Introduction yo In Vitro Cytotoxicology: Mechanisms and Methods 9780367225384, 0367225387, 9780429275487

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Introduction to

In Vitro Cytotoxicology Mechanisms

and Methods

Introduction to

In Vitro Cytotoxicology Mechanisms and Methods

Frank A. Barile, Ph.D. Department of Natural Sciences Health Professions Division City University of New York at York College Jamaica, New York

Boca Raton London New York

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PREFACE Cell culture technology, and accompanying in vitro cytotoxicology, is an important, newly developing discipline of modern toxicology and is gaining increasing acceptance in the field. This acceptance has been accomplished in part through the inevitable turn of events in society which have forced research scientists and regulatory toxicologists to assume more ethical and humane approaches to toxicity testing. Public demands have compelled administrators and legislators to impose stricter regulations governing the use of animals in biomedical sciences. As a result, an increasing number of applied toxicologists, such as those with traditional training in cell biology, physiology, analytical chemistry and biochemistry, have adapted their animal toxicity testing protocols to in vitro techniques. These procedures are having a profound influence in toxicity testing and cannot be ignored, as has been the tendency in the past. Because of the notoriety that in vitro cytotoxicity testing has been enjoying, a basic knowledge of cell culture and cytotoxicity testing methods in toxicology courses taken by health professional and biomedical students, including those encountered in schools of toxicology, environmental health, pharmacy, and human, dental, and veterinary medicine, is necessary. Consequently, this text is useful as part of general and upper level toxicology courses which incorporate cell culture methodology, mechanisms of cytotoxicity, and procedures used in toxicity testing. In addition, the text serves as a useful tool for academic, industrial, and regulatory toxicologists, cell biologists, pharmacologists, and animal welfare organizations. In general, the text introduces in vitro cytotoxicology for the benefit of toxicologists and other professionals. Some misunderstandings and concerns regarding in vitro toxicity assays will be reduced and the potential value of the model systems will be appreciated as alternative methodologies develop. There are many toxicologists today who consider cell tests for general toxicity to be immature analogues to the mechanistic short-term genotoxicity tests. In reality, the general toxicity cell tests are neither typically short-term nor mechanistic, but more often are empirically based quantitative tests of cell injury due to a host of various undefined mechanisms. In addition, the explanations developed to understand certain toxic phenomena are usually more precise when cell tests are employed than with the traditional animal experiments. Consequently, the text presents the prevailing mechanisms which underlie in vitro cytotoxicology and cellular toxicity. The chapter on cell culture methods is a useful reference for students and toxicologists who wish to become familiar with the terminology and general principles. It can also serve as a guide for researchers interested in establishing a cell culture laboratory. The background, techniques, and use of cellular tests are described in detail in some areas where the procedures are widely used or have withstood the test of time and are only summarized in other areas. This serves to introduce certain well-known assays, while detailed descriptions of procedures which are currently in use but not universally accepted may not be practical. The methods are presented to the fledgling investigator as a starting point and general guide for further investigation, rather than as a detailed flowchart for particular procedures. Thus, the information furthers understanding of the principles of cytotoxicity. The ability of the tests for predicting or screening for human toxicity is presented according to the current advantages and limitations which formulate the scientific and regulatory basis for humanriskassessment. Thus the optimum utilization of certain tests for specific groups of chemicals is discussed in light of the validation programs in progress. As a result, continuous evaluation of assays for general and organ-specific toxicity, as well as tests for genotoxicity and carcinogenicity, is currently being investigated by several toxicology groups throughout the world. The results of these efforts are anxiously awaited by the biomedical and toxicological communities, and by public interest groups. Frank A. Barile, Ph.D.

THE AUTHOR Frank A. Barile, Ph.D., is Associate Professor in the Health Professions Division, Department of Natural Sciences, at the City University of New York at York College. Dr. Barile received his B.S. in Pharmaceutical Sciences in 1977, his M.S. in Pharmacology in 1980, and his Ph.D. in Toxicology in 1982 from the College of Pharmacy at St. John's University, New York. After doing a post-doctoral fellowship in Pulmonary Pediatrics at the Albert Einstein College of Medicine, Bronx, New York, Dr. Barile moved to the Department of Pathology, Columbia University, St. Lukes/Roosevelt Hospital, New York, as a Research Associate. In these positions he investigated the role of pulmonary toxicants on collagen metabolism in cultured lung cells. In 1984, he was appointed as Assistant Professor in the Department of Natural Sciences at CUNY and became Associate Professor in 1991. Dr. Barile holds memberships in several professional associations, including the American Association for the Advancement of Science, Tissue Culture Association, Sigma Xi National Scientific Honor Society, Rho Chi National Pharmaceutical Honor Society, New York Academy of Sciences, Scandinavian Society of Cell Toxicologists, American Association of University Professors, Italian-American Institute of CUNY, and the CUNY Academy of Sciences. He has been appointed as a Consultant Scientist with several clinical and industrial groups, including the Department of Pediatrics, Pulmonary Division, of the Long Island Jewish Medical Center in New Hyde Park, New York, and Pharmacon International, New York. Dr. Barile has been the recipient of research grants from the National Institutes of Health, including awards from the Minority Biomedical Research Support (MBRS) program, the Minority High School Student Research Apprentice (MHSSRAP) program, and the Research Foundation of CUNY. Dr. Barile has authored and co-authored approximately 50 papers in various peerreviewed biomedical and toxicology journals along with distinguished international investigators. He also lectures regularly to undergraduate students in the health professions in toxicology and biomedically related courses. Dr. Barile continues fundamental research on the cytotoxic effects of therapeutic drugs and environmental chemicals in cultured mammalian lung cells.

