Since 1996, the Methods in Molecular Medicine series has provided medical researchers and laboratory scientists with reliable, stepbystep protocols used to discover new approaches to combating and treating diseases, such as cancer, arthritis, and cardiovascular disease. Each protocol is provided in readilyreproducible stepbystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice
i Human Cell Culture Protocols METHODS IN MOLECULAR MEDICINE TM John M Walker, SERIES EDITOR 113 113 Multiple Myeloma: Methods and Protocols, edited by Ross D Brown and P Joy Ho, 2005 112 Molecular Cardiology: Methods and Protocols, 112 edited by Zhongjie Sun, 2005 111 111 Chemosensitivity: Volume 2, In Vivo Models, Imaging, and Molecular Regulators, edited by Rosalyn D Blumethal, 2005 110 Chemosensitivity: Volume 1, In Vitro Assays, 110 edited by Rosalyn D Blumethal, 2005 109 109 Adoptive Immunotherapy, Methods and Protocols, edited by Burkhard Ludewig and Matthias W Hoffman, 2005 108 Hypertension, Methods and Protocols, 108 edited by Jérôme P Fennell and Andrew H Baker, 2005 107 107 Human Cell Culture Protocols, Second Edition, edited by Joanna Picot, 2005 106 Antisense Therapeutics, Second Edition, 106 edited by M Ian Phillips, 2005 105 105 Developmental Hematopoiesis: Methods and Protocols, edited by Margaret H Baron, 2005 104 Stroke Genomics: Methods and Reviews, edited 104 by Simon J Read and David Virley, 2005 103 103 Pancreatic Cancer: Methods and Protocols, edited by Gloria H Su, 2005 102 102 Autoimmunity: Methods and Protocols, edited by Andras Perl, 2004 101 101 Cartilage and Osteoarthritis: Volume 2, Structure and In Vivo Analysis, edited by Frédéric De Ceuninck, Massimo Sabatini, and Philippe Pastoureau, 2004 100 Cartilage and Osteoarthritis: Volume 1, 100 Cellular and Molecular Tools, edited by Massimo Sabatini, Philippe Pastoureau, and Frédéric De Ceuninck, 2004 99 99 Pain Research: Methods and Protocols, edited by David Z Luo, 2004 98 98 Tumor Necrosis Factor: Methods and Protocols, edited by Angelo Corti and Pietro Ghezzi, 2004 97 97 Molecular Diagnosis of Cancer: Methods and Protocols, Second Edition, edited by Joseph E Roulston and John M S Bartlett, 2004 96 96 Hepatitis B and D Protocols: Volume 2, Immunology, Model Systems, and Clinical Studies, edited by Robert K Hamatake and Johnson Y N Lau, 2004 95 95 Hepatitis B and D Protocols: Volume 1, Detection, Genotypes, and Characterization, edited by Robert K Hamatake and Johnson Y N Lau, 2004 94 94 Molecular Diagnosis of Infectious Diseases, Second Edition, edited by Jochen Decker and Udo Reischl, 2004 93 93 Anticoagulants, Antiplatelets, and Thrombolytics, edited by Shaker A Mousa, 2004 92 92 Molecular Diagnosis of Genetic Diseases, Second Edition, edited by Rob Elles and Roger Mountford, 2004 91 Pediatric Hematology: Methods and Protocols, 91 edited by Nicholas J Goulden and Colin G Steward, 2003 90 90 Suicide Gene Therapy: Methods and Reviews, edited by Caroline J Springer, 2004 89 The Blood–Brain Barrier: Biology and 89 Research Protocols, edited by Sukriti Nag, 2003 88 88 Cancer Cell Culture: Methods and Protocols, edited by Simon P Langdon, 2003 87 Vaccine Protocols, Second Edition, edited by 87 Andrew Robinson, Michael J Hudson, and Martin P Cranage, 2003 86 86 Renal Disease: Techniques and Protocols, edited by Michael S Goligorsky, 2003 85 Novel Anticancer Drug Protocols, edited by 85 John K Buolamwini and Alex A Adjei, 2003 84 84 Opioid Research: Methods and Protocols, edited by Zhizhong Z Pan, 2003 83 83 Diabetes Mellitus: Methods and Protocols, edited by Sabire Özcan, 2003 82 82 Hemoglobin Disorders: Molecular Methods and Protocols, edited by Ronald L Nagel, 2003 81 81 Prostate Cancer Methods and Protocols, edited by Pamela J Russell, Paul Jackson, and Elizabeth A Kingsley, 2003 iii METHODS IN MOLECULAR MEDICINE TM Human Cell Culture Protocols Second Edition Edited by Joanna Picot Southampton, UK Humana Press Totowa, New Jersey iv © 2005 Humana Press Inc 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com 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 Methods in Molecular Medicine™ is a trademark of The Humana 