, Kutty G, Hooks JJ, Chader GJ, Wiggert B. Increased expression of heme oxygenase-1 in human retinal pigment epithelial cells by transforming growth factor-beta. J Cell Physiol. 1994;159:371–378. 94. Lee  DY, Lee  S, Nam  JH, Byun  Y. Minimization of immunosuppressive therapy after islet transplantation: combined action of heme oxygenase-1 and PEGylation to islet. Am J Transplant. 2006;6:1820–1828. 95. Plesner  A, Liston  P, Tan  R, Korneluk  RG, Verchere  CB. The X-linked inhibitor of apoptosis protein enhances survival of murine islet allografts. Diabetes. 2005;54:2533–2540. 96. Kin  T, Senior  P, O’Gorman  D, Richer  B, Salam  A, Shapiro  AM. Risk factors for islet loss during culture prior to transplantation. Transpl Int. 2008;21:1029–1035. 97. Pankov  R, Yamada  KM. Fibronectin at a glance. J Cell Sci. 2002;115:3861–3863. 98. Hirabaru  M, Kuroki  T, Adachi  T, et  al. A method for performing islet transplantation using tissue-engineered sheets of islets and mesenchymal stem cells. Tissue Eng Part C Methods. 2015;11:1205–1215. 99. Matsushima H, Kuroki T, Adachi T, et al. Human fibroblast sheet promotes human pancreatic islet survival and function in vitro. Cell Transplant. 2016;25:1525–1537. 100. Imamura  H, Adachi  T, Kin  T, et  al. An engineered cell sheet composed of human islets and human fibroblast, bone marrow-­derived mesenchymal stem cells, or adipose-derived mesenchymal stem cells: an in  vitro comparison study. Islets. 2018;10:e1445948.

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C H A P T E R

35 Regulation for regenerative medicine-based therapies Giovanni Migliaccio⁎,† *European Advanced Translational Research Infrastructure in Medicine—EATRIS, Amsterdam, The Netherlands † Center for Biological and Pharmacological Evaluation CVBF, Bari, Italy

O U T L I N E Regulatory approach to regenerative medicine in the EU 499 General considerations 499 A brief historical perspective of the use of cells 500 Advent of cell and gene therapies 500

Therapeutic use of pancreatic cells Pancreas transplant Pancreatic islet transplant—Encapsulated cells RM—In vitro creation of pancreatic cells

501 501 501 502

Differences between United States and European Union regulatory framework Classification procedure

Conclusion

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References

502

500 501

Regulatory approach to regenerative medicine in the EU General considerations The term “regenerative medicine” (RM) has been introduced in the common language to indicate procedures intended to repair or substitute damaged or failing body parts.1 In general, it has a quite large usage in lay and practitioner language with meanings that are often contaminated by hype or commercial purposes.2 For the regulatory agencies, the meaning of RM is quite different as it indicates a specific mode of action for products based on cells. The underlying purpose of regulatory guidance and prescriptions is to ensure the safety of the products offered by health practitioners and industry alike to the public. Two major gatekeeping steps are in place, the first allowing to test the products in humans and the second to market them. The application of these general guidance documents to pancreatic cells use depends on a combination of the risk/benefit evaluation for the specific preparation and on the local legal environment. Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 2 https://doi.org/10.1016/B978-0-12-814831-0.00035-X

As for all medicinal products, efforts are in place to harmonize the risk/benefit evaluation and the procedures leading to the authorization for the marketing. In a simplistic description, RM applications are based on obtaining cells, preparing them, and administering to the recipients to obtain a therapeutic effect through the regeneration of failing endogenous tissue/organs. This means that the operational stages which need to be analyzed for the risk/benefit ratio, before marketing authorization, are three: (a) procurement, (b) manipulation and storage, and (c) administration and follow-up. As guidance for this evaluation, practitioners can turn to different sources which increasing prescriptive value depending on the legal status of the procedure which they need to perform. Noncommercial, laboratory research activities require only the authorization usually applied to the use of animal models and the informed consent of the donors for the human material involved. On the other hand, clinical studies in humans require an understanding of the cumulative risk posed by the product procurement, manipulation, and administration procedures which is not always straightforward.

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A brief historical perspective of the use of cells Historically, the path taken by the blood transfusion procedure in the 1800s and by bone marrow transplants during the 1940s was of trial and error in patients with little hope of survival due to terminal illnesses. This led to an understanding of the ABO and HLA system in time, and a slow increase in efficacy and safety, but both practices were never considered commercial. Blood transfusion, which is perceived as a semi-industrial process due to the large-scale operation involved, is regulated separately from the drugs with a focus on risk reduction for two main factors: the transmission of infective disease and mismatching of the ABO antigens.3 The legal ban to the selling of living human tissues and organs also increased the noncommercial aspect of these practices. Similar considerations led the transplant of organs to be considered a medical practice not subject to commercial regulation as an industrial product. A different script was played for chemical drugs. The thalidomide-related public health debacle of the 1950s,4 primed the governments for the creation of a complex system of preclinical and clinical testing for medicinal products commercially produced and distributed. The current process requires the production of preclinical data sufficient to have an evaluation of the mode of action, identity, potency, and predicting the safe initial dose before authorizing experimentation on human subjects. A final evaluation based on the risk/benefit ratio assessed during the clinical trials leads to the authorization to marketing the medicinal product and the definition of the conditions to do so. This evaluation is separated from the cost/benefit assessment and price negotiation for the final product payment. This system has been in place for more than 60 years, presiding over a sustained increase in the average life-span in the first world society, and an increased infant survival in the third world. The appearance of products derived from living organism (antibodies and growth factors) in the 1980s,5 blurred the line between drugs and living cells. However, the final products were still identifiable by a chemical formula albeit with additional data needed to clarify the sequence and posttranscriptional modification like glycosylation and protein folding. These characteristics and the large-scale industrial manufacturing led to the creation of the biologicals class of medicinal products but did not require changes in the regulatory approach.

Advent of cell and gene therapies The technological advances in the identification and separation of cells based on their phenotype,6, 7 the identification of numerous growth factors,8–10 the ability to obtain modification of the cell genome,11–13 and the development of defined culture media6, 14–16 allowed the

creation of cell populations manipulated in  vitro and intended to exert a therapeutic function in  vivo.17, 18 From a regulatory point of view, these products overlap with existing cell-based preparations intended for transplant (hemopoietic stem cells and pancreatic islets) and blood-derived products (RBC and platelets) with additional safety concerns, including between others somatic mutations and increased oncogenic potential, due to the in  vitro modification linked to cell culture media and procedures. These considerations led to the ruling that placed extensively manipulated cells, gene vectors, and genetically modified cells into a new class of biological medicinal products by the FDA.19 The EU took a similar approach introducing the advanced therapy medicinal products (ATMPs) with the Regulation 1394/2007/EC.20 With this regulation in addition to cell and gene therapies, a third category defined as "tissue engineering products" (TEP) was introduced. This last class of medicinal products was defined as acting through the “regeneration, repair, or replacement” of the target tissue or function. This was the first time that the term regeneration was referred to for a medicinal product in a European legislative document.

Differences between United States and European Union regulatory framework In the United States, as well in Japan, a central authority deals with all the stages of drug development, from the preclinical to clinical and marketing authorization. In Europe, the European Medicine Agency (EMA) is delegated by the European Commission to the evaluation of the Market Authorization Applications (MAA) for all the novel chemical entities and all biologicals, including ATMPs.21 The formal market authorization is by the European Commission and it is valid for all the Member States at once. However, Member States have maintained the competence on the Clinical Trials authorization and surveillance, manufacturing sites (Good Manufacturing Practices, GMP) and testing facilities (Good Laboratory Practices, GLP) inspection and authorization. A treaty between the EU Member States allows the mutual recognition of the facilities and the validity of the data produced for the clinical trials and the marketing authorization procedures. Similar treaties are in place with other non-EU countries. Recently, with the Regulation 536/2014 the coordination of the clinical trial authorization process for multinational trial has been given to the EMA to foster a faster turnaround of the applications. However, the evaluation's experts are members of the different national agencies, while the legal authorization remain a competence of the States. Similarly, a set of three Directives regulate

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Therapeutic use of pancreatic cells

the procurement, storage and distribution of human cell and tissues for therapeutic purposes.22 The application and supervision of these directives are relayed to the national authorities.

Classification procedure Currently, the sponsors in the United States may contact the FDA Office of Combination Products, which can provide formal or informal input regarding which of the medical product centers may have into jurisdiction (CDER, CBER, or CDRH) or contact the relevant jurisdiction officer directly.23 In the EU, the correct authority for each stage of development depends on the classification (is it a medicinal product or a transplant?) and on the location (where it is produced and clinically tested?). The Regulation 1394/2007/EC gives the Committee for Advanced Therapies (CAT), at EMA, the power to decide if a product is an Advanced Therapy20 allowing the developers a clear pathway for their product development strategy. It also allows the CAT to express an opinion of the validity of the preclinical data for the MAA through the certification procedure. In a simplified way, the following general rules are applied: • For a cell population, like pancreatic cells, to be included in the cell therapy medicinal products, the cells need to be extensively manipulated or to be used to exert a function which is not normally expected in vivo. As an example, the administration at nonphysiological concentration, or in a novel location in the body, is a mechanism exploited to exert a function normally not expected. The expansion of the cell number, or inducing maturation, in vitro is considered an extensive manipulation. In the Annex I of the Regulation 1394/2007, a list of manipulation which are considered not extensive is provided. • To be included in the gene therapy medicinal product class, they need to be genetically modified by mean of a recombinant nucleic acid or the therapeutic action is directly performed through the product of a recombinant nucleic acid. • To be included in the tissue engineering products, their mode of action must be exerted through a regeneration, replacement, or repair action or the cell population is administered in combination with a medical device. A reflection paper24 and public summary of previous classification procedures are available on the EMA website allowing the sponsors to rapidly identify if their product falls in a category already discussed and defined.

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Therapeutic use of pancreatic cells Pancreas transplant Diabetes type I is the most common pathology requiring the replacement of the pancreas, with nearly 50,000 procedures performed worldwide from the first attempt in 1966 at the University of Minnesota. However, as amply discussed elsewhere in this book (Vol. 1), the transplant of the pancreas is not devoid of the complication and side effects either from the immune suppression regimes required to avoid graft rejection and challenges linked to the lack of suitable donors. The number of pancreas transplant performed yearly is decreasing steadily with alternate approaches taking its place.25 As discussed in the introduction, organ transplants fall in the domain of the medical practice and are covered as best practices by guidelines from scientific societies. Regulatory aspects focus on safety issues linked to sourcing and donor screening.

Pancreatic islet transplant—Encapsulated cells The use of pancreatic islet for transplantation was introduced by the Edmonton group in 1988.26 It has less morbidity linked to the surgery and an overall performance similar to the pancreas transplant.25 In addition, the procedure of isolation of the pancreatic islet allows the use of marginal donors unsuitable for the whole pancreas donation. The use of the pancreatic islet does not reduce the need for immunosuppression and the related long-term morbidity. The current status of the pancreatic islets transplant is also amply discussed elsewhere in this book (Vol. 1). In the EU, the preparation of pancreatic islets according to the Edmonton protocol is not considered an extensive manipulation as the functional unit of the pancreas (i.e., the pancreatic islet) is not disrupted.27 From an EU regulatory perspective, encapsulated pancreatic islets might be a combined TEP27 and fall under the Regulation 1394/2007/EC on the basis of the combination of a medical device with living cells and on the extensive manipulation of the cells itself. The encapsulation of allogeneic or xenogeneic cell population in a shell of permeable material allows the exchange of nutrients and small proteins (including insulin) but do not expose the cells to the immune detection of the host bypassing the need for immunosuppression. A full disclosure, of the characteristic of both encapsulating device/matrix and of the pancreatic islets, is required for the clinical studies and the marketing authorization. An example of the possible parameters required could be: (a) the average number of pancreatic islets for single capsule, (b) the size of the pores and exclusion limit for the matrix, (c) its stability in time, and (d) the length of pancreatic cell survival and the response to glucose challenge in vivo. If the matrix or device used

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has a CE marking, the EMA can consult with the Notified Body involved in the certification for the relevant issues on its safety characteristics. From the point of view of the manufacturing, the creation of droplets containing the pancreatic islets in a reliable and consistent manner would be one of the major challenges for the scalability of the process and for the expansion of the manufacturing in multiple sites. Overall, for this particular class of medicinal products, the risk factors to be assessed are mostly linked to the encapsulating matrix and to the possible release of the pancreatic cells due to breakage for mechanical stress. The cells released could theoretically transmit infective diseases or rise an immune response undermining the lesser concerns due to their isolation to start with.

RM—In vitro creation of pancreatic cells RM approaches involving the use of pancreatic cells are limited by the paucity of donors and the risk of transmission of unrelated pathologies as infective agents or precancerous elements. The appearance of technologies like the embryonic stem cells (ESCs) or the induced pluripotent stem cells (iPSCs) opened the possibility to obtain stable sources of matched donor or autologous cell lines which could be differentiated in the desired element for the reconstruction of the pancreatic functions. To maintain and expand ESC and iPS cell lines to the amount where a commercial use can be planned is not an easy task. Immortalization, proliferation, and differentiation in  vitro are all process which are associated with an increased risk of senescence and oncogenic transformation. The steps required to obtain pure cell population are only partially understood and analytical tools capable to assess single cells genomic alteration are only now becoming feasible. Proliferation and differentiation, in an artificial environment under a proliferative pressure, are bound to result in some form of modification from donated pancreatic cells. It is unclear at the moment if such modifications are relevant for their ability to deliver the desired functions and if they pose any significant risk. Items that the regulatory agencies identified as safety concerns where the presence of antigens derived from the culture media on the cells surface, senescence of the final cells after a fast proliferation required to expand the population to the desired cell number, presence of nondifferentiated, possible cancerous, cells in the “final product,” lack of consistency in the cell population obtained from different donors, lack of three-dimensional arrangement of the cells obtained in vitro. These were all factors identified in addition to the possibility to carry infective agents, as virus, bacteria or mycoplasma.

Conclusion Pancreatic cell use is increasing rapidly following the capacity to expand primary cells ex vivo and to obtain progenitors which could be expanded and maintained for a long time. The rapid progression of the field is hampered by the lack of knowledge on the long-term safety of the cell population used. However, novel analytical tool allowing a genomic and proteomic scanning of single cells are now coming of age and will allow the regulatory science to better define the identity and function of cells as the active principle of this novel class of drugs.

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adipose-derived stem cells—a review. Biotechnol Adv. 2018;36(4): ­ 1111–1126. https://doi.org/10.1016/j.biotechadv.2018.03.011. Lavon  N, Zimerman  M, Itskovitz-Eldor  J. Scalable expansion of pluripotent stem cells. Adv Biochem Eng Biotechnol. 2018;163:23–37. https://doi.org/10.1007/10_2017_26. Jacobson EF, Tzanakakis ES. Human pluripotent stem cell differentiation to functional pancreatic cells for diabetes therapies: innovations, challenges and future directions. J Biol Eng. 2017;11:21. https://doi.org/10.1186/s13036-017-0066-3. Sordi  V, Pellegrini  S, Krampera  M, et  al. Stem cells to restore insulin production and cure diabetes. Nutr Metab Cardiovasc Dis. 2017;27(7):583–600. https://doi.org/10.1016/j.numecd.2017.02.004. FDA. Guidance for human somatic cell therapy and gene therapy. https://www.fda.gov/downloads/BiologicsBloodVaccines/ GuidanceComplianceRegulatoryInformation/Guidances/ CellularandGeneTherapy/ucm081670.pdf; 1998. EUR-Lex-02007R1394-20071230-EN-EUR-Lex. https://eur-lex. europa.eu/legal-content/EN/TXT/?uri=CELEX%3A02007R139420071230. Accessed November 28, 2018. Regulation (EC) No 726/2004 of the European Parliament and of the Council of 31 March 2004 Laying down community procedures for the authorisation and supervision of medicinal products for human and veterinary use and establishing a European Medicines Agency (text

22.

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25. 26.

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with EEA relevance); vol. 136; 2004. http://data.europa.eu/eli/ reg/2004/726/oj/eng. Accessed November 28, 2018. Directive 2004/23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells; vol. OJ L; 2004. http://data.europa. eu/eli/dir/2004/23/oj/eng. Accessed November 28, 2018. Witten CM, McFarland RD, Simek SL. Concise review: the U.S. Food and Drug Administration and regenerative medicine. Stem Cells Transl Med. 2015;4(12):1495–1499. https://doi.org/10.5966/sctm.2015-0098. Reflection paper on classification of advanced therapy medicinal products. https://www.ema.europa.eu/en/documents/scientific-guideline/reflection-paper-classification-advanced-therapy-medicinalproducts_en-0.pdf 2015. Accessed July 14, 2019. Dean PG, Kukla A, Stegall MD, Kudva YC. Pancreas transplantation. BMJ. 2017;357:j1321. https://doi.org/10.1136/bmj.j1321. Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW. Automated method for isolation of human pancreatic islets. Diabetes. 1988;37(4):413–420. Scientific recommendation on classification of advanced therapy medicinal products. https://www.ema.europa.eu/en/human-regulatory/marketing-authorisation/advanced-therapies/ advancedtherapy-classification. Accessed July 12, 2019.

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36 Catalyzing beta-cell replacement research to achieve insulin independence in type 1 diabetes: Goals and priorities Esther Latres JDRF International, New York, NY, United States

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novel or repurposed therapies being developed for type 2 diabetes and other diseases as adjunctive to insulin treatType 1 diabetes (T1D) results from an immune-­ ment has further improved glycemic control and overmediated loss of functional beta cells. Children and adults all metabolic homeostasis in T1D.3, 4 While therapies for living with T1D must monitor their glucose levels multi- treating T1D with current insulin analogs, technological ple times each day and rely on nonphysiologic exogenous approaches, and diligent self-care have been shown to insulin therapy to try and maintain a glycemic range improve diabetes outcomes, the percentage of people not close to physiological levels to prevent the risks or con- reaching their glycemic targets, the incidence of severe sequences of extreme blood glucose excursions. Insulin hypoglycemia and diabetic ketoacidosis (DKA), as well therapy to manage T1D is achieved by using short- and as the risk of acute and chronic end-stage diabetic comlong-acting insulin analogs administered via subcutane- plications, remain unacceptably high.5, 6,6a T1D is a chronic autoimmune disease that proous injections or infusion pumps. The use of devices that provide continuous blood glucose monitoring (CGM) gresses through distinct stages. The ability to stage the has significantly contributed to improvements in man- progression of the disease, from the initial presence of agement of the disease and demonstrated improvements ­beta-cell autoimmunity with no signs of dysglycemia, to in glucose control,1, 2 however adoption of these technol- the occurrence of diabetic complications due to a long-­ ogies remains a barrier. Over the past 10  years, several standing symptomatic disease, provides the opportuapproaches have been developed to combine CGMs with nity to develop diagnostic tools and multiple strategies glucose management algorithms and insulin-infusion for therapeutic interventions.7, 8 JDRF International pumps to generate automated insulin delivery (AID) was founded in 1970 by parents of children with T1D or artificial pancreas (AP) systems. Moreover, the use of with the goal of finding a cure for T1D. Today, JDRF’s

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36.  Catalyzing beta-cell replacement research to achieve insulin independence in type 1 diabetes: Goals and priorities

goal remains the same but has expanded its research commit­ment to preventing the disease and delivering new therapies or devices to improve outcomes and reduce the risks and burdens of T1D. The vision of JDRF is a world without T1D and the mission is to accelerate breakthroughs and development of therapies to cure, prevent, and treat T1D and its long-term complications. Since its inception, JDRF has invested over $2 billion worldwide in research projects and actively influenced policy and the commercial landscape to further its mission. As a patient driven organization, the main focus is to improve outcomes by allocating funds to promising research at all stages of development, from exploratory and translational research to human studies to validate clinically meaningful therapies. Given the cost and challenges of developing therapeutic products, it is critical to establish and potentiate strategic collaborations with the pharmaceutical and biotechnology industries, as well as other funding organizations, which can provide expertise and resources throughout the development stages to make products available to people with T1D. Moreover, to facilitate and accelerate the development of therapies across our pipeline, JDRF scientists work closely with the Advocacy team to ensure continued federal support of T1D research through the Special Diabetes Program (SDP), and to ensure reasonable regulatory, reimbursement and clinical adoption pathways exist to support development of beta-cell replacement (BCR) therapies (Fig.  1). To complement the development of new therapies, efforts to define clinically

DISCOVERY RESEARCH

TRANSLATIONAL RESEARCH

meaningful outcome measures beyond the established glycated hemoglobin (HbA1c) is a priority for JDRF and the T1D community. The prioritization of clinically meaningful outcomes not captured by HbA1c alone such as hypoglycemia or time in a desired glucose range (TIR) may be relevant to assess the safety and efficacy of therapies and technologies.9 For example, the significant improvements in health-related quality of life that have been recently reported in T1D patients with severe hypoglycemic events (SHE) after islet transplantation, reveals the impact and burden that hypoglycemia represents in this population.10

Beta-cell replacement While understanding the pathogenesis of T1D will contribute to future attempts to prevent and reverse the course of the disease, in the absence of a cure that halts autoimmunity and restores beta cells, replacing beta-cell function via cell therapy remains the only approach with a clinical proof of concept that demonstrates insulin independence can be achieved in long-standing T1D. Importantly, islet transplantation can reverse SHE and hypoglycemia unawareness, a serious consequence of T1D, as well as halt or stabilize other complications associated with the disease.11 While pancreatic islet transplantation is an available strategy, its viability for the vast majority of individuals living with T1D is limited by the number of organ donors and the need for ­long-term

REGULATORY APPROVAL

HEALTHCARE COVERAGE

Moving scientific discoveries from the laboratory to clinical trials Engage commercial commitment

Landscape analysis to identify gap-filling strategies

CLINICAL ADOPTION

Expanding access to the latest T1D therapies through education Accelerate development timelines and access via regulatory and policy activities Ensuring treatments

Put together appropriate teams and resources to succeed

accessible Develop business model and economic analysis

FIG. 1  JDRF’s ultimate metric and success are the improved outcomes for all ages and stages of T1D. JDRF supports R&D efforts throughout the development pipeline, ensuring access, and adoption of therapies.

