Introduction to Cell andTissue Culture

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Introduction to Cell andTissue Cultureto,Cell,cell


    Chapter 1: Introduction 1

    Chapter 2: Setting Up a Cell Culture Laboratory 9

    Chapter 3: The Physical Environment 25

    Chapter 4: Media 41

    Chapter 5: Standard Cell Culture Techniques 63

    Chapter 6: Looking at Cells 89

    Chapter 7: Contamination: How to Avoid It, Recognize It, and Get Rid of It 117 Chapter 8: Serum-Free Culture 129

    Chapter 9: Primary Cultures 151

    Chapter 10: Establishing a Cell Line 165

    Chapter 11: Special Growth Conditions 175

    Chapter 12: Cell Culture for Commercial Settings 195

    Glossary Appendix Index 205


    It is a pleasure to contribute the foreword to Introduction to Cell and Tissue Culture: Theory and

    Techniques by Mather and Roberts. Despite the occasional appearance of thoughtful works devoted to

    elementary or advanced cell culture methodology, a place remains for a comprehensive and definitive

    volume that can be used to advantage by both the novice and the expert in the field. In this book, Mather

    and Roberts present the relevant methodology within a conceptual framework of cell biology, genetics,

    nutrition, endocrinology, and physiology that renders technical cell culture information in a

    comprehensive, logical format. This allows topics to be presented with an emphasis on troubleshooting

    problems from a basis of understanding the underlying theory.

    The material is presented in a way that is adaptable to student use in formal courses; it also should be

    functional when used on a daily basis by professional cell culturists in academia and industry. The

    volume includes references to relevant Internet sites and other useful sources of information. In addition

    to the fundamentals, attention is also given to modem applications and approaches to cell culture

    derivation, medium formulation, culture scale-up, and biotechnology, presented by scientists who are

    pioneers in these areas. With this volume, it should be possible to establish and maintain a cell culture

    laboratory devoted to any of the many disciplines to which cell culture methodology

is applicable.


    We would like to thank Dr. David Phillips for all we have learned about looking at cells during many

    years of collaboration. Thanks also to Dr. Phillips for providing the scanning and transmission electron

    micrographs used throughout the book. Our thanks to Dr. David Barnes for many interesting discussions

    on the nature of cells, from worms to man, over many years. We would also like to thank Dr. Barnes,

    Dr. Monique LeFleur, Amy McMurtry, and Patricia Kaminsky for their careful reading of draft versions

    of the volume and their suggestions for corrections and clarifications. We would also like to thank

    Aldona Kallok for helping in many ways with the preparation of the manuscript. We would also especially like to thank Alicia Byer, Dr. Lin-Zhi Zhuang, Dr. Virgilio Perez-Infante,

    Mary Tsao, Robert Shawley, Diana Stocks, Dr. Margaret Roy, Dr. Yossi Orly, Dr. Teresa Woodruff, Dr.

    Alison Moore, Dr. Rong-hao Li, Dr. Jean-Philippe Stephan, Dr. Vidya Sundaresan, Terri Restivo,

    Marcel Zocher, Kathy King, Glynis McCray, and the other past and present members of our laboratory.

    It is impossible to overestimate the contributions of these friends and colleagues who have, in the course

    of their work and studies in the Mather Laboratories over the years, added greatly to our knowledge and

    the fun of cell culture. Finally, we would like to thank Dr. Gordon Sato, who introduced us to the joy of

    cell culture and the infinite variety of interesting things to do with cells. A note on the figures: The graphs and tables presented throughout the book are drawn from actual

    experimental data generated in the Mather Laboratories over the last 20 years. We have chosen those

    experiments that best illustrate the point being discussed in the text and have not necessarily provided all

    the experimental details for each figure.

    We would also like to thank the following vendors for their help in discussions of their equipment and,

    where noted, in providing photographs or data for the figures and tables: James Quach, Instrument

    Services, Genentech, BRL Life Technologies, Corning Corporation, Falcon (Becton Dickinson), The

Baker Company, Mike Alden of Coulter Electronics, E. Braun Biotech International,

    The Edge

    Scientific Instrument Co., Altair Gases, Sara Ferrer and Technical Instrument Company,

    and Brent

    Kolhede of Lab Equipment Company.


