Basic Cell Culture Protocols

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MET H 0 () SIN MOL E C U l ARB I () lOG Y 1\1

Basic Cell CultureProtocols

Third Edition

Edited by

Cheryl D. HelgasonDepartment ofCancer Endocrinology, British Columbia Cancer

Agency, Vancouver, British Columbia, Canada

Cindy L. MillerStemCell Technologies Inc., Vancouver, British Columbia, Canada

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Basic cell culture protocols.-- 3rd ed. 1edited by Cheryl D. Helgason,Cindy L. Miller.

p. ; cm. -- (Methods in molecular biology; 290)Includes bibliographical references and index.ISBN 1-58829-284-3 (hardcover: alk. paper) -- ISBN 1-58829-545-1 (pbk. : alk. paper)1. Cell culture--Laboratory manuals. 2. Tissue culture--Laboratory manuals.[DNLM: 1. Cell Culture--methods--Laboratory Manuals. 2. Tissue

Culture--methods--Laboratory Manuals. QS 525 B297 2005] I. Helgason, Cheryl D.II. Miller, Cindy L. III. Series: Methods in molecular biology(Clifton, N.J.); v. 290.

QH585.2.B375 2005571.6'38--dc22

2004011235

Preface

Tissue culture techniques were first developed at the beginning of the 20thcentury and have undergone dramatic changes and improvements since thattime. They are invaluable tools for the exploration of numerous biological ques­tions related both to cellular processes and to the signaling mechanisms thatregulate them. At some point in their careers, virtually every scientist, techni­cian, and many medical professionals have a need to utilize cell culturesystems, regardless of their area of specialization.

Our objective in preparing this book was to provide the novice cell culturistwith sufficient information to perform the basic techniques, to ensure the healthand identity of their cell lines, and to be able to isolate and culture specializedprimary cell types. It is not the intent to educate cell culturists on any specificcell type or organ system, but rather to offer clear methodologies pertinent tocurrent areas of investigation, as well as provide a valuable resource book foryears to come. It is anticipated that many readers will have a solid backgroundin the fundamentals of anatomy, histology, and biochemistry, but little or noexperience in cell culture. We anticipate that this book will prove a usefulresource for technicians, graduate students, postdoctoral fellows, as well as tothe research leaders (both basic scientists and clinicians) and those cell cultureexperts who are moving toward the use of new model systems.

The chapters that follow provide step-by-step instructions for the isolation andgrowth of various primary cell types. In addition, they illustrate the techniquesrequired for defining the properties of various types of cells, as well as for celldifferentiation and expansion of cultures for large-scale experimentation.

Finally, we wish to extend our sincerest appreciation to all the contributorswho willingly took their time to share their expertise and knowledge, and tothe many individuals who assisted us in the preparation of this book. Specialthanks go to Christine Kelly who was instrumental in maintaining organizationamidst the chaos.

Cheryl D. HelgasonCindy 1. Miller

v

Contents

Preface v

Contributors ix

Value-Added eBook/PDA xii

Culture of Primary Adherent Cells and a Continuously GrowingNonadherent Cell Line

Cheryl D. Helgason ....•.•.••.....•.•••••••.......•.•.••..........•.•............................• 1

2 Detection of Mycoplasma ContaminationsCord C. Uphoff and Hans G. Drexler 13

3 Eradication of Mycoplasma ContaminationsCord C. Uphoff and Hans G. Drexler 25

4 Authentication of Scientific Human Cell Lines:Easy-to-Use DNA Fingerprinting

Wilhelm G. Dirks and Hans G. Drexler 35

5 Cytogenetic Analysis of Cell LinesRoderick A. f. Macleod and Hans G. Drexler 51

6 Human and Mouse Hematopoietic Colony-Forming Cell AssaysCindy L. Miller and Becky Lai .••..........•••.••••...........•.•.......................... 71

7 Isolation and Culture of Murine MacrophagesJohn Q. Davies and Siamon Gordon 91

8 Isolation and Culture of Human MacrophagesJohn Q. Davies and Siamon Gordon 105

9 Development of T-Lymphocytes in Mouse Fetal ThymusOrgan Culture

Tomoo Ueno, Cunlan Liu, Takeshi Nitta,and Yousuke Takahama •••.•.••..............••.•................•...............•.•••• 117

10 In Vitro Generation of Lymphocytes From Embryonic Stem CellsRenee f. de Pooter, Sarah K. Cho,

and Juan Carlos Zuiiiga-Pfliicker 135

11 Hematopoietic Development of Human Embryonic Stem Cellsin Culture

Xinghui Tian and Dan S. Kaufman 149

VII

viii Contents

12 Generation of Murine Stromal Cell Lines: Modelsfor the Microenvironment of the Embryonic MouseAorta-Gonads-Mesonephros Region

