Human Cell Culture Protocols (Methods In Molecular .

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iHuman Cell Culture Protocols

METHODS IN MOLECULAR MEDICINETMJohn M. Walker, SERIES EDITOR113.113 Multiple Myeloma: Methods and Protocols,edited by Ross D. Brown and P. Joy Ho, 2005112 Molecular Cardiology: Methods and Protocols,112.edited by Zhongjie Sun, 2005111.111 Chemosensitivity: Volume 2, In Vivo Models,Imaging, and Molecular Regulators, edited byRosalyn D. Blumethal, 2005110 Chemosensitivity: Volume 1, In Vitro Assays,110.edited by Rosalyn D. Blumethal, 2005109.109 Adoptive Immunotherapy, Methods andProtocols, edited by Burkhard Ludewig andMatthias W. Hoffman, 2005108 Hypertension, Methods and Protocols,108.edited by Jérôme P. Fennell and AndrewH. Baker, 2005107.107 Human Cell Culture Protocols, SecondEdition, edited by Joanna Picot, 2005106 Antisense Therapeutics, Second Edition,106.edited by M. Ian Phillips, 2005105.105 Developmental Hematopoiesis: Methodsand Protocols, edited by Margaret H. Baron,2005104 Stroke Genomics: Methods and Reviews, edited104.by Simon J. Read and David Virley, 2005103.103 Pancreatic Cancer: Methods and Protocols,edited by Gloria H. Su, 2005102.102 Autoimmunity: Methods and Protocols, editedby Andras Perl, 2004101.101 Cartilage and Osteoarthritis: Volume 2,Structure and In Vivo Analysis, edited byFrédéric De Ceuninck, Massimo Sabatini,and Philippe Pastoureau, 2004100 Cartilage and Osteoarthritis: Volume 1,100.Cellular and Molecular Tools, edited byMassimo Sabatini, Philippe Pastoureau, andFrédéric De Ceuninck, 200499.99 Pain Research: Methods and Protocols, editedby David Z. Luo, 200498.98 Tumor Necrosis Factor: Methods and Protocols,edited by Angelo Corti and Pietro Ghezzi, 200497.97 Molecular Diagnosis of Cancer: Methods andProtocols, Second Edition, edited by Joseph E.Roulston and John M. S. Bartlett, 200496.96 Hepatitis B and D Protocols: Volume 2,Immunology, Model Systems, and ClinicalStudies, edited by Robert K. Hamatake andJohnson Y. N. Lau, 200495.95 Hepatitis B and D Protocols: Volume 1,Detection, Genotypes, and Characterization,edited by Robert K. Hamatake and JohnsonY. N. Lau, 200494.94 Molecular Diagnosis of Infectious Diseases,Second Edition, edited by Jochen Decker andUdo Reischl, 200493.93 Anticoagulants, Antiplatelets, andThrombolytics, edited by Shaker A. Mousa,200492.92 Molecular Diagnosis of Genetic Diseases,Second Edition, edited by Rob Elles andRoger Mountford, 200491 Pediatric Hematology: Methods and Protocols,91.edited by Nicholas J. Goulden and Colin G.Steward, 200390.90 Suicide Gene Therapy: Methods and Reviews,edited by Caroline J. Springer, 200489 The Blood–Brain Barrier: Biology and89.Research Protocols, edited by Sukriti Nag,200388.88 Cancer Cell Culture: Methods and Protocols,edited by Simon P. Langdon, 200387 Vaccine Protocols, Second Edition, edited by87.Andrew Robinson, Michael J. Hudson, andMartin P. Cranage, 200386.86 Renal Disease: Techniques and Protocols,edited by Michael S. Goligorsky, 200385 Novel Anticancer Drug Protocols, edited by85.John K. Buolamwini and Alex A. Adjei, 200384.84 Opioid Research: Methods and Protocols,edited by Zhizhong Z. Pan, 200383.83 Diabetes Mellitus: Methods and Protocols,edited by Sabire Özcan, 200382.82 Hemoglobin Disorders: Molecular Methodsand Protocols, edited by Ronald L. Nagel, 200381.81 Prostate Cancer Methods and Protocols,edited by Pamela J. Russell, Paul Jackson,and Elizabeth A. Kingsley, 2003