CONTRIBUTORS Thomas W. Sawyer, Ph.D., D.A.B.T. Research Toxicologist Biomedical Defence Section Defence Research Establishment Suffield Medicine Hat Alberta, Canada Bjorn Ekwall, M.D., Ph.D. Assistant Professor Department of Toxicology University of Uppsala Biomedical Center Uppsala, Sweden

Dedicated to my parents, Mr. and Mrs. Frank Barile, Sr., for their encouragement, advice, and support throughout the years.

ACKNOWLEDGMENTS Appreciation is extended to the National Institute of General Medical Sciences (NIGMS), National Institutes of Health (U.S.), for the years of grant support for the research performed in my laboratory. Several individuals and colleagues, whose interests, efforts, and concerns for the development of cell culture models to replace animals in toxicity testing, have enabled me to carry my work to completion and to make this book a reality. Among them, words of thanks are in order to Professors Robert Macholow and Hope Young, Ms. Myra James, Ms. Lolita Borges, and to my children, Frank, Lauren, and Matthew. Finally, my thanks are extended to my friends and colleagues: Dr. Thomas Sawyer, for his contribution to the project, and Dr. Bjorn Ekwall, without whose extensive discussions of the fundamentals of cytotoxicology and encouragement over the years, this book may not have materialized.

CONTENTS Chapter 1 Cell Culture Methodology and Its Application to In Vitro Cytotoxicology I. Introduction A. In Vitro Toxicology B. Test Systems

1 1 1

II. History A. Tissue Expiants B. Recent Developments

2 2 2

III. Equipment IV. The Cultured Cells A. Primary Cultures B. Finite Cell Lines C. Continuous Cell Lines D. Cell Strains E. Clonal Growth and Maintenance Cultures F. Criteria for Identification 1. Karyotypic Analysis 2. Aging in Culture 3. Anchorage-Dependent Cultures 4. Contact Inhibition V. Media A. Chemically Defined Media 1. Temperature 2. pH 3. Carbon Dioxide Tension 4. Buffering 5. Osmolality 6. Water Requirement 7. Glassware Maintenance B. Serum-Free Media VI. Procedures A. Static and Perfusion Cell Systems B. Dispersion of Tissues C. Subculturing D. Measurement of Growth and Viability E. Freezing Cells F. Cell Identification G. Sources of Cell Lines References

3 3 3 4 6 7 7 9 9 10 11 11 12 12 12 14 14 15 16 16 17 17 19 19 20 21 22 23 25 25 26

Chapter 2 Mechanisms of Cytotoxicology I. Introduction

27

IL Classification of Cellular Toxicity A. Functional Classification 1. Basal Cell Functions 2. Origin-Specific Cell Functions 3. Activities Destined for Extracellular Functions B. Cytotoxic Classification 1. Basal Cytotoxicity 2. Organ-Specific Cytotoxicity 3. Organizational Toxicity

28 28 28 28 29 29 30 31 32

III. Mechanisms of General Toxicity A. LD 50 Test in Animals B. Assumptions Concerning the Use of Cell Lines C. Use of Cell Lines for Assessing General Toxicity 1. Continuous Cell Lines 2. Acute vs. Chronic Cell Tests D. Applications of Basal Cytotoxicity Data 1. In Human Risk Assessment 2. What do Methods Using Cell Lines Measure? 3. When are Cell Line Methods Used for Assessing Acute Toxicity?

33 33 34 35 35 36 37 37 40 40

IV. Mechanisms of Organ-Specific Toxicity A. Use of Primary Cultures for Organ-Specific Toxicity B. Specialized Functions of Primary Cultures

41 41 42

V. Summary References

43 44

Chapter 3 Cellular Methods of General Toxicity I. Use of Differentiated Cells A. Introduction B. Comparisons to Mutagenicity and Carcinogenicity Testing II. Methods Using Cell Lines A. General Toxicity Criteria B. Neutral Red Uptake Assay C. MTT Assay D. MIT-24 Assay References

47 47 47 48 48 53 54 55 57

Chapter 4 Cellular Methods of Target Organ Toxicity I. Primary Cultures of Specialized Cells A. Establishment of Primary Cultures of Hepatocytes 1. In Situ Liver Perfusion 2. Primary Hepatocyte Enrichment 3. Hepatocyte Cell Culture 4. Functional Markers of Primary Hepatocyte Cultures

59 60 64 64 65 65

B. Establishment of Primary Cultures of Renal Cells 1. Isolation of Renal Cortical Cells 2. Use of Continuous Renal Cell Lines 3. Characterization of Renal Cells C. Establishment of Primary Cultures of Lung Cells 1. Isolation and Culture of Epithelial Cells 2. Isolation and Culture of Clara Cells 3. Isolation and Culture of Alveolar Macrophages 4. Isolation and Culture of Endothelial Cells 5. Characterization of Lung Cells II. General Functional Markers III. Differential Toxicity References

65 65 67 68 68 69 72 74 75 80 80 80 85

Chapter 5 Cellular Methods of Local Toxicity I. In Vitro Cell Modeling Systems II. Testing of Dental Materials

89 89

III. Testing of Ocular Irritants

90

IV. Testing of Local Irritants

95

References

96

Chapter 6 Cellular Methods of Teratogenicity I. The Classical Animal Methods II. In Vitro Embryotoxicity Testing A. Whole Embryo Culture B. Cell Culture Methods 1. Preimplantation Methods 2. Continuous Cell Culture Methods 3. Organ Culture Methods References

101 102 102 102 102 103 103 104

Chapter 7 Cellular Methods of Immune Function I. Introduction to Immunocytotoxicity Testing II. Structure and Function of the Immune System A. Natural Immunity B. Acquired Immunity

107 107 107 108

III. Natural Killer Cells in In Vitro Toxicology A. Isolation and Collection of NK Cells B. Monitoring NK Cell Activity

109 109 109

IV. Monocytes/Macrophages in In Vitro Toxicology A. Macrophages B. Isolation and Collection of Macrophages C. Monitoring Macrophage Activity for Toxicity Testing