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i­mmunosuppression therapy required to prevent rejection and the recurrence of autoimmunity. Therefore, JDRF’s BCR program has prioritized moving beyond the proof of concept provided by islet transplantation toward commercialization of a cell therapy product consisting of an unlimited insulin-producing cell source capable of restoring glucose control and delivering long-term insulin independence without the need for chronic systemic immunosuppression. The availability of safe and effective BCR therapies would restore the ability of people living with T1D to achieve significantly better blood-glucose control with little or no effort, thereby eliminating the excessive burden of managing T1D, and avoiding many of the life-threatening complications of the disease. There is extensive research in the field on what constitute the most functional source of insulin-producing cells whether is a complete islet-like structure or beta cells alone, but for the purpose of this review the terminology of beta cells will refere to all insulin-producing sources under validation. JDRF’s commitment to the development of BCR therapies is reflected by the funding resources allocated to the program. While most of the funding has been assigned to support research and clinical grants from academic institutions or research centers, significant investments have been made through other mechanisms that are key to the advancement of the program. These mechanisms include industry partnerships, postdoctoral training and early career awards for researches at relatively early stages of their independent career, and grant proposals for highly innovative research (Fig. 2).

Islet transplantation The history of islet and pancreas transplantation used to treat T1D has demonstrated the apparent utility of these procedures in improving the clinical outcomes in a subset of individuals with T1D.12–14 At first, the surgical risk of pancreas transplantation and the complexity to process islets restricted their use to patients who needed other life-supporting organ transplantation.

This equipoise has changed in the past three decades as improvements in islet production, engraftment strategies, and immunosuppressive therapy, have resulted in the introduction of donor islet transplantation as a minimally invasive approach to restore glucose control in people living with T1D. Over the years, clinical outcomes have improved and patients with life-­threatening hypoglycemia unawareness are now considered a population that can benefit from these procedures. JDRF funding for basic science research significantly contributed to pave the pathway into the clinic, and has played a role in many of the steps that have brought human cadaver islet transplantation to people with T1D. Most notably, following the first publication of what became known as the “Edmonton Protocol” for human cadaver islet transplantation, JDRF funded a number of centers internationally to catalyze the evaluation of this procedure. These studies contributed to the approval of this procedure in many countries and stimulated the funding by the NIH of a multicenter phase 3 trial to attain registration of a cadaveric islet cell preparation as a product in the United States.11 The phase 3 clinical data recently reported by the Clinical Islet Transplantation (CIT) Consortium have demonstrated durable near-­ normal glycemic control and restoration of hypoglycemia unawareness after islet transplantation for 2 years. Additional data from the Collaborative Islet Transplant Registry (CITR) shows up to 44% of recipients remain insulin independent after 3 years15 and as immunosuppression protocols are optimized the transplant survival rates appear to be increasing. While islet transplantation procedures are presently used to treat individuals with life-threatening hypoglycemia unawareness and increased incidence of hypoglycemic events, the excitement and clinical impact of these approaches triggered JDRF to commit further funding in a variety of areas to make them relevant to as many individuals with T1D as possible. The first priority identified was the need to create and validate alternative sources of insulin-producing cells

73%

Research Grants

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FIG. 2  Distribution of beta-cell replacement commitments from fiscal years 2014 to 2017 by grant funding mechanism.

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36.  Catalyzing beta-cell replacement research to achieve insulin independence in type 1 diabetes: Goals and priorities

that did not involve the limited resource of human cadaver pancreatic islets. Supporting basic research to understand the signals involved in pancreatic development was a key area of focus that led to the ability to generate functional beta cells or islet-like clusters containing additional cell types of pancreatic islet, from a variety of stem cell sources using in vitro protocols. These studies have resulted in the establishment of commercial entities like Viacyte that are already in clinical trials. Others are now scaling up and further optimizing the differentiation and maturation process of stem cells into functional islet-like clusters capable of restoring glucose control in models of T1D.16 JDRF has supported preclinical and clinical studies to validate porcine islets as a potential source of insulin-producing cells for human transplantation, ­ and contributed to the past and current developments in the field.17 The second priority identified was to develop strategies to protect the implanted cells from alloimmune, ­xenogeneic, and autoimmune rejection, as the impediment to broad application of BCR is the use of chronic immunosuppressive drugs. Lastly, the third priority was to optimize the local microenvironment of the transplant site for the long-term graft function. Over the years, JDRF has invested research funding into a number of areas including cell encapsulation approaches, local immunomodulation strategies, and genome editing to make the transplanted cells less immunogenic or more robust to adverse conditions. However, the significant advances seen in this space would not have been possible without the dedication of the BCR researchers and additional funding organization such as the Helmsley Charitable Trust and National Institutes of Health (NIH). Today, while a commercially available BCR product remains a distant prospect, there are a variety of approaches in preclinical validation and early clinical evaluation. One can expect to see more in the coming years and impact each other as we learn, collaborate, and bring BETA CELL SOURCE Stem Cells

Porcine Islet

Development and Optimization

together organizations in the regenerative medicine field facing the same challenges to fill the gaps.

BCR strategies Current lines of investigation and commercialization of BCR therapies have focused on the development of encapsulation approaches to evade autoimmune, allogeneic, and xenogeneic rejection of insulin-producing cell sources. However, in the long run there may be alternative strategies one should consider in the design of future generation products. As such, while optimizing the protocols to generate beta-cell sources, the validation of strategies for providing immunoprotection of these cells is currently a major priority (Fig. 3). For example, genome editing could be applied cells to mitigate immune recognition and promote tolerance so that less or no immunosuppression or encapsulation would be required. Another potential strategy involves induction of immune tolerance toward transplanted cells and may be an approach that would direct the host immune system to accept grafts without the use of chronic systemic immune suppression, obviating or reducing the requirement of a fully encapsulated system. At the current stage of investigation, parallel efforts are being pursued by JDRF-funded investigators, as one should consider that the most effective BCR therapy may be comprised of several components, such as a genetically modified cell source, an implantable delivery device, and a drug-based therapy to provide local immunosuppression or enhance cell survival. The significant advancements and limitations of the several components involved in a BCR therapy have been extensively reviewed in outstanding research publications and reviews, so only a brief overview is provided. Alternative cell sources: Recent advances in cell therapy have positioned stem cell-derived islet clusters and porcine islets as the most promising replenishable alternative sources of insulin-producing cells. The ultimate goal is to provide clinical proof of concept for

CELL SURVIVAL & PROTECTION Encapsulation

Provide Complete Immunoisolation

CLINICAL TRANSLATION

Scaffolds and Deviceless Strategies

Rely on Cell Engineering or Other Forms of Immune Tolerance

FIG. 3  Schematic representation of JDRF Beta-Cell Replacement priority areas.

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Identify Favorable Implantation Sites and Accelerate Promising Approaches



Beta-cell replacement

the d ­ evelopment of a renewable insulin-producing cell source from human stem cells or porcine islets.16, 18–28 a) Allogeneic human stem cells (hSCs): Progress in stem cell biology, pancreatic development, and betacell differentiation and maturation has resulted in protocols for deriving human pancreatic endocrine cell progenitors and surrogate beta cells from human embryonic stem cells (hESC) and human-induced pluripotent stem cells (iPSC). The jury is still out on whether the optimal commercial cell therapy product would incorporate a pancreatic progenitor cell population or a fully mature beta-cell population. Both cell sources have advantages and challenges. Current stem-cell derived preparations that are already in clinical testing generate insulin-producing cells contain polyhormonal cell populations that are not fully functional at the time of transplantation, and it remains to be determined whether additional non-β endocrine cells from pancreatic islets are beneficial and required to constitute the most efficacious cell therapy product for T1D. Development of stem cell-derived therapies also requires long-term safety assessment, such as the risk of uncontrolled growth and formation of teratomas, and establishment of effective manufacturing processes. The yield, purity, and consistency of these cell preparations will need to be optimized and scaled up under good manufacturing practice (cGMP) quality defined by regulatory agencies with responsibility for the review and approval of medicinal products. Over recent years, companies that have evaluated the safety and optimized manufacturing processes are poised to develop hSC-derived pancreatic progenitors and functional surrogate beta cells as potential commercial BCR products. Although using autologous iPS derived stem cell populations have been demonstrated to make beta-cell progenitor populations in vitro, the JDRF strategic approach remains focused on allogeneic sources as the need to control autoimmune destruction still present and there may be manufacturing benefits to a single source product. b) Xenogeneic islets isolated from pathogen-free pigs: As pig insulin has been known to be efficacious in humans, pigs are considered ideal candidates to provide islets for xenotransplantation. Xenotransplantation of porcine islets has shown some success in restoring glucose control in T1D model systems, and is gaining acceptance as an alternative readily available cell source.17 Key to the success of porcine islets as a source for BCR therapies will be to establish which developmental stage (neonatal, juvenile, or adult) will provide the best outcome, as well as overcoming the human

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immune response to porcine islets and concerns about transmission of porcine endogenous retroviruses (PERVs) from the pig genome. Further understanding of the xenogeneic immune response and infectivity potential, as well as the ability to eradicate targeted sequences using genome editing, make xenotransplantation a promising option.29–32 JDRF has supported the advancement of xenotransplantation by funding clinical studies to test the efficacy of encapsulated pig islets, and currently funding several preclinical projects aimed at validating approaches for xenotransplantation using encapsulation devices and gene editing of pancreatic pig islets (check JDRF.org for JDRF Funded Research). Strategies to protect implanted cells: Advances in biomaterial research, 3D medical printing, immunomodulation, and drug delivery strategies, as well as preclinical models to assess fibrosis and allogeneic responses have allowed development of both device and device-less approaches to protect beta cells after implantation.33 a) Encapsulation Technologies: Developing effective encapsulation approaches for immune protection of cell sources to circumvent the use of immunosuppression is currently a major area of investigation.34, 35,35a Immune protection of islet cells via encapsulation could overcome allogeneic, xenogeneic, and/or autoimmune responses against the foreign tissue. Encapsulation technologies use biomaterials to create an immunoprotective physical barrier around islet cells and are thereby designed to limit, and ideally eliminate, undesirable immunological responses to the foreign graft. A permselective biocompatible material allows for exchange of small molecules such as oxygen, glucose, insulin, and selected nutrients in and out of the device via diffusion, while blocking larger molecules, such as immune cells and antibodies. Devices under investigation differ by biomaterials, shape configuration, and methods used in fabrication. Several synthetic polymer and natural materials including alginate, agarose, polysulfone, and polyethylene glycol (PEG) are or have been used to encapsulate islets. Encapsulation schemes can be broadly categorized into macroencapsulation devices—one device containing a large mass of islet cells, and microcapsules—each capsule containing single islets or small groups of islets. Newer technologies under development aim at further reducing the thickness of the capsule wall: conformal coating uses novel coaxial flow apparatus to achieve uniform but thin coverage of islets; nano-encapsulation typically uses chemical and electrostatic interactions to deposit biomaterials

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36.  Catalyzing beta-cell replacement research to achieve insulin independence in type 1 diabetes: Goals and priorities

via layer-by-layer assembly at the nanometer scale. Micro- and macro-encapsulation technologies offer different advantages and challenges. Due to the reliance on passive transport for nutrient, glucose, and insulin exchange, the distance between the native tissue’s blood supply and the cell graft, and the limited ability to maintain a nutrient and oxygen rich environment, poses a limitation on cell survival and proper glucose response. This limitation is expected to be greater for macroencapsulation devices.36 Microcapsules provide a larger surface area/volume ratio, maximizing diffusion of oxygen and nutrients, but as currently employed they typically do not allow for complete graft retrieval, whereas macroencapsulation devices enable retrievability of the entire graft, which may be a desirable feature for products using hESC/iPSC-derived cells. At this time, JDRF is supporting both design approaches to better understand the potential benefits and risks of each approach. b) Scaffolds: A second approach to improve islet engraftment, survival, and function, and thus transplant outcomes, consist of the use of scaffolds. Several noteworthy reviews have been recently published on this subject.37–39 As encapsulation is already covered above, this section will address the use of “open” or macroporous scaffolds which do not provide a physical barrier (membranes or capsules) to protect the transplanted cells but allow and facilitate direct integration of the implanted construct with the host. Scaffolds can be synthesized from both natural and synthetic materials and can be used to accomplish various objectives, the most common being delivery of the islets where they facilitate islet distribution within the implant and can provide physical space.39–48 While most scaffolds are not designed to be immunoprotective, the ability to confer bioactive properties or use genetically modified cells is a feature that makes the use of scaffolds very appealing, as they allow researchers to engineer the transplant site in a highly defined manner. The significance of islet interactions with extra cellular matrix (ECM) has been widely described by several groups. The incorporation of ECM or synthetic ECM cell adhesion motifs (e.g., tripeptide Arg-Gly-Asp or RGD) into scaffolds to recapitulate the native islet 3D niche has been shown to improve islet survival and function, and reduced the time to achieve euglycemia in animal models of islet transplantation.49–56 Scaffolds can also be used to deliver oxygen or growth factors that induce angiogenesis and promote vascularization at the transplant site, thus enhancing islet viability and function.57–63 Finally, scaffolds can serve as a platform for controlled and localized delivery of drugs and immunomodulatory factors to protect transplanted cells from the host immune response, and

potentially induce tolerance or immunomodulation without the adverse effects of systemic delivery.64–67 Moving forward, one can envision using a combinatorial approach where selected modifications are employed, perhaps in combination with encapsulation or genetic engineering strategies, to develop an effective BCR product. c) Device-less approaches: Alternative approaches that include the combination of islet transplantation with various types of immunotherapies are being explored to obviate the need for systemic immunosuppression, and to provide a proof of concept for future BCR strategies. Induction of mixed chimerism in the recipient by hematopoietic stem cell transplantation has shown promise in the transplantation of solid organs,68 but some associated risks including the toxicity associated with the procedure and the potential for the development of graft-versus-host disease (GVHD), may outweigh the benefits given other therapeutic options currently available in the T1D population,.69–71 An alternative to this approach is the use of regulatory T-cell (T-reg) therapy, which has already produced promising results in the treatment of early onset T1D.72 Although there are caveats to implementing such an approach in the context of transplantation such as the attenuation of effector T-cell responses, it is an avenue of research that holds promise.73–76 To circumvent some of those caveats and simplify the delivery of T-regs, others have also explored genetic engineering approaches to improve the homing of T-regs to the graft by introducing chimeric antigen receptors (CARs).77 Additional efforts in the field of genetic engineering also include the genetic modification of cells, either islets or stem-cell derived insulin-producing cells as well as accessory cells that can be co-delivered, to modulate the immune response via repression of recognition surface markers such as MHC molecules and expression of checkpoint inhibitors.78 Finally, several strategies to reprogram immune cells in vivo to induce antigen-specific tolerance through the targeting of antigen-presenting cells (APC) and lymphocytes for delivery of antigen and tolerogenic signals are also under development.79 This area is advancing quickly and each approach may be simplified and made more applicable to T1D over the coming years.

Clinical trials in beta-cell replacement JDRF has been a driving force behind research that could make BCR a widely available option for people with T1D. While most opportunities and the largest commitment have been placed in exploratory research,

B. Bioengineering and regeneration of the endocrine pancreas



Beta-cell replacement

preclinical validation of a renewable beta-cell source and approaches to protect transplanted cells from immune rejection, our efforts have contributed to the launching of human clinical trials to test the safety and efficacy of replacement therapies. ViaCyte Inc., a San Diego-based regenerative medicine company, developed and tested its VC-01 device (also known as the PEC-Encap) in a phase I/II clinical trials. The combination product, comprised of the company’s Encaptra capsule filled with a subtherapeutic dose of its proprietary line of PEC-01 beta-cell precursors, is the first to use human stem cell-­ derived beta cells intended to treat T1D. While testing so far has provided a great deal of learning, including firsttime testing of PEC-01 (pancreatic endoderm) cells in humans, detection of further differentiation of the starting cell population and demonstration that the device was safe and immunoprotective, the response has v ­ aried across participants in the trial. In many participants, the efficacy of PEC-Encap appears to be inhibited by the foreign body response. This well-described response to foreign implants is observed in some individuals and limits the establishment of an effective vascular network surrounding the device, and potentially hindering the survival of the transplanted cells. As the PEC-Encap optimization progresses, the company is testing a second device known as PEC-Direct, which uses the same precursor cells as the PEC-Encap but has an open design, encouraging infiltration of blood vessels to support direct vascularization of the cells. As the device requires the use of traditional transplantation immunosuppression, the PEC-Direct device is designed for use by people with a critical unmet need, severe hypoglycemic unawareness, preferably those who already had a kidney transplant and are under immunosuppression therapy. It is hoped that the insights gained from the new clinical trial combined with the optimization of PEC-Encap will inform the development of immunoprotective product candidates going forward for all people with T1D. Israeli biomedical company Beta O2 has also tested its βAir encapsulation device in phase 1 clinical trials following encouraging preclinical data.80, 81 The βAir system is composed of a macroencapsulation device implanted subcutaneously in which oxygen is periodically supplied through a subcutaneous port to support survival and function of the islets. Implantation of the βAir system in humans demonstrated a good initial safety profile and successfully prevented immunization and rejection of the transplanted allogenic islets, allowing the survival of beta cells for several months. While further development to enhance the function of transplanted cells needs to occur, data from testing devices in humans provide crucial and valuable information that preclinical models fail to. The University of Miami’s Diabetes Research Institute (DRI)’s BioHub is testing an islet transplant scaffolding

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system that has so far enabled one participant to restore euglycemia and reach insulin independence using human cadaver islets.82 To overcome the limitations of intrahepatic islet transplantation, the biologic scaffolding system mixes donor islets with a biodegradable gel matrix generated from thrombin and autologous plasma and is laparoscopically implanted onto the recipient’s omentum, a layer of densely vascularized tissue covering the abdominal organs. The biodegradable gel matrix slowly dissolves, leaving the islet cells intact and functional. Transplant recipients must be on immunosuppressive therapy, but in addition to the benefits provided to the T1D population with hypoglycemia unawareness or severe hypoglycemia events, it generates a proof of concept for future combinations of immunoprotective strategies. JDRF is actively funding research for porcine islet transplantation, and in 2009 supported the phase I/II clinical trial of Diabecell in unstable type 1 diabetic subjects, which comprises encapsulated neonatal porcine islets for transplantation without immunosuppression.83 Diabecell, which is manufactured by Living Cell Technologies, was implanted in 16 subjects at doses ranging from 5000 and 20,000 IEQ/kg injected into the peritoneal cavity. While data from the JDRF funded study, as well as a follow up clinical study of the Diabecell,84 was safe and showed some level of clinical benefit, further investigation and optimization of the approach is needed.

Beta-cell replacement: Key considerations It is expected that BCR products using either hSC-­ derived cell products or porcine islet cells will evolve over a multistage therapeutic concepts. Each subsequent iterative product is anticipated to show improved features (renewable cell source, immunosuppression strategy, longevity, or surgery requirements), delivery of better glycemic outcomes, and broader clinical applicability within the T1D population (more details on roadmap and therapeutic concepts at http://grantcenter. jdrf.org/information-for-applicants/research-­priorityareas/). While discovery research, preclinical, and most importantly clinical data, will allow improvement over previous versions, the challenges, realistic deliverables and requirements for developing a therapeutic product should be considered from early stages.85 For example, (i) how to deliver a cell dose and packing density with an optimal insulin kinetics that is safe and clinically relevant, (ii) ensuring that early stage materials can translate into clinical-grade products, and (iii) how to protect grafts from allogeneic or xenogeneic rejection and recurrence of autoimmunity by tailoring cell sources to a specific cell delivery strategy whether in an encapsulation device, scaffold, or a device-less approach. As extensively reviewed in many research publications and

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TABLE 1  Considerations for the development of beta-cell replacement therapies TECHNICAL CONSIDERATIONS • Identifying the best cell source and composition • Cost of goods for manufacturing stem cell-derived products or porcine islets • Determining the best method to protect the cells: balance between encapsulation systems with limited host/graft integration, and scaffolds that rely on other forms of immune protection or use genome engineered cell sources • Inflammation, scarring, fibrosis, and oxygenation hurdles • Methods for measuring oxygen or vascularization levels in the implantation site including imaging technologies are not sufficient to predict graft function or outcome VARIOUS FIELDS OF EXPERTISE/COLLABORATION • Understanding that beta-cell replacement is at minimum a cell biology, immunology, endocrinology, and bioengineering challenge, with the potential for additional expertise such as modeling-simulation and others • Multi-disciplinary collaboration from a number of scientific disciplines is required to achieve a successful beta replacement cell product. For example, a better understanding of the key immunology challenges that could be combined with innovative bioengineering tools • Devices are being developed independently of cell sources, slowing optimization of a final device-cell combination product • Teams use different models, protocols, and experimental designs that complicate evaluation and comparisons. Limited standardization and reproducibility across laboratories • Traditional islet transplant centers are motivated to develop beta-cell replacement programs but additional medical programs and expertise that deal with related challenges such as vascularization and surgical implantation of biomaterials may be of benefit CLINICAL TRANSLATION • Define success criteria and identify preclinical animal models most predictive of human responses o Limited translatability of rodent and large animal studies, increasing the risk for success o Cost, complexity, and expertise needed for large animal studies o Lack of large animal model of autoimmune diabetes o Availability, variability, and cost of high-quality human cadaver islets as a comparator for cell quality assessment • Movement of translational research to clinical opportunities requires product development plans o Costs of later stage research is higher o Need to engage key industry players with commercial capabilities o Define potential opportunities for therapeutic concepts within relevant patient segments and their treatment paradigms. Design Target Product Profiles (TPP) that reflect the product features and balance the current therapeutic options with the anticipated next-generation products REGULATORY CONSIDERATIONS • Nonclinical and clinical trial data package requirement will be based on several factors including cell source, site of implantation, and need for immunosuppression, encapsulation, or alternative strategy to protect cells • Need to maintain close interaction with regulatory agencies throughout the research and development process

landscape analysis,86, 87 key challenges remain and must be addressed in order to make BCR therapies applicable to a wider percentage of individuals with T1D. As summarized in Table 1, key considerations can fall into four categories, (i) technical development, (ii) requirement for various scientific and clinical expertise to develop a complete product, (iii) clinical translation, and (iv) regulatory consideration for product development.