iiiMETHODS IN MOLECULAR MEDICINETMHuman CellCulture ProtocolsSecond EditionEdited byJoanna PicotSouthampton, UKHumana PressTotowa, New Jersey

iv 2005 Humana Press Inc.999 Riverview Drive, Suite 208Totowa, New Jersey 07512www.humanapress.comAll rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in anyform or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise withoutwritten permission from the Publisher. Methods in Molecular Medicine is a trademark of The Humana PressInc.The content and opinions expressed in this book are the sole work of the authors and editors, who havewarranted due diligence in the creation and issuance of their work. The publisher, editors, and authors are notresponsible for errors or omissions or for any consequences arising from the information or opinions presentedin this book and make no warranty, express or implied, with respect to its contents.This publication is printed on acid-free paper. hANSI Z39.48-1984 (American Standards Institute)Permanence of Paper for Printed Library Materials.Cover illustration: Figure 1 from Chapter 1, Establishment and Maintenance of Normal HumanKeratinocyte Cultures, by Claire Linge.Cover design by Patricia F. Cleary.For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; Email: humana@humanapr.com; or visit our Website: www.humanapress.comPhotocopy Authorization Policy:Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US 25.00 per copy is paid directly to theCopyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that havebeen granted a photocopy license from the CCC, a separate system of payment has been arranged and isacceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [1-58829222-3/05 25.00].Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1E-ISBN 1-59259-861-7Library of Congress Cataloging in Publication DataHuman cell culture protocols.— 2nd ed. / edited by Joanna Picot.p. ; cm. — (Methods in molecular medicine ; 107)Includes bibliographical references and index.ISBN 1-58829-222-3 (alk. paper)1. Human cell culture—Laboratory manuals.[DNLM: 1. Cells, Cultured—Laboratory Manuals. 2. Cytological Techniques—LaboratoryManuals. QS 525 H9175 2005] I. Picot, Joanna. II. Series.QH585.2.H85 2005616'.0277—dc222004007186

vPrefaceSince the publication of the first edition of Human Cell Culture Protocols,the field of human cell culture has continued to expand and increasing numbers ofresearchers find they need to dip their toes into the world of tissue culture, even ifthis is not the main focus of their work. Today, not only does a whole industrysupply all the materials necessary for tissue culture, there is a growing number ofcompanies specializing in the supply of a diverse range of human cells. For thosewithout existing links to clinicians and hospital departments, the purchase of cellsmay be an attractive starting point, although this can be an expensive option. Alternatively, many researchers find an essential element in success is buildingclose links with clinicians and hospital departments from which human tissuesamples are obtained. In particular, close collaboration can enable cell cultureto be initiated with the very minimum of delay, which is often the key to establishing a viable culture. Before any experimental work takes place, however,researchers must ensure that patients have provided informed consent and thatlocal and/or national ethical and other guidelines for the procurement and use ofhuman tissue are met. In addition, the tissues received are potentially biohazardous, possibly harboring infectious agents such as HIV, hepatitis, and tuberculosis, so appropriate safety measures must be in place.A quick search of any of the literature databases reveals the breadth ofuses that human cells are put to. Cell culture is the starting point for so manyapplications. Microarray technology continues to develop, helping to elucidate patterns of gene expression within cells. A wide range of techniques isavailable to help researchers identify and understand the complex web of protein–protein interactions within and between cells. Cell cultures are used totest approaches to gene therapy and to gain an understanding of the cell cycle,particularly in relation to the development of cancers. The construction of 3-Dcell cultures and the field of tissue engineering are the subjects of many othertexts and take us far beyond the scope of this volume. Advances in microscopyrefine our ability to image live cells in culture. Ultimately, the pooling of manystrands of knowledge over time allows the development of new therapeuticapproaches for human disease.The first edition of Human Cell Culture Protocols was published in 1996.Now in this second edition, the collection of chapters has been revised to bringthe methods up to date. As in the first edition, it has not been possible to coverv