Ill 111 112 112

V. Neutrophils in In Vitro Toxicology VI. Lymphocytes in In Vitro Toxicology A. Lymphocytes and Cell-Mediated Immunity B. Isolation and Collection of Lymphocytes C. Continuous Lymphocyte Cultures VII. Tier Testing in Mice References

113 114 114 115 115 116 116

Chapter 8 Pharmacokinetic Studies in Cellular Systems I. Introduction II. Metabolic Studies in Cell Culture III. In Vitro Studies of the Absorption, Distribution, and Elimination of Chemicals IV. Biotransformation of Chemicals in Cell Culture A. Enzymatic Metabolism B. The S9 Mixture References

121 121

122 123 123 124 125

Chapter 9 Cellular Methods of Genotoxicity and Carcinogenicity Thomas W. Sawyer I. Introduction A. History 1. Benzo[a]pyrene 2. The Polycyclic Aromatic Hydrocarbons 3. Initiation, Promotion, and Progression B. Validation and Predictive Ability of Short-Term Tests 1. Short-Term Test Batteries 2. Tier Testing 3. Decision Point Approach II. Structure-Activity Relationship Modeling

127 127 129 130 133 133 135 136 136 137

III. Metabolic Activation Systems A. Introduction B. The S9 Activation System 1. Preparation of S9 Fractions 2. Precautions Using the S9 System C. Intact Cellular Activation Systems

139 139 139 140 141 142

IV. Bacterial Mutagenesis A. The Ames Test 1. History 2. Experimental Protocol

145 145 145 145

V. Mammalian Mutagenesis A. Basis of Mammalian Cell Tests B. Factors that Influence Performance of the Tests C. The Cell Lines 1. CHO and V79 Cells 2. Mouse Lymphoma L5178Y Cells VI. In A. B. C. D.

Vitro Cytogenetic Testing Introduction Chromosomal Aberrations Sister Chromatid Exchange Detection of Micronuclei

VII. Unscheduled DNA Synthesis A. Introduction B. Radioactive Quantitative Methods C. Autoradiographic Methods VIII. Cell Transformation A. Introduction B. Experimental Models 1. The BHK Soft Agar Transformation Assay 2. The Focus Transformation Assay 3. Transformation Assays Using SHE Cells 4. Viral-Chemical Transformation Methods IX. Summary References

147 147 147 148 148 149 150 150 150 151 153 153 153 154 155 157 157 159 160 160 162 162 163 163

Chapter 10 Experimental Design and Statistics I. Practical Considerations A. Experimental Setup B. Incubation Medium and Test Chemical Incompatibility 1. Micronization 2. Use of Solvents to Improve Solubility 3. Sonication 4. Use of Paraffin (Mineral) Oil Overlay

175 175 176 176 177 177 178

C. Determination of Dosage Range D. Determination of Inhibitory Concentrations II. Statistical Methods A. Hypothesis Testing B. Parametric Tests 1. Regression Analysis 2. Correlation Analysis 3. Hypothesis Test for (3 = Zero 4. Analysis of Variance (ANOVA) and the F Test 5. Student's r-test C. Nonparametric, Goodness-of-Fit Tests 1. Chi-Square (x2) 2. Wilcoxon Rank Sum 3. Kruskal-Wallis Nonparametric ANOVA References

178 179 180 181 182 182 182 184 185 186 186 186 187 187 188

Chapter 11 Standardization and Validation Bjom Ekwall and Frank A. BarHe I. Introduction A. Development of Test Methods B. Standardization of Test Methods II. Single Laboratory Validation A. Definitions B. The Process of Validation by an Individual Laboratory 1. Relevance and Reliability 2. Selection of Chemicals 3. In Vivo Data 4. Comparison to Other In Vitro Methods 5. Comparison to Analogous In Vivo Data

189 189 190 191 191 191 191 193 194 195 196

III. Organized Multilaboratory Validation A. Comparison to Single Laboratory Validation B. Types of Validation Processes 1. Validation Based on the Reliability of a Few Methods 2. Multi laboratory, Multimethod Programs a. The CFTA Program b. The MEIC Program C. A Comparison of the Methodologies

196 197 198 198 200 200 201 201

IV. Proposed Validation Programs A. Introduction B. The CAAT/ERGAAT Document 1. Overview 2. Vital Components of a Validation Program

203 203 203 203 204

References

206

Chapter 12 Cell Culture or Animal Toxicity Tests? Or Both? I. Animal Experimentation and Animal Rights A. Animal Regulations B. Relationship of In Vitro Tests to Animal Experiments II. Practical Applications of Cytotoxicity Tests A. Applications of In Vitro Tests 1. As Part of a Battery of Tests 2. As Screening Protocols 3. For the Determination of Risk Assessment B. Applications of In Vitro Tests in Combination with In Vivo Data III. Future Perspectives Index

209 209 209 211 211 211 211 212 212 212 213

Chapter 1

CELL CULTURE METHODOLOGY AND ITS APPLICATION TO IN VITRO CYTOTOXICOLOGY I. INTRODUCTION A. IN VITRO TOXICOLOGY The techniques to grow animal and human cells and tissues on plastic surfaces or in suspension have contributed significantly to the development of biomedical science. Thus the term in vitro refers primarily to the handling of cells and tissues outside of the body under conditions which support their growth, differentiation, and stability. Cell culture techniques (and for the purposes of this book, cell culture and tissue culture are used interchangeably) are fundamental for understanding and performing the procedures in cellular and molecular biology and have been used with increasing frequency in medicinal chemistry, pharmacology, genetics, reproductive biology, and oncology. Cell culture technology has improved throughout the decades, partly as a result of the interest in the methods rather than through its applications. Much of the mystery originally surrounding the techniques has waned, but the basic principles underlying the establishment of the methodology remain. That is, tissue culture represents a tool where scientific questions in the biomedical sciences can be answered through the use of isolated cells or tissues, without the influence of other organ systems. With this understanding, the use of isolated cells does not purport to represent the whole human organism, but can contribute to our understanding of the workings of its components. B. TEST SYSTEMS The use of cell culture techniques in toxicological investigations is referred to as in vitro cytotoxicology, or in vitro toxicology, the latter term including non-cellular test systems such as isolated organelles. The reasons for the popularization of these techniques in toxicology are numerous: 1. 2. 3.

