Cell and tissue culture : laboratory procedures in biotechnology

CHAPTER 1 THE CELL: SELECTION AND STANDARDIZATION 1.1 Overview References 1.2 Cell Lines for Biotechnologists Introduction Cell line CHO dhfr- Cell line Sf9 Cell line Schneider-2 Cell lines COS 1/COS 7 Cell line NIH3T3 Cell line HeLa Cell line J558L Cell line Vero Myeloma cell lines Hybridomas Cell line MRC-5 Cell line WI-38 Cell line Namalwa Cell line BHK-21 Cell line MDCK Cell line GH3 Cell line 293 Cell line !VCRE/!VCRIP References Master and Working Cell Banks Scale and composition of cell banks Extended cell bank The cell banking environment and procedures Features required for GLP procedures Conclusion References 1.3 xiii xviii XiX xx 1 3 4 5 5 5 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 13 15 15 18 20 20 20 21 22 24 v1 CELL AND TISSUE CULTURE 1.4 1.5 1.6 1.7 1.8 1.9 CHAPTER 2 2.1 2.2 2.3 Identity Testing - An Overview Cytogenetic analysis Isoenzyme analysis DNA fingerprinting and DNA profiling References DNA Fingerprinting PRELIMINARY PROCEDURE: Probe preparation PROCEDURE: Hybridization Discussion References Detection of Mycoplasma PROCEDURE: DNA stain ALTERNATIVE PROCEDURE: Use of indicator cell lines SUPPLEMENTARY PROCEDURE: Microbiological culture SUPPLEMENTARY PROCEDURE: Elimination of contamination Discussion Ref ereiices Mycoplasma Detection Methods using PCR PROCEDURE: Amplification SUPPLEMENTARY PROCEDURE: Analysis of amplified samples Discussion References Bacteria and Fungi PROCEDURE: Detection of bacteria and fungi in cell cultures Discussion References Elimination of Contamination PROCEDURE: Eradication Discussion References CELL QUANTIFICATION Overview References Haemocytometer Cell Counts and Viability Studies PROCEDURE: Haemocytometer cell count Discussion MTT Assay PROCEDURE: MTT assay - suspension or monolayer cells ALTERNATIVE PROCEDURE: MTT assay - immobilized cells References 25 25 26 26 27 29 29 30 31 34 35 36 39 39 40 40 41 42 43 44 45 46 47 47 49 49 50 50 50 52 53 55 56 57 58 59 62 62 63 64 t CONTENTS vii 2.4 2.5 2.6 Neutral Red (NR) Assay PROCEDURE: Neutral red assay SUPPLEMENTARY PROCEDURE: Protein assay SUPPLEMENTARY PROCEDURE: Bioactivation SUPPLEMENTARY PROCEDURE: UV radiation References LDH Assay PROCEDURE: Measurement of LDH activity Discussion References Miniaturized Colorimetric Methods for Determining Cell Number PRELIMINARY PROCEDURE: Pretreatment of cells PRELIMINARY PROCEDURE: 96-Well cell growth or toxicity assays PRELIMINARY PROCEDURET: rypan blue exclusion method for cell viability estimation PROCEDURE: Colorimetric assays: general introduction Discussion References CHAPTER 3 CULTURE ENVIRONMENT 3.1 Overview 3.2 Serum-free Systems References Elimination of serum Serum substitution Discussion References PRELIMINARY PROCEDURE: Method for selecting serum PRELIMINARY PROCEDURE: Method for selecting nutrient medium PRELIMINARY PROCEDURE: Types of serum-free media Modifying the nutrient medium PROCEDURE: Method for adapting cells to serum-free medium Discussion References Amino Acid Metabolism PROCEDURE: Amino acid analysis Case study References 3.3 Adaptation to Serum-free Culture 3.4 65 66 68 68 69 70 71 71 73 74 76 76 77 77 78 80 80 83 85 86 87 88 88 90 91 92 93 94 94 95 96 97 98 100 101 106 107 ... CELL AND TISSUE CULTURE Vlll 3.5 3.6 3.7 4.3 4.4 Tissue Culture Surfaces The treatment process St ability Bioactivity Surface choice and comparison Microcarriers Porous membrane systems Discussion References Plastic and Glass Tissue Culture Surfaces PROCEDUREA: simple procedure for coating surfaces References Three-dimensional Cell Culture Systems Spheroids Microcarriers Filterwells Matrix sponges or three-dimensional gels and matrix sandwiches Microcontainers Simulated microgravity Conclusion References CHAPTER 4 4.1 Overview 4.2 BIOCHEMISTRY OF CELLS IN CULTURE Quantitative Analysis of Cell Growth, Metabolism and Product Formation Errors in calculations Cell growth and death rates Cell metabolism Product formation Concluding remarks Acknowledgements References Modelling Background for the modelling of mammalian cell cultures Method