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www.humanapress.com Photocopy 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 the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923 For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable 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 E-ISBN 1-59259-861-7 Library of Congress Cataloging in Publication Data Human 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) Human cell culture—Laboratory manuals [DNLM: Cells, Cultured—Laboratory Manuals Cytological Techniques—Laboratory Manuals QS 525 H9175 2005] I Picot, Joanna II Series QH585.2.H85 2005 616'.0277—dc22 2004007186 v Preface Since the publication of the first edition of Human Cell Culture Protocols, the field of human cell culture has continued to expand and increasing numbers of researchers find they need to dip their toes into the world of tissue culture, even if this is not the main focus of their work Today, not only does a whole industry supply all the materials necessary for tissue culture, there is a growing number of companies specializing in the supply of a diverse range of human cells For those without existing links to clinicians and hospital departments, the purchase of cells may be an attractive starting point, although this can be an expensive option Alternatively, many researchers find an essential element in success is building close links with clinicians and hospital departments from which human tissue samples are obtained In particular, close collaboration can enable cell culture to 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 that local and/or national ethical and other guidelines for the procurement and use of human 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 of uses that human cells are put to Cell culture is the starting point for so many applications Microarray technology continues to develop, helping to elucidate patterns of gene expression within cells A wide range of techniques is available to help researchers identify and understand the complex web of protein–protein interactions within and between cells Cell cultures are used to test 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-D cell cultures and the field of tissue engineering are the subjects of many other texts and take us far beyond the scope of this volume Advances in microscopy refine our ability to image live cells in culture Ultimately, the pooling of many strands of knowledge over time allows the development of new therapeutic approaches 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 bring the methods up to date As in the first edition, it has not been possible to cover v vi Preface the vast array of distinct cell types in one volume I have, however, kept to the ideals of the first edition in trying to ensure that protocols are provided for a selection 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 use cell culture methods for the first time with the detailed knowledge and helpful pointers they need It should enable them to achieve success quickly and with the minimum of difficulty Even those familiar with cell culture techniques may find this book a useful resource Finally, I would like to thank Gareth E Jones whose success in bringing together the first edition gave me a wonderful foundation Grateful thanks to the many authors who agreed to update their chapters from the first edition, and to those authors who have contributed for the first time Thanks also to Professor John Walker and the staff at Humana Press who were always extremely prompt in responding to any and every enquiry and who were also patient when my replies to them were less than punctual Joanna Picot vii Contents Preface v Contributors ix Establishment and Maintenance of Normal Human Keratinocyte Cultures Claire Linge Cultivation of Normal Human Epidermal Melanocytes in the Absence of Phorbol Esters Mei-Yu Hsu, Ling Li, and Meenhard Herlyn 13 Isolation and Culture of Human Osteoblasts Alison Gartland, Katherine A Buckley, Jane P Dillon, Judith M Curran, John A Hunt, and James A Gallagher 29 Human Osteoclast Culture from Peripheral Blood Monocytes: Phenotypic Characterization and Quantitation