Bridging the gap: Priorities and opportunities As discussed above, the goal of the JDRF BCR portfolio is to drive the development of therapeutic concepts and products that will safely restore glycemic control while reducing or eliminating the burden for people with T1D. A BCR “product” that will address these issues is unique in the realm of product development due to the specific requirements for an unlimited supply of highly functioning beta cells or islet-like clusters, a strategy that will protect the implanted cells from auto-

immunity and allogeneic or xenogeneic rejection while finding an ­implantation site that provides a nurturing niche in which beta cells respond with physiologic insulin kinetics to fluctuating blood glucose levels. Another key area of investigation is the durability of functional response—a factor that will play a critical role in determining adherence, cost, and effectiveness. Since the inception of the program, JDRF’s main priority has been to eliminate the barriers that hinder open collaboration. In order to advance discoveries and accelerate development in strategic areas of BCR, JDRF established a consortium in 2013 with the main purpose of identifying the missing gaps and addressing the key challenges that impede progress in the field (Fig. 4). By creating a research network and encouraging collaborations among multidisciplinary team of cell biologists, immunologists, bioengineers, chemists, immunologists, and transplant surgeons, as well as adopting protocol standardization and standards to evaluate reagents, the consortium has built a robust technology and preclinical

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Beta-cell replacement

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FIG. 4  JDRF beta-cell replacement (BCR) strategy develops in part through a comprehensive consortium to foster innovation, collaboration, and accelerate development of product concepts and prototypes.

pipeline. Since JDRF launched the Beta Cell Replacement Consortium, multidisciplinary research teams from academic institutions and industry partners have made significant progress in developing cell sources and novel engineering concepts tailored for BCR therapies. The consortium is trying things that would have never happened if JDRF had not brought everyone together, and while most collaborations are at the exploratory stages, a long-term milestone for JDRF will be for select teams to develop and translate these concepts into building a product that is safe and efficacious, and can provide a therapy to a broader set of patients with T1D than is currently an option today. Given the limited resources of the foundation, a second priority has been to encourage full participation by commercial entities into the space. JDRF’s role in spurring innovation and development in the field is to fund a select number of projects both in academia and industry that will generate proof-of-concept data in order to attract additional funding. JDRF has successfully worked with companies using a direct project financing mechanism (Industry Discovery & Development Partnership— IDDP) and through equity investments via the JDRF T1D

Fund (http://www.jdrf.org/about/t1dfund/). In 2014, JDRF was funding only two industry partners, ViaCyte and Beta O2, in this area. Today JDRF is supporting collaborative work or has investments through the JDRF T1D Fund in several different companies performing studies that go from early discovery to first-in-human trials. The financial incentives and the promise it holds for much improved outcomes in people with insulin requiring diabetes favor and drive the participation of industry in this potentially lucrative area. It is believed that a product that demonstrates competitive market opportunity and pricing will be able to capture a significant portion of the total addressable market estimated at 5–6 million (all T1D and insulin-requiring T2D in the United States). Given the increase in T1D incidence, the global T1D market alone is expected to be ~$10 billion by 2025. Nevertheless from a commercialization standpoint, the challenges facing companies attempting to develop a beta-cell replacement product are tied to scientific, regulatory, and reimbursement considerations. According to BioWorld, capital markets are investing at record pace in early-stage technologies. The first 5 months of 2018 have seen a total of $2.6 billion raised in seed, Series A and

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other types of investments. Despite the renewed interest from the financial markets in early stage companies, there have been few signs of increased investments in the BCR field with some notable examples that include stem cell and encapsulation biotechnology companies and the engagement of the large pharmaceutical industry with interest in cell therapies. These may signal a greater enthusiasm for investments in this area. While many challenges remain, the ability to advance discovery, research, and clinical trials in this area so efficiently is due to the talented community. JDRF will continue identifying opportunities that bring together the foremost public and private-sector researchers and resources to address the technical challenges in developing effective BCR therapies for T1D. We are at an interesting crossroads with several promising strategies in our portfolio and have an unprecedented opportunity to bring therapies into clinical trials and, ultimately, make them accessible to people with T1D. What’s striking is that even if the first generation of a BCR product is not the “holy grail therapy” for T1D with complete insulin independence, it will likely provide significant advantages over current standards of care and better outcomes, which will then translate into longer-term reductions in diabetes-related complications. Looking ahead, our future research priorities, as defined by our roadmap strategy will focus on three areas (i) identifying the best cell source and composition. As differences between beta cells from adult pancreatic islets and beta cells derived from porcine sources or human stem cells still persist, strategies to improve cell function, access to vascularization, and contact with other cell types remain to be explored; (ii) cell survival and immune protection. Areas of priority include the development of bioengineered delivery devices (encapsulation, scaffolds, and hydrogels), approaches to mitigate fibrosis and increase oxygenation, explore beta-cell survival agents, and strategies that incorporate immunomodulation or cell modification by genome editing to reduce or eliminate the requirements for encapsulation devices; and (iii) accelerating clinical translation by establishing functional cores to test and standardize components and reagents, identifying preclinical animal models most indicative of how these therapies are going to perform in humans, and leveraging early clinical trial data to improve next generation products.

Additional considerations When developing BCR therapies, one must also take into account the successful technical advancements and lessons learned from other areas that will impact the landscape of therapeutic options available to people with T1D. A relevant example has been the road map

to replicate islet physiology and blood glucose management with continuous glucose monitors (CGMs) and AID devices or closed-loop AP technologies. Early CGMs were successful in alerting hyperglycemic episodes but not accurate enough to reliably measure blood glucose in the hypoglycemic range.88 Over time, the accuracy of glucose sensors improved dramatically enabling users to significantly lower their HbA1c levels without increasing hypoglycemia, as demonstrated by the JDRF CGM Trial.1 Importantly, the JDRF-funded CONCEPTT trial has recently demonstrated the benefits of CGM use during pregnancy, which resulted in improved health outcomes for both mothers and babies.89 The improvement in GCM technology opened the doors to the development of glucose sensors that could guide insulin dosing via the use of infusion pumps. The gradual improvements of continuous insulin pumps over two decades have contributed to make closed-loop systems a commercial reality, and a breakthrough in the space has been the highly launch of the first hybrid closed-loop system in 2016. Another example of progress in the space is the approval of CGMs which obviates the need for fingerstick calibrations using blood glucose meters and have extended periods of use. Regardless of the great success of the first commercial products, the road map continues for the AP program to better achieve glycemic control and improved outcomes while reducing burden for users. Approaches to introduce hormones like glucagon, incorporate signals to detect meals and exercise to enhance automation, extend the wear period, make smaller devices, and more user friendly are undergoing. This is another area that JDRF has led and championed for over a decade, including advocating for regulatory guidelines and reimbursement pathways for all ages with T1D. We intend to emulate this success in the BCR arena, to ensure people with diabetes have the best treatments available, and more importantly, “choices” when it comes to a decision point.89a While all the progresses and data demonstrating the benefits of CGM and continuous insulin pumps,90, 91 large registry of insulin-dependent patients shows 30% of the total population use CGM and 60% use insulin pumps.6a An important consideration to keep in mind while developing novel therapies is understanding the potential barriers to adoption, which in the case of AP systems include on body burden, and cost of treatment. As for future BCR products, and the closer they get to a commercialization phase, it will be imperative to allocate resources and expertise to understand what the market opportunities are, as well as how to maximize patient access to these therapies.92, 93 JDRF remains committed to supporting multiple approaches to impact the lives of people with T1D and make sure they have therapeutic choices that meet their personal needs at different points in the life with T1D.

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References

Conclusion Shortly, the research community and the many lives affected by T1D will be celebrating the 100th anniversary of the discovery of insulin. While advancements in insulin replacement therapy have improved glycemic outcomes over the years, the number of people with T1D not achieving glycemic goals and suffering from long-term complications remains high. Among major research and advocacy undertaking in the T1D area, JDRF has been a driving force behind the scientific development of what could make BCR a widely available option for people with T1D. The concept of BCR has been considered for decades, and needs to be realistic when setting up nearand long-term expectations as many technical challenges remain. Lessons from past failures and future advancement in stem cell biology, immunology, and biomaterial engineering may contribute to overcome the roadblocks that impede progress and further improve the practical strategies required for making cell therapies a commercial reality. While insulin independence is the long-term goal, there are clinically impactful results, such as decrease serious hypoglycemia events, improve time in range, and reduce disease burden that might come from first generation products that fall short of that but are a clinical win for people with T1D. JDRF funding alone will not be able to bring these therapies to a commercial product, but is committed to allocate the research and advocacy resources to drive it. Looking forward, and when these early stages of development are proven to be successful, the field will need additional and larger entities to support a healthy commercial ecosystem. Even if BCR therapies come to fruition, one must be cognizant of other therapeutic options that will be available to people living with T1D and ensure we achieve a satisfactory balance of risk, efficacy, cost, and burden.

Acknowledgments The author thanks Drs Jaime Giraldo, Julia Greenstein, Sanjoy Dutta, Marjana Marinac, and Maria Luisa Candelore for valuable assistance with content and manuscript editing.

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47. Berman DM, Molano RD, Fotino C, et al. Bioengineering the endocrine pancreas: intraomental islet transplantation within a biologic resorbable scaffold. Diabetes. 2016;65(5):1350–1361. 48. Smink AM, Hertsig DT, Schwab L, et al. A retrievable, efficacious polymeric scaffold for subcutaneous transplantation of rat pancreatic islets. Ann Surg. 2017;266(1):149–157. 49. Cheng  JY, Raghunath  M, Whitelock  J, Poole-Warren  L. Matrix components and scaffolds for sustained islet function. Tissue Eng B Rev. 2011;17(4):235–247. 50. Stendahl JC, Kaufman DB, Stupp SI. Extracellular matrix in pancreatic islets: relevance to scaffold design and transplantation. Cell Transplant. 2009;18(1):1–12. 51. Lim DJ, Antipenko SV, Anderson JM, et al. Enhanced rat islet function and survival in vitro using a biomimetic self-assembled nanomatrix gel. Tissue Eng Part A. 2011;17(3-4):399–406. 52. Mirmalek-Sani SH, Orlando G, McQuilling JP, et al. Porcine pancreas extracellular matrix as a platform for endocrine pancreas bioengineering. Biomaterials. 2013;34(22):5488–5495. 53. Katsuki Y, Yagi H, Okitsu T, et al. Endocrine pancreas engineered using porcine islets and partial pancreatic scaffolds. Pancreatology. 2016;16(5):922–930. 54. Zhang  Y, Jalili  RB, Warnock  GL, Ao  Z, Marzban  L, Ghahary  A. Three-dimensional scaffolds reduce islet amyloid formation and enhance survival and function of cultured human islets. Am J Pathol. 2012;181(4):1296–1305. 55. Salvay  DM, Rives  CB, Zhang  X, et  al. Extracellular matrix ­protein-coated scaffolds promote the reversal of diabetes after extrahepatic islet transplantation. Transplantation. 2008;85(10): 1456–1464. 56. Yap  WT, Salvay  DM, Silliman  MA, et  al. Collagen IV-modified scaffolds improve islet survival and function and reduce time to euglycemia. Tissue Eng Part A. 2013;19(21-22):2361–2372. 57. Hlavaty  KA, Gibly  RF, Zhang  X, et  al. Enhancing human islet transplantation by localized release of trophic factors from PLG scaffolds. Am J Transplant. 2014;14(7):1523–1532. 58. Smink  AM, Li  S, Swart  DH, et  al. Stimulation of vascularization of a subcutaneous scaffold applicable for pancreatic islet-­ transplantation enhances immediate post-transplant islet graft function but not long-term normoglycemia. J Biomed Mater Res A. 2017;105(9):2533–2542. 59. Marchioli G, Luca AD, de Koning E, et al. Hybrid polycaprolactone/alginate scaffolds functionalized with VEGF to promote de novo vessel formation for the transplantation of islets of langerhans. Adv Healthc Mater. 2016;5(13):1606–1616. 60. Gebe  JA, Preisinger  A, Gooden  MD, D’Amico  LA, Vernon  RB. Local, controlled release in  vivo of vascular endothelial growth factor within a subcutaneous scaffolded islet implant reduces early islet necrosis and improves performance of the graft. Cell Transplant. 2018;27(3):531–541. 61. Mao D, Zhu M, Zhang X, et al. A macroporous heparin-­releasing silk fibroin scaffold improves islet transplantation outcome by promoting islet revascularisation and survival. Acta Biomater. 2017;59:210–220. 62. Pedraza  E, Coronel  MM, Fraker  CA, Ricordi  C, Stabler  CL. Preventing hypoxia-induced cell death in beta cells and islets via hydrolytically activated, oxygen-generating biomaterials. Proc Natl Acad Sci U S A. 2012;109(11):4245–4250. 63. Lee  EM, Jung  JI, Alam  Z, et  al. Effect of an oxygen-­generating scaffold on the viability and insulin secretion function of porcine neonatal pancreatic cell clusters. Xenotransplantation. 2018;25(2):e12378. 64. Jiang K, Weaver JD, Li Y, Chen X, Liang J, Stabler CL. Local release of dexamethasone from macroporous scaffolds accelerates islet transplant engraftment by promotion of anti-inflammatory M2 macrophages. Biomaterials. 2017;114:71–81.

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Further reading

65. Frei AW, Li Y, Jiang K, Buchwald P, Stabler CL. Local delivery of fingolimod from three-dimensional scaffolds impacts islet graft efficacy and microenvironment in a murine diabetic model. J Tissue Eng Regen Med. 2018;12(2):393–404. 66. Liu JMH, Zhang J, Zhang X, et al. Transforming growth f­ actor-beta 1 delivery from microporous scaffolds decreases inflammation post-implant and enhances function of transplanted islets. Biomaterials. 2016;80:11–19. 67. Zhu K, Dong L, Wang J, et al. Enhancing the functional output of transplanted islets in diabetic mice using a drug-eluting scaffold. J Biol Eng. 2018;12:5. 68. Strober S. Stable mixed chimerism and tolerance to human organ transplants. Chimerism. 2015;6(1-2):27–32. 69. Guo Z, Wu T, Sozen H, et al. A substantial level of donor hematopoietic chimerism is required to protect donor-specific islet grafts in diabetic NOD mice. Transplantation. 2003;75(7):909–915. 70. Lee  BW, Lee  JI, Oh  SH, et  al. A more persistent tolerance to islet allografts through bone marrow transplantation in minimal nonmyeloablative conditioning therapy. Transplant Proc. 2005;37(5):2266–2269. 71. Hsu BR, Fu SH, Wang AYL. Prolonged survival of subcutaneous allogeneic islet graft by donor chimerism without immunosuppressive treatment. Int J Endocrinol. 2017;2017:7057852. 72. Bluestone JA, Buckner JH, Fitch M, et al. Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Sci Transl Med. 2015;7(315). 315ra189. 73. Krzystyniak  A, Golab  K, Witkowski  P, Trzonkowski  P. Islet cell transplant and the incorporation of Tregs. Curr Opin Organ Transplant. 2014;19(6):610–615. 74. Takemoto N, Konagaya S, Kuwabara R, Iwata H. Coaggregates of regulatory T cells and islet cells allow long-term graft survival in liver without immunosuppression. Transplantation. 2015;99(5):942–947. 75. Ezzelarab  MB, Thomson  AW. Adoptive cell therapy with tregs to improve transplant outcomes: the promise and the stumbling blocks. Curr Transplant Rep. 2016;3(4):265–274. 76. Tang Q, Vincenti F. Transplant trials with Tregs: perils and promises. J Clin Invest. 2017;127(7):2505–2512. 77. Pierini  A, Iliopoulou  BP, Peiris  H, et  al. T cells expressing chimeric antigen receptor promote immune tolerance. JCI Insight. 2017;2(20). 78. Sneddon  JB, Tang  Q, Stock  P, et  al. Stem cell therapies for treating diabetes: progress and remaining challenges. Cell Stem Cell. 2018;22(6):810–823. 79. Pearson RM, Casey LM, Hughes KR, Miller SD, Shea LD. In vivo reprogramming of immune cells: technologies for induction of ­antigen-specific tolerance. Adv Drug Deliv Rev. 2017;114:240–255. 80. Carlsson PO, Espes D, Sedigh A, et al. Transplantation of macroencapsulated human islets within the bioartificial pancreas betaAir to patients with type 1 diabetes mellitus. Am J Transplant. 2017. 81. Ludwig B, Ludwig S, Steffen A, et al. Favorable outcome of experimental islet xenotransplantation without immunosuppression in

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a nonhuman primate model of diabetes. Proc Natl Acad Sci U S A. 2017;114(44):11745–11750. 82. Baidal DA, Ricordi C, Berman DM, et al. Bioengineering of an intraabdominal endocrine pancreas. N Engl J Med. 2017;376(19):1887–1889. 83. Matsumoto  S, Tan  P, Baker  J, et  al. Clinical porcine islet xenotransplantation under comprehensive regulation. Transplant Proc. 2014;46(6):1992–1995. 84. Matsumoto  S, Abalovich  A, Wechsler  C, Wynyard  S, Elliott  RB. Clinical benefit of islet xenotransplantation for the treatment of type 1 diabetes. EBioMedicine. 2016;12:255–262. 85. Skyler  JS. Hope vs hype: where are we in type 1 diabetes? Diabetologia. 2018;61(3):509–516. 86. Castro-Gutierrez R, Michels AW, Russ HA. beta Cell replacement: improving on the design. Curr Opin Endocrinol Diabetes Obes. 2018;25(4):251–257. 87. Korsgren  O. Islet encapsulation: physiological possibilities and limitations. Diabetes. 2017;66(7):1748–1754. 88. Diabetes Research in Children Network Study Group. Accuracy of the GlucoWatch G2 Biographer and the continuous glucose monitoring system during hypoglycemia: experience of the Diabetes Research in Children Network. Diabetes Care. 2004;27(3):722–726. 89. Feig DS, Donovan LE, Corcoy R, et al. Continuous glucose monitoring in pregnant women with type 1 diabetes (CONCEPTT): a multicentre international randomised controlled trial. Lancet. 2017;390(10110):2347–2359. 89a. Latres E, Finan DA, Greenstein JL, et al. Navigating two roads to glucose normalization in diabetes: automated insulin delivery devices and cell therapy. Cell Metab. 2019;29(3):545–563. https://doi. org/10.1016/j.cmet.2019.02.007. 90. Bally L, Thabit H, Hartnell S, et al. Closed-loop insulin delivery for glycemic control in noncritical care. N Engl J Med. 2018;379(6):547–556. 91. Heinemann L, Freckmann G, Ehrmann D, et al. Real-time continuous glucose monitoring in adults with type 1 diabetes and impaired hypoglycaemia awareness or severe hypoglycaemia treated with multiple daily insulin injections (HypoDE): a multicentre, randomised controlled trial. Lancet. 2018;391(10128):1367–1377. 92. Archibald  PR, Williams  DJ. Using the cost-effectiveness of allogeneic islet transplantation to inform induced pluripotent stem cell-derived beta-cell therapy reimbursement. Regen Med. 2015;10(8):959–973. 93. Wallner K, Shapiro AM, Senior PA, McCabe C. Cost effectiveness and value of information analyses of islet cell transplantation in the management of ‘unstable’ type 1 diabetes mellitus. BMC Endocr Disord. 2016;16:17.

Further reading 94. GlobalData. Type 1 Diabetes—Global Drug Forecast and Market Analysis to 2023; 2015. 95. BioWorld Update, May 18, 2018. Clarivate Analytics.

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37 Regenerative medicine technologies applied to beta cell replacement: The industry perspective William Rust Seraxis, Inc., Germantown, MD, United States O U T L I N E Can a “replenishable” (stem cell-derived) beta cell therapy mimic islet transplant therapy?

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The current standard of care for insulin-dependent diabetes involves daily intensive disease management with exogenous insulin and, eventually, treatment for morbidity associated with chronic hyperglycemia.1 Despite advances made in understanding the initiation and progression of disease, and the long-term efficacy of insulin treatment regimens, there remains little standardization of disease management and wide variability in patient outcomes.2 A therapy to address the root cause of disease, the loss of insulin-secreting beta cells, could cause a dramatic transformation of patient care and upend entire industries focused on diabetes management and treatment for diabetes-related complications. Several beta cell replacement therapies are in preclinical and clinical development.3–5 These promising preclinical data have justifiably generated excitement among researchers and investors alike. But how close is the industry to a commercial beta cell replacement technology? And when it arrives, how will it impact diabetes care? These are the questions that are defining the interest of industrial players in this nascent field of regenerative medicine.

Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 2 https://doi.org/10.1016/B978-0-12-814831-0.00037-3

There is no doubt that there is a sentiment among investors and established pharmaceutical companies that beta cell replacement technology is a credible future alternative to the standard of care. Publicly disclosed investments in companies developing beta cell replacement technologies from pluripotent stem cells (PSC) have totaled more than 550 million dollars globally (Fig. 1).6–9 Investments in companies developing porcine sources of islets have totaled more than 90 million dollars.10–12 Companies developing technology to deliver replacement beta cells within implantable devices have raised more than 100 million dollars.13–15 Many of the investments were made from the investment arms of large pharmaceutical companies.7, 13, 16, 17 In addition, many established pharmaceutical companies have invested in internal development of replacement beta cells and implantable devices.17, 18 These investment trends also signal that both alternative cell sources and implantable devices are considered relevant to the industry. Many of the technologies involved in developing replacement beta cell therapies are novel clinical products.

