Methods in Molecular Biology

Volume 5

Animal Cell Culture

Methods in Molecular Biology Edited by John M. Walker

Volume I: Proteins, 1984 Volume I1: Nucleic Acids, 1984 Volume II1: New Protein Techniques, 1988 Volume IV: New Nucleic Acid Techniques, 1988 Volume V: Animal Cell Culture, edited by Jeffrey W. Pollard

and John M. Walker, 1989 Volume VI: Plant Cell and Tissue Culture, edited by Jeffrey W.

Pollard and John M. Walker, 1990

Animal Cell

Culture Edited by

Jeffrey W. Pollard Albert Einstein College of Medicine, Bronx, New York

and

John M. Walker The Hatfield Polytechnic, Hatfield, Hertsfordshire, UK

H umana Press • Clifton, New Jersey

© 1990 The Humana Press Inc. Crescent Manor PO Box 2148 Clifton, New Jersey 07015

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 per- mission from the Publisher.

Printed in the United States of America

Library of Congress Cataloging in Publication Data Main entry under title:

Methods in molecular biology.

(Biological methods) Includes bibliographies and indexes. Contents: v. I. Proteins---v. 2. Nucleic acids--v. 3. New protein techniques--v. 4. New

nucleic acid techniques--v. 5. Animal cell culture techniques.

1. Molecular biology--Technique. I. Walker, John M., 1948-

QH506.M45 1984 574.8'8'078

ISBN 0-89603-062-8 (v. 1) ISBN 0-89603-064-4 (v. 2) ISBN 0-89603-126-8 (v. 3) ISBN 0-89603-127-6 (v. 4) ISBN 0-89603-150-0 (v. 5) ISBN 0-89603-161-1 (v. 6)

II. Series.

84-15696

P r e f a c e

For many years I performed tissue culture in large scientific institu- tions that had a great deal of infrastructure. When I set up a tissue lab- oratory outside such an infrastructure, however, I found there was a shortage of easily accessible information about the basic needs, reagents, and techniques for establishing such a facility. Much had to be done by trial and error or gleaned from originalpapers. Consequently, I felt that a methods book covering a wide variety of techniques from basic culture to the most sophisticated cell analysis would be a very valuable addit ion to the scientific literature. In the interim, several useful books (listed in Chapter I of this volume) did appear, but none entirely fitted the bill and some are now somewhat dated. Then, in 1984, the first of the Methods in Molecular Biology volumes from Humana Press was publ ished with its step-by-step recipe approach. This format appealed to me, and so I con- tacted John Walker, the series editor, about including cell culture in this series. The result was that we embarked upon a single volume covering both plant and animal cell culture. Such was the richness of the material that this project soon divided itself into separate volumes on animal cell (Volume 5) and plant cell (Volume 6) culture. In this volume (Volume 5), therefore, we have a imed to describe a variety of basic techniques and culture conditions for a range of cell types. We have included descrip- tions of traditional mammal i an fibroblastic cell cultures, as well as cul- ture conditions for cells of epithelial, neuronal, and hematopoietic ori- gin. In these chapters, particular emphasis has been placed upon the diversity of culture media (growth factors, and so forth) and extracellu- lar matrixes required to maintain the cell's differentiated function. Following this section are descriptions of techniques for analysis of both l iving and fixed cells. Here we have l imited the volume's scope to in- clude only those methods applicable to unfractioned cells. These chap- ters range from cinematographic analysis of l iving cells, through in situ mRNA hybridizat ion and immunofluorescence to immunoelectron microscopy. Modern genetic analysis requires the alteration of the cell's genotype as well as the testing of cloned gene's expression in vivo. Thus,

~d

vi Preface

we have devoted a substantial section to somatic cell hybridization and DNA transformation. Included here is the description of insect cell cul- ture and methods for introducing and expressing genes in these cells-- a technique that appears to have important new biotechnological appli- cations. Of course, one of the most important recent biotechnological and research advances involving the alteration of a cell's genotype is the creation of hybridoma cell lines, and thus the latter part of the volume covers all the major monoclonal antibody techniques and includes a few novel, specialist applications. Finally, there is an appendix listing the most commonly used culture media. John Walker and I hope that the coverage of this volume is comprehensive enough to provide not only beginning cell culturists with workable basic methods, but also experi- enced workers with new information enabling them to enhance their culture methodology.