Since the growth of the first mammalian cells were described in capillary glass tubes, the technology has progressed and has been extensively refined. Based on in vitro cytotoxicological methods, the direct interaction of chemicals with human and animal cells in vivo are responsible for the toxic effects demonstrated. The necessity to determine the toxic effects of industrial chemicals and pharmaceuticals that are developed and marketed at rapid and unprecedented rates has provoked the need for fast, simple, and effective test systems.

1

2

Introduction to In Vitro Cytotoxicology

4.

Although the normal rate of progression of any scientific discipline is determined by the progress within the scientific community, some areas have received more encouragement than others.

Specifically, the recent arguments and open protests of animal rights activists have forced researchers and regulatory agencies to direct research initiatives toward the development of alternative methods of toxicity testing. To understand the possibilities for, and more importantly, the limitations of cell culture methods in toxicology, it is necessary to be acquainted with the main features of the techniques to culture cells and tissues. Unlike other types of biomedical research, continuous care of the cells in culture is required for extended periods, which necessitates planning.

II. HISTORY A. TISSUE EXPLANTS In the fledgling years of cell culture, U.S. scientists removed tissue expiants from animals and allowed them to adhere to glass cover slips or placed them in capillary tubes in clots formed from lymph or plasma. It was discovered early on that the tissue or organ, which was once an intact biological specimen, would exhibit a breakdown in the supporting matrix with the migration of individual cells from the main body of the specimen as a consequence.1 With the addition of serum or whole blood, the resulting clot formed a hanging drop, making it possible to look at these cells through an ordinary light microscope. With some refinement, the expiants, with outgrowing cells, were cultivated in small glass flasks in plasma or embryonal extract. Cells were transferred from one flask to another by scraping and loosening the cells with a rubber "policeman". In the 1940s synthetic media were developed (Earle, Parker, Eagle) which were used together with various serum additives. The main problem encountered was bacterial contamination which, because of the more rapid rate of mitosis, usually outpaced the growth of the mammalian cells. This contamination generally resulted in overwhelming bacterial cell growth and disintegration of the cell cultures. In the 1950s, this setback was largely overcome by the addition of liquid antibiotics to the media.2 Later on, the development of better aseptic techniques, such as the incorporation of sterile, disposable glassware, autoclave units, and laminar air flow hoods, made antibiotics superfluous in most cases. With an increase in the understanding of the influence of pH, buffers, and ambient environment, and the incorporation of chemically inert plastics and microprocessor controlled incubators came the realization of the full potential of in vitro technology. B. RECENT DEVELOPMENTS Today, cell biologists have further developed culture techniques to aid in the understanding of cellular/extracellular interactions, such as mesenchymalepithelial relationships and epithelial-cell matrix interactions. These in

Cell Culture Methodology

3

vitro studies have materialized largely through the development of additions to accepted protocols, such as chemically defined cell culture media, addition of cellular substrata to culture flasks, porous membranes which allow for the passage of low molecular weight soluble materials, treated plastic surfaces, and incubation of cells with cocultures.

III. EQUIPMENT In the simplest case, cell cultivation only requires a sterile air flow work station, a temperature-controlled incubator, an autoclave, a hemacytometer, custom gas tanks, a source of ultrapure water, and pipettors. Biological safety cabinets will reduce bacterial and fungal contamination, and will also protect the operator from exposure (Figure 1). Automatic glassware washing facilities, an electronic cell counter, an incubator which monitors humidity and environment, a rate-controlled peristaltic pump, and camera equipment will facilitate experiments and allow for less troublesome, routine operations (Figure 2). The establishment of tissue culture laboratories as part of toxicological investigations has taken on a more sophisticated and dedicated role. In general, for reasons of maintaining sterility, viability, and identification of cultured cells, most tissue culture laboratories are dedicated to this function. However, complete separation of all functions is usually not necessary. For instance, an analytical balance, pH meter, hot plate, and a magnetic stirrer can be used in other laboratory areas. Media preparation equipment and glassware are generally reserved for the cell culture lab. Most supplies and plasticware used in handling cell cultures on a daily basis are sterile and disposable. These include borosilicate glass pipettes, tissue culture flasks and bottles, petri dishes, polypropylene and polyethylene centrifuge tubes, and filter units.

IV. THE CULTURED CELLS A. PRIMARY CULTURES It is easier to grow cells from embryonal tissue than from adult donors. In general, the younger the donor, the more replications and faster replication time can be expected from the resulting cells. Some cells derived from neoplastic tissue are, in this respect, similar to embryonal cells. Ideally, cell cultivation begins with the aseptic removal of an expiant (1/2 x 1 mm) from an animal or human organ. The cells of the expiant are mechanically or enzymatically separated from the matrix and are allowed to grow in medium in contact with the bottom surface of the culture vessel. While the central core of the expiant often atrophies due to less favorable diffusion of nutrients from the medium, the peripheral cells migrate and multiply. This establishes a primary culture where two parallel processes occur: (1) The differentiated cells of the original

4

Introduction to In Vitro Cytotoxicology

Figure 1. Photograph of biological safety cabinet used for tissue culture experiments. Most cabinets are equipped with HEPA filters which provide sterile air flow as well as operator protection from hazardous materials. (Courtesy of Labconco Inc.)