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FIG.  1  Publicly disclosed investment in companies up to 2018 (millions).

For example, in the United States there are no approved therapies derived from PSCs, there are no approved implanted medical devices that deliver and house allogeneic or xenogeneic tissues, and there are no approved therapies that effectively block or insulate an implant from the host immune system. Because of the novelty, the potential impact of these new technologies on the industry is not easily understood by comparison to past transformational technologies. It is clear that the potential for these technologies to generate revenue is considered significant. It is also likely that the market is large enough to accommodate the development of several alternative technological strategies in parallel. To understand the potential impact of the known technologies being developed on the market for diabetes care therapies, it is useful to benchmark the technologies against the current standard for beta cell replacement therapy, or the “Edmonton protocol,” developed by Dr. Shapiro at the University of Alberta in Edmonton, Canada.19 Following this experimental procedure, islets of Langerhans are harvested from (deceased) donor pancreases and infused via the hepatic portal vein to the microvasculature of the liver. Some of the entrapped islets establish residency in the liver and can, in a subset of patients, mediate total independence from insulin therapy for years. However, pharmacologic immune suppression is required for the transplanted islet survival. The implanted islets are capable of maintaining blood glucose within thresholds that lead to normal levels of glycosylated hemoglobin (HbA1c) and arrest or slow diabetes-associated morbidities such as cardiovascular disease.20, 21 Nonetheless, islet transplantation is not recommended for most patients due to the risk of severe complications associated with immune suppression, and is reserved for patients whose hypoglycemic episodes are life threatening. Availability of this therapy is also limited due to the scarcity of good quality donor pancreas and expertise in islet procurement and preparation. The patient population currently recommended for islet transplant in combination with immune suppression is a relatively small proportion of insulin-­ dependent diabetics—those who have frequent episodes of hypoglycemic unawareness, which can be as high as 40% of type 1 diabetics (Fig. 2).22–24 For risk:benefit reasons, islet transplantation, however, is generally limited to p ­ atients

FIG. 2  Worldwide prevalence of diabetes in 2014. Insulin-dependent diabetics with frequent episodes of hypoglycemic unawareness represent an estimated maximum of 4% of the total.

who, in addition to hypoglycemic episodes, have difficulty controlling blood sugar, wide swings of blood glucose, and at least one complication from diabetes.25 Patients who have received a kidney transplant are also eligible, as they have already assumed the risk of systemic immunosuppression. The prevalence of end-stage kidney disease among type 1 diabetics has been dropping but is still as much as 10% over the long term.26 The impact of a novel beta cell replacement technology can be estimated by how well the technology can: (A) mimic the success of the Edmonton protocol in eliminating the need for exogenous insulin for a sustained period of time, and lower glycosylated hemoglobin, and/or; (B) expand the patient population that benefits from beta cell replacement therapy. Expansion of the patient population can be accomplished by making the therapy more widely available, and minimizing or eliminating the need for immune suppression.

Can a “replenishable” (stem cell-derived) beta cell therapy mimic islet transplant therapy? To generate beta cells from PSCs, the cells are guided in the laboratory along developmental pathways to mature into the pancreatic phenotype.3–5 These lab-grown beta cells can secrete insulin in response to glucose but lack other features of mature islets. The insulin secretion kinetics is not identical to freshly harvested islets and the total amount of insulin secreted per cell is lower than that from a normal islet. To maintain glucose levels within the tight ranges required to eliminate the effects of chronic hyperglycemia, the insulin secretion kinetics should mimic the native islet as closely as possible. It is possible

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Beta cells within devices

that the ­insulin secretion kinetics of lab-grown beta cells might catch up to that of a mature islet with further maturation after implant, using the normal physiologic cues that cannot be captured in the lab. It has been shown that even immature pluripotent cell-derived pancreatic cells can continue to mature after implant to a rodent host, assuming progressively more normal beta cell structure and function.27, 28 This evidence suggests that PSC-derived beta cells will be able to eliminate the need for exogenous insulin, and will lower HbA1c in human patients. A key caveat here, however, may be the need for the implanted cells to be closely associated with the host tissue, with access to vasculature that is similar to native islets. As with all therapies derived from PSCs, it is possible that potentially tumorigenic undifferentiated stem cells persist within the population of transplanted differentiated cells.29 Therefore, the clinical safety of cell replacement therapies derived from PSCs requires that tumorigenic potential of the implanted cells be eliminated.30 For target diseases that are not acutely lethal, such as type 1 diabetes, even a small tumorigenic risk from the implant will cause an unfavorable risk/reward profile and the therapy will not advance through the regulatory process to the clinic.31 Further, immunosuppression increases the risk of tumorigenesis from the grafted cells. Therefore, the benefit of lab-grown islets over freshly harvested mature islets would be increased supply at the expense of increased patient risk. Strictly as a replacement for freshly harvested islets from donor pancreases (using the Edmonton protocol), the patient population that can be treated with this therapy remains small compared to the patient population in need. As such, development of this type of therapy is currently a stepping-stone toward a more widely applicable therapy that would substantially increase the population that can benefit from a beta cell replacement therapy. Islets harvested from porcine sources are mature and functional at the time of implant and are capable of mediating normoglycemia and normalizing HbA1c in humans and nonhuman primates.32–34 However, there is a risk that endogenous retroviruses found in all pig genomes could be transmitted to a human host and subsequently between humans.35–38 Efforts are underway to inactivate retroviruses from porcine stock through extensive genetic manipulation, creating a safer xenogeneic source of transplantable organs, including porcine islets.38a

technology.38b But, considering the time and expense (as well as risk of epigenetic manipulation) required to generate each individual graft, this strategy is not practical. The most widely published method to avoid immune suppression is to deliver an allogeneic therapeutic cell population within a device that forms a barrier between the grafted cells and the host immune system.39, 40 Another method in development is to “cloak” the cells by creating a local site of immune privilege wherein allogeneic cells are tolerated by the host.41 Another approach is transfusion of T regulatory cells that mediate transplant tolerance, and this strategy is currently in clinical trials for islet transplant recipients.42 And finally, another recent approach is to create a pluripotent cell line that, through genetic manipulation, lacks antigenic determinants that would stimulate an immune reaction.43, 44 This method can be enhanced by causing the expression of proteins that would induce tolerance.

Beta cells within devices Several physical devices that act as a barrier between the transplanted cells and targeted host immune destruction are in development.18, 39, 40, 45, 46 The devices should also prevent egress of the transplanted cells outside the device. Unfortunately the literature is characterized by a lack of convincing preclinical evidence of a device that facilitates long-term therapy for insulin-dependent diabetes. Questions about the devices are centered on the device size, access to host vasculature, and the stimulation of fibrosis (that could block gas and nutrient exchange across the device) as a result of the host foreign body response. Another concern that may influence clinical acceptance and practicality is easy retrievability and replaceability of the proposed device (Fig. 3).

Immune suppression To treat every patient who would benefit from islet transplantation, a strategy to eliminate the need for immune suppression is essential. Autologous cells that do not require an immune suppression strategy can be created from donor tissue with induced PSC reprogramming

FIG.  3  Implantable devices: The obstacles to overcome for a practical, effective, and safe device. The size must be sufficient to carry a therapeutic dose, but not encumber the patient. The device must have access to blood components to maintain blood glucose within physiological limits. The device must avoid the stimulation of fibroses. The device must be retrievable and replaceable.

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Device size The Edmonton protocol suggests that a therapeutic islet “dose” required to achieve normoglycemia in a 70-kg patient can be as much as 700 million cells.19–21 It can be argued that if a high percentage of the transplanted cells remain viable after transplant, a lower dose may be sufficient, but this contention is not yet clinically proven. It can conversely be argued that inefficient cell differentiation from PSC sources produces a population of cells for transplant that are not 100% therapeutically relevant. In this case, the cell dose required to achieve normoglycemia will be greater.3, 4, 27, 28 The device design must provide grafted cells easy access (short distance) to the host blood supply. A square planar device housing 700 million beta cells in a chamber that allows the cells to be packed 100 cells deep would have a surface area of 564 m2 (assuming a cell volume of 1000 μm3 and a cell thickness of 12.4 μm). It is relevant therefore to question the design of the device necessary to enable a therapeutic dose of cells to be surgically implanted without unacceptable complications. As the device gets larger, the risk increases. The device must also not be susceptible to physical damage during usual activity or cause discomfort. Because device size is limiting, clinical trials attempted to date have used relatively small solid devices containing subtherapeutic islet doses.47, 48 If miniaturization of the device enables only a subtherapeutic dose to be implanted, it is relevant to question the value to the patient. Will the cost and risk to the patient be justified by the value of only reducing the need for exogenous insulin, or only reducing the frequency of glycemic excursions? This question requires careful design of clinical trial end points for evaluating the true risk:benefit ratio to patients.

Access to blood supply The normal location of pancreatic islets enables their tight control of blood glucose. The pancreas is highly vascularized, with close contact between pancreatic capillaries and endocrine cells. The gastrointestinal hormone glucagon-like peptide-1 (GLP-1) helps control the timing of insulin secretion by beta cells as it is stimulated by the ingestion of food. The secreted endocrine hormones (insulin, glucagon) drain to the splenic, mesenteric, and portal veins. The liver then rapidly responds to these signals to either store or release glucose. Beta cell-containing devices have been implanted subcutaneously and into the peritoneal cavity. 47–51 Subcutaneous implantation is surgically accessible and provides a large surface area for implant. The deficit of this site is the paucity of blood vessels compared to the pancreas, and the lack of direct drainage to the portal circulation. Consequently, methods to increase the

­ascularization to subcutaneous pockets that would v house the transplanted beta cells have been studied.52, 53 It is reasonable, however, to question whether the kinetics of glucose control from beta cells housed subcutaneously would be as effective in reducing hyper- and hypoglycemic episodes as standard insulin therapy. If the insulin distribution patterns from subcutaneous secretion are suboptimal, the therapy may lessen the burden of daily diabetes care with exogenous insulin but not entirely remove periodic hyperglycemia or consistent mild hyperglycemia. Reduction in diabetes-­ associated morbidity would in this case be expected to be also suboptimal. These kinds of assessments must be done as new stem cell-based encapsulated islet therapies are studied in clinical trials. The peritoneal cavity is an alternate site for ­device-encapsulated islets. In this case the grafted cells need to receive glucose and supply insulin to the blood by passive diffusion through three barriers: the device barrier, the peritoneal fluid, and the peritoneal tissue barrier, and so, it seems reasonable that implants into the peritoneal cavity would demonstrate delayed glucose control. In mice, however, islets transplanted into the peritoneal cavity were sufficient to restore normal glucose control.46 An alternative strategy in development is to implant the cells/device onto the greater omentum.54, 55 This omentum is vascularized similar to the pancreas. Clinical trials aimed at evaluating this site for islet transplantation in immune-suppressed patients are ongoing.56–58

Stimulation of fibroses Artificial or engineered implants can stimulate the foreign body response, which is an effort to encapsulate and isolate foreign bodies that cannot be eliminated.59 The result of the foreign body response is deposition of collagens, which prevents the implant from being connected to host vasculature. This fibrosis has caused failure of implantable encapsulating devices.60 A device intended to house beta cells must evade starvation resulting from the host foreign body response. Chemical modification of alginate microbeads with triazole-­containing analogs was shown to reduce the attachment of host macrophages with appropriate size modifications of the encapsulating microbeads.61 Stem cell-­derived beta cells encapsulated within these beads and implanted in the peritoneal cavity were capable of restoring normoglycemia to mice with chemically induced diabetes for more than 6 months.46 Ultrapure alginate, in a string shape, alone was also sufficient to avoid attachment of host macrophages and eliminate the foreign body response.18 Islets within this device were capable of restoring normoglycemia without a foreign body response for 3 months.

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Regulatory approval

Can the therapy be removed and replaced? Assuming that the technical issues above can be addressed, a remaining consideration is the ability of the transplant to be removed and replaced, if the cells are not functioning well or causing a problem. This analysis also assumes that the proposed therapies may not last a lifetime, and so it is preferable that the implant be replaceable. Surgically implanted devices, such as subcutaneous implants, necessarily modify the tissue environment surrounding the implant, and can cause localized tissue damage, and increase the risk of removal compared to implantation. Microcapsules that float freely in the peritoneal cavity are not entirely retrievable. Efforts to create a niche for transplanted islets using the greater omentum also necessarily destroy a portion of that tissue, though the omentum is not an essential organ. When considering the practicality of a therapy intended for a broad patient population that should last the lifetime of the patient, retrievability and replaceability are important parameters.

Can the therapy be mass-produced? Assuming that replacement beta cells are differentiated from PSC populations, another concern among industry players is the ability of the therapy to be mass-produced. A modest lot of manufactured cells comprising 100 doses could be as many as a trillion cells. For pluripotent cell sources, the manufacturing methods are long, complex, and expensive.3–5 The differentiation protocols last at least several weeks in duration and employ many dozens of highly pure ingredients that each need appropriate quality controls. The cells pass through stages during differentiation that can each require separate release tests in addition to usual quality controls. Adding to this complexity, cell behavior in culture can be inconsistent even when cell culture conditions are tightly controlled. In the United States, manufacturing must comply with current Good Manufacturing Practice (cGMP) and be compliant with 21 Code of Federal Regulations (CFR) parts 210 and 211. A relevant question, therefore, is whether manufacturing of such a therapy is scalable. The most widely reported cell manufacturing methods involve stirred suspension cultures in bioreactors.62, 63 These large-scale bioreactor methods are difficult to optimize, and the cell culture conditions can change the growth and maturation of the cells. The alternative is to produce many fewer doses per batch using small-scale cell culture techniques. This method could preserve the quality of the cells produced but obviate any cost savings due to economies of scale. Another concern is that accepted lot release tests are often culture-based and require s­ everal

weeks to complete.64, 65 The cell therapy product must persist in culture long enough for approved release testing to be completed. These time-related constraints could be removed by cryopreserving the intermediate or final products. For this reason, progress has been made to achieve high viability and function of islets after cryopreservation.66 Storage and distribution are yet more technical hurdles that pose new challenges for the manufacture of beta cell replacement technologies. The shelf life and storage requirements of living cells might require that manufacturing sites be close to transplant centers. Distribution of living or cryopreserved therapies will require specialized shipping and monitoring to ensure product quality. Point of care sites will need specialized facilities and equipment to enable thaw and recovery of the therapy before implant. Likewise the personnel will need specific training on the receipt, storage, recovery, handling, and finally, implantation of the living cell therapy.

Regulatory approval Industry analysts debate the time-to-market for pluripotent cell-based therapies. A key determinant is the path to regulatory approval. This path is not easily evaluated because of the lack of precedents and little regulatory guidance to specifically address the clinical evaluations of safety and efficacy for pluripotent cellbased products. For example, a central safety criteria for PSCs is lack of tumorigenicity.67 PSCs are immortal, a quality essential in developing a replenishable source of beta cells, but the trade-off is tumorigenicity.68 An accepted protocol to evaluate the tumorigenicity of a pluripotent cell-based implant is not yet established.69 In addition, safety tests specific to cells, to devices, and to drugs would all need to be performed to evaluate the safety of the beta cell replacement therapy. Examples include assessing biodistribution of potentially escaped cells away from the delivery site, safety of extractables and leachables from the device, and safety of endocrine hormones released under control of the implant. In the United States, the guidelines that would apply to a cell-based therapy within an implantable device would be drawn from two FDA agencies: the newly formed Office of Tissues and Advanced Therapies (OTAT) and the Center for Devices and Radiological Health (CDRH). OTAT is itself formed from the merger in 2016 of the Office of Blood Research and Review (OBRR), the Office of Vaccines Research and Review (OVRR), and the Office of Cellular, Tissue, and Gene Therapies (OCTGT). Perhaps in response to the complexity of these novel cell therapies, there are efforts among regulators to create a framework specific for the growing field of cell therapy that may shorten the time to market. For example,

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the FDA created a Regenerative Medicine Advanced Therapy designation to accelerate approval of cell therapies that show clinical evidence of addressing an unmet need for a life-threatening condition. Japan enacted in 2014 the Act on the Safety of Regenerative Medicine and the Act on Pharmaceutical and Medical Devices to ensure regulation of regenerative medicine practices and to expedite approval of potential breakthrough cell therapies. The lack of a clear regulatory path suggests the need for preclinical cell therapy developers to engage early with regulators in order to identify the most applicable, expeditious regulatory path.

Patient acceptance With a therapy potentially right around the corner, how ready are doctors and patients for novel encapsulated stem cell-derived therapy? Because of the novelty, an effort to educate doctors, patients and patient advocates, and caregivers on the benefit of the therapy will be needed to drive acceptance in the marketplace. If embryonic stem cell sources are used, some patients may not accept the therapy on religious grounds.

Conclusion Cost and reimbursement A beta cell replacement therapy that is available to a patient in a doctor’s clinic will undoubtedly carry a high price tag. A quick analysis of the catalog prices of the various ingredients used to grow and differentiate stem cells to the beta cell phenotype reveals that a single human dose could cost more than $35,000 in reagents alone. Adding a 6-week manufacturing process, specialty equipment and facilities, and high personnel qualification and training requirements, and the cost easily approaches some of the most expensive medical therapies available.70 Pricing strategy will consider those costs as well as the anticipated efficacy and its duration. A high cost will be more easily justified for a therapy that delivers insulin independence over a therapy that reduces the frequency of hyper- and hypoglycemic events. For this analysis, cost of comorbidities, cost of lost productivity, and quality of life must all be considered in developing a rationale for a price that is significantly higher than disease management with exogenous insulin. Considering that the islet transplant therapy is a single application, is surgically implanted, and is intended to be long term, the most similar cost model and reimbursement structure can be derived from the solid organ transplant experience. Organ transplants costing hundreds of thousands of dollars are considered reasonable because they mediate long-term disease control or cures. Total billing for a pancreas transplant in the United States in 2017 was $347,000 on average.71 Kidney, liver, and heart transplants were billed at $414,800, $812,500, and $1,382,400, respectively. An organ transplant, however, is usually the only option for patients with endstage disease. Insulin-dependent diabetics have the options of exogenous insulin therapy or, potentially, the use of an artificial pancreas. Will the advantages of the implant over those technologies justify the extra cost? The answer can be yes, but only if the transplant provides a cure or significantly better glucose homeostasis such that long-term diabetic complications and mortality are reduced.

If a manufactured islet implant can truly deliver insulin independence and normalize HbA1c levels without immune suppression for insulin-dependent diabetics, there will be a shift in the entire treatment paradigm. It will be interesting to observe how industries that are currently focused on diabetes care and management will adapt. To get there, however, significant issues related to the practicality of the therapy, safety and cost, and the appropriate patient population need to be addressed. These issues are: efficacy of the cells in reproducing native islet function; immune suppression requirements; the tolerability of an immune-protective device; a complex path to regulatory approval; rationale for a high-priced therapy when less expensive options are available; and patient and doctor acceptance. A stepwise transformation in diabetes patient care is possible rather than instantly transformational, once the technological hurdles needed for the therapy to benefit the majority of insulin-dependent diabetics are overcome. In light of the amount of investment, the breadth of efforts to achieve a practical and effective therapy, and the initiation of the first clinical trials, it is clear that there is a strong sentiment within industry that a successful stem cell-derived islet therapy is within reach, and that the therapy will be marketable and beneficial to patients.

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61. Vegas AJ, Veiseh O, Doloff JC, et al. Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates. Nat Biotechnol. 2016;34(3):345–352. 62. Dang SM, Gerecht-Nir S, Chen J, Itskovitz-Eldor J, Zandstra PW. Controlled, scalable embryonic stem cell differentiation culture. Stem Cells. 2004;22(3):275–282. 63. Krawetz R, Taiani JT, Liu S, et al. Large-scale expansion of pluripotent human embryonic stem cells in stirred-suspension bioreactors. Tissue Eng Part C Methods. 2010;16(4):573–582. 64. FDA points to consider in the characterization of cell lines used to produce biologicals; 1993. 65. Guidance for Industry; Characterization and qualification of cell substrates and other biological materials used in the production of viral vaccines for infectious disease indications. U.S. Department of Health and Human Services. Food and Drug Administration. Center for Biologics Evaluation and Research. 66. Kojayan GG, Alexander M, Imagawa DK, Lakey JRT. Systematic review of islet cryopreservation. Islets. 2018;10(1):40–49. 67. Ben-David  U, Benvenisty  N. The tumorigenicity of human embryonic and induced pluripotent stem cells. Nat Rev Cancer. 2011;11(4):268–277.

68. Gordeeva  O, Khaydukov  S. Tumorigenic and differentiation potentials of embryonic stem cells depend on TGFβ family signaling: lessons from teratocarcinoma cells stimulated to differentiate with retinoic acid. Stem Cells Int. 2017;7284872. 69. Turinetto  V, Orlando  L, Giachino  C. Induced pluripotent stem cells: advances in the quest for genetic stability during reprogramming process. Int J Mol Sci. 2017;18(9):1952–1970. 70. https://www.health.harvard.edu/blog/the-11-most-expensivemedications-201202094228. 71. Bentley  TS, Phillips  SJ. Milliman Research Report: 2017 U.S. organ and tissue transplant cost estimates and discussion; 2017.

Further reading 72. https://www.fiercebiotech.com/partnering/updated-j-j-goes-allviacyte-hands-over-betalogics-assets-hunt-for-diabetes-cure.