viPrefacethe vast array of distinct cell types in one volume. I have, however, kept to theideals of the first edition in trying to ensure that protocols are provided for aselection of the major tissue groups. New to this edition are chapters on fibroblasts, Schwann cells, gastric and colonic epithelial cells, and parathyroid cells.This collection of protocols will provide researchers who are starting to usecell culture methods for the first time with the detailed knowledge and helpfulpointers they need. It should enable them to achieve success quickly and withthe minimum of difficulty. Even those familiar with cell culture techniquesmay find this book a useful resource.Finally, I would like to thank Gareth E. Jones whose success in bringingtogether the first edition gave me a wonderful foundation. Grateful thanks tothe many authors who agreed to update their chapters from the first edition, andto those authors who have contributed for the first time. Thanks also to Professor John Walker and the staff at Humana Press who were always extremelyprompt in responding to any and every enquiry and who were also patient whenmy replies to them were less than punctual.Joanna Picot

viiContentsPreface . vContributors . ix1 Establishment and Maintenance of Normal HumanKeratinocyte CulturesClaire Linge . 12 Cultivation of Normal Human Epidermal Melanocytesin the Absence of Phorbol EstersMei-Yu Hsu, Ling Li, and Meenhard Herlyn . 133 Isolation and Culture of Human OsteoblastsAlison Gartland, Katherine A. Buckley, Jane P. Dillon,Judith M. Curran, John A. Hunt, and James A. Gallagher . 294 Human Osteoclast Culture from Peripheral Blood Monocytes:Phenotypic Characterization and Quantitation of ResorptionKatherine A. Buckley, Benjamin Y. Y. Chan, William D. Fraser,and James A. Gallager . 555 Human Chondrocyte Cultures as Models of Cartilage-SpecificGene RegulationMary B. Goldring . 696 Human Myoblasts and Muscle-Derived SP CellsGrace K. Pavlath and Emanuela Gussoni . 977 Cell Cultures of Autopsy-Derived FibroblastsVolker Meske, Frank Albert, and Thomas G. Ohm . 1118 Primary Culture and Differentiation of Human AdipocytePrecursor CellsVanessa van Harmelen, Thomas Skurk, and Hans Hauner . 1259 Human Mononuclear Phagocytes in Tissue CultureYona Keisari . 13710 Purification of Peripheral Blood Natural Killer CellsBice Perussia and Matthew J. Loza . 14711 Human Fetal Brain Cell CultureMark P. Mattson . 163vii

viiiContents12 Culturing Human Schwann CellsVictor J. Turnbull .13 Well-Differentiated Human Airway Epithelial Cell CulturesM. Leslie Fulcher, Sherif Gabriel, Kimberlie A. Burns,James R. Yankaskas, and Scott H. Randell .14 Isolation and Culture of Human Alveolar Epithelial CellsCarsten Ehrhardt, Kwang-Jin Kim, and Claus-Michael Lehr .15 A New Approach to Primary Culture of Human Gastric EpitheliumPierre Chailler and Daniel Ménard .16 Isolation and Culture of Human Colon Epithelial CellsUsing a Modified Explant Technique Employinga Noninjurious ApproachHamid A. Mohammadpour .17 Isolation and Culture of Human HepatocytesMartin Bayliss and Graham Somers .18 Glomerular Epithelial and Mesangial Cell Cultureand CharacterizationHeather M. Wilson and Keith N. Stewart .19 Isolation and Culture of Human Renal Cortical Cellswith Characteristics of Proximal TubulesGabrielle M. Hawksworth .20 Culture of Parathyroid CellsPer Hellman .21 Long-Term Culture and Maintenance of Human Isletsof Langerhans in Memphis Serum-Free MediaDaniel W. Fraga, A. Osama Gaber, and Malak Kotb .22 Primary Culture of Human Antral Endocrine and Epithelial CellsSusan B. Curtis and Alison M. J. Buchan .23 Conjunctiva Organ and Cell CultureMonica Berry and Marcus Radburn-Smith .24 Establishment, Maintenance, and Transfection of In Vitro Culturesof Human Retinal Pigment EpitheliumMartin J. Stevens, Dennis D. Larkin, Eva L. Feldman,Monte A. DelMonte, and Douglas A. Greene .Index .173183207217237249269283291303313325343353

