Cell and Tissue Culture Laboratory Procedures in Biotechnology

by Doyle, Alan; Griffiths, J. Bryan

• ISBN13: 9780471982555
• ISBN10: 0471982555
• Edition: 1st
• Format: Paperback
• Copyright: 1998-11-18
• Publisher: Wiley

Summary

Cell and Tissue Culture: Laboratory Procedures in Biotechnology Edited by Alan Doyle Centre for Applied Microbiology & Research, Porton Down, Salisbury, UK. and J. Bryan Griffiths Scientific Consultancy & Publishing, Porton, Salisbury, UK. Cell and Tissue Culture: Laboratory Procedures in Biotechnology introduces the reader to animal cell culture methods describing the key cells, core techniques, how to scale up the culture for commercial production, and regulatory aspects. This book provides easy to follow, step-by-step protocols, with trouble-shooting tips and notes on time considerations. Alternative procedures, background information and references supplement the main procedures described. Other features include: * Experimental examples to indicate expected results; * Quick reference symbols such as safety icons with warning notes; and, * A list of suppliers is provided to allow easy access to laboratory products. Written by a team of international scientists, Cell and Tissue Culture: Laboratory Procedures in Biotechnology will be of interest to researchers, technicians and process engineers using cell culture within the biotechnology, biomedicine and pharmaceutical industries.