Editors of books always have many debts of gratitude. First, I would like to thank those who taught me tissue culture and particularly somatic cell genetics. The principal influence was Dr. Cliff Stanners, who at that time was at the Ontario Cancer Institute in Toronto. Gratitude must also go to all the other members of the Toronto somatic cell genetics group, and especially to Drs. Lou Siminovitch and Vic Ling. Finally, but not least, I would like to thank Ms. Margaret Hudgell and Ms. Rita Romita for their expert and cheerful secretarial help, often at times of crisis.

Jeffrey Iv. Pollard

In keeping with the successful format of earlier volumes, we have again applied a step-by-step approach for describing each technique, al- though in some cases, particularly those involving sophisticated instru- mentation, this format has necessarily been modified somewhat. Our aim, as with earlier volumes, is to provide clearly defined protocols that should allow the new worker to obtain successful results with the first attempt. We hope we have been successful in this endeavor! I would like to express my gratitude to Jeff Pollard for suggesting this volume, for his considerable efforts in helping to find appropriate authors, and for his diligent editing. Finally, as ever, my sincere thanks to my wife Jan, who once again without complaint and often at short notice, took on the role of typist, proofreader, and unofficial editor.

John M. Walker

C o n t e n t s

Preface ................................................................................................................ v Contributors .................................................................................................... xi

CH. 1. Basic Cell Culture, Jeffrey W. Pol lard ........................................... 1 CH. 2. Establishment, Maintenance, and Cloning of Human

Primary Cell Strains, Gareth E. Jones ...................................... 13 CH. 3. Aging of Cultured Human Skin Fibroblasts,

Calvin B. Har ley .......................................................................... 25 CH. 4. Separation and Maintenance of Primary T and B

Lymphocytes, Derek Kinchington and Eleanor Berrie ......... 33 CH. 5. Establishment of Lymphoblastoid Cell Lines, A. Doy le .......... 43 CH. 6. Scale-Up of Suspension and Anchorage-Dependent

Animal Cells, J. Bryan Grif f i ths ............................................... 49 CH. 7. Mycoplasma Detection, J. M. M o w I e s ......................................... 65 CH. 8. Short-Term Chorionic Villi and Amniotic Fluid Cultures,

M. L. Murer-Orlando and V. M. McGuire .............................. 75 CH. 9. Keratinocyte Culture, Yvonne Bar low and Richard J. Pye ..... 83 CH. 10. Establishment of Mouse Epithelial Lung Cell Strains

and Cell Lines, Garry J. Smi th .................................................. 99 CH. 11. Culture of Human Brain Tumors on an Extracellular Matrix

Derived from Bovine Corneal Endothelial Cells and Cultured Human Glioma Ceils, Manfred Wes tpha t , Mar ianne HiinseI, Hildegard Nausch, Eva Rohde, and Hans-Die t r ich Herrmann ................................................. 113

CH. 12. Embryonic Rodent Brain Cells in Culture, Narayan R. B h a t ........................................................................ 133

CH. 13. Human Thyroid Epithelial Cells, D. W. W i l l i a m s and Dav id W y n f o r d - T h o m a s .................................................. 139

CH. 14. Preparation of Isolated Rat Liver Hepatocytes, Bjarn Quistorf f , John Dich, and Niels Grunnet .................. 151

CH. 15. Primary Cultures of Rat Hepatocytes, John Dich and Nie ls Grunnet ..................................................................... 161

vii

viii Contents

CH. 16. Preparation of Isolated Periportal or Perivenous Hepatocytes from Rat Liver, Bjarn Quis tor f f ...................... 177

CH. 17. Primary Kidney Cells, M a r y Taub ............................................. 189 CH. 18. Adipocytes, Shinobu Gamou, Yoshiko Shimizu,

and Nobuyosh i Sh imizu ........................................................... 197 CH. 19. Tissue Culture of Skeletal Muscle, Terry A. Partridge ........... 209 CH. 20. Derivation and Maintenance of Embryonic Stem Cell

Cultures, Elizabeth J. Robertson ............................................ 223 CH. 21. Defining Hormone and Matrix Requirements for

Differentiated Epithelia, Lola M. Reid .................................. 237 CH. 22. Culturing Primitive Hemopoietic Cells: Long-Term Mouse