ixContributorsFRANK ALBERT Charité, Department: Klinische Zell und Neurobiologie,Institute for Anatomie, Berlin, GermanyMARTIN BAYLISS GlaxoSmithKline, Stevenage, Hertfordshire, UKMONICA BERRY Division of Ophthalmology, Bristol Eye Hospital, Universityof Bristol, UKALISON M. J. BUCHAN Department of Physiology, University of BritishColumbia, Vancouver, CanadaKATHERINE A. BUCKLEY Human Bone Cell Research Group, Department ofHuman Anatomy and Cell Biology, The University of Liverpool, UKKIMBERLIE A. BURNS Cystic Fibrosis/Pulmonary Research and TreatmentCenter, The University of North Carolina at Chapel Hill, NC, USAPIERRE CHAILLER CIHR Group on the Functional Development andPhysiopathology of the Digestive Tract, Department of Anatomy andCell Biology, Université de Sherbrooke, Québec, CanadaBENJAMIN Y. Y. CHAN Department of Clinical Chemistry, Royal LiverpoolUniversity Hospital, UKJUDITH M CURRAN UK Centre for Tissue Engineering, Department ofClinical Engineering, University of Liverpool, UKSUSAN B. CURTIS Department of Physiology, University of British Columbia,Vancouver, CanadaMONTE A. DELMONTE Sierra Sciences Inc., Reno, NV, USAJANE P. DILLON Human Bone Cell Research Group, Department of HumanAnatomy & Cell Biology, University of Liverpool, UKCARSTEN EHRHARDT Department of Pharmaceutics and PharmaceuticalTechnology, Trinity College, Dublin, IrelandEVA L. FELDMAN Sierra Sciences Inc., Reno, NV, USADANIEL W. FRAGA Islet Transplant Laboratory, University of Tennessee,Memphis, TN, USAWILLIAM D. FRASER Department of Clinical Chemistry, Royal LiverpoolUniversity Hospital, UKM. LESLIE FULCHER Cystic Fibrosis/Pulmonary Research and TreatmentCenter, The University of North Carolina at Chapel Hill, NC, USAA. OSAMA GABER Islet Transplant Laboratory, University of Tennessee,Memphis, TN, USAix