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	xiii	(5)
Foreword	xviii	(1)
Preface	xix	(1)
Safety	xx
CHAPTER 1 THE CELL: SELECTION AND STANDARDIZATION	1	(52)
1.1. Overview	3	(2)
References	4	(1)
1.2 Cell Lines for Biotechnologists	5	(13)
Introduction	5	(1)
Cell line CHO dhfr	5	(1)
Cell line Sf9	6	(1)
Cell line Schneider-2	7	(1)
Cell lines COS 1/COS 7	7	(1)
Cell line NIH3T3	8	(1)
Cell line HeLa	8	(1)
Cell line J558L	9	(1)
Cell line Vero	9	(1)
Myeloma cell lines	10	(1)
Hybridomas	10	(1)
Cell line MRC-5	11	(1)
Cell line WI-38	11	(1)
Cell line Namalwa	12	(1)
Cell line BHK-21	12	(1)
Cell line MDCK	13	(1)
Cell line GH3	13	(1)
Cell line 293	13	(2)
Cell line PSI CRE/PSI CRIP	15	(1)
References	15	(3)
1.3 Master and Working Cell Banks	18	(7)
Scale and composition of cell banks	20	(1)
Extended cell bank	20	(1)
The cell banking environment and procedures	20	(1)
Features required for GLP procedures	21	(1)
Conclusion	22	(2)
References	24	(1)
1.4 Identity Testing - An Overview	25	(4)
Cytogenetic analysis	25	(1)
Isoenzyme analysis	26	(1)
DNA fingerprinting and DNA profiling	26	(1)
References	27	(2)
1.5 DNA Fingerprinting	29	(6)
PRELIMINARY PROCEDURE: Probe preparation	29	(1)
PROCEDURE: Hybridization	30	(1)
Discussion	31	(3)
References	34	(1)
1.6 Detection of Mycoplasma	35	(7)
PROCEDURE: DNA stain	36	(3)
ALTERNATIVE PROCEDURE: Use of indicator cell lines	39	(1)
SUPPLEMENTARY PROCEDURE: Microbiological culture	39	(1)
SUPPLEMENTARY PROCEDURE: Elimination of contamination	40	(1)
Discussion	40	(1)
References	41	(1)
1.7 Mycoplasma Detection Methods using PCR	42	(5)
PROCEDURE: Amplification	43	(1)
SUPPLEMENTARY PROCEDURE: Analysis of amplified samples	44	(1)
Discussion	45	(1)
References	46	(1)
1.8 Bacteria and Fungi	47	(3)
PROCEDURE: Detection of bacteria and fungi in cell cultures	47	(2)
Discussion	49	(1)
References	49	(1)
1.9 Elimination of Contamination	50	(3)
PROCEDURE: Eradication	50	(1)
Discussion	50	(2)
References	52	(1)
CHAPTER 2 CELL QUANTIFICATION	53	(30)
2.1 Overview	55	(2)
References	56	(1)
2.2 Haemocytometer Cell Counts and Viability Studies	57	(5)
PROCEDURE: Haemocytometer cell count	58	(1)
Discussion	59	(3)
2.3 MTT Assay	62	(3)
PROCEDURE: MTT assay - suspension or monolayer cells	62	(1)
ALTERNATIVE PROCEDURE: MTT assay - immobilized cells	63	(1)
References	64	(1)
2.4 Neutral Red (NR) Assay	65	(6)
PROCEDURE: Neutral red assay	66	(2)
SUPPLEMENTARY PROCEDURE: Protein assay	68	(1)
SUPPLEMENTARY PROCEDURE: Bioactivation	68	(1)
SUPPLEMENTARY PROCEDURE: UV radiation	69	(1)
References	70	(1)
2.5 LDH Assay	71	(5)
PROCEDURE: Measurement of LDH activity	71	(2)
Discussion	73	(1)
References	74	(2)
2.6 Miniaturized Colorimetric Methods for Determining Cell Number	76	(7)
PRELIMINARY PROCEDURE: Pretreatment of cells	76	(1)
PRELIMINARY PROCEDURE: 96-Well cell growth or toxicity assays	77	(1)
PRELIMINARY PROCEDURE: Trypan blue exclusion method for cell viability estimation	77	(1)
PROCEDURE: Colorimetric assays: general introduction	78	(2)
Discussion	80	(1)
References	80	(3)
CHAPTER 3 CULTURE ENVIRONMENT	83	(46)
3.1 Overview	85	(2)
References	86	(1)
3.2 Serum-free Systems	87	(5)
Elimination of serum	88	(1)
Serum substitution	88	(2)
Discussion	90	(1)
References	91	(1)
3.3 Adaptation to Serum-free Culture	92	(8)
PRELIMINARY PROCEDURE: Method for selecting serum	93	(1)
PRELIMINARY PROCEDURE: Method for selecting nutrient medium	94	(1)
PRELIMINARY PROCEDURE: Types of serum-free media	94	(1)
Modifying the nutrient medium	95	(1)
PROCEDURE: Method for adapting cells to serum-free medium	96	(1)
Discussion	97	(1)
References	98	(2)
3.4 Amino Acid Metabolism	100	(9)
PROCEDURE: Amino acid analysis	101	(5)
Case study	106	(1)
References	107	(2)
3.5 Tissue Culture Surfaces	109	(7)
The treatment process	109	(1)
Stability	110	(1)
Bioactivity	111	(1)
Surface choice and comparison	111	(1)
Microcarriers	112	(1)
Porous membrane systems	113	(1)
Discussion	114	(1)
References	114	(2)
3.6 Plastic and Glass Tissue Culture Surfaces	116	(5)
PROCEDURE: A simple procedure for coating surfaces	118	(2)
References	120	(1)
3.7 Three-dimensional Cell Culture Systems	121	(8)
Spheroids	122	(1)
Microcarriers	123	(1)
Filterwells	124	(1)
Matrix sponges or three-dimensional gels and matrix sandwiches	124	(1)
Microcontainers	124	(1)
Simulated microgravity	125	(1)
Conclusion	125	(1)
References	125	(4)
CHAPTER 4 BIOCHEMISTRY OF CELLS IN CULTURE	129	(90)
4.1 Overview	131	(2)
4.2 Quantitative Analysis of Cell Growth, Metabolism and Product Formation	133	(27)
Errors in calculations	134	(1)
Cell growth and death rates	134	(11)
Cell metabolism	144	(12)