of Resorption Katherine A Buckley, Benjamin Y Y Chan, William D Fraser, and James A Gallager 55 Human Chondrocyte Cultures as Models of Cartilage-Specific Gene Regulation Mary B Goldring 69 Human Myoblasts and Muscle-Derived SP Cells Grace K Pavlath and Emanuela Gussoni 97 Cell Cultures of Autopsy-Derived Fibroblasts Volker Meske, Frank Albert, and Thomas G Ohm 111 Primary Culture and Differentiation of Human Adipocyte Precursor Cells Vanessa van Harmelen, Thomas Skurk, and Hans Hauner 125 Human Mononuclear Phagocytes in Tissue Culture Yona Keisari 137 10 Purification of Peripheral Blood Natural Killer Cells Bice Perussia and Matthew J Loza 147 11 Human Fetal Brain Cell Culture Mark P Mattson 163 vii viii Contents 12 Culturing Human Schwann Cells Victor J Turnbull 13 Well-Differentiated Human Airway Epithelial Cell Cultures M Leslie Fulcher, Sherif Gabriel, Kimberlie A Burns, James R Yankaskas, and Scott H Randell 14 Isolation and Culture of Human Alveolar Epithelial Cells Carsten Ehrhardt, Kwang-Jin Kim, and Claus-Michael Lehr 15 A New Approach to Primary Culture of Human Gastric Epithelium Pierre Chailler and Daniel Ménard 16 Isolation and Culture of Human Colon Epithelial Cells Using a Modified Explant Technique Employing a Noninjurious Approach Hamid A Mohammadpour 17 Isolation and Culture of Human Hepatocytes Martin Bayliss and Graham Somers 18 Glomerular Epithelial and Mesangial Cell Culture and Characterization Heather M Wilson and Keith N Stewart 19 Isolation and Culture of Human Renal Cortical Cells with Characteristics of Proximal Tubules Gabrielle M Hawksworth 20 Culture of Parathyroid Cells Per Hellman 21 Long-Term Culture and Maintenance of Human Islets of Langerhans in Memphis Serum-Free Media Daniel W Fraga, A Osama Gaber, and Malak Kotb 22 Primary Culture of Human Antral Endocrine and Epithelial Cells Susan B Curtis and Alison M J Buchan 23 Conjunctiva Organ and Cell Culture Monica Berry and Marcus Radburn-Smith 24 Establishment, Maintenance, and Transfection of In Vitro Cultures of Human Retinal Pigment Epithelium Martin J Stevens, Dennis D Larkin, Eva L Feldman, Monte A DelMonte, and Douglas A Greene Index 173 183 207 217 237 249 269 283 291 303 313 325 343 353 ix Contributors FRANK ALBERT • Charité, Department: Klinische Zell und Neurobiologie, Institute for Anatomie, Berlin, Germany MARTIN BAYLISS • GlaxoSmithKline, Stevenage, Hertfordshire, UK MONICA BERRY • Division of Ophthalmology, Bristol Eye Hospital, University of Bristol, UK ALISON M J BUCHAN • Department of Physiology, University of British Columbia, Vancouver, Canada KATHERINE A BUCKLEY • Human Bone Cell Research Group, Department of Human Anatomy and Cell Biology, The University of Liverpool, UK KIMBERLIE A BURNS • Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, NC, USA PIERRE CHAILLER • CIHR Group on the Functional Development and Physiopathology of the Digestive Tract, Department of Anatomy and Cell Biology, Université de Sherbrooke, Québec, Canada BENJAMIN Y Y CHAN • Department of Clinical Chemistry, Royal Liverpool University Hospital, UK JUDITH M CURRAN • UK Centre for Tissue Engineering, Department of Clinical Engineering, University of Liverpool, UK SUSAN B CURTIS • Department of Physiology, University of British Columbia, Vancouver, Canada MONTE A DELMONTE • Sierra Sciences Inc., Reno, NV, USA JANE P DILLON • Human Bone Cell Research Group, Department of Human Anatomy & Cell Biology, University of Liverpool, UK CARSTEN EHRHARDT • Department of Pharmaceutics and Pharmaceutical Technology, Trinity College, Dublin, Ireland EVA L FELDMAN • Sierra Sciences Inc., Reno, NV, USA DANIEL W FRAGA • Islet Transplant Laboratory, University of Tennessee, Memphis, TN, USA WILLIAM D FRASER • Department of Clinical Chemistry, Royal Liverpool University Hospital, UK M LESLIE FULCHER • Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, NC, USA A OSAMA GABER • Islet Transplant Laboratory, University of Tennessee, Memphis, TN, USA ix 344 Stevens et al Bovine calf serum (defined and iron-supplemented) and fetal calf serum (FCS) (defined) are supplied by Hyclone Laboratories (Logan, UT), sterilely divided into 50-mL aliquots