Cell and Tissue Culture for Medical Research

by Doyle, Alan; Griffiths, J. Bryan

• ISBN13: 9780471852131
• ISBN10: 0471852139
• Edition: 1st
• Format: Paperback
• Copyright: 2000-08-22
• Publisher: WILEY

Summary

Cell and tissue culture is a technique in which plant or animal cells are grown under controlled conditions in the laboratory. This is then used for the analysis of the cells themselves, the assessment of the cell's response to chemicals, or as a tool to produce cellular-derived protein products. This book is a collection of fundamental and specific applied procedures in cell and tissue culture which form the basis of the new medical techniques of tissue engineering and gene therapy. It combines both detailed laboratory procedures and informative overviews. * Provides step-by-step protocols with troubleshooting tips and notes on time considerations. * Main procedures are supplemented by alternative procedures, background information and references. * Experimental examples indicate expected results.

Author Biography

Alan Doyle is the director of the Fountain House Institute. He is a former assistant director for occupational education for a regional school system on Long Island, has served as the federal liaison for the Massachusetts State Education Commissioner, and attended Fordham University and the Harvard Graduate School of Education.

Table of Contents

Contributors	xv
Preface	xxiii
Safety	xxiv
Basic Laboratory Set-Up and Procedures	1	(44)
General Introduction	3	(2)
References	4	(1)
Cryopreservation: Basic Techniques and Storage	5	(7)
Basic techniques	5	(1)
Cryopreservation using controlled-rate freezing apparatus	6	(1)
Long-term storage	7	(1)
Thawing	8	(1)
Mechanical freezing and two-stage freezing	8	(1)
Discussion	9	(1)
Literature review	10	(1)
References	11	(1)
Haemocytometer Cell Counts and Viability Studies	12	(5)
Haemocytometer cell count	12	(4)
Discussion	16	(1)
Prophylactic Use of Antibiotics in Cells and Tissues with a High Risk of Microbial Contamination	17	(10)
Determining the cytotoxicity of antibiotics in cell and tissue culture	19	(1)
Cytotoxicity test in lymphoblastoid cell cultures	20	(1)
Stability of antibiotics in cell and tissue culture medium	21	(1)
Antibiotic assays	21	(1)
Antibiotic solution	22	(1)
Preparation of assay plates	22	(1)
Diffusion assay	22	(2)
Discussion	24	(1)
References	25	(2)
Enzymic Techniques for Tissue Dissociation	27	(11)
Tissue types	28	(1)
Enzymes	29	(2)
Preparation and storage of enzymes	31	(1)
Discussion: Optimization of cell dissociation	32	(4)
References	36	(2)
Routine Subculturing	38	(7)
General	38	(1)
Subculture-attached cells	39	(1)
Serum-free cultures	40	(1)
Mechanical removal of cells	40	(1)
Discussion: Alternative Proteases	41	(1)
Subculture--suspension cells	42	(1)
Subculture by dilution	43	(1)
Discussion: Serum-free cultures	43	(1)
References	44	(1)
Specialized Techniques	45	(46)
Cell Quantification -- An Overview	47	(4)
Haemocytometer cell counts	48	(1)
Colony-forming efficiency	48	(1)
MTT assay	49	(1)
LDH activity	49	(1)
Neutral Red (NR) assay	49	(1)
Choice of method	49	(1)
References	50	(1)
Background reading	50	(1)
Mycoplasma	51	(1)
Detection of Mycoplasma	51	(7)
DNA stain	52	(2)
Use of indicator cell lines	54	(1)
Microbiological culture	55	(1)
Elimination of contamination	56	(1)
Discussion	56	(1)
