Abbreviations |
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ix | |
Preface |
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xi | |
Chapter 1 Introduction: The use of animal cell culture |
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1 | (10) |
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1. Tissue culture, organ culture and cell culture |
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1 | (1) |
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2. Why grow animal cells in culture? |
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1 | (1) |
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3. The advantages and disadvantages of using cell culture |
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2 | (1) |
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4. The early history of cell culture |
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3 | (2) |
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5. How long will primary cultures survive? |
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5 | (1) |
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6 | (1) |
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7. The first products of animal cell technology |
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7 | (4) |
Chapter 2 Characteristics of cells in culture |
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11 | (16) |
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1. Where to obtain cells? |
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11 | (1) |
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2. Cells from tissue: a primary culture |
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11 | (2) |
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13 | (1) |
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4. How to select a particular cell type |
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14 | (1) |
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5. What is a 'normal' cell? |
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15 | (1) |
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16 | (1) |
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7. The culture of differentiated cells |
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17 | (1) |
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18 | (1) |
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19 | (1) |
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20 | (1) |
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11. Cells from a culture collection |
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21 | (3) |
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Protocol 2.1: Preparing a primary cell culture of chick embryo fibroblasts |
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24 | (2) |
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Protocol 2.2: Isolation of lymphocytes from a blood sample |
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26 | (1) |
Chapter 3 Basic equipment and laboratory design: what you need to get started in cell culture |
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27 | (20) |
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27 | (1) |
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27 | (2) |
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3. Washing re-usable glassware |
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29 | (1) |
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4. Biosafety/laminar flow cabinets |
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30 | (1) |
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31 | (3) |
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6. Laboratory-scale culture flasks |
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34 | (7) |
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41 | (1) |
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42 | (1) |
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9. Liquid nitrogen storage |
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42 | (3) |
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45 | (2) |
Chapter 4 Growth and maintenance of cells in culture |
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47 | (20) |
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1. How to culture cells and what to expect |
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47 | (4) |
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2. The importance of aseptic techniques |
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51 | (3) |
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54 | (1) |
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55 | (3) |
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58 | (2) |
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6. Cell metabolism during culture |
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60 | (4) |
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Protocol 4.1: Harvesting anchorage-dependent cells with trypsin |
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64 | (1) |
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Protocol 4.2: Adaptation of cells to a serum-free medium |
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65 | (1) |
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Protocol 4.3: The trypsinization of cells in serum-free medium |
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66 | (1) |
Chapter 5 Cell line and culture monitoring |
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67 | (32) |
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1. Cell counting and monitoring |
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67 | (3) |
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2. Indirect methods of cell determination |
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70 | (2) |
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3. Viability measurements |
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72 | (2) |
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4. Cell line identification |
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74 | (4) |
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5. Analysis of the cell cycle |
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78 | (5) |
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Protocol 5.1: Determination of the concentration of viable cells in a suspension by hemocytometer counting |
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83 | (1) |
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Protocol 5.2: Determination of the nuclei concentration in a culture sample |
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84 | (1) |
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Protocol 5.3: Determination of cell concentration by a Coulter counter |
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85 | (1) |
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Protocol 5.4: Determination of the protein concentration of a cell suspension |
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86 | (1) |
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Protocol 5.5: Determination of the DNA concentration of a cell suspension |
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87 | (2) |
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Protocol 5.6: Determination of the glucose concentration in culture |
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89 | (3) |
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Protocol 5.7: Determination of the viable concentration of cells by dye exclusion: the tetrazolium assay |
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92 | (1) |
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Protocol 5.8: Determination of cell viability by lactate dehydrogenase determination |
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93 | (1) |
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Protocol 5.9: Determination of the intracellular energy charge |
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94 | (2) |
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Protocol 5.10: Determination of the cellular rate of protein synthesis |
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96 | (1) |
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Protocol 5.11: Cell characterization by Giemsa banding of chromosomes (karyotype analysis) |
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97 | (2) |
Chapter 6 Genetic engineering of animal cells in culture |
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99 | (14) |
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99 | (1) |
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2. The need for mammalian cells |
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100 | (1) |
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3. How to get DNA into mammalian cells |
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100 | (3) |
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4. How to select and amplify the genes of transfected cells |
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103 | (2) |
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5. How to get genes to express proteins |
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105 | (2) |
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6. Regulation of gene expression |
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107 | (2) |
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Protocol 6.1: Transfection of CHO-K1 cells by lipofection |
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109 | (1) |
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Protocol 6.2: Transfection by electroporation |
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110 | (1) |
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Protocol 6.3: Development and isolation of methotrexate-resistant CHU cells |
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111 | (2) |
Chapter 7 The glycosylation of proteins in cell culture |
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113 | (22) |
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113 | (1) |
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2. Glycan structures present in glycoproteins |
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114 | (1) |
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3. Assembly and processing of glycans on proteins |
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115 | (4) |
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119 | (1) |
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5. Why mammalian cells are chosen for glycoprotein production |
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120 | (2) |
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6. Control of glycan processing in mammalian cell culture |
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122 | (5) |
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7. Genetic engineering of mammalian cells to modify glycosylation |
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127 | (3) |
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130 | (3) |
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Protocol 7.1: Analysis of glycans from in-gel release of protein bands |
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133 | (2) |
Chapter 8 Hybridomas - sources of antibodies |
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135 | (20) |
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135 | (1) |
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2. Antibody production in vivo |
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135 | (1) |
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3. The structure of antibodies |
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136 | (1) |
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4. Glycosylation of antibodies |
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137 | (1) |
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138 | (1) |
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138 | (7) |
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7. Assay of monoclonal antibodies |
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145 | (2) |
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8. Human monoclonal antibodies |
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147 | (1) |
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9. Recombinant antibodies |
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148 | (3) |
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10. Potential alternative methods of production |
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151 | (1) |
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11. The importance of glycosylation to therapeutic antibodies |
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152 | (1) |
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153 | (2) |
Chapter 9 Scaling up animal cell culture |
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155 | (20) |
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1. Why scale-up cultures? |
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155 | (1) |
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2. The stirred tank reactor (STR) |
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155 | (10) |
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165 | (1) |
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4. Alternative types of bioreactors |
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166 | (9) |
Chapter 10 Modes of culture for high cell densities |
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175 | (20) |
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175 | (4) |
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179 | (1) |
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180 | (7) |
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187 | (6) |
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Protocol 10.1: Establishment and monitoring of a microcarrier culture |
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193 | (2) |
Chapter 11 Production from cell culture |
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195 | (10) |
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1. Why use animal cell culture for production? |
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195 | (1) |
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2. How to produce biologicals from cell culture |
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195 | (4) |
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3. How to purify the final product |
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199 | (1) |
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4. The efficiency and productivity of a culture system |
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200 | (3) |
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5. Cost of the cell culture process |
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203 | (2) |
Chapter 12 Mammalian cell products: established and potential |
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205 | (28) |
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205 | (1) |
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205 | (4) |
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209 | (2) |
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4. Therapeutic recombinant glycoproteins from mammalian cells |
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211 | (10) |
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5. Risks associated with cell culture products |
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221 | (2) |
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223 | (1) |
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224 | (3) |
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227 | (6) |
Glossary |
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233 | (8) |
Index |
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241 | |