Robert A. J. Oostendorp, Kirsty Harvey,and Elaine A. Dzierzak 163

13 Culture of Human and Mouse Mesenchymal CellsEmer Clarke 173

14 Isolation, Purification, and Cultivation of Murine and HumanKerati nocytes

Frizell L. Vaughan and Ludmila I. Bernstam 187

15 Isolation and Culture of Primary Human HepatocytesEdward L. LeCluyse, Eliane Alexandre,

Geraldine A. Hamilton, Catherine Viollon-Abadie,D. James Coon, Summer Jolley, and Lysiane Richert 207

16 Primary Kidney Proximal Tubule CellsMary Taub 231

17 Enzymatic Dissociation and Culture of Normal HumanMammary Tissue to Detect Progenitor Activity

John Stingl, Joanne T. Emerman, and Connie J. Eaves 249

18 Generation and Differentiation of Neurospheres From MurineEmbryonic Day 14 Central Nervous System Tissue

Sharon A. Louis and Brent A. Reynolds 265

19 Isolation and Culture of Skeletal Muscle Myofibers as a Meansto Analyze Satellite Cells

Gabi Shefer and Zipora Yablonka-Reuveni 281

20 Adult Ventricular Cardiomyocytes: Isolation and CultureKlaus-Dieter SchWter and Daniela Schreiber 305

21 Isolation and Culture of Primary Endothelial CellsBruno Larrivee and Aly Karsan 315

22 Studying Leukocyte Rolling and Adhesion In VitroUnder Flow Conditions

Susan L. Cuvelier and Kamala D. Patel 331

23 Isolation and Characterization of Side Population CellsMargaret A. Goodell, Shannon McKinney-Freeman,

and Fernando D. Camargo 343

24 Scalable Production of Embryonic Stem Cell-Derived CellsStephen M. Dang and Peter W. Zandstra 353

Index 365

Contributors

ELIANE ALEXANDRE· Laboratoire de Chirurgie Experimentale, FondationTransplantation, Strasbourg, France

LUDMILA I. BERNSTAM • Department ofEnvironmental Health Sciences,University ofMichigan School ofPublic Health, Ann Arbor, MI

FERNANDO D. CAMARGO· Center for Cell and Gene Therapy, Celland Molecular Biology Program, Baylor College ofMedicine, Houston, TX

SARAH K. CHO • Division ofBiological Science, University of California,San Diego, La Jolla, CA

EMER CLARKE • StemCell Technologies Inc., Vancouver, British Columbia,Canada

D. JAMES COON • CellzDirect Inc., Pittsboro, NCSUSAN L. CUVELIER • Immunology Research Group, Department ofPhysiology

and Biophysics, University of Calgary, Calgary, Alberta, CanadaSTEPHEN M. DANG • Institute ofBiomaterials and Biomedical Engineering,

Department of Chemical Engineering and Applied Chemistry, Universityof Toronto, Toronto, Ontario, Canada

JOHN Q. DAVIES • Sir William Dunn School ofPathology, UniversityofOxford, Oxford, UK

RENEE F. DE POOTER • Department ofImmunology, University of Toronto,Sunnybrook & Women's College Health Sciences Centre, Toronto,Ontario, Canada

WILHELM G. DIRKS • DSMZ - German Collection ofMicroorganismsand Cell Cultures, Braunschweig, Germany

HANS G. DREXLER • DSMZ - German Collection ofMicroorganisms and CellCultures, Braunschweig, Germany

ELAINE A. DZIERZAK • Department of Cell Biology and Genetics, ErasmusUniversity Medical Center, Rotterdam, the Netherlands

CONNIE J. EAVES • Terry Fox Laboratory, British Columbia Cancer Agency,Vancouver, British Columbia, Canada

JOANNE T. EMERMAN· Department ofAnatomy, University ofBritishColumbia, Vancouver, British Columbia, Canada

MARGARET A. GOODELL • Center for Cell and Gene Therapy, Departmentof Immunology and Cell and Molecular Biology Program, Baylor CollegeofMedicine, Houston, TX

SIAMON GORDON • Sir William Dunn School ofPathology, UniversityofOxford, Oxford, UK

ix

x Contributors

GERALDINE A. HAMILTON • CellzDirect Inc., Pittsboro, NCKIRSTY HARVEY • Department of Cell Biology and Genetics, Erasmus

University Medical Center, Rotterdam, the NetherlandsCHERYL D. HELGASON • Department ofCancer Endocrinology, British Columbia