References	57	(1)
Detection of Mycoplasma by DNA Amplification (PCR)	58	(4)
Amplification	59	(1)
Discussion	60	(1)
References	61	(1)
Bacteria and Fungi	62	(3)
Detection of bacteria and fungi in cell cultures	62	(2)
Discussion	64	(1)
References	64	(1)
Elimination of Contamination	65	(3)
Eradication	65	(1)
Discussion	65	(2)
References	67	(1)
Removal of Fibroblasts	68	(7)
Choice of method	68	(1)
Preparation of dishes for `panning'	69	(1)
Preparation of cells for `panning'	69	(1)
`Panning'	70	(1)
Complement and antibodies	70	(1)
Rapid Procedure: EDTA/trituration	70	(1)
Other techniques	71	(1)
Immunoological techniques	72	(1)
Literature review	73	(1)
References	73	(2)
Identity Testing -- An Overview	75	(4)
Cytogenic analysis	75	(1)
Isoenzyme analysis	76	(1)
DNA fingerprinting and DNA profiling	76	(1)
References	77	(2)
Scale-up of Laboratory Cultures	79	(12)
Roller Bottle Culture	79	(1)
Extended Surface Area Roller Bottle (ESRB)	80	(1)
Spinner flask culture	81	(2)
Microcarrier culture	83	(3)
Siliconization	86	(1)
Microcarrier culture	87	(1)
Discussion	88	(1)
References	89	(1)
Background reading	90	(1)
Cell Characterization and Analysis	91	(56)
Techniques for Cell Characterization and Analysis -- An Overview	93	(2)
Image Cytometry--An Overview	95	(6)
Goals of medical microscopic imaging	95	(1)
Diversity of microscopic imaging	95	(3)
Laboratory requirements	98	(1)
Major ergonomic weakness	99	(1)
Comment	99	(2)
Fluorescent Image Cytometry	101	(5)
Image acquisition	102	(2)
Discussion: Critical parameters	104	(1)
References	104	(2)
Immunocytochemical Labelling and Analysis for Light Microscopy	106	(9)
Cell culture	106	(1)
Detection method	107	(7)
Alternative detection methods	114	(1)
Controls	114	(1)
References	114	(1)
Flow Cytometry -- An Overview	115	(7)
Instrumentation	115	(2)
Data analysis	117	(2)
Cell sorting	119	(1)
Applications	119	(2)
Choice of instrument	121	(1)
Further reading	121	(1)
Electron Microscopy -- An Overview	122	(17)
Instrumentation	122	(6)
Preparation techniques	128	(9)
References	137	(2)
Cell Death in Culture Systems (Kinetics of Cell Death)	139	(8)
Morphological characterization of cell death	140	(1)
Biochemical characterization of cell death	140	(4)
Purification of apoptotic cells	144	(1)
Discussion	144	(1)
References	145	(2)
Cell Line Derivation Methods	147	(182)
Cell Line Derivation -- An Overview	149	(5)
Human cell lines	152	(1)
Risk assessment	152	(1)
References	153	(1)
Cell Lines for Medical Research	154	(11)
Introduction	154	(1)
VERO	155	(1)
A2780	156	(1)
NCI-H460	156	(1)
NCI-H596	156	(1)
MRC-5	157	(1)
MCF-7	157	(1)
FDCP-mix clone A4	158	(1)
HIT-T15	158	(1)
C6 Glial Cells	158	(1)
SK-Mel-1	159	(1)
WiDr	159	(1)
K-562	159	(1)
A6	160	(1)
HUV-EC-C	160	(1)
PtK2 (NBL-5)	161	(1)
References	162	(3)
Explant Cultures	165	(7)
Preparation of human tissue explants	165	(2)
Preparation of animal tissue explants	167	(2)
Culture vessels	169	(1)
Procedure A	169	(1)
Procedure B	170	(1)
Discussion	170	(1)
References	171	(1)
Haemopoetic Cells in Culture: Human B and T Lymphocytes	172	(1)
Introduction	172	(1)
EBV transformation	173	(6)
Separation of mononuclear cells from peripheral blood	173	(1)
Preparation of EBV-containing supernatant	174	(1)
Titration of EBV supernatant using cord blood mononuclear cells	175	(1)
Establishment of human B lymphoblastoid cell lines from peripheral blood mononuclear cells	176	(2)