Marrow Cultures and the Establishment of Factor- Dependent (FDCP-Mix) Hemopoietic Cell Lines, Elaine Spooncer and T. Michael Dexter ................................ 277

CH. 23. High Proliferative Potential Colony Forming Cells, T. R a y Bradley, George S. Hodgson, and Ivan Bertoncello ............ 289

CH. 24. Murine Bone Marrow-Derived Macrophages, E. Richard S tanley ..................................................................... 299

CH. 25. Long-Term B Lymphoid Cultures from Murine Bone Marrow: Establishment and Cloning by Using Stromal Cell Line AC 6.21, Cheryl A. Whi t lock and Christa E. Muller-Sieburg ........................................................................... 303

CH. 26. CFU-C in Agar, Vincent Praloran and A n n a Bartocci ........... 323 CH. 27. Human Long-Term Bone Marrow Culture, A r m a n d Keat ing

and Paul Toor ............................................................................ 331 CH. 28. In Vitro Clonal Culture of Human Hematopoietic Progenitor

Cells, A r m a n d Keat ing and Paul Toor .................................. 339 CH. 29. Flow Sorting for Isolating CFU-E, Suzanne M. W a t t

and John M. Davis .................................................................... 347 CH. 30. Production of Human and Murine Eosinophils In Vitro

and Assay for Eosinophil Differentiation Factors, Malco lm Strath, Elaine J. Clutterbuck, and Colin J. Sanderson ....... 361

CH. 31. Clonogenic Assays for Hematopoietic and Tumor Cells Using Agar-Containing Capillaries, H. Rainer Maurer and Christine Echarti ............................................................... 379

CH. 32. Cytogenetic Techniques for Human Leukemias, G. John Swansbury .................................................................... 395

CH. 33. Time-Lapse Cinemicroscopy, Peter N. Riddle ......................... 415 CH. 34. Transmission and Scanning Electron Microscope

Preparation of Whole Cultured Ceils, Josef Neumiil ler ..... 447

Contents ix

CH. 35. Double Indirect-Immunofluorescent Labeling of Cultured Cells, Christ ine A. Boocock ..................................................... 473

CH. 36. Gene Mapping to Chromosomes by Hybridization In Situ, John R. Gosden ........................................................................... 487

CH. 37. In Situ Hybridization with Radiolabeled cRNA Probes, Using Tissue Sections and Smears, Carolin A d a m s , Qu tay ta A. Hamid , and Julia M. Po lak ................................ 501

CH. 38. Immunogold Labeling for Electron Microscopy, John Pacy .................................................................................... 515

CH. 39. Human Chromosome Analysis and Sorting, Judith A. Fantes and Daryl I K. Green ..................................................... 529

CH. 40. Flow Cytometry, Michael G. Ormerod and Patr ick R. Imrie ................................................................. 543

CH. 41. Fluorescence-Activated Cell Sorting According to Receptor Density: Application for Isolating Transfected Cell Lines, StetIa Clark ..................................................................... 559

CH. 42. Transfection and Transformation of Human Thyroid Epithelial Cells, Nicholas Rober t Lemoine and Dav id W y n f o r d - T h o m a s .................................................. 565

CH. 43. Expression of Foreign Genes in Cultured Insect Cells Using a Recombinant Baculovirus Vector, Mar t in J. Page and Vivienne F. M u r p h y ............................... 573

CH. 44. Chloramphenicol Acetyltransferase as a Reporter in Mammalian Gene Transfer, Chris topher D. Corsico and Bruce H. H o w a r d ............................................................... 589

CH. 45. Immunizing Schedules for Hybridoma Production, Bjiirn Gus ta f s son ....................................................................... 597

CH. 46. Fusion Protocol for the Production of Mouse Hybridomas, Bjf~rn Gus ta f s son ....................................................................... 60I

CH. 47. Cloning of Hybridomas, Bjf~rn Gus ta f sson ............................ 609 CH. 48. Enzyme-Linked Immunosorbent Assay for Screening

of Antibodies in Hybridoma Supernatants, Bjb'rn Gus ta fsson ....................................................................... 613

CH. 49. Cryopreservation of Hybridomas, Bjf~rn Gus ta f s son ............ 619 CH. 50. Isotype Determination of Monoclonal Antibodies

by Immunodiffusion, Bjf~rn Gus ta f s son ............................... 623 CH. 51. Isoelectric Focusing and Immunofixation of Monoclonal

