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Concise introduction to a major technique of cell biology laboratories for those new to the field.
This volume discusses the requirements, advantages, and limitations of studying cell culture. The chapters in this book cover topics such as in vitro blood-brain barrier functional assays in human iPSC-based models; neuron-glia interactions examines with in vitro co-culture; epigenetic changes in cultures neurons and astrocytes; rat brain slices; brain punching technique; and using microRNA for in vitro neurotoxicity testing and related disorders. In Neuromethods series style, chapters include the kind of detail and key advice from the specialists needed to get successful results in your laboratory. Authoritative and cutting-edge, Cell Culture Techniques, Second Edition is a valuable resource for students and experiences researchers who are interested in learning more and making risk decisions in this evolving field.
Now completely revised and updated from the original, much-acclaimed and bestselling first edition, Basic Cell Culture Protocols, 2nd ed. offers today's most comprehensive collection of easy-to-follow, cutting-edge protocols for the culture of a wide range of animal cells. Its authoritative contributors provide explicit, step-by-step instructions, along with extensive notes and tips that allow both experts and beginners to successfully achieve their desired results. Topics range from basic culture methodology to strategies for culturing previously uncultured cell types and hard-to-culture differentiated cells. Methods are also provided for the analysis of living cells by FACS, video microscopy, and confocal microscopy. Like the first edition, this book should be in every cell culture laboratory and be of use to all who use cell cultures in research.
Uniquely integrates the theory and practice of key experimental techniques for bioscience undergraduates. Now includes drug discovery and clinical biochemistry.
Cell culture techniques allow a variety of molecular and cell biological questions to be addressed, offering physiological conditions whilst avoiding the use of laboratory animals. In addition to basic techniques, a wide range of specialised practical protocols covering the following areas are included: cell proliferation and death, in-vitro models for cell differentiation, in-vitro models for toxicology and pharmacology, industrial application of animal cell culture, genetic manipulation and analysis of human and animal cells in culture.
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xl II INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I STERILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Aseptic Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Physical manipulations • Use of the sterile cabinet (hood) Sterilization Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Heat • Radiation • Toxic gas • Filtration • Antibiotics Quality Control of Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Routine labeling Suggested Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 vi CONTENTS ROUTINE CELL CULTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Feeding Schedules and Media Components . . . . . . . . . . . . . . . . . . . . 29 General properties of media and salt solutions • water as a reagent· Establishingfeeding schedules Subcultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Solutions and methods for adherent cells • Common enzyme solutions • Inoculating (seeding) the cultures Cell Enumeration and Cell Viability . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Hemocytometer • Particle counter • Cell viability Putting Routine Methods to Work . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Normal cell growth characteristics Detecting and Disposing of Contamination . . . . . . . . . . . . . . . . . . . . 66 Bacteria and fUngi • Fungi • Mycoplasma • Viruses • Dealing with contamination Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Inadequate cell growth • Recurrent contamination • When to call your vendor Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Biological hazards • Chemical hazards Suggested Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Problem Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 EXPERIMENTS IN CULTURE . . . . . . . . . . . . . . . . . . . . . . . . . . 91 II Alterations of the Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Serum • Treatments of serum • Plasma-derived serum • Serum-free and low-protein media Substrata. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Coatingplasticware with solutions • Alterations with polymers • Using cells to coat the plasticware • Culturing cells on microcarriers Altering the Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Temperature changes • Gaseous changes Problem Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 CONTENTS vii PRIMARY CELL CULTURE. . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Dissection • Enzymatic dissociation methods • Nonenzymatic isolation • Purification of cell suspensions • Consideringyield and survival Chatacterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
This is a comprehensive research guide that describes both the key new techniques and more established methods. Every chapter discusses the merits and limitations of the various approaches and then provides selected tried-and-tested protocols, as well as a plethora of good practical advice, for immediate use at the bench. It presents the most accessible and comprehensive introduction available to the culture and experimental manipulation of animal cells. Detailed protocols for a wide variety of methods provide the core of each chapter, making new methodology easily accessible. This book is an essential laboratory manual for all undergraduates and graduates about to embark on a cell culture project. It is a book which both experienced researchers and those new to the field will find invaluable.
It is a pleasure to contribute the foreword to Introduction to Cell and Tissue Culture: The ory and Techniques by Mather and Roberts. Despite the occasional appearance of thought ful 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 method ology within a conceptual framework of cell biology, genetics, nutrition, endocrinology, and physiology that renders technical cell culture information in a comprehensive, logical for mat. 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 a- demia and industry. The volume includes references to relevant Internet sites and other use ful sources of information. In addition to the fundamentals, attention is also given to mod ern 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 devot ed to any of the many disciplines to which cell culture methodology is applicable.
This volume provides complete and thorough coverage of the classical and state-of-the-art methods used in cell culture. It also includes basic principles used in the selection of cells for specific scientific study, as well as analytical and procedural techniques.Key Features* Reviews basic principles of cell culture* Gives options and techniques on how to look at cells