Culture of Animal Cells

A Manual of Basic Technique and Specialized Applications

R. Ian Freshney

• Publisher: Wiley-Blackwell
• Year: 2015
• Print ISBN: 9781118873656
• eBook ISBN: 9781118873649
• Edition: 7
• Topic: Biological Sciences
• Subtopic: Cell Biology
• Index: Biological Sciences

Since the publication of the sixth edition of this benchmark text, numerous advances in the field have been made – particularly in stem cells, 3D culture, scale-up, STR profiling, and culture of specialized cells. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Seventh Edition is the updated version of this benchmark text, addressing these recent developments in the field as well as the basic skills and protocols.

This eagerly awaited edition reviews the increasing diversity of the applications of cell culture and the proliferation of specialized techniques, and provides an introduction to new subtopics in mini-reviews. New features also include a new chapter on cell line authentication with a review of the major issues and appropriate protocols including DNA profiling and barcoding, as well as some new specialized protocols. Because of the continuing expansion of cell culture, and to keep the bulk of the book to a reasonable size, some specialized protocols are presented as supplementary material online.

Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Seventh Edition provides the most accessible and comprehensive introduction available to the culture and experimental manipulation of animal cells. This text is an indispensable resource for those in or entering the field, including academic research scientists, clinical and biopharmaceutical researchers, undergraduate and graduate students, cell and molecular biology and genetics lab managers, trainees and technicians.

CHAPTER 1
Introduction

Tissue culture, in its various different guises, has been around now for more than a century. As the technique evolves, it is possible to discern three distinct phases: (a) the first period, of approximately 40 years, which was dependent mostly on explantation and studying the behavior of the outgrowth from explants as pioneered by Harrison, Carrel, and others [Harrison 1907; Carrel 1912]; (b) the second period, of approximately 50−60 years, which was dependent of the development of cell lines, both finite, such as WI-38 [Hayflick & Moorhead 1961] and MRC-5 [Jacobs 1970], and continuous, such as the L cell [Earle et al. 1943], HeLa [Gey et al. 1952] and many others; and (c) the third, current period, exploring the analysis and manipulation of gene expression of cell lines and freshly isolated cells. The field has moved from being exploratory research into how to grow cells in vitro, conducted by a few individuals, to a major tool in cell and molecular biology, virology, cellular pathology, bioengineering, and industrial pharmaceutics on a scale never envisioned by the early workers.

With a dramatic increase in the understanding of the role of transcription factors on gene expression, genetic manipulation of commonly and easily cultured cells, such as skin fibroblasts, can convert them into pluripotent stem (iPS) cells, capable of differentiating into a range of different cell types [Lewitzky & Yamanaka 2007; Nakagawa et al. 2007; Yu et al. 2007]. Coupled with the isolation of stem cells from many different tissues in addition to the embryo and the use of chemical inducers of transcriptional changes in the genome (such as valproic acid) the possibility of creating iPS cells by epigenetic manipulation, rather than genetic intervention, becomes a real possibility. The whole concept of irreversible commitment to a particular path of differentiation has had to be revisited as it has proved to be possible to induce transdifferentiation from one lineage to another via reversion to a precursor cell [Kondo & Raff 2000; le Douarin et al. 2004] and even to persuade cells to change lineage without reversion to a more primitive cell [Vierbuchen et al. 2010] first proposed in eye regeneration many years ago [Pritchard et al. 1978; Okada 1983]. The implications of these developments are far-reaching; instead of the need for complex selective culture techniques, simple culture procedures may be used to initiate a cell line and epigenetic manipulation used to convert it into a new phenotype, directly or via regression to a stem cell or other progenitor cell. The possibilities that this opens up for the study of the regulation of differentiation, the determination of errors that occur in abnormal differentiation [Ebert et al. 2009] and malignancy, the provision of screening systems for diagnosis and drug development with cell lines from known pathologies, and the creation of autografts by tissue engineering, promises a further expansion of tissue culture technology and usage comparable to the biotechnology boom of the turn of the last century.

While the development of cell lines has been pivotal to most of the advances in cell culture over the past 50 years, it has brought with it a number of unforeseen problems. The observation that differentiated properties were lost in culture alerted early workers in the field to the problems of dedifferentiation and selection and the fact that cultured cells may be genetically, as well as phenotypically, unstable. What was not predicted was that the generation of cell lines would (1) provide an ideal substrate for the growth of mycoplasma and (2) extend the risks of cross-contamination and misidentification already familiar to microbiologists. It does not count to the credit of present-day cell culturists that both of these problems are often overlooked despite having been recognized in the 1960s. Hence many of the subsequent pages will reinforce the need for good cell culture practice [Balls et al. 2006; Geraghty et al. 2014] and emphasize the need for stringent quality control to minimize the risks of microbial contamination, misidentification, and cross-contamination.

1.1 HISTORICAL BACKGROUNd

Tissue culture was initially devised at the beginning of the twentieth century [Harrison 1907; Carrel 1912] (Table 1.1) as a method for studying the behavior of animal cells free of systematic variations that might arise in vivo both during normal homeostasis and under stress due to experimental cond...

Table of contents

• Cover
• Titlepage
• Copyright
• Dedication
• List of Color Plates
• List of Protocols
• List of Minireviews
• Preface and Acknowledgments
• Abbreviations
• About the Companion Website
• CHAPTER 1 Introduction
• CHAPTER 2 Biology of Cultured Cells
• CHAPTER 3 Laboratory Design and Layout
• CHAPTER 4 Equipment and Materials
• CHAPTER 5 Aseptic Technique
• CHAPTER 6 Safety, Bioethics, and Validation
• CHAPTER 7 Culture Vessels and Substrates
• CHAPTER 8 Defined Media and Supplements
• CHAPTER 9 Serum-Free Media
• CHAPTER 10 Preparation and Sterilization
• CHAPTER 11 Primary Culture
• CHAPTER 12 Subculture and Cell Lines
• CHAPTER 13 Authentication and Validation
• CHAPTER 14 Microbial Contamination
• CHAPTER 15 Cryopreservation and Banking
• CHAPTER 16 Cloning and Selection
• CHAPTER 17 Cell Sorting
• CHAPTER 18 Cell Line Characterization
• CHAPTER 19 Differentiation
• CHAPTER 20 Three-Dimensional Culture
• CHAPTER 21 Scale-up and Automation
• CHAPTER 22 Senescence, Immortalization, and Transformation
• CHAPTER 23 Quantitation
• CHAPTER 24 Cytotoxicity
• CHAPTER 25 Culture of Specific Cell Types
• CHAPTER 26 Stem Cells
• CHAPTER 27 Training Programs
• CHAPTER 28 Problem Solving
• CHAPTER 29 In Conclusion
• Appendix I Calculations and preparation of reagents
• APPENDIX II Glossary
• Index
• Links
• Supplementary Images
• EULA