References	178	(1)
Cryopreservation of human blood for B-cell immortalization	179	(8)
Cryopreservation and immortalization procedure	181	(3)
Discussion	184	(2)
References	186	(1)
T-cells and thymocytes	187	(18)
Sources of T-cells	188	(1)
Procedures for establishing T-cells reactive with exogenous soluble antigen	189	(3)
Assay for the antigen specificity of T-cells reactive with exogenous soluble antigen	192	(1)
Cloning of T-cells reactive with exogenous soluble antigen	193	(1)
Improved method for the expansion of human T-cell clones	194	(2)
Procedures for establishing T-cells reactive with endogenous cell surface antigen	196	(1)
Assay for the antigen specificity of T-cells reactive with cell tissue surface antigen	197	(1)
Cloning of T-cells reactive with endogenous cell surface antigen	198	(1)
Mass long-term cultures of human T-cells	199	(2)
Mass cultures of CD4+ and CD8+ cells	201	(1)
Discussion	202	(1)
References	203	(2)
Establishment of Human Leukaemia Cell Lines	205	(14)
Establishment of human leukaemia cell lines: Basic protocol	205	(5)
Maintenance of human leukaemia cell lines: Basic protocol	210	(3)
Storage of human leukaemia cell lines: Basic protocol	213	(2)
Commentary	215	(3)
Background reading	218	(1)
Derivation and Subculturing of Human Diploid Cell Strains	219	(5)
Derivation and subculturing	219	(3)
Theory of population increase by subcultivation	222	(1)
References	223	(1)
Rapid Initiation of Adherent Monolayer Cell Cultures: The `Cold Cocktail' Method	224	(11)
Introduction	224	(1)
Materials	225	(1)
Reagents, solutions and media	226	(1)
Procedure	227	(4)
Troubleshooting	231	(1)
Alternative protocols	231	(2)
Background	233	(1)
Acknowledgement	234	(1)
References	234	(1)
Cultures of Human Keratinocytes	235	(10)
Skin biopsies	235	(1)
Explant culture	236	(1)
Dispersed epidermal cell culture	237	(3)
Reconstructed epidermis	240	(2)
References	242	(2)
Background reading	244	(1)
The Isolation and Maintenance of Human Gastric Epithelial Cells in Primary Culture	245	(6)
Gastric epithelial cell adhesion requirements	245	(3)
Processing a human gastric biopsy sample	248	(1)
Dissociation of human gastric epithelial cells	248	(1)
Maintenance of human gastric epithelium in long-term culture	249	(1)
Propagation of cells	249	(1)
Discussion	250	(1)
Acknowledgements	250	(1)
References	250	(1)
Replicative and Functional Cultures of Normal Human Hepatocytes	251	(18)
Introduction	251	(1)
Equipment and material	252	(1)
Basic procedure	253	(7)
Characterization of hepatic function	260	(4)
Discussion	264	(2)
References	266	(3)
Embryonic Kidney in Organ Culture	269	(6)
The preparation of Nuclepore filters and pancreatin--trypsin	270	(1)
Pre-treatment of Nuclepore filters	271	(1)
Setting up the cultures	271	(3)
Discussion	274	(1)
References	274	(1)
The Culture of Amniocytes, Chorionic Villi, and Human Foetal and Placental Tissue	275	(16)
Culture of amniocytes	277	(3)
Culture of human foetal and placental tissues for chromosome analysis	280	(3)
Culture of chorionic villi for prenatal chromosome analysis	283	(3)
In situ amniocyte cell culture and harvesting	286	(2)
Discussion	288	(2)
References	290	(1)
Endothelial Cells (from the Umbilical Cord Vein)	291	(8)
Endothelial cell extraction	293	(1)
Cell cultures	294	(2)
Complementary Techniques	296	(1)