    Chapter 1: Introduction 1

    The History of Tissue and Organ Culture 1 The Practice and Theory of Tissue Culture 3 Primary Culture 4

    Established Cell Lines 6

    The Physical and Chemical Environment 6 Complex versus Defined Culture Environments 7 Further Information 7

    References 8

    Chapter 2: Setting Up a Cell Culture Laboratory 9 Space Requirements 9

    Equipment 11

    The Teaching Laboratory 11

    The Standard Tissue Culture Laboratory 13 The Optimal Tissue Culture Laboratory 19 Plasticware and Glassware 20

    Maintaining the Laboratory 21

    Daily Tasks 21

    Weekly Tasks 23

    Monthly Tasks 23

    Chapter 3: The Physical Environment 25 Temperature 25

    pH 28

    Osmolality 30

    CO2, Oxygen, and Other Gases 31

    Surfaces and Cell Shape 32

    Adherent versus Nonadherent Cells 33 Plastic Different Types for Different Purposes 34 Glass 35

    Cell Shape 36

    Basement Membrane and Attachment Factors 36 Artificial Membranes 36

    Stress 37

    pH, Temperature, and Osmolality 39 Mechanical 39

Toxic Chemicals and Heavy Metals 39

    Proteases 40

    References 40

Chapter 4: Media 41

    What Does the Medium Do? 41

    Matching the Incubator Settings and the Medium 47 How to Select the Appropriate Medium 48 Media Preparation 51

    Preparing Medium 52

    Filter Sterilization 52

    Serum Treatment 53

    Testing Media and Components?ªQuality Control 54

    Troubleshooting Medium Problems 55

    Altering Commercial Media for Special Uses 56 Medium Optimization 57

    References 61

    Chapter 5: Standard Cell Culture Techniques 63 Subculturing 63

    Subculturing Adherent Cells 64

    Subculturing Suspension Cultures 65

    Growth Curves and Measuring Cell Growth 66 Using the Hemacytometer or Electronic Particle Counter 67

    Electronic Particle Counting 68

    Generating a Growth Curve 70

    Secondary Endpoint Assays for Proliferation 71 [3H]Thymidine Incorporation Assay for DNA Synthesis 72 High-Throughput Assays for Secondary Endpoints for Cell Number 73

    Measuring Cell Viability 73

    Acridine Orange-Ethidium Bromide Viability Determination 74

    Plating Efficiency 74

    Conditioning Medium 76

    Cloning 76

    Cloning by Picking Colonies of Attached Cells 77 Cloning in Serum-Free Media 78

    Cloning by Limiting Dilution 79

    Freezing and Thawing Cells 80

    Freezing 81

    Temporary Freezing of Large Numbers of Clones 82 Thawing 83

    Frozen Cell Storage 85

    Record Keeping 85

    Summary 86

References 87

Chapter 6: Looking at Cells 89

    Just Look at the Dish 89

    The Light Microscope Level 89

    Phase Contrast 91

    Hoffman or Nomarski Optics 95

    Care and Handling of the Phase Contrast Microscope 96 Fluorescence Microscopy 97

    Labeling Cells with a Fluorescent Viable Cell Dye 101 Cell Preparation, Fixation, and Antibody Binding 102 Bright Field 104

    Dark Field 104

    Adding the Third Dimension 106

    Confocal Microscopy 106

    Adding the Fourth Dimension 107

    Real-Time Video 107

    Time-Lapse Video 107

    High-Speed Video 110

    Looking More Closely 110

    Scanning Electron Microscopy 112

    Transmission Electron Microscopy 112

    References 114

Chapter 7: Contamination: How to Avoid It, Recognize It, and Get Rid of It 117

    Strings, Wigglies, and Pretty Balls of Fluff 117 Things You Cannot See Can Hurt You 119

    Mycoplasma 119

    Method for Fluorescent Detection of Mycoplasma 121 Virus 123

    Cross-Culture Contamination 123

    What Can You Do to Prevent Contamination? 125 What Can You Do to Get Rid of Contamination in Cultures? 126 References 127

Chapter 8: Serum-Free Culture 129

    The Substitution of Defined Components for Serum 131 Preparation and Selection of Medium 135

    Water 136

    Preparing and Testing Hormones and Growth Factors 137 Stock Preparations 137

    Sterilization 140

    Subculture and Setting Up Experiments 141 Reducing or Eliminating Serum 145

    Freezing Cells in Serum-Free Medium 146 Carrying Cell Lines in Serum-Free Medium 146 Setting Up a Serum-Free Growth Experiment 146 References 149