expiant (if there were any) usually do not divide, and with time, will successively lose some of their specialized functions (dedifferentiation); and (2) less specialized cells, e.g., fibroblasts, divide rapidly, and will eventually outnumber the specialized cells. B. FINITE CELL LINES If the primary culture is transferred to a new culture vessel with new, fresh medium, it is designated as a cell line. The subcultivation (passage) is made by detaching the cells from the glass or plastic using chemical (Versene, a calcium chelator) or enzymatic (trypsin) methods and then the cells are dispersed and inoculated in a number of new vessels (Figure 3).3 Because of selective survival of viable cells, the cell line will be more homogenous and dedifferentiated with time. A culture which is demonstrating adequate growth is harvested every 3 to 7 days and subdivided to several new flasks, indicating a doubling rate of 48 to 72 hours. After 40 to 50 cell divisions (also referred to as population doubling level and may require months in culture depending on growth rate), most cell lines stop growing and ultimately die, possibly due to the timing of the genetic program (Figure 4). Such a finite cell line has a constant number of chromosomes (diploid number) and exhibits an orderly

Cell Culture Methodology

5

Figure 2a. Photograph of single and dual chamber water-jacketed C0 2 incubators, with electronic temperature control and safety alarm systems with digital display.

Figure 2b. Photograph of disposable plastic and glass and reusable glass supplies used in routine cell culture laboratories (from Coming® Corp., Coming, NY). For cell culture, the disposable plastic apparatus includes multiple well plates, roller bottles, tissue culture flasks and plates, and plastic tubes. Disposable pipettes, cryogenic vials, centrifuge tubes, filtration units, and vacuum filter systems are available for liquid handling, storage, and separation.

6

Introduction to In Vitro Cytotoxicology

Figure 3. Phase contrast micrograph of epithelial cells emerging from explanted lung tissue in culture. Note the dark contrast of the expiant tissue and the change in phenotypic appearance as cells migrate away from the source. (Magnification x 100.)

orientated growth pattern, including inhibition of growth of individual cells by contact with its neighbor cells (Figure 5a and 5b, Figure 6). C. CONTINUOUS CELL LINES A small percentage of cell lines will not die out with time, but are transformed to continuous cell lines, with a growth pattern often referred to as immortal. Transformation occurs either spontaneously or as a result of incubation with viruses or chemicals. The exact mechanism of the transformation is still undefined, but a continuous cell line has acquired a set of characteristics, such as varied chromosome number (heteroploidy), and loss of contact inhibition. Moreover, continuous cell lines are able to form colonies in soft agar media, i.e., without help of glass or plastic contact, and induce tumors if implanted in immunologically nude animals. In spite of this, some highly differentiated functions, which mimic those of specialized cells, will persist in continuous cell lines. These differentiated functional markers are used to characterize the line and are relied upon to monitor the progress of the cells in culture.4-5 They are also often used as the parameters for assessing in vitro toxicity. Table 1 presents some cell lines which are often used in toxicological studies.

Cell Culture Methodology

7

Figure 4. Phase contrast micrograph of fibroblasts emerging from explanted lung tissue in culture. Note their spindle shaped appearance, with rounded cells sparsely populating the culture. (Magnification x 100.)

D. CELL STRAINS It is possible to select a single cell in a culture and subcultivate this cell to form a clonal cell line. Cloning allows for the selection of one type of cell in a culture (e.g., cells with specific functional markers) and the establishment of a subculture in which the desired characteristic has been passed on to the progeny (identified as a cell strain). E. CLONAL GROWTH AND MAINTENANCE CULTURES By taking specific measures, the highly differentiated parenchymal (epithelial) cells of the expiant can be maintained in primary culture. Such measures include the use of serum-free medium, arginine-free medium, coating of the plastic culture surfaces with cell-specific matrix (laminin or fibronectin) which promotes growth and adhesion of either epithelial or mesenchymal cells, respectively, and/or separation of the cell types by clonal growth ox differential

8

Introduction to In Vitro Cytotoxicology

Figure 5a. Electron micrograph of cultured lung epithelial cells. Dense electron opaque granules, corresponding to phospholipids, and numerous microvilli, characterize this cell type. The presence of condensed nuclei and limited amount of heterochromatin suggests mitotically active cells. (Magnification x 3630.)

adhesion. The last technique separates epithelial cells from fibroblasts based on the ability of the latter cells to settle and adhere to the surface more quickly. One to three hours after an initial inoculation of the culture with the cell suspension, the medium is removed and the epithelial cells, which have not adhered, can be cultured in a separate vessel. The primary culture of the specialized cells now serves as a maintenance culture. Normal primary cultures of specialized cells, including those derived from adult liver, may be kept for weeks, while cells derived from embryonal liver, neuronal, or lung epithelial cells are viable for months. There is always a tendency of the established culture to lose specific functions with time, but this process may be counteracted by culturing the specialized cells on a feeder layer

Cell Culture Methodology

9

Figure 5b. Electron micrograph of cultured lung epithelial cells. The formation of junctional complexes along the length of their cytoplasmic borders is a hallmark of these cells. (Magnification x 23,100.)

of other cell types or by using serum-free medium, Such measures sometimes may even induce specialized functions in less differentiated epithelial cells, including cell lines. Isolation of primary cultures, however, requires repeated use of animals for the extraction and establishment of expiants, which entails expense and is labor intensive. F. CRITERIA FOR IDENTIFICATION Several criteria are used for the identification and classification of cells in culture. These methods are employed when the cell line is established and periodically to monitor the genetic purity of the cells.6 The criteria include: 1.

Karyotypic Analysis — For a cell line designated as being derived from normal tissue, the chromosome complement should be identical to the parent cell or the species of origin. In this way, the cell line may be classified as diploid. Any other designation would classify the cells as

10

Introduction to In Vitro Cytotoxicology

Figure 6. Electron micrograph of cultured lung fibroblasts. The spindle shaped morphology, granular cytoplasm, and limited amount of rough endoplasmic reticulum, are characteristic of these cells in culture. (Magnification x 9900.)