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C H A P T E R

38 Pancreas whole organ engineering Catalina Pineda Molina⁎,†, Yoojin C. Lee⁎,‡, Stephen F. Badylak⁎,†,‡ ⁎

McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States †Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, United States ‡ Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States

O U T L I N E Introduction

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Fundamental concepts of tissue development Whole organ engineering

527 528

Three-dimensional bioscaffolds for whole organ pancreas engineering Whole organ decellularization

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Pancreatic whole organ decellularization Effects of pancreatic organ decellularization on ECM composition, structure, and mechanics

529 530

Challenges to current approaches

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References

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Introduction

Fundamental concepts of tissue development

Both the endocrine and exocrine functions of the pancreas are essential, and therefore, pancreatic disease has significant morbidity and can be life-threatening. The incidence of acute pancreatitis (AP) has been increasing since 1988, and currently affects approximately 300,000 people per year in the United States alone.1, 2 Although chronic pancreatitis (CP) and pancreatic cancer have a lower incidence than other major organs, the combined exocrine and/or endocrine insufficiencies that typically accompany cancer contribute to the high mortality rate relative to most other types of cancer.2 Currently, the most effective treatment option for pancreatic failure is organ transplantation. Pancreatic transplants, however, are limited by the number of available donors and organ rejection.3 Alternative therapeutic options for pancreatic disease are badly needed. This chapter reviews some of the strategies for creating functional pancreatic tissue, the advances that have been achieved, and the remaining challenges.

The basic unit of all tissues and organs is the cell. All cells respond to their immediate microenvironment; that is, the microenvironmental niche, which includes the molecular signals delivered by cytokines and chemokines, the physical signals delivered through mechanical forces, and nonphysical factors such as oxygen and carbon dioxide concentration and pH. These cell-extracellular interactions influence cell phenotype and behavior in both unicellular organisms and multicellular organisms during all stages of life, including fetal development, postnatal growth, adult homeostasis, and during states when normal (i.e., healthy) conditions are disrupted by trauma, infectious agents, or preneoplastic and neoplastic disease. The tissue organization field theory (TOFT) posits that changes in microenvironment are largely responsible for normal and abnormal tissue and organ growth, and for the initiation of neoplastic processes.4, 5 These principles of TOFT are relevant because they are being applied to the field of tissue engineering/­regenerative

Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 2 https://doi.org/10.1016/B978-0-12-814831-0.00038-5

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© 2020 Elsevier Inc. All rights reserved.

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38.  Pancreas whole organ engineering

medicine, in which ex  vivo or in  vivo creation of functional tissues and organs is attempted. The microenvironment of multicellular organisms is largely embodied in the extracellular matrix (ECM). The ECM is manufactured and secreted by the resident cells of each tissue and organ, which as stated above, are influenced by the ECM itself. The ECM consists of a complex and constantly changing mixture of functional and structural molecules arranged in a ­tissue-specific ultrastructure. Although extrinsic factors that contribute to the microenvironmental niche such as mechanical forces, oxygen and CO2 concentration, and humidity are not components of the ECM, their influence on cells must be transmitted through the ECM. Simply stated, the ECM is a critical determinant of tissue and organ morphogenesis, including the development of the pancreas. During the past 20 years, methods for harvesting ECM from source tissues such as small intestine, dermis, urinary bladder, and pancreas have been developed.6–11 The harvested ECM has been used as a bioscaffold for numerous clinical applications such as hernia repair,12, 13 muscle regeneration,14, 15 and cardiac repair,16 among others. These ECM-based products, typically regulated by the FDA as devices, have proven to be safe and effective. These same materials are now being investigated for their potential use in whole organ engineering, including engineered pancreatic tissues.

Whole organ engineering Progress in the basic science and the clinical translation of tissue engineering/regenerative medicine (TE/RM) has made the concept of whole organ replacement possible (i.e., whole organ engineering). Advances in the understanding of stem/progenitor cell biology, ­bioscaffold-cell interactions, molecular signals that influence cell behavior, bioreactor technology, and three-­ dimensional (3D) printing have all contributed to current strategies for whole organ engineering. This chapter will review these various strategies.

Three-dimensional bioscaffolds for whole organ pancreas engineering The most commonly investigated approach to whole organ engineering, including pancreatic engineering, involves decellularization of a xenogeneic (usually porcine) pancreas to create a three-dimensional organ bioscaffold composed of organ-specific ECM, followed by recellularization with site appropriate autologous cells within an ex  vivo bioreactor environment. Following variable periods of time to allow for cell proliferation,

maturation, and organization, the cell-bioscaffold construct would then, theoretically, be implanted in the recipient for further maturation and integration with the host. The various steps of this approach are described in more detail below.

Whole organ decellularization The stroma, or ECM, is a complex network of structural and functional molecules secreted by resident cells within all tissues and organs. As stated above, the ECM is largely responsible for the microenvironmental niche and plays an essential role in tissue development,17, 18 postnatal homeostasis,19, 20 and response to injury.21, 22 More than 300 different structural and functional molecules, which include collagens, glycoproteins, glycosaminoglycans, proteoglycans, growth factors, cryptic peptides, and matrix bound nanovesicles,23 are present within the ECM, and account for both the biochemical and biomechanical properties that distinguish each tissue from its neighbor.24 The cellular and nuclear antigens present in allogeneic organ transplants are recognized as foreign molecules by a recipient, and are largely responsible for the adverse immune response and subsequent rejection of the implanted allogeneic organ or tissue.25 In contrast, ECM components are highly conserved across different species24, 26 and although recognized as nonself, do not elicit an adverse immune response. Thus, ECM harvested from allogeneic and xenogeneic tissue sources can, and has been, used for a variety of regenerative medicine applications. Removal of cells (i.e., decellularization) from a healthy organ results in a three-­ dimensional ECM structure that logically should be an ideal substrate for organ-specific cells, or stem/progenitor cells. Repopulation of the three-dimensional ECM construct with such cells could potentially recreate a functional organ.27, 28 The ultimate goal of organ decellularization is to remove cellular and nuclear components, while preserving the macro-, micro-, and ultrastructural architecture of the native organ ECM, as well as the biological activity of its components.29 Decellularization can be accomplished by a combination of physical, chemical, and enzymatic methods, followed by disinfection and sterilization processes. ECM scaffold materials from a variety of tissues, including urinary bladder, small intestinal submucosa, skin, nerves, among others, have been successfully used for the repair and replacement of less complex tissues without the prior addition of cells.14, 15 Instead, endogenous cells of the recipient effectively infiltrate the ECM scaffold materials and form functional tissue. However, whole organ engineering will require apriori addition of appropriate cell types.

B. Bioengineering and regeneration of the endocrine pancreas



Three-dimensional bioscaffolds for whole organ pancreas engineering

The most commonly used decellularization methods are presented below. Physical methods • Freeze-thaw cycles: The technique of repeated freezethaw cycles provides an effective method for decellularization. The formation of intracellular ice crystals promotes cell membrane disruption and therefore cell lysis. When the rates of freezethaw cycles are properly adjusted for each tissue type, and extracellular cryoprotectants are added, the ultrastructure of the ECM without the need of detergents is preserved.30, 31 The use of freeze-thaw cycles needs to be complemented with additional processes (e.g., thorough rinsing, mechanical disruption) to remove the lysed cells from the ECM.25 • Agitation: The process of agitation within the decellularization process is usually performed on immersed tissues (e.g., immersion in enzymatic or chemical solutions). Agitation provides a mechanical disruption of the tissue that facilitates the infiltration of the solution, and therefore the decellularization process. Variable factors such as aggressiveness of agitation and length of time are usually adjusted for each tissue to be decellularized.31 • Pressure: The use of pressure gradients throughout the tissue improves the efficiency of decellularization by promoting the delivery of decellularizing solution and removal of cell debris out of the tissues/organs. Chemical methods • Ionic, nonionic, and zwitterionic detergents: The use of detergents in a decellularization process helps solubilize proteins. Differential effects can be obtained from ionic, nonionic, and zwitterionic detergents. Whereas ionic detergents are implicated in cell membrane disruption and strong protein denaturation, nonionic and zwitterionic detergents protect the nature and enzymatic activity of the solubilized proteins, and therefore have shown less effectiveness in cell removal of dense tissues. A harsh decellularization method with ionic detergents, however, has been implicated in loss of mechanical properties and removal of ECM components such as GAG, and growth factors, affecting the final bioactivity of the produced ECM scaffold.25, 32 Commonly used detergents are: ionic (sodium dodecyl sulfate (SDS), sodium deoxycholate, and Triton X-200), nonionic (Triton X-100), and zwitterionic (3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate). The use of detergents should be followed by extensive washes to remove residuals, especially when SDS is employed. Residual SDS

529

has been shown to have adverse effects, promoting cytotoxicity, within the ECM-derived scaffolds.33, 34 • Acid and basic solutions: Both acid and basic solutions catalyze the hydrolytic degradation of nucleic acids and are associated with an increased efficacy of cell removal. Commonly used acid solutions include acetic acid, peracetic acid, and deoxycholic acid, all of which are effective in decellularizing thin tissues, such the urinary bladder, and small intestinal submucosa. Bases, on the other hand, are frequently used in dense tissues such as dermis. Some of the common basic solutions employed include ammonium hydroxide, sodium hydroxide, and calcium hydroxide. All these solutions exhibit distinctive effects on the ECM components, and therefore should be carefully used. Some of the common effects involve collagen degradation, removal of GAG, and growth factors, among others.31, 32, 35 • Alcohols: Alcohols such as glycerol, methanol, ethanol, and chloroform are used to dehydrate cells and extract lipids from tissues/organs. These solutions, however, can alter the ECM ultrastructure by cross-linking collagen fibrils and fibers and can increase the stiffness of the final product.31 Enzymatic methods • Proteases: The use of enzymes, such as trypsin, and various collagenases are commonly used to disrupt cell-matrix interactions. Dispase, which targets the protein components of the basement membrane, separates epithelial cells from subjacent structures. Phospholipase A2 is used to hydrolyze phospholipids from the tissues. These enzymes should be used only sparingly to avoid disruption of ECM structural proteins and the inevitable detrimental effects on the mechanical strength of the remaining ECM.35, 36 • Nucleases: Endo- and exonucleases are used to degrade nucleic acids and facilitate their removal from the tissue through subsequent rinsing. DNAase in particular is an effective enzyme for the removal of the residual DNA following cell lysis.36

Pancreatic whole organ decellularization Antegrade or retrograde vascular perfusion with decellularizing solutions has shown to be an effective method for maintaining the microarchitecture of an organ or tissue while efficiently removing the cells.36, 37 The use of physiologic pressure to perform the perfusion takes advantage of the close proximity between the capillaries and cells to widely disperse the decellularizing solutions, disrupt cells, and remove the cellular debris.38 Decellularization of whole organs, such as the pancreas, is usually intended to be followed by recellularization with organ appropriate cells. Therefore, maintenance of

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38.  Pancreas whole organ engineering

the organ microstructure and ultrastructure is important. Table 1 provides an overview decellularization protocols applied to the pancreas in different species. All protocols utilize vascular perfusion as a preferred method of decellularization. The specific solutions used and the temporal sequence of delivery, however, differ among the various protocols.11, 39, 41, 42 Nonionic (Triton X-100) and ionic (SDS) detergents were commonly used as decellularizing solutions. Decellularization times ranged from 1 day for rat pancreas to 2–3 days for mice pancreas, 6 days for pig pancreas, and 8 days for human tissue.11, 39–43

Effects of pancreatic organ decellularization on ECM composition, structure, and mechanics The ultimate goal of a decellularization process is to reduce or eliminate any adverse immunologic response following implantation of the resulting ECM scaffold.44 The objective therefore is the removal of cellular antigens such as the α-Gal epitope.45 Every decellularization method modifies/alters the structural and compositional integrity of the ECM to some degree. Time-offlight secondary ion mass spectrometry (ToF-SIMS) of ECM bioscaffolds has been used to characterize the surface modifications that occur with various methods of decellularization.46, 47 Thus, a balance between cell/nuclear debris removal and preservation of ECM constituents is desirable to create a functional three-dimensional ECM organ bioscaffold.28 Likewise, every decellularization method has an effect on the mechanical properties (e.g., stiffness) of the produced organ scaffolds, resulting in alterations of cell adherence and differentiation during the recellularization process.48 An important factor of consideration in whole organ decellularization is the preservation of the microvasculature, which is essential for recellularization purposes and/or maintenance of oxygen demands within the organ. In addition, maintenance of anisotropic or isotropic properties of the ECM should be considered to facilitate the appropriate orientation of reseeded cells.33 Bioreactors vs in vivo pancreatic organ engineering The three-dimensional scaffolds produced either by the assembly of polymeric molecules (e.g., 3D printing) or through whole organ decellularization need to be repopulated with cells to generate functional bioartificial organs.49 The use of decellularized organs (i.e., 3D ECM organ bioscaffolds) provides distinct advantages over the use of 3D printed scaffolds comprising synthetic polymers. These advantages include a preserved three-dimensional structure of the native tissue, bioactive factors that guide the functions of infiltrating cell, and a vascular system that enhances the recellularization process and the survival of the cells within the tissue.50 Contrary to tissue engineering applications that use

thin, two-dimensional ECM bioscaffolds that are r­ eadily populated by host cells following implantation, the complexity of decellularized organs requires a directed apriori recellularization process to be effective.51 The identification of appropriate progenitor cells able to differentiate and perform the functions required for the indicated organ is required for successful pancreatic organ engineering. To date, different sources of autologous cells with potential for clinical application have been evaluated, including mesenchymal stem cells, induced pluripotent cells, endothelial cells, and to a less extent, tissue-specific cells.28, 49 Methods of cell delivery, induction factors required, and long-term functional evaluation are also important considerations when developing engineered organs. Static and dynamic seeding strategies have been reported for a broad group of engineered organs.3, 28, 52–55 Advances in the design of bioreactors expanded the possibilities and strategies for whole organ engineering. Dynamic bioreactors facilitate the perfusion of a population of cells through the vascular system, allowing their migration into the parenchyma, and assuming a broad distribution.56, 57 Variables such as the number of seeding injections, perfusion pressure, and types of cell culture media used as the perfusate have been widely studied. The seeding efficiency has proven to be increased when repeated cell injections are employed in comparison to a single injection of a concentrated cell population.58 Progress in pancreas whole organ engineering has been significant in recent years. Different approaches to recellularize whole pancreas are being investigated.11, 39, 42, 43 Pancreatic cell lines or primary islets have been used in preclinical studies to determine the ability of decellularized matrices to sustain the phenotype of the cells and support the exocrine and endocrine function of the pancreas. Table  2 summarizes the different strategies that have been employed to recellularize pancreas-derived 3D ECM bioscaffolds. An alternative approach to in vitro bioreactors is now being explored. Hashemi et al. have recently reported an in vivo recellularization of a decellularized pancreas to generate a complex recellularized organ that can mimic the normal pancreas. Rat-derived pancreas-ECM matrices were implanted in normal rat recipients between the pancreas and the omentum. Increased cellular infiltration, angiogenesis, and upregulation of pancreatic-­ specific genes of interest (Ngn3, Pdx1, and insulin) were evident within the construct after 3 months.59 This study provided the proof of concept that 3D ECM bioscaffolds can recruit and instruct repopulating cells to appropriately proliferate and differentiate toward a complex organ. This study also provided an example of the critical role that the immune system has in regulating a response to the implanted matrix and that contributes to an outcome of engraftment.48 Additional studies are required to evaluate the capability of these constructs to supply

B. Bioengineering and regeneration of the endocrine pancreas

TABLE 1  Pancreas decellularization protocols

B. Bioengineering and regeneration of the endocrine pancreas

Species

ICR mice

C57BL/6J mice

Rat

Pig

Pig

Human

Form

Whole pancreas

Whole pancreas

Whole pancreas

Whole pancreas

Whole pancreas

Whole pancreas

Method for delivery of decellularization agent

Retrograde perfusion through hepatic portal vein. Flow rate: 8 mL/min

Retrograde perfusion through hepatic portal vein. Flow rate: 4 mL/min

Comparison study of perfusion through the pancreatic duct, portal vein, or abdominal aorta. Variable flow rate

Perfusion through pancreatic duct and superior mesenteric vein. Flow rate: 12.5 mL/min

Perfusion through Perfusion through pancreatic duct, pancreatic duct. Flow superior mesenteric artery, and rate: 20 mL/min splenic artery. Variable flow rate

Decellularization • 0.5% SDS. 5.4 h protocol • Deionized water. 15 min • 1% Triton X-100. 15 min • 90 U/mL benzonase. 15 min • PBS containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin/ streptomycin solution. 48 h

• Wash with distilled water • Freeze-thaw cycle (−80°C). 1 day • PBS containing 1% Triton X-100 and 0.1% ammonium hydroxide. 4 h • PBS

• 1% Triton X-100. 1 h. 10 mL/min • 0.5% SDS. 2 h. 10 mL/min • 1% Triton X-100. 15 min. 10 mL/min • Wash with PBS. 4 h. 2 mL/min

• Wash with PBS containing 10 U/mL sodium heparin • PBS containing 1% Triton X-100 and 0.1% ammonium hydroxide. 24 h • PBS. 5 days

• Wash with PBS. 3–4 h • Freeze-thaw cycle (−80°C). 1 day • Wash with PBS. 6 h • 0.05% Trypsin. 6 h • Wash with PBS. 3 h • 0.1% Triton X-100 and 0.05% EGTA. 24–36 h • Wash with PBS. 12 h

Evaluation of • Absence of nuclei on decellularization H&E stained sections efficacy • Absence of major pancreatic constituents (C-peptide and amylase) by immunofluorescence • DNA concentration: less than 50 ng/ mg dry weight decellularized pancreas

• Absence of nuclei on H&E stained sections • Vasculature evaluation by trypan blue dye infusion • DNA concentration: less than 50 ng/ mg dry weight decellularized pancreas • Presence of type I collagen by immunofluorescence

• Absence of nuclei on H&E stained sections • Vasculature evaluation by digital subtraction angiography with contrast agent • Histologic assessment by Picrosirius red and Alcian blue • Characterization of ECM proteins by immunolabeling: collagen type IV, fibronectin, laminin • Total DNA: less than 70 μg • Quantification of sGAG and hydroxyproline • SEM to evaluate microstructure

• Absence of nuclei on • Absence of nuclei H&E and Masson’s in H&E stained trichrome stained sections sections • Vasculature • Vasculature evaluation by evaluation by corrosion casting perfusion of • Characterization fluorescein of ECM isothiocyanate proteins by (FITC)-labeled immunolabeling: dextran particles collagen type • Histologic IV, fibronectin, assessment by laminin Alcian blue and Sirus red stained sections • Characterization of ECM proteins: collagen type IV, elastin, laminin by immunolabeling

• Absence of nuclei in H&E stained sections • Vasculature evaluation by angiography with contrast agent • Histologic assessment by Masson’s trichrome, Picrosirius red, elastic Van Gieson and Alcian blue • Absence of HMC I and II • Characterization of ECM proteins: laminin, collagen type I, collagen type IV, fibronectin • DNA concentration: less than 50 ng/mL decellularized pancreas

Reference

39

40

41

43

11

42

Preparation: • Wash with saline solution containing 10% v/v betadine • Wash with saline solution containing 10% penicillin/ streptomycin solution • Wash with PBS • Store at 4°C Perfusion: • PBS containing 10 U/mL heparin. 60 min. 6 mL/min • 1% Triton X-100 and 0.1% ammonium hydroxide. 48 h. 12.5 mL/min • Rinse with DNAse and 0.0025% magnesium chloride • Saline solution. 5 days. 6 mL/min

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TABLE 2  Methods of pancreas recellularization ECM-derived organ origin

Murine

Murine

Porcine

Human

Cell types

• Endocrine β-cell line (MIN-6) • Exocrine acinar cell line (AR42J)

• Endocrine β-cell line (MIN-6)

• Primary porcine pancreatic islets

• Primary human pancreatic endothelial cells

Recellularization method

Multistep injection technique: three injections of 10 × 106 cells each time. With 20 min intervals between injections. Both cell types were seeded simultaneously: MIN-6 cells injected through the hepatic portal vein and AR42J cells through the pancreatic duct Recellularized pancreas were maintained on static culture for 5 days

Multistep injection technique: three injections of 1 × 107 cells each time through the hepatic portal vein. With 20 min intervals between injections Recellularized pancreas were maintained on static culture for 5 days

Multistep injection technique: four injections of 1 × 105 IEQ islets each time through the hepatic portal vein. With 1 h interval between injections Recellularized pancreas were maintained on dynamic culture (perfusion chamber) for 4 days

Recellularization process performed on a small portion of the pancreas-ECM One-step injection technique: 1 × 107 endothelial cells through the superior mesenteric artery and the splenic artery. After 2 h of incubation, perfusion culture was maintained for 6 days

Functional outcomes

Cell adhesion and functionality were evaluated by immunolabeling: C-peptide expression was identified in MIN-6 cells and amylase expression was identified in AR42J cells. Appropriate location of the two cell types was identified Expression of the genes ins 1 and ins 2 were increased in MIN-6 cells seeded on pancreas-ECM compared to other scaffolds

Cell adhesion was evaluated by H&E staining and scanning electron microscopy (SEM) Cell activity was evaluated by both gene expression and immunohistochemistry against insulin

Cell adhesion was evaluated by H&E Cell activity was evaluated by measuring the insulin secretion over time, and immunohistochemistry against insulin and glucagon after 4 days of culture

Cell adhesion was evaluated by H&E localizing the cells at the lumen of the vessels Expression of CD31 and K167 were evaluated by immunohistochemistry