for kinetic model construction Use of the model for the evaluation of rate-limiting factors Discussion References Background reading Cell Death in Culture Systems (Kinetics of Cell Death) PROCEDURE: Morphological characterization of cell death PROCEDURE: Biochemical characterization of cell death 109 109 110 111 111 112 113 114 114 116 118 120 121 122 123 124 124 124 125 125 125 129 131 133 134 134 144 156 157 159 159 160 160 163 174 175 178 175 179 180 182 CONTENTS ix 4.5 4.6 4.7 4.8 SUPPLEMENTARY PROCEDURE: Purification of apoptotic cells Discussion References Detoxification of Cell Cultures PROCEDURE: Detoxification by dialysis gel filtration Discussion References Oxygenation PROCEDURE: Measurement of oxygen transfer coefficient and oxygen uptake rate SUPPLEMENTARY PROCEDURE: Oxygenation methods References Mixing Assessing cell damage Parameters used to correlate cell damage due to agitation and/or air sparging Cultures of freely suspended cells Anchorage-dependent cells (microcarrier cultures) References Mechanical Protection PRELIMINARY PROCEDURE: Additive preparation PROCEDURE: Testing before using an additive Additives for freely-suspended cells Additives for microcarrier cultures References ALTERNATIVE PROCEDURE: Detoxification by CHAPTER 5 CULTURE PROCESSES AND SCALE-UP 5.1 Overview Scale-up factors Scale-up strategies General principles Monolayer and suspension culture Culture modes Biological factors Summary References Roller Bottle Culture PROCEDURE: Roller bottle culture of animal cells Comment Supplementary procedures Discussion Background reading 5.2 183 184 185 187 187 188 189 189 190 191 194 198 202 202 203 204 206 208 210 21 1 21 1 212 216 216 219 221 222 222 224 224 225 225 226 227 228 228 229 229 230 230 X CELL AND TISSUE CULTURE 5.3 5.4 5.5 5.6 5.7 5.8 5.9 Spinner Flask Culture PROCEDURE: Culture of suspension cells in a spinner flask Discussion Background reading Pilot-scale Suspension Culture of Hybridomas - an Overview Pilot- and large-scale in vitro systems for hybridomas Cultivation modes References Pilot-scale Suspension Culture of Human Hybridomas PROCEDURE: Optimization of culture parameters and scaleup Inoculuni preparation and optimization of parameters Discussion References Chemostat Culture Equipment Method Discussion References Growth of Human Diploid Fibroblasts for Vaccine Production Multiplate Culture PROCEDURE: Propagation and subcultivation of human diploid cells in 150-cm’ plastic culture vessels PROCEDURE: Seeding, cultivation, trypsinization and infection of a Nunc 6000-cm2 multiplate unit Discussion References Background reading Microcarriers - Basic Techniques PRELIMINARY PROCEDURE: Siliconization PROCEDURE: Growth of cells on microcarriers Discussion References Background reading Porous Microcarrier and Fixed-bed Cultures PRELIMINARY PROCEDURE: Initial preparation and calibration of equipment PROCEDURE: System set-up PROCEDURE: Inoculation and maintenance of culture system consumption and production rates in the fixed-bed porous-glass-sphere culture system PROCEDURE: Assembly Of culture vessels SUPPLEMENTARY PROCEDURE: Analysis Of 23 1 231 234 234 235 235 237 238 240 240 241 243 245 246 248 248 251 251 254 254 255 259 261 261 262 264 265 266 266 267 268 212 272 274 274 275 CONTENTS xi 5.10 SUPPLEMENTARY PROCEDURE: Termination Of culture and determination of cell numbers Discussion References Control Processes Basic process control Enhanced control of physicalkhemical parameters Enhanced control of cell metabolism Discussion References CHAPTER 6 REGULATORY ISSUES 6.1 Regulatory Aspects of Cells Utilized in Biotechnological Processes Cell line derivation Recombinant cells Cell characterization studies References CONCLUDING REMARKS Productivity APPENDIX k TERMINOLOGY Some aspects of the problem Solutions to the problem Terminology associated with cell, tissue and organ culture, molecular biology and molecular genetics References APPENDIX 2: COMPANY ADDRESSES APPENDIX 3: RESOURCE CENTRES FOR BIOTECHNOLOGISTS 277 278 280 282 282 285 288 290 290 293 295 296 297 298 299 301 301 305 305 306 306 313 315 325 Index 329