Chapter 9: Primary Cultures 151

    Tumors 152

    Setting Up a Primary Culture from a Tumor 153 Primary Culture of Normal Rodent Tissues 155 Primary Culture of 20-Day-Old Rat Sertoli Cells 157 Primary Culture of Nonciliated Lung Epithelial Cells 158

    Serum versus Serum-Free Media 161

    Special Considerations for Human Tissues 162 References 163

    Chapter 10: Establishing a Cell Line 165 Transformed Cell Lines 165

    Tumor Tissue 166

    Transforming Normal Cells in Vitro 166 Cell Lines from Normal Tissues 167

    Rodent Cells in Serum-Free Culture 167 Human Cells-Limited Life Span 167

    Crisis and Senescence 170

    Karyotyping 170

    Establishing Sterility 171

    Confirming Identity 171

    "Banking" the Line 172

    References 173

    Chapter 11: Special Growth Conditions 175 Methods for High-Throughput Assays for Secondary Endpoints Correlating

    with Cell Number


    Growth of Cells in 96-Well Plates 177 Calcein-AM 178

    MTT Reduction 178

    Other Dye Reduction Colorimetric Methods 179 [3H]Thymidine Incorporation Assay for DNA Synthesis 179

    Alamar Blue 179

    Crystal Violet 180

    Acid Phosphatase 181

    Growth Configurations for Scaling-Up Attachment-Dependent Cells 181

    Suspension-Adapting Cells 181

    Scaling-Up Suspension-Adapted Cells 183

Roller Bottles 184

    Microcarrier Beads 185

    Growing Cells on Microcarriers 186

    Growing Cells in Hollow Fibers 187

    Special Substrates for Cell Culture 187

    Growth in Semisolid Media 187

    Collagen Gels 189

    Cell-Cell Interaction 190

    Protocol to Create Feeder Layers Using Mitomycin C 192 Transwell Dishes 192

    Summary 193

    References 193

    Chapter 12: Cell Culture for Commercial Settings 195 The Cell as Industrial Property 196

    Engineering Cells for Specific Properties 196

    Preparing, Characterizing, and Storing Cell Banks 197 Very Small Scale 198

    Very Large Scale 198

    References 204

Glossary 205

    Appendix 1: Formula for Calculating Osmolarity 213

    Appendix 2: Time-Lapse Photomicrography: Assembling Equipment 215 Appendix 3: Pituitary Extract Preparation 219

    Appendix 4: Siliconization of Glassware 221

    Appendix 5: Suppliers 223

    Index 239

Chapter 1


    The History of Tissue and Organ Culture

    Tissue culture as a technique was first used almost 100 years ago to elucidate some

    of the most basic

    questions in developmental biology. Ross Harrison at the Rockefeller Institute, in

    an attempt to observe

    living, developing nerve fibers, cultured frog embryo tissues in plasma clots for

    1 to 4 weeks (Harrison,

    1907). He was able to observe the development and outgrowth of nerve fibers in these

    cultures. In 1912,

    Alexis Carrel, also at the Rockefeller Institute, attempted to improve the state of

    the art of animal cell

    culture with experiments on the culture of chick embryo tissue:

    The purpose of the experiments . . . was to determine the conditions under which the active life of a tissue

    outside the organism could be prolonged indefinitely. It might be supposed that senility and death of cultures,

    instead of being necessary, resulted merely from preventable occurrences; such as accumulation of catabolic

    substances and exhaustion of the medium . . . . It is even conceivable that the length of life of a tissue outside the organism could greatly exceed its normal duration in the body. (Carrel, 1912, p. 9)

    Carrel succeeded in expanding the possibilities of cell culture by keeping fragments of chick embryo

    heart alive and beating into the third month of culture and growing chick embryo connective tissue for

    over 3 months. Using apparatus such as that shown in Fig. 1.1, Carrel reported growing chick embryo

    tissue for many years in vitro, and thus helped convince the scientific community that in vitro cultures

    were useful experimental systems.

    The next important advance in the conceptualization and technology of cell culture was the

    demonstration by Katherine Sanford and co-workers (1948) that single cells could be grown in culture.