Reference

11

39

42

43

functional needs in diseased models of pancreas, as well as scalability in large animals. Bioprinting of pancreas Bioprinting has received significant attention in the past decade but its effectiveness in whole organ engineering has yet to be shown. The ultimate goal of bioprinting is to develop tissues/organs analogs for in vitro and in  vivo applications, such as in  vitro pharmacokinetic studies and drug screening, and in  vivo tissue engineering applications.60 Bioprinting involves the assembly of polymeric molecules, cells, and bioactive factors in a layer-by-layer fashion to produce tissue-like or organ-like structures. Bioprinting provides high precision control over the spatial distribution of the injected components, pore size, and three-dimensional structure formed.61 Therefore, bioprinting could potentially overcome some of the current problems of recellularization of complex three-dimensional structures.62 Successful application of bioprinting requires the identification and understanding of the three-dimensional structure that is to be replicated. Computerized tomography or magnetic resonance imaging can provide the required three-dimensional structural information.63 However, the ultrastructural spatial arrangement of the

various ­molecular components of the ECM is not known in sufficient detail to print a replicate ECM.60 The use of ECM-derived hydrogels as the bioink substrate may address some of these limitations but has yet to be investigated for the creation of functional pancreatic tissue.64 The bioprinting approach also requires the creation of a vascular system either by preassembling a vascular network or directly printing it into the construct. There are, however, limitations on the formation of capillaries, and mature vessels able to support the required strength for clinical transplantation.65 It should be noted that the bioprinting process must include a postprinting maturation stage within bioreactors to ensure cell engraftment and viability. Although, the generation of complex, highly ­oxygen-demanding structures, such as pancreas, are still a challenge, the potential of the bioprinting technology to create three-dimensional tissue and organ constructs has awakened the interest of researchers working in this field, since it provides a promising approach for creating both exocrine and endocrine pancreas.66 Pancreatic tissue engineering Initial efforts of pancreatic tissue engineering have focused on the development of pancreatic islets to partially restore the endocrine function of the pancreas, specifically

B. Bioengineering and regeneration of the endocrine pancreas



Three-dimensional bioscaffolds for whole organ pancreas engineering

by inducing the secretion of insulin. These approaches have provided short-term functionality but have been associated with increased risk of thrombosis, since the islets are delivered by infusion via the portal vein.67 In all cases, the islets are harvested from allogeneic sources, and therefore immunosuppression is required. More recent approaches are investigating methods to protect the pancreatic islets by encapsulating them within a variety of materials. Islet encapsulation provides mechanical strength, prevents interaction of the islet cells with the immune system, and attempts to preserve diffusion of oxygen and nutrients.68–70 Depending on the biomaterial the functional outcomes have a high variability. The encapsulation with biomaterials such as PLLA, PLGA, or alginate negatively affects islet function, with patients typically remaining dependent on insulin. In contrast, islet encapsulation with ECM or ECM components has been shown to improve β-cell survival and differentiation, as well as increase insulin secretion.69 The clinical use of pancreatic islets is limited to the endocrine function of the pancreas, providing only a limited solution after pancreatic failure. Successful clinical applications that include pancreatic tissue engineering of both endocrine and exocrine functions have yet to be realized. As illustrated in Tables 1 and 2, and summarized in Fig. 1, most studies are developing appropriate methods to obtain decellularized pancreas (i.e., pancreatic ECM) preserving its microstructure,71

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and defining the cell types and recellularization strategies that will provide the best functional outcomes. Goh et al. investigated perfusion-decellularization of a whole pancreas to effectively remove cellular and nuclear material while preserving the three-dimensional architecture of crucial components such as the ECM, vasculature, and ductal network.11 The decellularized pancreas was then recellularized with different pancreatic cell types and showed successful cellular engraftment upregulation of insulin that supported in vitro pancreatic cell functionality. In another study, Napierala et al. infused the decellularized pancreas with whole islets of Langerhan cells in three different perfusion routes—arterial, venous, and ductular—which presents an opportunity for generating an implanted decellularized pancreas with functional islets.40 More recently, Guruswamy Damodaran and Vermette evaluated the functionality of pancreatic ECM reseeded with pancreatic islets, and showed the ability of these cells to secrete insulin after glucose induction. The study utilized a β-like cell line (GFP-transfected INS-1 cells) and kept these cells in culture for up to 120 days to evaluate the cytocompatibility and the ability of the scaffold to induce the spontaneous formation of islet-like structures.68 Experience derived from pancreatic transplants and pancreatic islet transplants suggests that tissue engineering approaches should induce a desirable host immune response, and avoid immunosuppression when

FIG.  1  Pancreas tissue engineering. (A) Perfusion decellularization of whole pancreas through the hepatic portal vein (blue arrows) or the ­pancreatic duct (red arrows). (B) Multistep recellularization of acinar cells and β-cells for the construction of a tissue engineered organ.

B. Bioengineering and regeneration of the endocrine pancreas

FIG.  2  The host immune response to tissue engineered pancreas. An ideal scenario of the host response to implanted tissue engineered pancreas will allow a transition from an initial proinflammatory response, led by the infiltration and activation of M1-like macrophages, into a proremodeling response, led by M2-like macrophages. Within the tissue engineered pancreas, the presence of cellular components (derived from the recellularization process), and depending on the time between the recellularization and the implant, necrotic cells and cell debris are expected to prolong the proinflammatory response, which could have a detrimental impact in the long-term functionality of the bioartificial organ.

FIG. 3  Challenges of tissue engineered pancreas.

B. Bioengineering and regeneration of the endocrine pancreas



References

­ ossible; an approach that has been associated with fap vorable outcomes when biomaterials are implanted in soft tissues14, 15, 72 (Fig. 2).

Challenges to current approaches A major challenge to pancreatic tissue engineering is the necessity for both functional endocrine and exocrine components (Fig. 3). Current approaches are focused on the endocrine function, specifically the secretion of insulin, with less consideration of the exocrine aspect of this organ.69, 73 Current pancreatic whole organ engineering approaches have yet to show in  vivo functionality and long-term viability in either small or large animal models.68, 74 Perhaps the greatest challenge is identifying the most appropriate cell type and cell concentration.27, 75 There has, however, been notable success in the isolation and decellularization of whole pancreas in spite of the complex vascular supply and exocrine ductular system.54 Whole organ engineering remains a distinct possibility as advances in cell harvesting and stem cell biology continue, biocompatible scaffold materials continue to be developed, and the ability to manipulate the host response improves.

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B. Bioengineering and regeneration of the endocrine pancreas

Index Note: Page numbers followed by f indicate figures and t indicate tables.

A Abdominal pain syndrome, 161 Acellular scaffold, 250–251, 333–334 Acetaminophen, 37, 143–144 Acid suppression, 144 Acinar cells, 12, 175 differentiation, 191 mutations in, 10 Activin A, 364 Act on Pharmaceutical and Medical Devices, 523–524 Act on the Safety of Regenerative Medicine, 523–524 Acute pancreatitis (AP), 6, 10–11, 13, 527 gallstones role in, 17 recurrent, 13 Adult pancreatic progenitors, 191–192 Adult pig islets advantages and disadvantages, 426–427, 427t after isolation, 427f Advanced therapy medicinal products (ATMPs), 417, 500 Agarose, 465 Agitation process, 529 Air-jet method, 277–278 Alcohol-induced chronic pancreatitis, 7–9 Alginate, 291–293, 465 Alginate-poly-L-lysine-alginate (APA), 316–317 Alginate polymers, 316 ALK5 inhibitor II (Alk5iII), 365–367 Allogeneic human stem cells (hSCs), 509 Allogeneic mouse models, 303–306 Allogeneic vs. autologous islet, 52–53, 53t Allograft, 80, 92–95, 96f, 452–453 All transretinoic acid (ATRA), 217 α-cell counter-regulatory response, 151–155 Alternatively activated macrophages (AAMs), 22 Aminopyrazine, 195 Analytical test gradient system (ATGS), 95, 96f Anoïkis, 259, 466–467 counteract, 259–261 decellularized gels, 260 hydrogels, 260–261 integrin activation, 259–260 mechanotransduction, 260 molecular approach, 259 organoid organization protects islet from, 259 scaffolds for islets, 260–261 Anti-CD154 monoclonal antibody (antiCD154mAb), 434 Anticoagulation, 145

Antigen-presenting cells (APCs), 371 AP. See Acute pancreatitis (AP) Aqueous-phase channel, 297f, 298 Arginine-glycine-aspartate (RGD), 308–309 Arteriovenous malformation (AVM), 129–130 Artificial beta cells, 414–416, 415f Artificial beta-mimetic cells, 416–417 Artificial pancreas (AP) systems, 505 Autograft, 78, 81, 94–95, 96f, 452 Autoimmune chronic pancreatitis, 35 Autoimmune pancreatitis, 7, 9–10 type 1, 10, 10t type 2, 10, 10t Autologous islet isolation, 97–98 Autologous skeletal myoblast transplantation, 489 Autologous vs. allogeneic islet, 52–53, 53t Automated insulin delivery (AID) devices, 505, 514 Autophagy, 224 Autophagy-related 7 (Atg7), 224 AVM. See Arteriovenous malformation (AVM)

B

βAir device, 340–341, 390–391, 511 Basement membrane (BM), 245 Basic fibroblast growth factor (bFGF), 383, 487–488 Baxter Health Care, 390 Beta cell delivery devices, 461 endothelial cells and, 204 extracellular matrix (ECM) coencapsulation function effect, 310–311 proliferation effect, 311 surface receptor, 310t survival effect, 309–310 function, normal levels of, 150–151 generation, synthetic biology, 410–414 glucose responsiveness of, 463–464 homeostasis and function, 216–218 macrophages and, 204–206 mitochondrial function, 368 regeneration, 399 replacement, 291, 384–388 stromal cells and, 206 Beta cell-containing devices, 521 access to blood supply, 522 device size, 522 stimulation of fibroses, 522 Beta cell differentiation cell sources, characterization, and requirements for, 363–367 protocols, 367

537

Beta-cell mass, 195, 202 Beta-cell proliferation cell-cell communications regulate, 204–206 extracellular matrix (ECM) coencapsulation, 311 molecular signaling pathways control, 206–208 Beta-cell replacement (BCR) therapy, 423–424, 438–439, 461, 505–507, 519–520, 523 challenges, 479–480, 523 cost and reimbursement, 524 Juvenile Diabetes Research Foundation (JDRF) (see Juvenile Diabetes Research Foundation (JDRF)) patient acceptance, 524 from pluripotent stem cells (PSCs), 519–521 publicly disclosed investment in companies, 519, 520f regulatory approval, 523–524 three-dimensional (3D) printing, 475–477 Beta-cell replication, 201–202, 348–349 neonatal period, 202–203 partial pancreatectomy, 203 postnatal beta-cell growth, 202–204 pregnancy, 203 Beta-mimetic cells, 416–417 Beta O2 Technologies, 340–341, 390–391, 511, 513–514 Biliary tree stem/progenitor cells (BTSCs), 193, 347–348, 350–352, 351f Biliopancreatic stem/progenitor cells, 193 Bioartificial pancreas (BAP), 333, 334f Biocompatibility of hydrogels, 464 silk matrices, 331–332 Biodegradability scaffold, 262 silk matrices, 332 Bioengineering, 478 Bioinks, 468, 477 Biological-origin materials, 488 Biological safety cabinets (BSCs), 70 Biomaterials, 329–330, 464 beta-cell replacement devices, 470f decellularized ECM (dECM)-based materials, 468 degradable, 470–471 hydrogels, 464 natural hydrogels, 464–468 nondegradable, 469–470 silk, 330–332 solid, 469 synthetic hydrogels, 464, 468–469

538 Index Biopolymers, 330 Bioprinting process, 532 extrusion-based, 465 inkjet, 475t laser-assisted, 475t micro extrusion printing, 475, 475t three-dimensional (3D), 466 decellularized ECM (dECM)-based materials, 468 gelatin hydrogel, 467 regenerative medicine, 475–477 Bioreactor large-scale, 523 vs. in vivo pancreatic organ engineering, 530–532 Biorep Technologies, 70 Bioresorbability, 332 BioWorld, 513–514 Blastocyst complementation, 448. See also Interspecies blastocyst complementation (IBC) Blastocyst polymerase chain reaction (PCR), 453–454 Blood glucose (BG), 215 Blood transfusion, 500 Bombyx mori, 330–332 Bone marrow (BM), 205 Bone marrow-derived MSC (BM-MSC), 492 Bone morphogenetic protein (BMP), 387 Bone morphogenetic protein 7 (BMP-7), 400 Bowel obstruction, 159 Branching morphogenesis, 172–174 Breeding schemes, 453–454 Brittle diabetes, 397–398 BSCs. See Biological safety cabinets (BSCs)

C Calcification, pancreatic, 21–22 Calcium-sensing receptor (CaSR), 11 Calorie restriction (CR), 216–217 Cancer pancreatic, 23–24 risk of, 146 Carbohydrates-reduced high-protein (CRHP) diet, 221 Carbonic anhydrase-II (CA-II), 349, 401 Cardiomyocyte cell sheets, 489 Cas9-based technology, 411–412, 412f CCK. See Cholecystokinin (CCK) Cell-cell interactions, 368–369, 413 Cell encapsulation technology, 340, 462–463, 473 categories of, 473 chitosan for, 465–466 hydrogels for, 464 three-dimensional (3D), 329–330 Cell Pouch System, 341–342, 390–391 Cell proliferation, 203 Cell sheet engineering, 488 in cell transplantation, 488–490, 489t subcutaneous islet transplantation using, 490–491, 490t, 491f Cell therapy, 500–501, 508–509 Cell-to-cell communication, 178, 204–206 Cell transplantation, 488–490, 489t

Cellular myelocytomatosis oncogene (c-MYC), 383, 391 Center for Devices and Radiological Health (CDRH), 523–524 Centers for Disease Control (CDC), 408 CFTR. See Cystic fibrosis transmembrane conductance regulator (CFTR) Children islet isolation and transplant in, 119–120 pancreatitis in, 117–119, 118t Chimera, 448 female, 454 gallbladder formation, 456 generation, 454 human-animal, 455 interspecies (see Interspecies blastocyst complementation (IBC)) pancreatic epithelium in, 448 Chitosan, 465–466 Cholangitis, 159 Cholecystectomy, 43 Cholecystokinin (CCK), 17–18 Chronic immunosuppressive therapy, 462–463 Chronic obstructive pancreatitis, 7 Chronic pain, 144 Chronic pancreatitis (CP), 6, 67–68, 89, 90f, 102, 117–118, 141–142, 397–398, 399f abdominal pain, 34, 34t alcohol-induced, 7–9 atrophy, 23 autoimmune, 35 Beger procedure and Berne modification, 44, 45f calcification, 21–22 cancer, risk of, 146 cholecystectomy, 43 classification of, 6 clinical evaluation, 33–36 clinical trials, 45–46 diagnosis of, 33–36, 36f dominant head mass, 43 endoscopic management, 37–40 benign biliary strictures management, 40 pancreatic duct stones management, 39–40, 40f panncreatic duct stricture management, 37–39, 38f endoscopic ultrasound examination of pancreas, 35–36 etiopathogenesis, 7 exocrine insufficiency, 19 Frey procedure, 45 hyperplasia in murine models, 352–353 incidence of, 527 medical and conservative management exocrine pancreatic insufficiency and malnutrition, 36–37 lifestyle modifications, 36 pain management, 37 mesenteric thrombosis, 145 molecular mechanisms in development of, 24 nutritional management, 144 optimal therapy of, 33 pain control, 143–144

pancreatic insufficiency, 34–35 pancreaticoduodenectomy, 43–44, 43f pathogenesis of, 6–7, 24 pathophysiology of, 8f, 18–21 patient selection and preoperative preparation, 142 postoperative management and complications, 142–146 preclinical models of, 8–9 prevalence of, 6 risk factors of, 6–7, 6f Roux-en-Y lateral pancreaticojejunostomy, 42 surgical management, 40–41 tobacco effect of, 18 total pancreatectomy for, 129 treatment of, 33–34, 36 tropical, 16 weight loss and malnutrition, 34 Chronic pancreatitis-related diabetes (CPRD), 201 Chronic systemic immune suppression, 508 Chylomicrons, 12 Chymotrypsin C (CTRC), 10–11, 13 Cigarette smoking, 18 Claudin 2 (CLDN2), 7 Clinical islet laboratories deviation in processing, 64 environment control, 58–59 equipment, 59–61, 60f establishment and maintenance of quality program, 56 facilities and environmental control clean room cost, 58, 59f clean room entry and exit, 58, 58–59f use of a clean room to manufacture islet cells, 57, 57t vulnerability of islet cells to contamination, 56–57 inspections, 64 labeling, 63 operating procedures, 62–64, 62t personnel, 61–62 process and process controls, 63 product tracking, 63 records, 63 recovery, 63 registration with Food and Drug Administration (FDA), 53, 55f registration with regulatory agencies, 56 reporting, 63–64 requirements for, 56 storage, 62–63 supplies and reagents, 62 telemedicine, 61, 61f training, 58 Clinical islet transplantation (CIT), 461, 474, 479–480, 507 alginate encapsulation, 465 immunoprotective barrier strategy, 462–463 micro extrusion printing, 475 success of, 361 for type 1 diabetes (T1D), 51 Clinical islet xenotransplantation, 437t Clinical trial authorization process, 500–501

Index 539

Closed-loop systems, 408 Clustered regularly interspaced short palindromic repeats (CRISPR), 411–412, 412f, 432, 454–455 Code of Federal Regulations (CFR), 523 Co-encapsulation, extracellular matrix (ECM) function effect, 310–311 proliferation effect, 311 surface receptor, 310t survival effect, 309–310 Collaborative Islet Transplant Registry (CITR), 315–316, 487, 507 Collagen, 245, 466 Collagenase, 92–93, 97–98 Committee for Advanced Therapies (CAT), 501 Completion pancreatectomy, 102, 107, 113 Composite islet-kidney grafts, 430 Computer-aided design (CAD), 475–477, 479–480 Conformal coating advantages of, 294f alginate, 291–293 allogeneic mouse models, 303–306 allotransplantation without immunosuppression, 305f beta cell replacement, 291 hydrogels, 291, 294–295 immunoisolation, 293 in vitro performance, 299–301 in vivo performance, 301–306, 302f islet niche, 295, 297f microencapsulation, 292f, 293 Plateau-Rayleigh instability, 295–298 refinement, 300f syngeneic mouse models, 301–303 system and process, 297f, 298–299 Continuous glucose monitoring (CGM), 272, 505, 514 Controlled islet distribution, microfabrication techniques, 472t microfluidics, 474 microthermoforming, 473–474 nanoencapsulation, 474–475 pillared wafer, 473 Conventional diet (CD), 221 Corneal transplantation, 488–489 Cotransplantation, 430 CP. See Chronic pancreatitis (CP) Cryopreservation, 523 Culture media, 500 Current Good Manufacturing Practice (cGMP), 523 Cyclin-dependent kinases inhibitors (CDKIs), 203 Cylindrical stainless mesh, 488 Cystic fibrosis, 7 Cystic fibrosis transmembrane conductance regulator (CFTR), 7, 11, 13, 118 Cytokines, 491–492 Cytoprotection, 491 Cytotoxic T lymphocyte antigen 4 fused with immunoglobulin (CTLA4-Ig), 372 Cytotoxic T-lymphocytes, 462–463

D

E

Damage-associated molecular patterns (DAMPs), 24 Decellularization technique, 247–249, 528–530, 531t biophysical properties of, 249 decellularized ECM (dECM)-based materials, 468 growth factor retention, 249 mechanical properties, 249 microarchitecture, 249 Decellularized gels, 260 Definitive endoderm (DE), 184, 362, 364, 385 Degradable biomaterials, 470–471 Delayed gastric emptying (DGE), 159–160 Delta-Notch pathway, 409 Dendritic cells (DC), 318–319 Destructive immunologic reactions, 427–428 Detergents in decellularization process, 529 Device-less approaches, 510 Dexmedetomidine, 143–144 Diabecell, 511 Diabetes mellitus, 408 clinical trials of stem cells for, 372–373 endocrine insufficiency and, 19–20 insulin-producing cells (IPCs) therapy, 362 long-term complications of, 163 management after TPIAT, 120–121 metabolic/bariatric surgery for, 233–235 nonbariatric metabolic surgery for, 235 pancreatogenic autologous islet transplantation to prevent, 129, 130t incidence of, 128–129, 128–129t prevalence, 520f Diabetes Research Institute (DRI), 474–475 DIC. See Disseminated intravascular coagulation (DIC) Diet fasting, 222–223 modifications to, 215 in type 1 diabetes (T1D), 218–220 in type 2 diabetes (T2D), 220–221 for women with gestational diabetes, 221–222 Disease modeling-in-a-dish, 383 Disseminated intravascular coagulation (DIC), 112 Dithiothreitol (DTT), 295 Donor islets, transplantation of, 201 Dorsal pancreas induction, 187 Doxycycline controllable promoter, 455 Doxycycline-inducible CRISPR platform (iCRISPR), 411–412 Drug toxicity-induced pancreatitis, 16–17 Dry spinning process, 472 Dual specificity tyrosine-phosphorylationregulated kinase 1A (DYRK1A) inhibitors, 195 Ductal cells, 11, 175 Ductal differentiation, 190–191 Ductal obstruction, pancreatic, 13–14 Ductal plexus, 188 Ductal progenitors, 191–192 Duct cells, 349 Dysmotility, 159–160

Edmonton protocol, 389, 487, 501, 507, 520–522 Electrospinning, 472–473 Embryonic pancreatic progenitors, 401 Embryonic pig islets, 426–427, 427t Embryonic stem cell (ESC), 202, 381–382, 382f, 408, 454, 502 for beta-cell replacement (BCR) therapy, 384–388 insulin-producing cells, 386 wild-type mouse, 374 Encapsulation. See also specific encapsulation devices, 339–340 pancreatic islet transplant, 501–502 pig islets xenotransplantation, 427–428, 428t technology, 509–510 Endocrine progenitor (EP), 365 Endocrine system development during late gestation, 177 differentiation, 189, 401–402 fate allocation, 175–177 insufficiency and diabetes mellitus, 19–20 pancreas during embryonic development, 385 in postnatal islets, 178 regeneration, modalities of, 191f specification, 189 subtype specification α- and β-cells, 176 δ, PP, and ε cells, 176–177 transplantation, 424–425 Endocrine transdifferentiation to endocrine transdifferentiation, 192 exocrine transdifferentiation to, 192 Endodermal patterning, 185–186 Endoderm formation conservation across vertebrates, 184 developing embryo, 185f Endogenous retroviruses, 521 Endoplasmic reticulum (ER) stress, 368 Endoscopic balloon dilation, 489–490 Endoscopic retrograde cholangiopancreatography (ERCP), 35, 39–40 Endoscopic submucosal dissection (ESD), 489–490 Endothelial cells, 204 End-stage kidney disease, 520 Entrapped islets, 520 Enzymatic digestion, 329 Enzymatic methods, 529 Epididymal fat pad (EFP), 303 Epithelial-mesenchymal transition (EMT), 369, 400 ERCP. See Endoscopic retrograde cholangiopancreatography (ERCP) ESWL. See Extracorporeal shock wave lithotripsy (ESWL) Ethanol, 8, 9f Ethisorb, 471 European Medicine Agency (EMA), 417, 500–501 European Society for Biomaterial (ESB), 464 Exendin-4, 385