xContributorsSHERIF GABRIEL Cystic Fibrosis/Pulmonary Research and TreatmentCenter, The University of North Carolina at Chapel Hill, NC, USAJAMES A. GALLAGHER Human Bone Cell Research Group, Department ofHuman Anatomy and Cell Biology, The University of Liverpool, UKALISON GARTLAND Human Bone Cell Research Group, Department ofHuman Anatomy & Cell Biology, University of Liverpool, UKMARY B. GOLDRING Beth Israel Deaconess Medical Center, Department ofMedicine, Division of Rheumatology, New England Baptist Bone andJoint Institute, Harvard Institutes of Medicine, Boston, MA, USADOUGLAS A. GREENE Sierra Sciences Inc., Reno, NV, USAEMANUELA GUSSONI Division of Genetics, Childrens Hospital, Boston, MA,USAHANS HAUNER Else-Kröner-Fresenius-Center for Nutritional Medicine ofthe Technical University of Munich, GermanyGABRIELLE M. HAWKSWORTH Department of Medicine & Therapeutics,University of Aberdeen, Scotland, UKPER HELLMAN Department of Surgery, University Hospital, Uppsala,SwedenMEENHARD HERLYN The Wistar Institute, Philadelphia, PA, USAMEI-YU HSU The Wistar Institute, Philadelphia, PA, USAJOHN A. HUNT UK Centre for Tissue Engineering, Department of ClinicalEngineering, University of Liverpool, UKYONA KEISARI Department of Human Microbiology, Sackler Faculty ofMedicine, Tel Aviv University, Tel Aviv, IsraelKWANG-JIN KIM Departments of Medicine, Physiology and Biophysics,Molecular Pharmacology and Toxicology, and Biomedical Engineering, andWill Rogers Institute Pulmonary Research Center, Schools of Pharmacy,Medicine, and Engineering, University of Southern California, Los Angeles,CA, USAMALAK KOTB Department of Surgery/Immunology, University of Tennessee,Memphis, TN, USADENNIS D. LARKIN Sierra Sciences Inc., Reno, NV, USACLAUS-MICHAEL LEHR Department of Biopharmaceutics and PharmaceuticalTechnology, Saarland University, Saarbrücken, GermanyLING LI The Wistar Institute, Philadelphia, PA, USACLAIRE LINGE The RAFT Institute of Plastic Surgery, Mount Vernon Hospital,Northwood, Middlesex, UKMATTHEW J. LOZA Department of Microbiology and Immunology, KimmelCancer Center, Thomas Jefferson University, Philadelphia, PA, USAMARK P. MATTSON NIA Gerontology Research Center, Baltimore, MD, USA

ContributorsxiDANIEL MÉNARD CIHR Group on the Functional Development andPhysiopathology of the Digestive Tract, Department of Anatomy andCell Biology, Université de Sherbrooke, Québec, CanadaVOLKER MESKE Department: Klinische Zell und Neurobiologie, Institute forAnatomie, Charité, Berlin, GermanyHAMID A. MOHAMMADPOUR Sierra Sciences Inc., Reno, NV, USATHOMAS G. OHM Department: Klinische Zell und Neurobiologie, Institutefor Anatomie, Charité, Berlin, GermanyGRACE K. PAVLATH Department of Pharmacology, Emory University,Atlanta, GA, USABICE PERUSSIA Department of Microbiology and Immunology, KimmelCancer Center, Thomas Jefferson University, Philadelphia, PA, USAJOANNA PICOT Southampton, UKMARCUS RADBURN-SMITH Division of Ophthalmology, Bristol Eye Hospital,University of Bristol, UKSCOTT H. RANDELL Cystic Fibrosis/Pulmonary Research and TreatmentCenter, The University of North Carolina at Chapel Hill, NC, USATHOMAS SKURK Else-Kröner-Fresenius-Center for Nutritional Medicine ofthe Technical University of Munich, GermanyGRAHAM SOMERS GlaxoSmithKline, Stevenage, Hertfordshire, UKMARTIN J. STEVENS Sierra Sciences Inc., Reno, NV, USAKEITH N. STEWART Department of Medicine & Therapeutics, University ofAberdeen, ScotlandVICTOR J. TURNBULL Van Cleef Centre for Neuroscience, Alfred Hospital,Melbourne, AustraliaVANESSA VAN HARMELEN Else-Kröner-Fresenius-Center for NutritionalMedicine of the Technical University of Munich, GermanyHEATHER M. WILSON Department of Medicine & Therapeutics, Universityof Aberdeen, ScotlandJAMES R. YANKASKAS Cystic Fibrosis/Pulmonary Research and TreatmentCenter, The University of North Carolina at Chapel Hill, NC, USA

1Establishment and Maintenance o

84. Opioid Research: Methods and Protocols, edited by Zhizhong Z. Pan, 2003 83. Diabetes Mellitus: Methods and Protocols, edited by Sabire Özcan, 2003 82. Hemoglobin Disorders: Molecular Methods and Protocols, edited by Ronald L. Nagel, 2003 81. Prostate Cancer Methods and Protocols, edited by Pamela J. Russell, Paul Jackson, and Elizabeth A .

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