in 50-mL tubes, and stored at –20°C L-Glutamine, penicillin/streptomycin, and sterile sodium bicarbonate solutions are supplied by Gibco and stored at –20°C Establishment of primary cultures of human RPE is accomplished with the following recipe: Ham’s F12 Nutrient Mixture, 16% fetal bovine serum (FBS), 0.02 mM L-glutamine, 100 U/mL penicillin/100 μg/mL streptomycin, 0.075% (w/v) sodium bicarbonate Trypsin-etheylenediaminetetraacetic acid (EDTA) is supplied by Gibco, sterilely divided into 10-mL aliquots in 15-mL tubes, and stored at –20°C Papain (cat no P3125) and L-cysteine-HCl (cat no C7880) are supplied by Sigma (St Louis, MO) Papain stock solution is prepared in advance in Ca2+- and Mg2+-free HBSS by adding mM L-cysteine HCl, mM EDTA, and 10 μL/mL papain The stock solution is stable for wk when stored at 4°C Acid-soluble collagen (Sigma type III) is supplied by Sigma and stored at –20°C Silane (cat no M6514) for treating pipets is supplied by Sigma Pipets are coated by rinsing in 0.2% silane, then chloroform, ethanol, and several water rinses, or by coating with silane vapor in a desiccator and then autoclaved to sterilize Methods 3.1 Preparation of Collagen-Coated Culture Vessels (see Note 1) Coating of dishes with acid-soluble type collagen is easily accomplished as follows: 0.5% solution of acid-soluble collagen in 0.1 M acetic acid is painted on the surface of the culture vessels with a fine brush The thin collagen coating is gelled by exposure to NH3 fumes from ammonium hydroxide for 12 h The excess ammonia is neutralized with buffered saline Dishes are sterilized by overnight exposure to UV light 3.2 Initial Dissection, Cell Isolation, and Primary Culture (see Note 2) Postmortem eyes are obtained from an eye bank within 96 h of death, and the anterior segment of the eye is removed by circumferential incision mm posterior to the limbus The vitreous and neurosensory retina are separated from the pigment epithelium, cut free at the optic nerve, and removed The eyecup is rinsed three times with HBSS without Ca2+ or Mg2+ It is important to keep the eyecup moist throughout the isolation procedure The eyecup is filled with stock HBSS/papain solution and incubated for 40 at 37°C or until the RPE cell layer appears marbled The papain reaction is stopped by the addition of 50 μL of FBS to the eyecup The HBSS/papain solution is removed from the eyecup and replaced with Ham’s F12-medium without serum Human RPE 345 Fig Dissecting microscope—view of harvesting human RPE from the posterior of the eye using a fire-polished Pasteur pipet Velvety appearing RPE (large arrow) is being gently vacuumed into the pipet (star) leaving behind the shiny Bruch’s membrane (small arrow) Optic disk is marked with O for orientation The RPE cells are dislodged and suspended with a stream of medium from a firepolished Pasteur pipet treated with 0.2% silane to prevent sticking Remaining adherent RPE is gently vacuumed free from Bruch’s membrane using the firepolished Pasteur pipet under direct observation with a dissecting microscope as shown in Fig The cell/papain solution is removed from the eye cup, placed in a sterile tube, and centrifuged at 50g for to pellet the cells The cells are resuspended and washed in HBSS 10 The washed pellet is resuspended in complete media consisting of Ham’s F12-M 346 Stevens et al Fig Primary explants of human RPE as examined by phase-contrast microscopy immediately after harvesting (×140) media supplemented with 16% FCS, 0.075% sodium bicarbonate, 0.02 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin 11 Cells are distributed evenly into sterile uncoated or extracellular matrix-coated 35-mm plastic culture dishes or the wells of 6- or 24-well culture plates Twentyfour-well PRIMARIA culture plates (Falcon Plastics) provide excellent cell adhesion and colony formation characteristics, and are recommended for most primary culture applications Other dishes or extracellular matrix coatings are fine for established cultures Fresh RPE explants consist of single or small clusters of polygonal, deeply pigmented epithelial cells, which appear much as they in vivo as seen in Fig 12 Cultures