Introduction to Cell and Tissue Culture Theory and Technique huangzhiman 2002.12.18 Foreword 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 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 2 , Oxygen, and Other Gases 31 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 [ 3 H]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 [...]... that cell cultures can be "compartmentalized," that is, primary cell cultures can be handled in a specifically designated hood(s), and kept in an incubator chamber separate from other cell lines being maintained in long-term culture Cell cultures coming into the laboratory, as frozen vials or as viable cultures, primaries, or established cell lines, should be quarantined in an incubator chamber and handled... 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... composite) and cleaned with a disinfectant before and after each use The Standard Tissue Culture Laboratory This may be a core facility, working in conjunction with other laboratories, or it may be the primary facility for a cell biology laboratory All basic cell culture studies can be done in this facility that would be adequate for cell culture use for most cell and molecular biology laboratories, as... entry and exit, for example, by having a refrigerator and freezer in the culture room or an airlock "entry room" so that there is no need to enter and exit the culture room during the course of an experiment to obtain reagents necessary for the culture work If space allows, an airlock can help to ensure a "clean" tissue culture room If it is not possible to have a separate room for the cell culture. .. teaching Page 10 laboratory, the standard research laboratory, and the optimal tissue culture laboratory In all cases, however, the laboratory or designated area should be designed with minimal and optimal flow of traffic This concept will affect placement of incubators, hoods, microscopes, freezers and refrigerators, and storage of sterile, disposable supplies, as well as positive and negative interaction... other) cell culture is to provide an environment that mimics, to the greatest extent possible, the in vivo environment of that specific cell type The cell culture incubator, the culture dish or apparatus, and the medium together create this environment in vitro They provide an appropriate temperature, pH, oxygen, and CO2 supply, surface for cell attachment, nutrient and vitamin supply, protection from toxic... sources of materials and equipment and references to both the primary literature and other methods volumes that describe specialized techniques and specific areas of interest in more detail For example, we will briefly mention how to recognize and measure apoptosis, or programmed cell death, as it occurs in cell cultures and how to differentiate it from necrotic cell death We will not go into great detail... that is of good quality and perhaps better suited to their needs, budget, or locale Primary Culture Several different types of culture are routinely performed These can be roughly divided into "primary culture" and "culture of established cell lines." Primary culture can consist of the culture of a complex organ or tissue slice, a defined mixture of cells, or highly purified cells isolated directly... ability to grow the cell, or its precursor, indefinitely in culture This can be accomplished using conditional transformation or by establishing the cell line from stem cell or precursor cells, which can then be induced to differentiate into a terminally differentiated cell type in culture These lines are generally more challenging to handle in vitro and will be covered in the last section The Physical and. .. 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 . 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. 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. 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