Antibodies, Bjf~rn Gus ta fsson ................................................. 627

x Contents

CH. 52. Large-Scale Production and Storage of Monoclonal Antibodies and Hybridomas, Stuar t A. Clark, J. Bryan Griffiths, and Christopher B. Morr@ ..................... 631

CH. 53. Purification of Murine Monoclonal Antibodies, Michael G. Baines, A n d r e w J. H. Gearing, and Robin Thorpe ............... 647

CH. 54. Rat x Rat Hybridomas, Christopher J. Dean ............................ 669 CH. 55. Monoclonal Antibodies Against Glycosphingolipids

(GSLs)-Gangliosides, Bruce Fletcher Clark .......................... 679

APPENDIX, Jeffrey W. Pol lard ..................................................................... 691

INDEX ................................................................................................................................................... 705

C o n t r i b u t o r s CAROLIN ADAMS • Royal Postgraduate Medical School, Hammersmith

Hospital, London, UK MICHAEL G. BAINES • National Institute for Biological Standards

and Control, Herts, UK YVONNE BARLOW • Addenbrooke's Hospital, Cambridge, UK ANNA BARTOCCI • Albert Einstein College of Medicine, Bronx, New

York ELEANOR BERRIE • British Bio-Tech, Oxford, UK IVAN BERTONCELLO • Peter McCaUum Cancer Institute, Melbourne,

Australia NARAYAN R. BHAT • College of Medicine, University of Kentucky,

Lexington, Kentucky C H R I S T I N E A . B O O C O C K • King's College, London, UK T. RAY BRADLEY • Peter McCallum Cancer Institute, Melbourne,

Australia BRUCE FLETCHER CLARK • Imperial Cancer Research Fund, London, UK STUART A. CLARK • Vaccine Research and Production Laboratory,

PHLS CAMR, Salisbury, UK STELLA CLARK • Ludwig Institute for Cancer Research, University

College Branch, London, UK ELAINE J. CLUTTERBUCK • National Institute for Medical Research,

London, UK CHRISTOPHER D. CORSICO • Howard Hughes Medical Institute, NIH,

Bethesda, MD J O H N M. DAVIS • Blood Products Laboratory, Elstree, Herts, UK CHRISTOPHER J. DEAN • Institute of Cancer Research, Surrey, UK T. MICHAEL DEXTER • Paterson Institute for Cancer Research,

Christie Hospital and Holt Radium Institute, Manchester, UK J O H N D I C H • The Panum Institute, University of Copenhagen,

Copenhagen, Denmark A. DOYLE • European Collection of Animal Cell Cultures, PHLS

CAMR, Salisbury, UK

xi

xii Contributors

CHRISTINE ECHARTI • Institut ffir Pharmazie der Freien Universit/it Berlin, Berlin, Germany

J U D I T H A. FANTES • MRC Clinical and Population Cytogenics Unit, Western General Hospital, Edinburgh, Scotland

SHINOBU GAMOU " Keio University School of Medicine, Tokyo, Japan A N D R E W J. H . G E A R I N G • National Institute for Biological Standards

and Control, Herts, UK J O H N R. GOSDEN • MRC Clinical and Population Cytogenics Unit,

Western General Hospital, Edinburgh, Scotland DARYLL K. GREEN • MRC Clinical and Population Cytogenics Unit,

Western General Hospital, Edinburgh, Scotland J. BRYAN GRIFFITHS • Vaccine Research and Production Laboratory,

PHLS CAMR, Salisbury, UK NIELS GRUNNET • The Panum Institute, University of Copenhagen,

Copenhagen, Denmark BjSRN GUSTAFSBON • Karolinska Institute and National Bacteriological

Laboratory, Stockholm, Sweden Q U T A Y L A A. HAMID • Royal Postgraduate Medical School, Hammer-

smith Hospital, London, UK MARIANNE HANSEL • University Hospital Eppendorf, Hamburg,

Federal Republic of Germany CALVIN B. HARLEY • McMaster University, Hamilton, Ontario, Canada HANS-DIETRICH HERRMANN • University Hospital Eppendorf, Ham

burg, Federal Republic of Germany GEORGE S. HODGSON • Peter McCallum Cancer Institute, Melbourne,