2.

aneuploid or heteroploid. Continuous cell lines derived from tumors, or transformed cells, become part of the latter categories. Aging in Culture — The life span of cells in culture is measured according to the population doubling level (pdl). This critérium refers to the number of times a cell undergoes mitosis since its original isolation in vitro. The general formula for assessing pdl is: pdl, = 3.32 (log F - log I) + pdl, where pdl, = the final pdl at the time of trypsinization or at the end of a given subculture; F = the final cell count; I = the initial cell number used

11

Cell Culture Methodology TABLE 1 Some Commonly Used Cell Lines Origin Human, cervix uteri Human, adult lung, epithelial Human, trachea Human, liver Human, epidermoid tumor Human, embryonal lung, fibroblast Mouse, embryo Mouse, lymphoma Mouse, neuroblastoma Rat, intestine Rat, lung epithelial Chinese hamster, ovary Hamster, kidney

Name HeLa A549 HEP 2 Chang liver KB HFL1, WI-38, IMR-90, MRC-5 3T3 L5178 CI 300 IEC-17 L2 CHO BHK21

Notes Continuous Continuous Continuous Continuous3 Continuous Finite Continuous Continuous Continuous3 Finite Continuous Continuous Continuous

Several functional markers persist.

3.

4.

as inoculum at the beginning of the subculture; pdlj = the doubling level of the inoculum used to initiate the subculture. Cells with finite life spans generally show signs of aging, such as loss of cell shape and increase in the cytoplasmic lipid content. The number of pdl that a particular finite cell line exhibits depends on the age of the organ of origin. Permanent cell lines are capable of indefinite continuous multiplication in vitro, provided they are maintained under optimum conditions. Anchorage-Dependent Cultures — Some cells are capable of growing in suspension culture, while others require attachment to a matrix. This matrix may take the form of polyethylene plastic, treated so that its electrostatic properties allow isolated cells to attach and migrate. Other attachment matrices include components of the extracellular matrix, such as laminin and fibronectin. These components mimic the in vitro attachment substrates normally present in the epithelial lining and the interstitium, respectively. Thus, they may be selective for cells of epithelial or mesenchymal origin. Still other support matrices use microporous filter membranes which not only allow for cellular attachment but also permit passage of macromolecules through the membranes to the basolateral surface of the cell layer. Contact Inhibition — Cells which exhibit contact inhibition will arrest their cell cycle in the G0 phase when the layer of cells completely occupies the surface available for proliferation, thus forming monolayer cultures. Cells which do not exhibit contact inhibition will grow in multilayers.

12

Introduction to In Vitro Cytotoxicology

Other methods of cell classification are not necessarily routinely performed. These include analysis of tissue-specific differentiated properties, such as secretion of macromolecules or the presence of intracellular enzymes, ultrastructural identification, fluorescent labeling for identifiable structural proteins, or the ability of the cells to form an invasive malignant tumor when injected into immunologically deficient {nude) mice.

V. MEDIA A. CHEMICALLY DEFINED MEDIA Cells are often cultured in a chemically defined medium after the addition of fetal or newborn calf serum. The medium is composed of a buffered saline solution containing soluble amino acids, carbohydrates, vitamins, minerals, and cofactors. Optional ingredients include a pH indicator, separate buffering systems, such as HEPES buffer, and some non-essential amino acids which may be used by particular cell types. The formulas for these media are listed in any company catalog which supplies the readily soluble powdered or liquid media. Some of the more commonly used media are modified Eagle's medium (MEM), basal medium Eagle (BSE), Dulbecco's modified Eagle's medium (DMEM, Table 2), and Ham's F12 medium. These media are generally designed for use with serum or serum proteins. Serum is a complex mixture of poorly defined biological components consisting of cell growth factors, transport proteins, hormones, trace metals, lipids, and biomatrix adhesion factors. Without the addition of a minimum percentage of serum (5 to 20%) as part of the growth formulation, cellular proliferation does not proceed favorably. In cell culture, balanced salt solutions function as washing, irrigating, transporting, and diluting fluids while maintaining intra- and extracellular osmotic balance. In addition, they provide cells with water and bulk inorganic ions essential for normal cell metabolism and are formulated to provide a buffering system to maintain the medium within the physiological pH range (7.2 to 7.6). The solutions may also be combined with a carbohydrate, such as glucose, thus supplying an energy source for cell metabolism. The most commonly used prepared salt solutions include Dulbecco's phosphate buffered saline, Earle's balanced salts, and Hank's balanced salt solution (HBSS, Table 3). In addition to satisfying nutrient requirements, the cells must be placed in an environment which mimics the in vivo situation. Table 4 summarizes the conditions which support survival and multiplication, and include temperature, pH, carbon dioxide tension, buffering, osmolality, and humidification. These parameters vary somewhat with the cell type, but are very similar when growing various mammalian cell lines. 1. Temperature For most mammalian cells, the optimum temperature for cell proliferation and differentiation is 37°C. Cells withstand falls in temperature more easily than rises in temperature. In fact, temperatures as low as 4°C may reduce

13

Cell Culture Methodology TABLE 2 Components of a Typical Cell Culture Growth Medium, Dulbecco's Modified Eagle's Medium Component Inorganic salts CaCl2 (anhyd.) Fe(N0 3 )-9H 2 0 KC1 MgS04 (anhyd.) MgS0 4 -7H 2 0 NaCl NaHC0 3 Na 2 HPÒ4H 2 0 Other components D-Glucose Phenol red Sodium pyruvate Amino acids L-Arginine HC1 L-Cystine L-Cystine-2HC1 i.-Glutamine Glycine L-Histidine HC1H 2 0 L-Isoleucine L-Leucine L-Lysine HC1 L-Methionine L-Phenylalanine L-Serine L-Threonine L-Tryptophan L-Tyrosine L-Tyrosine-2Na-2H20 i.-Valine Vitamins Ca pantothenate Choline chloride Folic acid /-Inositol Niacinamide Pyridoxal HC1 Riboflavin Thiamine HC1

1 x Liquid (mg/L)

200.00 0.10 400.00 97.67 200.00 6400.00 3700.00 125.00 1,000.00 15.00 110.00 84.00 48.00 63.00 584.00 30.00 42.00 105.00 105.00 146.00 30.00 66.00 42.00 95.00 16.00 72.00 104.00 94.00 4.00 4.00 4.00 7.20 4.00 4.00 0.40 4.00

Courtesy of Grand Island Biological Company (GIBCO), Grand Island, NY.