Cancer Agency, Vancouver, British Columbia, CanadaSUMMER JOLLEY • Department ofDrug Delivery and Disposition, University

ofNorth Carolina, School ofPharmacy, Chapel Hill, NCALY KARSAN· Department ofPathology and Laboratory Medicine, University

ofBritish Columbia and Departments ofMedical Biophysics and Pathologyand Laboratory Medicine, British Columbia Cancer Agency, Vancouver,British Columbia, Canada

DAN S. KAUFMAN • Stem Cell Institute and Department ofMedicine,University ofMinnesota, Minneapolis, MN

BECKY LAI • StemCell Technologies Inc., Vancouver, British Columbia,Canada

BRUNO LARRIVEE • Department ofMedicine, University ofBritish Columbia,and Department ofMedical Biophysics, British Columbia CancerAgency, Vancouver, British Columbia, Canada

EDWARD L. LECLUYSE· Department ofDrug Delivery and Disposition,University ofNorth Carolina, School ofPharmacy, Chapel Hill, NC

CUNLAN LIU • Division ofExperimental Immunology, Institute for GenomeResearch, University ofTokushima, Tokushima, Japan

SHARON A. LOUIS • StemCell Technologies Inc., Vancouver, British Columbia,Canada

RODERICK A. F. MACLEOD • DSMZ - Department ofHuman and Animal CellCultures, German Collection ofMicroorganisms and Cell Cultures,Braunschweig, Germany

SHANNON McKINNEy-FREEMAN • Center for Cell and Gene Therapy,Department of Immunology, Baylor College ofMedicine, Houston, TX

CINDY L. MILLER • StemCell Technologies Inc., Vancouver, British Columbia,Canada

TAKESHI NITTA • Division ofExperimental Immunology, Institute for GenomeResearch, University of Tokushima, Tokushima, Japan

ROBERT A. J. OOSTENDORp· The Stem Cell Physiology Laboratory, III,Medizinische Klinik, Klinikum Rechts der Isar, Technical University,Munich, Germany

KAMALA D. PATEL • Immunology Research Group, Department ofPhysiologyand Biophysics, University ofCalgary, Calgary, Alberta, Canada

BRENT A. REYNOLDS • StemCell Technologies Inc., Vancouver, BritishColumbia, Canada

Contributors xi

LYSIANE RICHERT· Laboratoire de Chirurgie Experimentale, FondationTransplantation, Strasbourg, France and Laboratoire de BiologieCellulaire, Faculte de Medecine et de Pharmacie, Besan~on, France

KLAUS-DIETER SCHLUTER • Physiologisches Institut, Justus-Liebig-UniversitiitGiessen, Giessen, Germany

DANIELA SCHREIBER • Physiologisches Institut, Justus-Liebig-UniversitiitGiessen, ·Giessen, Germany

GABI SHEFER • Department ofBiological Structure, School ofMedicine,University of Washington, Seattle, WA

JOHN STINGL • Terry Fox Laboratory, British Columbia Cancer Agencyand StemCell Technologies Inc., Vancouver, British Columbia, Canada

Y OUSUKE T AKAHAMA • Division ofExperimental Immunology, Institutefor Genome Research, University of Tokushima, Tokushima, Japan

MARY T AUB • Biochemistry Department, State University ofNew Yorkat Buffalo, Buffalo, NY

XINGHUI TIAN • Stem Cell Institute and Department ofMedicine, UniversityofMinnesota, Minneapolis, MN

TOMOO UENO • Division ofExperimental Immunology, Institute for GenomeResearch, University ofTokushima, Tokushima, Japan

CORD C. UPHOFF • DSMZ - German Collection ofMicroorganisms and CellCultures, Braunschweig, Germany

FRIZELL L. VAUGHAN • Department ofEnvironmental Health Sciences,University ofMichigan School ofPublic Health, Ann Arbor, MI

CATHERINE VIOLLON-ABADIE • Laboratoire de Biologie Cellulaire, Facultede Medecine et de Pharmacie, Besanron, France

ZIPORA YABLONKA-REUVENI • Department ofBiological Structure, SchoolofMedicine, University of Washington, Seattle, WA

PETER W. ZANDSTRA • Institute ofBiomaterials and Biomedical Engineering,Department of Chemical Engineering and Applied Chemistry, Universityof Toronto, Toronto, Ontario, Canada

JUAN CARLOS ZUNIGA-PFLUCKER • Department ofImmunology, Universityof Toronto, Sunnybrook & Women's College Health Sciences Centre,Toronto, Ontario, Canada

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