are incubated at 37°C in a humidified 95% air, 5% CO2 atmosphere After d, the media are aspirated and replaced Cell attachment and initiation of colony formation are monitored by phase contrast microscopy 13 Alternatively, at step 6, medium with serum is added to the eyecup, and small patches of cells are dislodged and plated directly into culture dishes or plates without the centrifugation step This technique is especially useful for obtaining cultures from specific locations of the globe, such as the macular area, midperiphery, or periphery, to study regional differences 3.3 Establishment and Maintenance of RPE Cell Lines (see Notes 3–6) The medium is aspirated, and the cells are rinsed with mL HBSS without Ca2+ or Mg2+ for Cells can then be removed from the dishes by addition of either mL 0.05% trypsin in 0.53 mM EDTA or mL of stock HBSS/ papain Human RPE 347 solution The cells are then incubated at 37°C for 2–15 until they are rounded and begin to detach from the plastic Trypsinization is terminated by addition of vol of medium supplemented with 10–20% calf serum The cell suspension is centrifuged at 50g for and resuspended in medium Established cell lines can be maintained in simpler media, such as DMEM with 16% FBS replaced by as low as 2–5% calf serum or even, for short defined periods of time, with defined serum-free medium Cells are subcultured at a density of 10,000–20.000 cells/cm2 in T-25 or T-75 tissue-culture flasks Cells are maintained at 37°C and humidified 95% air, 5% CO2 atmosphere with fresh medium changes three times weekly 3.4 Transient Transfection of RPE Cells (see Notes 7–10) When transfecting RPE cells, it is necessary to transfect cell cultures which are in exponential growth phase to maximize DNA uptake Seeding density should be adjusted to allow cultures to reach approx 80–90% confluency at time of harvest Therefore, our laboratory seeds cells at a density of 40,000 cells per well in 24-well cluster plates (Costar cat no 3524) containing 0.5 mL MEM, 5% bovine calf serum (BCS) It is important to exclude the use of antibiotics, to avoid increased toxicity or cell death caused by excess intracellular uptake of antibiotics during the DNA uptake period Cultures are transfected 16 h post-seeding with the minimum amount of DNA necessary to produce reliably measurable luciferase activity In our laboratory, we have obtained good results using lipofectamine 2000 (Invitrogen cat no 52758) and transfecting 180 ng test DNA and ng control DNA to normalize for variations in transfection efficiency On the day of the transfection, combine test DNA and control DNA in 50 μL Opti-MEM (Gibco, cat no 31985-062) per well to be transfected Control DNA should be 1/50 of test DNA or less (1–20 ng) to minimize potential interference by competing promoters Per well: Dilute μL of lipofectamine 2000 (LF2000) into 50 μL opti-MEM Incubate at room temperature Combine diluted LF2000 with the diluted DNA and allow to complex 20 at room temperature Seed 100 μL of LF2000/DNA complex per well Cells should be re-fed after 6–8 h with standard medium with antibiotics Alternatively, cells can be re-fed 16 h post-transfection, but increased toxicity may result Allow 24–48 h expression prior to assay Notes Cell attachment and colony formation are enhanced, especially in eyes from older donors and those obtained later after death, by coating the culture vessels with collagen or other artificial extracellular matrix (10,14) However, with the develop- 348 Stevens et al Table Donor Factors Influencing Human RPE Culture Success Donor characteristics Success/attempts % 3/3 6/7 6/8 1/3 100 85 75 33 6/8 10/13 75 77 5/5 9/10 2/4 0/2 100 90 50 Age 80 Sex Male Female Time from death, h 72 Adapted from ref 13 with permission ment of specially treated tissue-culture plastics, such as PRIMARIA (Falcon Plastics), cell adhesion and colony formation are excellent Therefore, the use of artificial extracellular matrix is usually unnecessary today unless required for specific experimental conditions Donor characteristics influence the establishment of successful cultures: The highest rates of viable cultures are found if patients are