Introduction to In Vitro Cytotoxicology

14

TABLE 3 Components of a Typical Cell Culture Physiological Salt Solution, Hank's Balanced Salt Solution7 6 mM Component Inorganic salts CaCl 2 (anhyd.) KC1 KH2PO4 MgCl 2 -6H 2 0 MgS04-7H 2 0 NaCl NaHC0 3 Na 2 HP0 4 Other components D-Glucose Phenol red

g/L

0.14 0.40 0.06 0.10 0.10 8.00 0.35 0.048 1.00 0.01

mM

1.3 5.0 0.3 0.5 0.4 138.0 4.0 0.3 5.6 0.03

Note: Formula for higher concentrations are also available. Courtesy of Grand Island Biological Company Catalog (GIBCO), Grand Island, NY.

metabolic activity, but will not irreversibly inhibit biological functions. Temperatures as high as 39°C may stimulate the formation of heat shock proteins resulting in irreversible ultrastructural and functional damage. 2. pH The pH range that supports optimum cellular multiplication is 7.2 to 7.4, with some cell growth at the extremes of 7.0 and 7.6. Some cells are more tolerant of slightly acidic conditions than corresponding basic conditions, and transformed cells are often more resistant to changes in pH than diploid cells. 3. Carbon Dioxide Tension Most culture media contain bicarbonate as part of the buffering system to prevent large and rapid changes in pH. At standard temperature and pressure in solution, bicarbonate and carbonic acid are in equilibrium as determined by the pH. At 37°C, however, carbonic acid equilibrates with gaseous carbon dioxide, thus driving the carbonic acid into the gaseous phase. This makes it impossible to maintain an adequate concentration of the acid and bicarbonate in the culture medium without also maintaining an increased partial pressure of carbon dioxide in the gas phase above the liquid. Balanced C0 2 tension and maintenance of pH is accomplished with modern water-jacketed C0 2 incubators, which adequately maintain the gaseous phase in the chamber at controlled levels. Therefore, most

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Cell Culture Methodology TABLE 4 Conditions Necessary for Support and Proliferation of Cultured Cells Criteria

Parameter

Culture conditions

Sterility pH Buffered growth medium Buffered wash medium Temperature Humidity Osmolality Time Ultrapure water

In vitro supplementation

Fetal bovine serum Addition of physiological substrata to culture vessels Culture vessel inserts Defined culture additives Pretreated plastic flasks (Primaria®) Antibiotics Co-cultures

Optional additives

pH indicators HEPES buffer

culture media are formulated for use with 2 to 10% C0 2 , so that the appropriate pH can be maintained throughout the life cycle of the cells. 4. Buffering When petri dishes or multiwell plates are removed from the incubation chamber, carbon dioxide necessarily escapes. This inevitably results in the inability of the pH to be adequately maintained in the absence of the gaseous phase above the liquid. This occurrence is not usually a problem when the cultures are removed during washing steps with physiological salt solutions or replacing the expended medium. When in vitro experiments necessitate long periods outside of the incubation chamber, however, a buffering system is required. Many laboratories now routinely incorporate organic buffers, such as HEPES, in the media formulations to prevent the rapid shift of pH when cultures are removed from the C0 2 incubator. Sufficient sodium hydroxide or sodium bicarbonate is added to reach the working pH. Alternatively, cells are grown in tissue culture flasks, which can be gassed with gas mixtures separate from the incubator, and specially formulated for the medium. The individual flasks are gassed and sealed with screw caps, thus

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Introduction to In Vitro Cytotoxicology

preventing escape of the gaseous phase. Although this method is tedious, it may also be helpful when volatile organic liquids or gases are tested in toxicity testing protocols. 5. Osmolality The optimum range of osmolality in the culture medium depends on the cell type, but it is generally between 250 and 325 milliosmoles per kilogram (mOsm/kg). The osmolality of the solution is established during the preparation of the powdered medium, and is standardized when the product is obtained. The concerns arise during the period of incubation when the relative humidity in the chamber is kept at near saturation. When the incubator door is opened, dry air enters and cools the chamber, thus releasing the humidified air by evaporation. An incubator which does not provide for adequate heat transfer will remain cool. If the liquid media in the cultures is at a higher temperature, the water in the media will evaporate, leaving behind a medium solution with a higher osmolality. To prevent evaporation and to maintain the osmolality of the culture medium, rapid equilibration of heat throughout the chamber is necessary. Most modern water-jacketed incubators are equipped with internal fans which distribute the heat. These instruments are also precisely insulated so as to prevent the formation of cool spots, which prevent the humidification of the chamber. In general, the chamber humidity is maintained by leaving an open trough of water, preferably with a large surface area. Adequate humidification is determined visually by the presence of condensation on the glass door of the incubator, but no condensation should be formed on the culture dishes. Alternatively, humidification is monitored by measuring the osmolality of control media incubated parallel to cell cultures. If the osmolality is too high, as compared with fresh medium at the start of a cell cycle, then the humidity is not enough and evaporation is occurring. 6. Water Requirement A fundamental requirement of cell culture systems, and one that is often overlooked in the initial steps of establishing the cell culture laboratory, is the quality of the water used to make the media and salt solutions. In general, distilled water or a single passage of water through a deionizing column is not sufficient to remove all of the impurities which remain from the feed water. Contaminants in the water may take the form of trace metals, organics, variable amounts of divalent cations such as magnesium or calcium, and metabolic products of microorganisms. Such contaminants will interfere with cell growth and metabolic processes. These impurities are essentially removed with systems that recirculate water through a series of deionizing and organic exchange columns. Most of these systems have a digital display which shows the purity of the collected water in milliosmoles. At present, water resistivity measuring at least 10 megaohms is suitable for culturing cells, and the limit of purification with many of the exchange columns is about 18 megaohms. In addition, some