Australia BRUCE H. HOWARD • National Cancer Institute, National Institutes

of Health, Bethesda, Maryland PATRICK R. IMRIE • Institute of Cancer Research, The Haddow Laborato

ries, Surrey, UK G A R E T H E , JONES • King's College, London, UK ARMAND KEATING • Toronto General Hospital, Toronto, Ontario, Canada DEREK KINCHINGTON • St. Mary's Hospital Medical School, London, UK NICHOLAS ROBERT LEMOINE • University of Wales College of Medicine,

Heath Park, Cardiff, UK V. M. McGuIRE • United Medical School of Guys and St. Thomas's

Hospital, London, UK H. RAINER MAURER • Institut ftir Pharmazie der Freien Universit/it Berlin,

Berlin, Germany CHRISTOPHER B. MORRIS • Vaccine Research and Production Laboratory,

PHLS CAMR, Salisbury, UK

Contributors xiii

J. M. MOWLES • PHLS CAMR, Salisbury, UK CHRISTA E. MULLER-SIEBURG " Lilly Research Laboratories, La Jolla,

California M. L. MURER-ORLANDO • United Medical School of Guys

and St. Thomas's Hospital, London, UK VIVIENNE F. MURPHY " Wellcome Biotech, Beckenham, Kent, UK I-IILDEGARD NAUSCH • University Hospital Eppendorf, Hamburg,

Federal Republic of Germany JOSEF N E U M O L L E R • Ludwig Boltzmann Institute of Rheumatology

and Balneology, Vienna-Oberlaa, Austria MICHAEL G. ORMEROD " Institute of Cancer Research, The Haddow

Laboratories, Surrey, UK J O H N P A C Y • King's College, University of London, London, UK MARTIN J. PAGE • Wellcome Biotech, Beckenham, Kent, UK TERRY A. PARTRIDGE • Charing Cross and Westminster Medical School,

London, UK J U L I A M. POLAK • Royal Postgraduate Medical School, Hammersmith

Hospital, London, UK JEFFREY W. POLLARD • Albert Einstein College of Medicine, Bronx,

New York VINCENT PRALORAN " Laboratoire D'Hematologie H6tel-Dieu, Nantes

Cedex, France RICHARD J. PYE • Addenbrooke's Hospital, Cambridge, UK B J O R N Q U I S T O R F F • The Panum Institute, University of Copenhagen,

Copenhagen, Denmark LOLA M. REID • Albert Einstein College of Medicine, Bronx, New York PETER N. RIDDLE • Imperial Cancer Research Fund, London, UK ELIZABETH J. R O B E R T S O N " Columbia University, New York, New York EVA ROHDE • University Hospital Eppendorf, Hamburg, Federal

Republic of Germany COLIN J. S A N D E R S O N " National Institute for Medical Research,

London, UK NOBUYOSHI SHIMIZU " Keio University School of Medicine, Tokyo,

Japan and University of Arizona, Tucson, Arizona YOSHIKO SHIMIZU • University of Arizona, Tucson, Arizona GARRY J. S M I T H " School of Pathology, University of New South Wales,

Kensington, Australia ELAINE SPOONCER • Paterson Institute for Cancer Research,

Christie Hospital and Holt Radium Institute, Manchester, UK E. RICHARD STANLEY " Albert Einstein College of Medicine, Bronx,

New York

xiv Contributors

MALCOLM STRATH • National Institute for Medical Research, London, UK G. J. SWANSBURY * The Royal Marsden Hospital, London, UK MARY TAUB * State University of New York at Buffalo, Buffalo, New York ROBIN THORPE • National Institute for Biological Standards and Control,

Hefts, UK PAUL TOOR • Toronto General Hospital, Toronto, Ontario, Canada J O H N M. WALKER • The Hatfield Polytechnic, Hatfield, Hertfordshire, UK SUZANNE M. WATT • Imperial Cancer Research Fund, St. Bartholomew's

Hospital, London, UK MANFRED WESTPHAL * University Hospital Eppendorf, Hamburg,

Federal Republic of Germany CHERYL A. WHITLOCK • Stanford University Medical Center, Palo Alto,

California D. W. WILLIAMS • University of Wales College of Medicine, Heath Park,

Cardiff, UK DAVID WYNFORD-THOMAS • University of Wales College of Medicine,

Heath Park, Cardiff, UK