Table Of ContentIntroduction to Cell and
Tissue Culture
Theory and Technique
huangzhiman 2002.12.18
Foreword
1
Chapter 1: Introduction
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
Page i
Introduction to Cell and Tissue Culture
Theory and Technique
Page ii
INTRODUCTORY CELL AND MOLECULAR BIOLOGY TECHNIQUES
SERIES EDITOR: Bonnie S. Dunbar, Baylor College of Medicine, Houston, Texas
INTRODUCTION TO CELL AND TISSUE CULTURE: Theory and Technique
Jennie P. Mather and Penelope E. Roberts
Page iii
Introduction to Cell and Tissue Culture
Theory and Technique
Jennie P. Mather and
Penelope E. Roberts
Genentech Inc.
South San Francisco, California
Page iv
Library of Congress Cataloging-in-Publication Data
Mather, Jennie P., 1948¨C
Introduction to cell and tissue culture : theory and technique /
Jennie P. Mather and Penelope E. Roberts.
p. cm.¡ª (Introductory cell and molecular biology
techniques)
Includes bibliographical references and index.
ISBN 0-306-45859-4
1. Cell culture. 2. Tissue culture. I. Roberts, Penelope E.
II. Title. III. Series.
[DNLM: 1. Tissue Culture¡ªmethods. 2. Microbiological Techniques.
QS 525M427i 1998]
QH585.2.M38 1998
571.5'38¡ªdc21
DNLM/DLC
for Library of Congress 98-27597
CIP
ISBN 0-306-45859-4
©1998 Plenum Press, New York
A Division of Plenum Publishing Corporation
233 Spring Street, New York, N.Y. 10013
http://www.plenum.com
10 9 8 7 6 5 4 3 2 1
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by
any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written
permission from the Publisher
Printed in the United States of America
Page v
To the memory of Dr. Izumi Hayashi,
whose life was as elegant as her experiments
Page vii
Foreword
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.
DR. DAVID BARNES
DEPARTMENT OF BIOCHEMISTRY AND BIOPHYSICS
OREGON STATE UNIVERSITY
Page ix
Acknowledgments
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.
Page xi
Contents
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
Page xii
CO , Oxygen, and Other Gases 31
2
Surfaces and Cell Shape 32
Adherent versus Nonadherent Cells 33
Plastic¡ªDifferent 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¨CEthidium 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