Product formation	156	(1)
Concluding remarks	157	(2)
Acknowledgements	159	(1)
References	159	(1)
4.3 Modelling	160	(19)
Background for the modelling of mammalian cell cultures	160	(3)
Method for kinetic model construction	163	(11)
Use of the model for the evaluation of rate-limiting factors	174	(1)
Discussion	175	(3)
References	178	(1)
Background reading	178	(1)
4.4 Cell Death in Culture Systems (Kinetics of Cell Death)	179	(8)
PROCEDURE: Morphological characterization of cell death	180	(2)
PROCEDURE: Biochemical characterization of cell death	182	(1)
SUPPLEMENTARY PROCEDURE: Purification of apoptotic cells	183	(1)
Discussion	184	(1)
References	185	(2)
4.5 Detoxification of Cell Cultures	187	(3)
PROCEDURE: Detoxification by dialysis	187	(1)
ALTERNATIVE PROCEDURE: Detoxification by gel filtration	188	(1)
Discussion	189	(1)
References	189	(1)
4.6 Oxygenation	190	(12)
PROCEDURE: Measurement of oxygen transfer coefficient and oxygen uptake rate	191	(3)
SUPPLIEMENTARY PROCEDURE: Oxygenation methods	194	(4)
Reference	198	(4)
4.7 Mixing	202	(8)
Assessing cell damage	202	(1)
Parameters used to correlate cell damage due to agitation and/or air sparging	203	(1)
Cultures of freely suspended cells	204	(2)
Anchorage-dependent cells (microcarrier cultures)	206	(2)
References	208	(2)
4.8 Mechanical Protection	210	(9)
PRELIMINARY PROCEDURE: Additive preparation	211	(1)
PROCEDURE: Testing before using an additive	211	(1)
Additives for freely-suspended cells	212	(4)
Additives for microcarrier cultures	216	(1)
References	216	(3)
CHAPTER 5 CULTURE PROCESSES AND SCALE-UP	219	(74)
5.1 Overview	221	(7)
Scale-up factors	222	(1)
Scale-up strategies	222	(2)
General Principles	224	(1)
Monolayer and suspension culture	224	(1)
Culture modes	225	(1)
Biological factors	225	(1)
Summary	226	(1)
References	227	(1)
5.2 Roller Bottle Culture	228	(3)
PROCEDURE: Roller bottle culture of animal cells	228	(1)
Comment	229	(1)
Supplementary Procedures	229	(1)
Discussion	230	(1)
Background reading	230	(1)
5.3 Spinner Flask Culture	231	(4)
PROCEDURE: Culture of suspension cells in a spinner flask	231	(3)
Discussion	234	(1)
Background reading	234	(1)
5.4 Pilot-scale Suspension Culture of Hybridomas-an Overview	235	(5)
Pilot- and large-scale in vitro systems for hybridomas	235	(2)
Cultivation modes	237	(1)
References	238	(2)
5.5 Pilot-scale Suspension Culture of Human Hybridomas	240	(6)
PROCEDURE: Optimization of culture parmeters and scale-up	240	(1)
Inoculum preparation and optimization of parameters	241	(2)
Discussion	243	(2)
References	245	(1)
5.6 Chemostat Culture	246	(8)
Equipment	248	(1)
Method	248	(3)
Discussion	251	(1)
References	251	(3)
5.7 Growth of Human Diploid Fibroblasts for Vaccine Production Multiplate Culture	254	(8)
PROCEDURE: Propagation and subcultivation of human diploid cells in 150-cm(2) plastic culture vessels	254	(1)
PROCEDURE: Seeding, cultivation, trypsinization and infection of a Nune 6000-cm(2) multiplate unit	255	(4)
Discussion	259	(2)
References	261	(1)
Background reading	261	(1)
5.8 Microcarriers - Basic Techniques	262	(6)
PRELIMINARY PROCEDURE: Siliconization	264	(1)
PROCEDURE: Growth of cells on microcarriers	265	(1)
Discussion	266	(1)
References	266	(1)
Background reading	267	(1)
5.9 Porous Microcarrier and Fixed-bed Cultures	268	(14)
PRELIMINARY PROCEDURE: Initial preparation and calibration of equipment	272	(1)
PROCEDURE: Assembly of culture vessels	272	(2)
PROCEDURE: System set-up	274	(1)
PROCEDURE: Inoculation and maintenance of culture system	274	(1)
SUPPLEMENTARY PROCEDURE: Analysis of consumption and production rates in the fixed-bed porous-glass-sphere culture system	275	(2)
SUPPLEMENTARY PROCEDURE: Termination of culture and determination of cell numbers	277	(1)
Discussion	278	(2)
References	280	(2)
5.10 Control Processes	282	(11)
Basic process control	282	(3)
Enhanced control of physical/chemical parameters	285	(3)
Enhanced control of cell metabolism	288	(2)
Discussion	290	(1)
References	290	(3)
CHAPTER 6 REGULATORY ISSUES	293	(8)
6.1 Regulatory Aspects of Cells Utilized in Biotechnological Processes	295	(1)
Cell line derivation	296	(1)
Recombinant cells	297	(1)
Cell characterization studies	298	(1)
References	299	(2)
CONCLUDING REMARKS	301	(4)
Productivity	301	(4)
APPENDIX 1: TERMINOLOGY	305	(10)
Some aspects of the problem	305	(1)
Solutions to the problem	306	(1)
Termonology associated with cell, tissue and organ culture, molecular biology and molecular genetics	306	(7)
References	313	(2)
APPENDIX 2: COMPANY ADDRESSES	315	(10)
APPENDIX 3: RESOURCE CENTRES FOR BIOTECHNOLOGISTS	325	(4)
Index	329