Culture dish coating	296	(1)
von Willebrand factor characterization	297	(1)
Conclusion	298	(1)
References	298	(1)
Dissociated Nerve Cells	299	(7)
Culture of dissociated nerve cells plated on poly-lysine-coated glass coverslips or smooth plastic surfaces	299	(4)
Culture of dissociated nerve cells on uncoated microporous membranes	303	(1)
Discussion	303	(1)
References	304	(2)
Neuronal and Glial Tumours IN VITRO -- An Overview	306	(15)
Introduction	306	(1)
Neuronal neoplasms	307	(1)
Glial neoplasms	307	(1)
Embryonal neoplasms	308	(1)
Growth of nervous system tumours in vitro	309	(1)
Nervous system tumours in culture	310	(7)
References	317	(4)
Colon Adenocarcinoma Cells (Primary/established Cultures of Human Colon Carcinomas)	321	(8)
Preparation of collagen-coated flasks	321	(1)
Preparation of Swiss 3T3 feeder cells	322	(1)
Establishment of cell lines from colorectal tumours	322	(4)
Discussion	326	(2)
References	328	(1)
Modern Clinical Applications of Cultured Cells	329	(96)
Immortalization Methods -- An Overview	331	(3)
References	332	(2)
Long-term Maintenance and Stability of Hybridomas	334	(11)
Endpoint cloning	334	(2)
Mini-cloning	336	(1)
Soft-agar cloning	336	(2)
Long-term maintenance	338	(3)
Monitoring of antibody secretion using quantitative ELISA	341	(4)
Organic Culture Techniques in Tissue Banking	345	(7)
Corneal structure and function	345	(1)
Corneal transplantation	346	(1)
Corneal storage by organ culture	346	(3)
Graft outcome	349	(1)
Future developments	349	(1)
References	350	(2)
Cell and Tissue Engineering	352	(12)
Three-dimensional cell culture systems	352	(5)
Tissue engineered skin	357	(1)
Applications of tissue engineering	357	(1)
Current advances	358	(2)
References	360	(4)
Gene Therapy and Recombinant Cells	364	(1)
Recombinant DNA cell lines	364	(6)
Overview	364	(3)
References	367	(3)
Adenovirus vectors	370	(18)
The adenovirus genome	370	(3)
Adenoviruses as expression vectors	373	(1)
Vector construction and rescue	374	(2)
Infection of cells with adenovirus and isolation of adenovirus DNA from infected cells	376	(2)
Rescue of adenovirus early replacement vectors	378	(7)
Discussion	385	(1)
References	385	(3)
Retroviruses and retroviral vectors	388	(5)
References	392	(1)
Retroviral-vector-mediated gene transfer	393	(9)
Selection of retrovirus helper cells and design of retroviral vectors	393	(2)
Construction of retroviral vectors with inserted gene sequences	395	(1)
Transfection of mammalian cells with retroviral vectors	396	(2)
Generation of helper cells stably producing a relatively homogeneous supply of vector virus	398	(1)
Infection of susceptible target cells with vector virus produced from helper cell lines	399	(1)
References	400	(2)
In Vitro Toxicity Testing	402	(23)
Toxicity of xenobiotics	402	(1)
The use of cultured cells in pharmaco-toxicological research	402	(1)
The choice of the in vitro biological model: cell lines versus primary cultures	403	(1)
Toxicity risk assessment in cultured cells	404	(14)
Applications of in vitro methods in biomedical toxicology	418	(1)
References	419	(6)
APPENDIX 1 TERMINOLOGY	425	(9)
Some aspects of the problem	425	(1)
Solutions to the problem	426	(1)
Terminology associated with cell, tissue and organ culture, molecular biology and molecular genetics	426	(7)
References	433	(1)
APPENDIX 2 COMPANY ADDRESSES	434	(5)
Index	439