Animal Cell Culture: A Practical Approach

Animal Cell Culture The Practical Approach Series Related Practical Approach Series Titles Membrane Transport Affinity Separations Flow Cytometry [3e] Subcellular Fractionation Apoptosis Platelets Cell Growth, Differentiation and Epithelial Cell Culture Senescence Neural Cell Culture Virus Culture Basic Cell Culture Eukaryotic DNA Replication Plant Cell Culture [2e] Growth Factors and Receptors Plant Cell Biology Cell Separation The Cell Culture Please see the Practical Approach series website at http://www.oup.co.uk/pas for full contents lists of all Practical Approach titles. Animal Cell Culture Third Edition A Practical Approach Edited by John R. W. Masters 3rd Floor Research Laboratories, University College London OXFORD UNIVERSITY PRESS OXFORD UNIVERSITY PRESS Great Clarendon Street, Oxford 0X2 6DP Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide in Oxford New York Athens Auckland Bangkok Bogota Buenos Aires Calcutta Cape Town Chennai Dar es Salaam Delhi Florence Hong Kong Istanbul Karachi Kuala Lumpur Madrid Melbourne Mexico City Mumbai Nairobi Paris Sao Paulo Singapore Taipei Tokyo Toronto Warsaw with associated companies in Berlin Ibadan Oxford is a registered trade mark of Oxford University Press in the UK and in certain other countries Published in the United States by Oxford University Press Inc., New York © Oxford University Press, 2000 The moral rights of the author have been asserted Database right Oxford University Press (maker) First Edition First Published 1989 Second Edition First Published 1992 Reprinted 1994, 1995 Third Edition First Published 2000 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Oxford University Press, or as expressly permitted by law, or under terms agreed with the appropriate reprographics rights organization. Enquiries concerning reproduction outside the scope of the above should be sent to the Rights Department, Oxford University Press, at the address above You must not circulate this book in any other binding or cover and you must impose this same condition on any acquirer British Library Cataloguing in Publication Data Data available Library of Congress Cataloguing in Publication Data Animal cell culture : a practical approach / edited by John R. W. Masters.—3rd ed. p. cm.—(Practical approach series; 232) Includes bibliographical references and index. 1. Cell culture. 2. Tissue culture. I. Masters, John R. W. II. Series. QH585.2 A55 2000 571.6'381—dc21 00-026267 1 3 5 79 10 8 6 42 ISBN 0 19 963797 0 (Hbk.) ISBN 0 19 963796 2 (Pbk.) Typeset in Swift by Footnote Graphics, Warminster, Wilts Printed in Great Britain on acid-free paper by The Bath Press, Avon Preface The use of cell culture is increasing exponentially, and is an essential tool in nearly all biological and medical research laboratories. New techniques are con- stantly being applied, and this new third edition of Animal Cell Culture builds on the superb foundation laid by Ian Freshney. Stem cell assays, apoptosis, genetic manipulation, and tissue engineering are areas of current interest that have been introduced, while all the other chapters from previous editions have been completely rewritten or updated. There is one message that has not yet penetrated the minds of the whole scientific community. Cross-contamination of cell lines continues to occur at a high rate and continues to be ignored. The use of cell lines which have not been checked and which are not what they are claimed to be amounts to fraud, and is not something which cannot be ignored as lightly as it has been in the past. Scientists must now authenticate their lines (see chapter by Robert Hay and col- leagues from the American Type Culture Collection) and editors should demand that cell lines are authenticated before publication. J.M. May 2000 V This page intentionally left blank Contents List of protocols page xiii Abbreviations xvii ± Introduction to basic principles R. Ian Freshney 1 Background 1 2 Biology of cells in culture 2 Origin and characterization 2 Differentiation 3 3 Choice of materials 3 Cell type 3 Source of tissue 4 Subculture 5 Selection of medium 7 Gas phase 8 Culture system 9 4 Procedures 10 Substrate 10 Medium 11 Cell culture 11 References 16 2 Scallng-up of animal cell cultures Bryan Griffiths 1 Introduction 19 2 General methods and culture parameters 20 Cell quantification 20 Equipment and reagents 21 Practical considerations 22 Growth kinetics 23 Medium and nutrients 23 pH 24 Oxygen 26 vii CONTENTS Types of culture process 30 Summary of factors limiting scale-up 32 3 Monolayer culture 32 Introduction 32 Cell attachment 34 Scaling-up 35 4 Suspension culture 48 Adaptation to suspension culture 49 Static suspension culture 50 Small scale suspension culture 50 Scaling-up factors 53 Stirred bioreactors 54 Continuous-flow culture 56 Airlift fermenter 57 5 Immobilized cultures 58 Immurement cultures 59 Entrapment cultures 62 Porous carriers 62 References 66 3 Cell line preservation and authentication R J. Hay, M. M. Cleland, S. Durkin, and Y. A. Reid 1 Introduction 69 2 Cell line banking 70 3 Cell freezing and quantitation of recovery 71 Equipment 72 Preparation and freezing 73 Reconstitution and quantitating recovery 74 4 Cell line authentication 78 Species verification 78 Tests for microbial contamination 81 Testing for intraspecies cross-contamination 94 Miscellaneous characterizations and cell line availability 101 Acknowledgements 102 References 102 4 Development of serum-free media Soverin Karmiol 1 Introduction 105 2 Role of serum and other undefined tissue extracts in cell culture systems 105 3 Response curves 106 Proliferating cultures 106 Non-proliferating cultures (hepatocytes) 111 4 Antimicrobials, phenol red, Hepes, and light 112 Phenol red 113 Gentamicin 114 viii CONTENTS Hepes 315 Light 116 5 Purity of components 117 6 Fatty acids 117 Acknowledgements 120 References 120 5 Three-dimensional culture L. A Kunz-Schughart and W. Mueller-Kleiser 1 Introduction 123 2 Multicellular tumour spheroids (MCTS) 124 MCTS monocultures 124 MCTS co-cultures 133 3 Experimental tissue modelling 139 Current research on tissue modelling 139 Tissue modelling of skin and mucosa 140 Embryoid bodies 143 References 143 6 Tissue engineering Robert A. Brown and Rebecca A. Porter 1 Introduction 149 2 Design stages for tissue engineering 150 Tissue engineered skin 152 Tissue engineered urothelium 152 Tissue engineered peripheral nerve implants 153 3 Cell substrates and support materials 154 4 Cell sources 157 5 Orientation 159 Mechanical cues 161 6 Protocols 163 Cell seeding of implantable materials 163 Slow release systems for local control of TE constructs or repair sites 165 Acknowledgements 168 References 168 7 Cytotoxlclty and viability assays Anne P. Wilson 1 Introduction 175 2 Background 176 3 Specific techniques 177 Culture methods 177 Duration of drug exposure and drug concentrations 179 Recovery period 182 ix