    This, along with Harry Eagle's (1955) demonstration that the complex tissue extracts, clots, and so forth

    previously used to grow cells could be replaced by " . . . an arbitrary mixture of amino acids, vitamins,

    co-factors, carbohydrates, and salts, supplemented with a small amount of serum protein . . . " (p. 50)

    opened up a new area of cell culture. A vast range of manipulations that had not been possible

    previously could now be performed with cells, including production of genetically altered cell lines through mutagenesis and cloning, direct comparison of cells from normal and transformed tissues, the study of cellular physiology and metabolism, and the growth of normal and transformed human cells in vitro (Hayflick and Moorehead, 1961; Leibovitz, 1963; Puck and Marcus, 1955).

    Arising out of this work was the demonstration that cells in culture could be established as cell lines that

    maintained, at least in part, the differentiated functions characteristic of their cell type of origin. Thus,

    the creation of cell lines that maintained some functional properties of adrenal cells, pituitary cells

    (Bounassisi et al., 1962), neurons (Augusti-Tocco and Sato, 1969), myocytes (Yaffe, 1968), and

    hepatocytes (Thompson et al., 1966) allowed the study not only of growth but of the response to

    hormones and other environmental factors and the production and secretion of hormones and other

    differentiated functions in vitro.

    The demonstration that each cell type has an optimal mix of nutrients that supports its function (Ham

    and McKeehan, 1979; Waymouth, 1981) has led to media derived to support specific cell types under

    specialized conditions. In parallel, the recognition that serum could be replaced by defined components

    such as attachment proteins, transport proteins, and hormones and growth factors (Barnes and Sato,

    1980a,b; Bottenstein et al., 1979; Mather and Sato, 1979) once again opened up new possibilities for the

    maintenance of specialized cells and tissues in culture, and thus the ability to address important

    biological questions in new ways.

    Finally, the advent of recombinant expression in mammalian cells and the creation of antibodyproducing

    hybridoma cell lines, coupled with the use of large-scale culture techniques for culturing

    mammalian cells, has created an important niche for industrial cell culture as a production system for

    recombinant proteins. The special considerations inherent in industrial production using large-scale

    cultures have further increased our understanding of the range of cell "behaviors," their inherent stability, the ability to genetically manipulate cell properties, and the technical challenges of growing mammalian cells in tanks large enough to have several atmospheres' difference in pressure from top to bottom (Fig. 1.2). Each of these insights and technical advances has brought new challenges, raised more questions, and

    widened our experience with that "microorganism" (see Puck, 1972), perhaps better defined as a "social

    organism," which is the mammalian cell in vitro.

The Practice and Theory of Tissue Culture

    This book is meant to serve as an introduction to cell culture both for students who have little or no

    experience of cell culture and for scientists who do have some experience with sterile

technique and

    mammalian cell culture and wish to set up a cell culture facility in their laboratory. Thus, each section

    on the techniques, space, and equipment will be divided into a "minimal," "standard," and "optimal," or

    "ideal" laboratory. The minimal facility is described as one that can be used for a teaching laboratory or

    for a laboratory where there is only an occasional use for tissue culture. The standard facility should be

    considered the desired level if tissue culture is an important and frequently used part of the research

    work (e.g., a laboratory that studies expression of recombinant proteins) but is not the central task of the

    laboratory. The optimal facility described is one that should be achieved if cell culture is of critical

    importance to the work done in the laboratory (e.g., new cell line development, in vitro studies of the

    regulation of gene function, etc.). One can, of course, mix equipment and space considerations based on

    the available space, equipment, and research goals.

    In parallel, the book will cover the concepts and technology of cell and tissue culture on several different

    levels. Cell culture consists of a few basic concepts and techniques that can and should be mastered at

    the student or introductory level in a few weeks or months. These include sterile technique, subculture of

    cells, freezing and thawing cells, cloning cells, measuring cell growth and viability, and starting primary

    cultures. With these few techniques the scientist or student can usually successfully handle many of the

    experiments performed with established cell lines, especially those lines that are relatively hardy.

    However, it is important for the scientist who makes tissue culture an important tool in his or her

    research to have a more complete understanding of the science and the years of experimentation behind

    these techniques. What does the medium do for the cells? How does the choice of incubator setting and

    medium interrelate? How can the environment be altered for optimal growth of cells at high density?

    How should the medium be changed for suspension culture? What does one do when the cells "just

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