540 Index Exocrine differentiation acinar differentiation, 191 ductal differentiation, 190–191 Exocrine system cell fate allocation, 175 dysfunction, 160 insufficiency, 19 pancreas-derived MSCs, 194–195 progenitors, 192 specification, 189 tissue, 6 transdifferentiation, 192 Extracellular matrix (ECM), 68, 307, 488, 528 anoïkis, 259 beta cell adhesion to, 244 collagens, 245 components, 329 decellularization, 246–247, 528–529 decellularized ECM (dECM)-based materials, 468 direct interaction, 244 fibrin, 246 fibronectin, 245 glycosaminoglycans, 245–246 growth factor retention, 249 integrins and mechanotransduction, 258–259 islet microenvironment, 257–258 laminins, 245 liver and kidney to pancreas, 251 macromolecules, 244 mechanical properties, 249 microarchitecture, 249 in pancreatic islets, 244–246 peptides and polysaccharides, 244 proteins, 361–362 tissue engineered islet scaffold incorporating, 246 whole organ decellularization, 247–249 whole organ engineering of pancreas, 246–251, 247f whole organ recellularization, 249–251 Extracellular matrix (ECM) co-encapsulation benefits of, 307–308 beta cell function effect, 310–311 proliferation effect, 311 surface receptor, 310t survival effect, 309–310 Extracorporeal shock wave lithotripsy (ESWL), 39–40, 102 Extrahepatic islet transplantation, 461–462 Extrapancreatic sources, 192–193 Extrusion-based bioprinting, 465

F Fabrication techniques, 464, 470–471, 478 electrospinning, 472–473 microencapsulation by droplet generators, 473 microfluidics, 474 microthermoforming, 473–474 nanoencapsulation, 474–475 particulate leaching, 471–472 phase separation, 472 pillared wafer, 473

Fasting clinical relevance of, 222–223 pancreas functional restoration, 223–225 Fasting-mimicking diet (FMD), 216–217, 223–225 Fatty infiltration, 22 Fetal pig islet xenotransplantation advantages and disadvantages, 426–427, 427t different sites for, 431t encapsulation vs., 427 preclinical studies, 428–429 Fibrin, 246, 261–262, 310, 466–467 Fibroblast sheet, islet with, 491–492, 491f Fibrocalculous pancreatic diabetes, 16 Fibronectin, 245, 263 Fibrosis, 6–7, 22 Film-casting technique, 473–474 First-in-human (FIH) trial, 383–384 Flavonoids, 217–218 Fluorescence-activated cell sorting (FACS), 349, 416 Food and Drug Administration (FDA), 51, 53 HCT/P establishment registration, 55f history of FDA's regulatory approach, 52t regulation, 61 Foregut-derived organogenesis, 186–187 Foregut endoderm compartmentalization, 172 Foxa2, 177, 184–185 FoxM1, 207 Freeze drying, 472 Freeze-thaw cycles, 529 Functional beta cell mass (FBM), 416

G Gabapentin, 143–144 Gallbladder dysfunction, 17–18 Ganciclovir, 369–370 Gastroduodenal artery (GDA), 42, 104 Gastrointestinal function outcomes, 159–160 Gelatin, 467–468 Gene editing, 391 Gene-knockout (GTKO) pig, 426–427, 432–434 Generalized extracellular molecule sensors (GEMS), 413 Generation of mechanically stable hydrogels (GelMA), 469 Gene-regulatory network (GRN), 409–410 Gene therapy, 500 approach, 401 medicinal products, 501 transforming growth factor β1 (TGF-β1), 491–492 Genetic lineage tracing models, 349, 402 Genetic manipulation-based methods, 350 Genetic modifications pig islets xenotransplantation, 432–435, 432t to prevent immune rejection, 371–372 Genetic pancreatitis, 10–12, 11f Genistein, 217–218 Genome editing, 508–510, 514 CRISPR/Cas9-based, 411–412 for therapeutic advantage, 369–370

Germ cells, in human, 455 Gestational diabetes mellitus (GDM), 221–222 Global Alliance for induced Pluripotent Stem Cell Therapies (GAiT), 390 Glucagon-like peptide-1 (GLP-1), 366, 522 Glucagon-producing alpha cells, 243 Glucose potentiation of arginine-inducted insulin secretion (GPAIS), 150–151, 151f Glucose-stimulated insulin release (GSIR), 293 Glucose-stimulated insulin secretion (GSIS), 177–178, 217, 300f, 306, 366–368, 413, 416 Glutaraldehyde (GTA), 467–468 Glycated hemoglobin (HbA1c), 219 Glycemia, 150–151 Glycemic index (GI), 218 Glycogen synthase kinase 3 beta (GSK3B), 195 Glycoprotein 2 (GP2), 392 Glycosaminoglycans (GAGs), 245–246 Glycosylated hemoglobin (HbA1c), 150–151 G-protein coupled receptor, 11 Graft-versus-host disease (GVHD), 510 Graves' disease, 408 Greater omentum, 270–275 Griffonia simplicifolia lectin 1 (GSL-1), 283 Growth factors (GFs), 262–263 Guide RNA, 454

H HA-collagen-derived (HA-COL) hydrogels, 467 Hairy Enhancer of Split-1 (HES-1), 188 Haplobank, 390 Healing process, 257 Heat shock protein 32, 491–492 HEK-293T cells, 414 Helmsley Charitable Trust, 508 Hematoxylin and eosin (H&E) staining, 490–491, 492f Hemorrhage, 145 Heparin-releasing silk fibroin (H-SF), 333, 334f Heparin sulfate proteoglycan (HSPG), 245–246 Hepatic disorders, 416–417 Hepatocyte cell sheet engineering, 490 Hepatocyte growth factor (HGF), 244, 317–318 Hereditary pancreatitis, 7, 12–13 diagnosis of, 12 pathophysiology of, 11f Herpes simplex thymidine kinase (HSV-TK) gene, 369–370 High-fat-diet (HFD) feeding, 204 Hormone-expressing cells, 178 Hormone-expressing endocrine cells (EN), 386 Human biliary tree stem/progenitor cells (hBTSCs), 347–348 Human embryonic stem cell (hESC), 363–365, 369, 509 immunogenicity, 370–372

Index 541

interspecies organogenesis, 373–374 in vitro timeline, 364f in vitro differentiation of, 179f Human endogenous retroviruses (HERV), 437 Human-induced pluripotent stem cell (hiPSC), 363–364, 509 clinical practice, 383–384 immunogenicity, 370–371 interspecies organogenesis, 373–374 Human intra-islet vasculature, 308–309, 308f Human islets, 93, 95 Humanized mice, immunogenicity, 372 Human leukocyte antigen (HLA) haplobanks, 371, 390 interspecies blastocyst complementation (IBC) allograft strategy, 452–453 and pig islets xenotransplantation, 435 Human pancreatic development, 409–410 Human pancreatic ductal cell line (HPDE), 400 Human pluripotent stem cell (hPSC), 359–360, 447, 455–456 insulin-producing cells (IPCs), 360 interspecies organogenesis, 373–374 types of, 363–364 Human stem cells (hSCs), 509 Human umbilical vein endothelial cells (HUVECs), 250, 277–278 Hyaluronic acid, 245–246, 467 Hydrogels, 260–261, 291, 464 coating, 294–295 immunoprotective and revascularization approaches for, 463f natural, 464–468 silk matrices, 332–333 synthetic, 464, 468–469 Hyperbranched polymers, 474–475 Hypercalcemia, 11–12, 16 Hyperlipidemia, 11 Hyperparathyroidism, 12 Hypersensitivity reactions, 16–17 Hypertriglyceridemia, 12, 16 Hypoglycemia, 163 following meals and exercise, 151–155 unawareness, 143

I IAT. See Islet isolation and autotransplantation (IAT) Idiopathic duct-centric pancreatitis (IDCP), 9–10 Idiopathic pancreatitis, 17 IL-6, 491–492 Immune system suppression, 520–521 through gene editing, 391 Immune tolerance, 372, 435–436 Immunogenicity, 370 autologous pluripotent stem cells (PSCs), 371 genetic modification to prevent immune rejection, 371–372 humanized mice, 372

human leukocyte antigen (HLA)-typed haplobanks, 371 immune tolerance, 372 induced pluripotent stem cell (iPSC), 388–389 interspecies blastocyst complementation (IBC), 450–451 Immunoisolation approach, 333–334 Immunomodulation, 332–333 Immunoprotective strategy, 461–462, 462f barriers of, 462–463 hydrogels, 463f Immunosuppression, 284, 315–316, 339, 509–510 induced pluripotent stem cell (iPSC), 389 insulin-producing cells, 361–362 pig islets xenotransplantation, 434, 435t Implantable devices beta-cell replacement (BCR) therapy, 519, 521, 521f cell-based therapy within, 523–524 encapsulation, 522 Implanted cells, 509 Implant fabrication techniques, 464 Induced pluripotent stem cell (iPSC), 202, 363–364, 408, 410f, 502 autologous, 371 for beta-cell replacement (BCR) therapy, 384–388 clinical practice, 383–384 clinical trials with, 384, 385t discovery of, 382–383 graft protection, 389–391 haplobank, 390 human leukocyte antigen (HLA)-typed haplobanks, 371 immunogenicity, 388–389 interspecies blastocyst complementation (IBC), 452 interspecies organogenesis, 373–374 number of publications, 384f retinal pigment epithelial (RPE) cells, 383–384, 389 safety issues of, 391–392 in vitro differentiation of, 179f Industry Discovery & Development Partnership (IDDP), 513–514 Inferior vena cava (IVC), 104 Inflammation, 6, 12, 17–18 acute, 13–14 pancreatic, 7 Inkjet bioprinting, 475t Innate immune system, 462–463 Inner cell mass (ICM), 381–382 Instant blood-mediated inflammatory reaction (IBMIR), 269–270, 361, 429f genetic modification, 432–434, 432t pharmacologic strategies to reduce, 428 Insulin-dependent diabetes, 362, 520–521, 524 prevalence, 520f standard of care for, 519 Insulin independence, 68, 84, 97–98 Insulin-independent islet transplantation, 487 Insulinoma-associated antigen 1 (INSM1), 400

Insulin-producing beta cells, 243–244, 307, 398–403 Insulin-producing cells (IPCs) embryonic stem cell (ESC), 386 glucose-stimulated insulin secretion (GSIS), 366–368 human pluripotent stem cells (hPSCs), 360 Insulin resistance, 204 Integrins, 258–259, 308–309 Intensive care unit (ICU), 143–144 Intercalated ducts, 353–355 Interlobular ducts, 353–355 Internal hernia, 159 International Pancreas and Islet Transplant Association and The Transplantation Society (IPITA-TTS), 424, 438–439 International Xenotransplantation Association (IXA), 436–439 Interpenetrating polymer network (IPN), 467–468 Interspecies blastocyst complementation (IBC), 447–448 advantages, 449 allograft strategy, 452–453 autograft strategy, 452 breeding schemes, 453–454 challenges in humans, 449 CRISPR gene editing system, 454–455 direct differentiation, 455 efficiency of, 453 ethical concerns, 455–456 human pancreas generation with, 448f induced pluripotent stem cell (iPSC), 452 islets transplantation, 449, 450f, 456 pancreas generation with, 448–449, 449f posttransplant immunogenicity, 451 pretransplant immunogenicity, 450 principles of, 448 solid organ formation, 448–449 suitability of large animal hosts, 455 trial-and-error approaches, 456 vasculature, 451–452 Interspecies organogenesis, 373–374 Interstitial matrix (IM), 245 Intrahepatic islet transplantation, 487, 511 Intraislet progenitors, 192 Intraperitoneal glucose tolerance test (IPGTT), 332 Intraportal delivery of cadaver islets, 361 In vitro pancreatic differentiation, 386f In vitro reprogramming, 399–401 In vivo studies, 401 Ionic detergents, 529 IPITA-TTS Executive Summary, 424 Islet amyloid polypeptide (IAPP), 341 Islet autologous transplantation (IAT), 90–91, 90–91f Islet autotransplantation (IAT), 84, 101–102, 127–128, 162, 398–399 after total pancreatectomy (TP), 397–398, 398f COBE purification process, 95 complications, 94, 112 enzyme combination, 94 enzyme dose and perfusion of pancreas, 93 enzyme selection and perfusion, 92–93

542 Index Islet autotransplantation (IAT) (Continued) indications for, 129–133, 131t interstitial perfusion, 93 islet cell isolation, 90–91 pancreatectomy and pancreas transport, 91 percutaneous infusion/transplantation of islets, 111 perfusion conditions, 93 post-distention trimming, 93 postoperative care, 111–112 purification process, 95–96 salvage pancreatectomy, 113 tissue digestion, 93–94 tissue recombination, 94–95 transplant preparation, 96–97 trimming and cannulation of pancreas, 91–92 Islet cells allogeneic vs. autologous, 52–53 allotransplantation, 102, 424, 487 contamination of, 112 culture, plasma scaffold for, 263 growth factors, 262–263 infusion and complications, 144–145 isolation, 90–91 purification, 79–81, 79f scaffolds for, 260–261 Islet delivery device, 471, 472t microfabrication techniques, 478 microfluidics, 474 microthermoforming, 473–474 nanoencapsulation, 474–475 pillared wafer, 473 minimally invasive ideal, 479f scaffold fabrication techniques, 464 electrospinning, 472–473 microencapsulation by droplet generators, 473 particulate leaching, 471–472 phase separation, 472 Islet-destructive cytokines, 462–463 Islet encapsulation, silk, 332t hydrogels, 332–333 scaffolds, 333–336 Islet extracellular matrix, 308–309 Islet graft function, 162–163 Islet isolation, 58, 59f, 61, 72–73, 72f collagenase, selection and dose, 75–76 digestion, 76–77 dilution and tissue collection, 77–79 islet cell purification, 79–81, 79f laboratory preparation and setup, 69–72, 71f organ procurement and transport, 68–69 pancreas cleaning, trimming, and cannulation, 73–74 pancreas perfusion, 74–75 preparation for administration, 81–84, 83f procedure, 258–259, 258f, 398f Ricordi isolation chamber, 70–71, 71f Islet isolation and autotransplantation (IAT), 67–68 Islet microenvironment, 257–258 Islet neogenesis, 399 Islet neogenesis-associated protein (INGAP), 400 Islets of Langerhans, 6, 243, 307, 520 Islet transcription factors (ITFs), 400

Islet transplantation (ITx), 51, 101, 329, 506–507, 520 baseline characteristics and metabolic data, 274t beta-cell replacement (BCR) therapy, 315–316 challenge, 284–285 clinical application of, 339 clinical experience, 271–275 co-encapsulation, 319–324 collagen, 466 encapsulation, 316–317 endothelialized collagen modules for, 278t, 279–280f, 283f extrahepatic sites for, 270 greater omentum, 270–275 with immune suppression, 520 inflammatory response, 285f insulin-independent, 487 interspecies blastocyst complementation (IBC), 449, 450f, 456 intrahepatic site for, 269–270 intravascular devices, 316 JDRF beta-cell replacement (BCR) therapy, 507–508 mesenchymal stromal cells and, 317–319 mixed with autologous plasma, 273f omental pouch model, 278–280 on omentum, 273f of pancreatic islets, 277–278 preclinical experience, 271 severe hypoglycemic events (SHE) after, 505–506 subcutaneous space, 488 subcutaneous transplantation, 280–284 type 1 diabetes mellitus and, 315–316 using biologic resorbable scaffold, 272f IVC. See Inferior vena cava (IVC)

J Jejunoileal bypass (JIB), 230–233 Juvenile Diabetes Research Foundation (JDRF) BCR program, 506–507 beta-cell replacement (BCR) therapy, 505–507 automated insulin delivery (AID) devices, 505, 514 clinical translation, 511–512, 512t clinical trials in, 510–511 continuous glucose monitoring (CGM), 505, 514 development in omprehensive consortium, 513f distribution, 507f islet transplantation, 507–508 metric and success, 506f priorities and opportunities, 512–514 priority areas representation, 508f regulatory considerations, 511–512, 512t strategies, 508–510 technical considerations, 511–512, 512t funded CONCEPTT trial, 514 T1D Fund, 513–514

K Keratinocyte growth factor (KGF), 364–365 Ketamine, 143–144 Krüppel-like factor 4 (KLF4), 383

L Laminins, 245 Large duct disease, 41–42 Laser-assisted bioprinting, 475t Late gestation, 177 Lateral inhibition, 409 Laxative therapy, 160 Layer-by-layer techniques, 474–475 Lineage-control networks, 410–411, 411f Lineage specification, 188–191 Lineage-tracing models, 348, 401–402 Living Cell Technologies, 511 Long-term islet graft function, 162–163 Lymphoplasmacytic sclerosing pancreatitis (LPSP), 9–10

M Macrocapsules, 390 Macroencapsulation, 340, 390–391, 509–510 βAir, 340–341 Cell Pouch, 341–342 of cells, 473 MAcroencapsulation of PANcreatic Islets (MailPan), 342 pouch-based, 340t ViaCyte, 341 MAcroencapsulation of PANcreatic Islets (MailPan), 342 Macrophages, 24, 204–206 Macroporous islet-delivery devices, 463–464 Macroporous revascularization device strategy, 461–462, 462f Madin-Darby canine kidney cells (MDCK) cells, 413 Magnetic retrograde cholangiopancreaticography (MRCP), 35 Major pancreatic duct (MPD), 188 Malabsorptive operations, 235–236 Market Authorization Applications (MAA), 500–501 Matrigel (MG), 303–305 Matrix metalloproteinases (MMPs), 244 Maturation, 367–368 Maturity onset diabetes of the young (MODY), 362 Mechanical stress, 461 Mechanotransduction, 258–260 Medicinal products, 500. See also specific medicinal products Mesenchymal stem cell (MSC), 193–195, 278, 465 cell sheet engineering, 491–492f, 492 co-encapsulation, 319–324, 320f, 323f cotransplantation, 430 cytokine/chemokine protein expression, 321f cytoprotective effects, 491 exocrine pancreas-derived, 194–195 and islet transplantation, 317–319 pancreatic islet-derived, 194 silk islets, 332 Mesenteric thrombosis, 145

Index 543

Metabolic/bariatric surgery, 229 biliopancreatic diversion, 232f for diabetes, 233–235 duodenal switch, 232f follow-up, 237 history of, 230–233 malabsorptive operations, 235–236 mechanisms, 235–237 mortality for, 237 nonbariatric metabolic surgery, 235 partial ileal bypass, 230f restrictive operations, 235–236 Roux-en-Y gastric bypass, 231f sleeve gastrectomy, 233f for type 2 diabetes (T2D), 237 vertical banded gastroplasty, 231f Metabolic disorders, 11–12, 414–416 Methacrylated compounds, 469 Methacrylated glycol-chitosan (MGC) biomaterial, 469 Microcapsules, 509–510 Microencapsulation, 292f, 293, 390–391, 473 Micro extrusion printing, 475, 475t Microfabrication techniques, 478 microfluidics, 474 microthermoforming, 473–474 nanoencapsulation, 474–475 pillared wafer, 473 Microfibers, 474 Microfluidics, 474 MicroRNA (miRNA), 387–388, 413 Microthermoforming, 473–474 Milan protocol, 132–133 Minimal change chronic pancreatitis (MCCP), 164–165 Minimally invasive ideal islet delivery device, 479f Mitochondrial function, β cells, 368 Mitogen-activated protein kinase (MAPK), 350, 414 Modular extracellular sensor architecture (MESA), 413 Modular tissue engineering, 277–278 Molecular cloning techniques, 335 Molecular signaling pathways, 206–208 Morphogen-induced signaling pathways, 409 Mouse embryonic fibroblasts (MEF), 381–382 Mouse embryonic stem cell (mESC), 374 Mouse insulin promoter 1-green florescent protein (MIP-GFP), 400 Mouse islet vascular basement membrane, 308–309, 308f MRCP. See Magnetic retrograde cholangiopancreaticography (MRCP) Muc1 lineage-labeled cells, 401–402 Mulberry silkworm, 330–331 Multicellular organisms, 528 Multilayer nanoencapsulation technique, 474–475 Multipotent pancreatic progenitor cells (MPCs), 172–174 Mus musculus, 448

N Nanoencapsulation, 473–475, 509–510 Nanomaterial-based drug delivery systems (NDDS), 295

National Institutes of Health (NIH), 508 Natural hydrogels, 464 agarose, 465 alginate, 465 chitosan, 465–466 collagen, 466 fibrin, 466–467 gelatin, 467–468 hyaluronic acid, 467 Natural islet environment, 261 Near total pancreatectomy, 67 Neonatal diabetes mellitus (NDM), 362 Neonatal period, beta-cell development, 202–203 Neonatal pig islets, 426–427, 427t Neoplastic benign disease, 131–132 Neoplastic malignant disease, 132 Neovascularization, 463–464, 488 Neurogenin 3 (Ngn3), 409 Neurog3 expression, 175–177, 365–367 Neutrophil-extracellular traps (NETs), 24 New enzyme mixture (NEM), 92–93 Nicotine, 18 NNK, 18 Nodal signaling, 184–185, 364 Nonbariatric metabolic surgery, 235 Nonbiodegradable semipermeable membrane (AN69), 342 Nondegradable biomaterials PEOT-PBT block copolymers, 470 polydimethylsiloxane (PDMS), 469 Nonhepatic transplantation sites, 155 Nonionic detergents, 529 Non-mulberry silkworms, 330–331 Nonneoplastic diseases, 129–130 Nonobese diabetic (NOD), 387 Notch signaling, 188, 365 Nuclear transfer-embryonic stem cells (NTESC), 382 Nucleases, decellularization, 529 Nutrition β-cell homeostasis and function, 216–218 deficiency, 160 management, 144