Cell Culture Methodology

17

water purifying systems can also yield water which is sterilized by gamma irradiation at the collecting end. Several water purifying systems are pictured in Figure 7. 7. Glassware Maintenance Another aspect of maintaining a cell culture laboratory at optimum efficiency is the manner in which the glassware is handled, washed, and stored. All glassware designated for use as part of the cell culture area should not be used for storage of other chemicals or for experiments not related to the growth of cells. Glassware should be clearly marked for cell or tissue culture only and should be washed in detergent that is compatible with the objectives of the lab. Essentially, the detergent should leave no residue adhering to the pores of the glass, and should be easily rinsed with deionized water. The glassware should also be stored separately from the rest of the equipment and materials kept in the research laboratory. B. SERUM-FREE MEDIA A trend in modern cell culture has been to replace serum by a mixture of synthetic or otherwise defined factors, such as hormones (insulin, transferrin), trace metals (selenium, manganese), growth factors (epidermal growth factor, fibroblast growth factor), and extracellular matrix components (laminin, fibronectin). These selective serum-free media facilitate adaptation of cells to the culture and allow for better standardization of experimental conditions. Serum is a complex and poorly characterized mixture, the composition of which may vary according to the commercial batch; the concentrations of some components essential for cell growth may vary. Serum may contain naturally occurring substances or microbiological contaminants (e.g., mycoplasma, viruses, endotoxins) that are toxic for certain types of cultures.1011 Serum-free medium facilitates the isolation of the desired cell type. When primary cell cultures are established, the use of serum-free medium virtually eliminates the overgrowth of fibroblasts that usually grow rapidly in serumsupplemented media. The advantages of serum-free (or low serum) media have been demonstrated in the establishment of differentiated rodent thyroid cells in hormone-supplemented medium containing only very small amounts of serum.8 Other cell lines form differentiated features which are not necessarily expressed in serum-supplemented media. For example, the MC845 line forms villus-like secretory structures,9 the HLE 222 human lung epidermoid carcinoma cells produce extensive keratinization,1 ' and rat granulosa cells synthesize large amount of progestins and estrogens after stimulation by follicle-stimulating hormone (FSH).12 Additional examples are reported by Barnes and Sato.510 Serum-free media must be individually prepared and adapted to each cell type. Such media have been described for use in maintenance cultures of many specialized cells, as well as some finite and continuous cell lines, such as human fetal lung fibroblasts and Chinese hamster ovary cells (WI-38, CHO,

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Introduction to In Vitro Cytotoxicology

Figure 7. Photograph of water purifying systems, (a) Water Prodigy™ benchtop model (Courtesy of Labconco Corporation, Kansas City, MO) contains a pretreatment cartridge, reverse osmosis membrane, granular activated carbon organic adsorption cartridge, mixed bed deionization cartridge and submicron and composite vent filters; (b) DF® System (Coming, Coming NY) contains a 5-(im prefilter, two deionization cartridges and a 0.2-u,m final filter; (c) MP-190 Water Purification system™ wall mounted model (Corning) contains pretreatment, dual bed deionizing and final filtration cartridges and can deliver up to 90 liters/hour of Type I reagent water.

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Cell Culture Methodology

Figure 7c.

lung epithelial cells). Thus, optimization of growth using serum free defined media reduces experimental variation which may be encountered using different batches of sera and also encourages standardization of test procedures. In experiments conducted in our lab (Table 5), different lots of serum used over the course of 3 years accounted for varying rates of collagen production in human fetal lung cells, HFL1.

VI. PROCEDURES A. STATIC AND PERFUSION CELL SYSTEMS In general, culture conditions during an experiment or assay differ from those used during routine growth and maintenance of stock cultures. All stock cultures, and almost all experiments, are static cell systems which entertain a change of cell medium before or during subcultivation. During normal incubation, depletion of nutrients and accumulation of excretion products occur in the medium, resulting in less favorable cell culture conditions. Cell types that are anchorage dependent are often cultured as monolayers on the bottom surface of a flask covered with medium, in addition to an overlying gas phase consisting of 5 to 20% carbon dioxide in air. Cells may also be anchored on millipore filters or polystyrene particles. Rotating flasks or tubes are used to increase the culture area by providing contact of the sides and tops of the vessel with media, which also permits better oxygenation of cells. Some cell types (mainly continuous cell lines) need not adhere to a substratum and may be cultured in magnetically stirred suspension cultures or roller bottles. In addition, perfusion cultures are more advanced compared to static cell cultures since they allow for uniform flow of fresh medium by continuous irrigation of the flask. In addition to the usual cell culture manipulations, this system also uses positive pressure

Introduction to In Vitro Cytotoxicology

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TABLE 5 Effect of PGE1 on Percentage Collagen Production in Human Fetal Lung Fibroblasts: Influence of Different Lots of Seraa Control (%) Experiment Experiment Experiment Experiment

1 2 3 4

6.6 ± 6.1 ± 10.0 ± 12.2 ±

0.6 0.6 2.0 1.2

PGE1 (%) 3.2 ± 1.2 3.4 ± 0.8 5.0 ± 1.0 6.2 ± 0.2

Pb