O Octamer-binding transcription factor 4 (OCT3/4), 382–383, 455 Office of Blood Research and Review (OBRR), 523–524 Office of Cellular, Tissue, and Gene Therapies (OCTGT), 523–524 Office of Tissues and Advanced Therapies (OTAT), 523–524 Office of Vaccines Research and Review (OVRR), 523–524 Omental pouch model, 278–280 Omentum islet transplantation, 270–275 Open-loop systems, 408 Opioid use, 144 Optogenetic designer cells, 414 Oral glucose tolerance test (OPGTT), 279, 281f Oral pancreatic enzyme replacement therapy, 36–37 Organogenesis, 171, 179

Organoid organization, 259 Oxygen diffusion, 264–265

P Pain long-term, 161 total pancreatectomy with islet autotransplantation (TPIAT), 161 Pancreas bioengineering, challenges in, 478 bioprinting of, 532 cannulation of, 91–92, 92f ductal cells of, 11 effects of ethanol on, 8, 9f functional restoration, 223–225 liver and kidney to, 251 preservation using two-layer method (TLM), 69 recellularization methods, 529–530, 532–533, 532t, 534f transdifferentiation in, 192 transplant, 501 transport, 91 trimming of, 91–92 whole organ engineering of, 246–251, 247f Pancreas development, 171–172, 362–363 adult pancreas, 171–172 anterior-posterior axis of the gut tube, 174f branching morphogenesis, 172–174 encapsulation device for cell-based β-cell replacement therapies, 179f endocrine cell subtype specification, 176–177 foregut endoderm compartmentalization, 172 late gestation, 177 pancreatic buds, 172–174 postnatal islets, 177–178 secondary transition, 175–177 timeline of mouse and human, 173f Pancreas divisum, 14, 15f Pancreas duodenum homeobox gene-1 (Pdx1), 348 Pancreas whole organ engineering bioreactor vs., 530–532 challenges to current approaches, 535 decellularization methods, 529–530 effects of, 530–535 protocols, 529–530, 531t fundamental concepts, 527–528 three-dimensional bioscaffolds for, 528–535 Pancreatectomy, 91, 113 Pancreatic acinar cells, 350 Pancreatic arteriovenous malformation (AVM), 129–130 Pancreatic buds, 172–174 generation of, 186–187 pancreatic progenitors from, 188 Pancreatic calcification, 21–22 Pancreatic cancer, 23–24, 527 Pancreatic cells therapeutic use of, 501–502 toxic effects of medication on, 16 Pancreatic diabetes (PD), 67 Pancreatic differentiation, 190f

544 Index Pancreatic ductal cells (PDCs), 398–399 beta cell regeneration, 399, 402f facultative progenitors, 399, 401 reprogramming to insulin-producing betalike cells, 398–403 in vitro reprogramming of, 399–401 Pancreatic ductal obstruction, 13–14 Pancreatic ductal progenitor cells, 401 Pancreatic duct epithelial cells, 349 Pancreatic duct glands (PDGs), 347–349, 353 hyperplasia in murine models, 352–353 as pancreatic progenitors, 353–355 Pancreatic duct ligation (PDL), 207, 400–402 Pancreatic endocrine cells, 193–195 Pancreatic endoderm (PE), 184–188, 385–386 Pancreatic enzyme replacement therapy, 119 Pancreatic inflammation, 7, 18–20 Pancreatic islet-derived MSC, 194 Pancreatic islets extracellular matrix in, 244–246 transplantation of, 277–278 Pancreatic islets regeneration, 348 acinar cells, 350 pancreatic duct epithelial cells, 349 residing role, 349 Pancreatic islet transplantation, 243–244, 506–507 encapsulated cells, 501–502 therapeutic approaches and challenge, 361–362 Pancreaticoduodenectomy, 43–44 Pancreaticojejunostomy, 42 Pancreatic precursors, 192–193 Pancreatic progenitor cells (PPCs), 352–353, 364, 409–410 Pancreatic Progenitor cells (PEC)-Direct device, 341, 373, 510–511 Pancreatic Progenitor cells (PEC)-Encap device, 341, 510–511 Pancreatic progenitors, 348 adult, 191–192 compartmentalization of, 188–191 lineage specification, 188–191 from pancreatic bud, 188 proliferation, 188–191 size control, 188 Pancreatic pseudocysts, 22–23 Pancreatic regeneration, 195 Pancreatic stellate cells (PSCs), 8–9f, 10, 15, 18–19 Pancreatic stem/progenitor cell, 350–352 Pancreatic stone formation, 10 Pancreatic tissue engineering, 532–535, 533f challenges, 534f, 535 host immune response to, 534f Pancreatic trauma, 15, 130 Pancreatic tubulogenesis, 188 Pancreatitis. See also specific pancreatitis in children, 117–119, 118t smoking and, 18 Pancreatogenic diabetes mellitus autologous islet transplantation to prevent, 129, 130t incidence of, 128–129, 128–129t Paracrine signals, 368–369 Parenchymal reconstruction, 249–250

Parkinson disease, 408 Partial pancreatectomy (PPX), 203, 401 Particulate leaching, 471–474 Patient-controlled analgesia (PCA) pump, 143–144 PD. See Pancreatic diabetes (PD) Peribiliary glands (PBGs), 347–348, 352, 354 Pericapsular fibrotic overgrowth (PFO), 316–317, 318f Peritoneal cavity, 522–523 Pharmacologic immunosuppression, 520 Phase separation, 472 PiggyBac, 391–392 Pig islets xenotransplantation. See also Pig-tononhuman primate (NHP) model advantages/disadvantages, 426–427, 427t composite islet-kidney grafts, 430 concept of, 425 cotransplantation, 430 determining efficacy, 438 encapsulation, 427–428, 428t establishing safety, 437–438 evolution of, 424 experience with clinical, 437t free islet, 427–429, 431t gene-knockout, 426–427, 432–434 genetic modifications, 432–435, 432t human IgM and IgG antibody to, 430f human leukocyte antigens and, 435 induction of immune tolerance, 435–436 modern era of, 425–426 patient selection, 438 porcine insulin functions, 426, 436 of porcine islets, 509 preclinical investigation, 436–437 research priorities, 438–439 sensitization to, 435 T cell response control, 434–435 Pig-to-nonhuman primate (NHP) model, 423–425. See also Pig islets xenotransplantation preclinical investigation, 426–427, 436, 438 T cell response, 434 wild-type vs. genetically-engineered pigs in, 432, 435t Pillared wafer, 473 Plasma, 257, 475–477 Plasma scaffold, 261–263 composition, 264 during hepatic implantation, 264f for islet culture, 263 for omental implantation, 264f oxygen diffusion in, 264–265 uses, 265 Plateau-Rayleigh instability, 295–298 Platelet-derived growth factor, 22 Pluripotency genes, 347–348 Pluripotent stem cells (PSCs), 381, 408 beta-cell replacement (BCR) therapy from, 519–521 embryonic stem cell (ESC), 381–382, 382f functional beta cell mass from, 416 fundamental steps in history of, 384f immunogenicity, 371 induced (see Induced pluripotent stem cell (iPSC))

interspecies organogenesis, 373–374 removal from differentiated cell product, 392 somatic cell nuclear transfer (SCNT), 382 therapy, 360f Pluronic, 468 Poly(ethylene glycol) (PEG), 468–469 Poly(lactic acid) (PLA), 470–471, 475–477 Poly(lactic acid-co-glycolic acid) (PLG), 470–471 Polybutylene terephthalate (PBT) block copolymers, 470 Polydimethylsiloxane (PDMS), 469 Polyethylene glycol (PEG) hydrogel, 291–295, 296f Polyethylene glycol-maleimide (PEG-MAL), 303–305 Polyethylene glycol maleimide (PEG-MAL) hydrogels, 468–469 Polyethylene glycol-oligoethylenesulfide (PEG-OES) nanofibers, 295 Polyethylene oxide terephthalate (PEOT) block copolymers, 470 Polyglactin 910, 471 Polyglycolic acid (PGA), 470–471, 488 Polyhormonal cells, 367 Polymerase chain reaction (PCR), 453–454 Polymer interpenetrating networks, 467–468 Poly-p-dioxanone (PDS), 471 Poly-vinyl alcohol (PVA), 330, 471 Porcine endogenous retroviruses (PERVs), 437, 509 Porcine insulin, 426, 436 Porcine islet transplantation, 511 Portal vein thrombosis, 112 Posterior foregut (PFG) cells, 364–365, 385 Postnatal beta-cell growth, 202–204 Postnatal islets α- and β-cell interactions, 178 endocrine cells in, 178 maturation of, 177–178 Posttransplant immunogenicity, 451 Pouch-based macroencapsulation devices, 340t POU domain class 5 transcription factor 1 (POU5F1), 382–383 Pregnancy, beta-cell development, 203 Pressure gradients, 529 Pretransplant immunogenicity, 450 Prevascularization strategy, 475 Primary hyperparathyroidism, 12 Primitive gut tube (PGT), 362, 385 Progenitor cells, 193 Programmed death ligand-1 (PD-L1), 372 Program on the Surgical Control of the Hyperlipidemias (POSCH), 229 Protease-activated receptors (PARs), 21 Protease chain (PC), 413 Proteases, decellularization, 529 Protein kinase C (PKC) signaling, 364–365 Proton-pump inhibitors (PPIs), 160 PRSS1 mutation, 10–11 PSCs. See Pancreatic stellate cells (PSCs) Pseudocysts, pancreatic, 22–23 Psoriasis, 408 Puestow procedure, 42

Index 545

Q Quality of life (QOL), 20, 161

R Radioimmunoassay technique, 408 Random islet distribution, scaffold fabrication techniques, 464, 472t electrospinning, 472–473 microencapsulation by droplet generators, 473 particulate leaching, 471–472 phase separation, 472 RAP. See Recurrent acute pancreatitis (RAP) Rat aortic endothelial cells (RAEC), 277–278 Reactive oxygen species (ROS), 366 Recellularization methods, 529–530, 532–533, 532t, 534f Receptors solely activated by synthetic ligands (RASSLs), 413 Recurrent acute pancreatitis (RAP), 13, 117–119 pain control, 143–144 patient selection and preoperative preparation, 142 Reendothelialization, 250–251 Refractory esophageal stricture, 489–490 Regenerative medicine (RM), 347–348, 499 cell and gene therapy, 500 classification procedure, 501 European Medicine Agency (EMA), 500–501 historical perspective of use of cells, 500 in vitro creation of pancreatic cells, 502 three-dimensional (3D) printing, 475–477 in United States, 500–501 Regenerative Medicine Advanced Therapy, 523–524 Rejuvenation therapy, 424–425 Reproductive cloning, 454 Reprogramming technique, induced pluripotent stem cell (iPSC), 387–388, 391 immunogenicity, 388 safety of, 391–392 somatic cells, 383 Yamanaka’s factors, 382 Retinal pigment epithelial (RPE) cells, 383–384, 389 Retinoic acid (RA), 186 Revascularization strategy, 463–464, 463f Ricordi isolation chamber, 70–71, 71f Roux-en-Y gastric bypass, 231, 231f Roux-en-Y lateral pancreaticojejunostomy, 42

S Salvage pancreatectomy, 113 SAPE hypothesis. See Sentinel acute pancreatitis event (SAPE) hypothesis Scaffolds, 510 biodegradable scaffold, 262 fabrication techniques, 464 electrospinning, 472–473 microencapsulation by droplet generators, 473 particulate leaching, 471–472 phase separation, 472 growth factor (GFs) of interest for islets, 262–263

silkworm silk, 333–334, 335f spider silk foams, 334–336 ubiquitous tunable scaffold, 261–263 Scanning electron microscopy (SEM), 249 Secondary transition, exocrine vs. endocrine development during, 175–177 Sefton lab, 277–278, 284 Self-condensation, 414 Semipermeable scaffolds, 329 Semma’s stem cell-derived insulin-secreting cells, 342 Sendai virus, 391 Sentinel acute pancreatitis event (SAPE) hypothesis, 13, 14f Sernova Cell Pouch, 341–342 Sertoli cells (SCs), 430 Severe combined immunodeficiency (SCI1D)/bg mice, 280–284, 282f Severe hypoglycemic events (SHE), 505–507 Sex-determining region Y (SRY)-box 2 (SOX2), 383 Signal transducer and activator of transcription 3 (STAT3), 350 Silicones, 469 Silk biocompatibility, 331–332 biodegradability and bioresorbability, 332 graft morphology, 333f hydrogels, 332–333 scaffolds, 333–336 silkworm, 330–331, 332t, 333–334, 335f spider, 330–331, 332t, 334–336, 335f structure of, 330–331 Silk fibroin (SF), 330–331, 331f Silk sericin (SS), 331 Silkworm silk, 330 islet encapsulation, 332t scaffolds, 333–334, 335f structure of, 330–331 Single-chain variable fragment (scFv), 413 slc30a8 gene, 217 Sleeping Beauty, 391–392 Sleeve gastrectomy (SG), 233, 233f, 237 Slow colonic transit, 159–160 SMAD, 207–208 SMAD7, 205, 207 Small-scale cell culture techniques, 523 Smoking cigarette, 18 and pancreatitis, 18 SMVs. See Superior mesenteric veins (SMVs) Sodium dodecyl sulfate (SDS), 248 Soft lithography, 474 Solid biomaterials, 469 Solid organ transplantation, 449, 452 Somatic cell nuclear transfer (SCNT), 382, 454 Sox17, 184–185 Special Diabetes Program (SDP) funding, 505–506 Sphincter of Oddi dysfunction, 14–15 Spider silk, 330 clusters formation, 335f foams, 334–336 islet encapsulation, 332t structure of, 331 Stearoyl-coA desaturase (SCD1), 392

Stellate cell activation, 23f Stem cell-based therapy, 408 Stem cell-derived beta cells (SC-βs), 363–368, 475–477, 520–521 Stem cell-derived insulin-producing cells (SCIPC), 361–362 Stem cell-derived therapy, 509 Stem cells, 381. See also specific stem cells Stromal cells, 206 Subcutaneous implantation, 475–477, 522–523 Subcutaneous islet transplantation, 488, 490–491, 490t, 491f Superior mesenteric veins (SMVs), 104 Swedish Obese Subjects (SOS) study, 234 Syngeneic mouse models, 301–303 Synthemax, 413–414 Synthetic biology, 407–408 beta cell generation CRISPR-Cas9-based technology, 411–412, 412f hanging-drop technology, 414 lineage-control networks, 410–411, 411f synthetic biomaterials, 413–414 synthetic messenger RNAs, 412–413 synthetic receptors, 413 clinical applications, 416–417 Synthetic biomaterials, 413–414 Synthetic hydrogels methacrylated compounds, 469 Pluronic, 468 poly(ethylene glycol), 468–469 Synthetic messenger RNAs, 412–413 Synthetic Notch (synNotch) receptor, 413

T Target chain (TC), 413 T cells, 24 T cells response, 434–435 TDEs. See Tissue dissociation enzymes (TDEs) Telomere shortening, 348 Temperature-responsive culture dishes, 488 Tetraploid complementation, 454 TheraCyte, 390 Therapeutic modulation, 195 Thermal quenching process, 472 Thermoforming, 464 Three-dimensional (3D) cell encapsulation, 329–330 Three-dimensional (3D)-ductal cysts, 399–400 Three-dimensional (3D) islet-like spheroid, 333, 335, 335f Three-dimensional (3D)-printed lithography, 474 Three-dimensional (3D) printing, 464–466, 469, 475–477 application of, 478 controlled method of fabrication, 478–479 decellularized ECM (dECM)-based materials, 468 gelatin hydrogel, 467 islets in macroporous alginate-based construct, 478f regenerative medicine, 475–477 tissue engineering purposes, 476f

546 Index Thrombosis mesenteric, 145 portal vein, 112 Thymoglobulin, 272 Time-of-flight secondary ion mass spectrometry (ToF-SIMS), 530 Tissue dissociation enzymes (TDEs), 92 Tissue engineered islet scaffold, 246 Tissue engineering hydrogels, 464 three-dimensional (3D) printing, 476f Tissue engineering products (TEP), 500–501 Tissue organization field theory (TOFT), 527–528 Tobacco effect, 18 Total pancreatectomy (TP), 67, 89–90, 90f, 102, 117, 127–128, 397–398 for chronic pancreatitis, 129 contraindications, 103, 103t delayed gastric emptying, 112 islet infusion/transplantation, 107–108, 108f islet isolation, 107 laparoscopic, 111 minimally invasive surgery, 108–111 operative procedure, 104–107, 105–107f outcomes, 113 patient selection, 102–103 preoperative preparation, 104 preoperative testing and assessment, 103–104 principles, 104 robotic, 108–111 salvage pancreatectomy, 113 Total pancreatectomy with islet autotransplantation (TPIAT), 102, 104, 108, 110f, 112–113, 119, 141–142 asplenia management after, 122 bowel obstruction, 159 cancer, risk of, 146 cholangitis, 159 C-peptide positive, 151f diabetes management after, 120–121, 123–124 favorable metabolic outcomes, 150–151 free of insulin treatment, 150f gastrointestinal function outcomes, 159–160 gastrointestinal (GI) management after, 121–122 hepatic changes on imaging, 145 history and rationale for, 149–150 infections, 145–146 internal hernia, 159 islet yield and glycemic management, 142–143 long-term outcomes data, 158t metabolic outcomes, 162–163 nonhepatic transplantation sites, 155 nutritional management, 144 outcomes, 157 pain, 161 pain management after, 121 pain relief and quality of life after, 122–123 patient selection and preoperative preparation, 142 plasma glucagon responses, 153f postoperative management and complications, 142–146

postoperative management of child after, 120–122 primary indication for, 157 quality of life (QOL), 113–114, 161 screening protocol for eligibility for, 119t serum insulin responses, 151f surgical complications, 146 surgical outcomes, 122, 158–159 survival and cost of care, 163–165 unfavorable metabolic outcomes, 151–155 University of Minnesota criteria for, 142t Transcription factors (TFs), 382, 400–403 Transdifferentiation exocrine to endocrine, 192 in pancreas, 192 Transforming growth factor beta (TGF-beta), 207–208, 385 Transforming growth factor β1 (TGF-β1) gene therapy, 491–492 Translationally controlled tumor-associated protein (TCTP), 204 Transmission electron microscopy (TEM), 249 Trauma, pancreatic, 15, 130 Trial-and-error approaches, 456 Triethanolamine (TEOA), 298 Tropical chronic pancreatitis, 16 Tropical pancreatitis, 7 Trypsinogen, 12, 21 Tumor necrosis factor (TNF), 107–108 Type 1 diabetes (T1D), 201, 215–216, 339, 408, 416, 461, 501 autologous cell replacement therapy, 387 beta-cell homeostasis and function, 216–218 chitosan-based encapsulation, 465–466 clinical islet transplantation for, 51 dietary intervention for, 218–220 holy grail therapy, 416, 514 insulin-producing cells (IPCs) therapy, 362 and islet transplantation, 315–316 JDRF beta-cell replacement (BCR) therapy, 505–507 automated insulin delivery (AID) devices, 505, 514 clinical translation, 511–512, 512t clinical trials in, 510–511 continuous glucose monitoring (CGM), 505, 514 development in omprehensive consortium, 513f distribution, 507f islet transplantation, 507–508 metric and success, 506f priorities and opportunities, 512–514 priority areas representation, 508f regulatory considerations, 511–512, 512t strategies, 508–510 technical considerations, 511–512, 512t long-term complications, 461 nsulin-producing beta-like cells for, 411 pathogenesis of, 506–507 stem cell-based therapy for, 408 Type 2 diabetes (T2D), 201, 215–216 β-cell homeostasis and function, 216–218 diet in, 220–221 insulin-producing cells (IPCs) therapy, 362 Type 3 diabetes (T3D), 201

U Ubiquitous tunable scaffold, 261–263 Ultrapure alginate, 522 University of Miami’s Diabetes Research Institute (DRI)’s BioHub, 511 Urocortin 3 (UCN3), 366–368

V Vascular endothelial growth factor (VEGF), 262–263, 271, 319, 487–488, 491 Vascular endothelial growth factor A (VEGF-A), 204 Vascular endothelial growth factor receptor I (VEGFR1), 205 Vasculature, 451–452 Ventral endoderm, 204 Ventral pancreas induction, 187–188 Very low-calorie diet (VLCD), 221 ViaCyte Inc., 341, 373, 386, 390, 392, 507–508, 510–511, 513–514 ViaCyte’s PEC-DirectTM product, 390 Viral gene therapy, 411 Vitamin C, 366 VitD nuclear receptor (VDR), 217 Volume ratio, 298

W Whipple procedure, 141–142 Whole organ decellularization, 247–249, 528 chemical methods, 529 enzymatic methods, 529 pancreas, 529–530 effects, 530–535 protocols, 529–530, 531t physical methods, 529 Whole organ engineering, 246–251, 247f Whole organ recellularization parenchymal reconstruction, 249–250 reendothelialization of acellular scaffold, 250–251 Wild-type pigs, 434, 435t Wnt3a culture, 364

X Xenogeneic model, 430 Xenotransfusion, 424 Xenotransplantation. See Pig islets xenotransplantation X-linked inhibitor of apoptosis protein, 491–492

Y Yamanaka’s factors, 382, 388, 392

Z Zinc transporter, 217 Zwitterionic detergents, 529