Human Cell Culture (eBook)

Table of Contents 6
Introduction 7
Hematopoietic Stem and Progenitor Cells 8
1. INTRODUCTION 8
1.1 Function and Organization of the Hematopoietic System 8
1.2 Stem Cell Self-Renewal 9
1.3 Hematopoietic Cell Growth Factors 11
1.4 The Bone Marrow Microenvironment 11
2. TISSUE PROCUREMENT AND PROCESSING 13
2.1 Human Hematopoietic Tissue Procurement and Shipping 13
2.2 Human Hematopoietic Tissue Processing 15
3. ASSAYS 16
3.1 Non-Biological Assays 16
3.2 Biological Assays 18
4. CULTURE TECHNIQUES 20
4.1 Historical Perspective: Long-Term Bone Marrow Culture ( LTBMC) 20
4.2 Current Culture Techniques 21
5. UTILITY OF HEMATOPOIETIC STEM AND PROGENITOR CELL CULTURES 5.1 Clinical Background 26
5.1 Clinical Background 26
5.2 Clinical Use of Hematopoietic Cell Cultures 27
5.3 Other Uses of Hematopoietic Cell Cultures 28
6. SUMMARY 28
LITERATURE CITED 29
In Vitro T-Lymphopoiesis 38
1. INTRODUCTION 38
2. PROTOCOLS FOR IN VITRO T-CELL DIFFERENTIATION 41
2.1 T-cell Differentiation Using Rhesus Thymic Stroma 41
2.2 T-cell Differentiation Using Human Thymic Stroma 43
2.3 Thymic Organ Culture 44
2.4 Reconstituted Human-SCID Mice Thymic Cultures 45
3. ANALYSIS OF CELL OUTPUT 3.1 Immunophenotyping of Cultured Cells by Flow Cytometry 45
3.1 Immunophenotyping of Cultured Cells by Flow Cytometry 45
3.2 RT-PCR Analysis 46
4. UTILITY OF THE SYSTEM 48
5. CONCLUDING REMARKS 49
T-Lymphocytes: Mature Polyclonal and Antigen- Specific Cell Culture 52
1. INTRODUCTION 52
1.1 Anatomy In Vivo 53
1.2 T-cell Activation 54
1.3 The Beginnings of T-Lymphocyte In Vitro Culture 57
2. TISSUE PROCUREMENT AND PROCESSING 57
2.1 Procurement 57
2.2 Enrichment/Purification from Whole Blood or an Apheresis Product 59
2.3 Selection of T-Cells 61
2.4 Cryopreservation 64
3. CULTURE TECHNIQUES 64
3.1 Media and Serum 65
3.2 Culture Vessels and Large-Scale Culture 66
3.3 Polyclonal Activation and Culture of T-Cells 68
3.4 Development of Ag-Specific T-cell Lines 75
4. ASSAY TECHNIQUES 81
4.1 Proliferation 81
4.2 Tracking Cell Division in T-cell Subsets 84
4.3 TCR Repertoire 86
4.4 Flow Cytometry and T-cell Culture 87
4.5 Cytokine Secretion and Intracellular Cytokine Flow Cytometry 88
4.6 CTL Activity 89
5. UTILITY OF THE SYSTEM 90
6. SUMMARY 92
ACKNOWLEDGEMENTS 93
REFERENCES 93
The Culture, Characterization and Triggering of BLymphocytes 108
1. INTRODUCTION 108
1.1 Origin of Normal B- Lymphocytes 109
1.2 Historical Aspects of the Culture of B- Lymphocytes 109
2. TISSUE PROCUREMENT AND PROCESSING 111
2.1 Sources of Normal B-Lymphocytes 111
2.2 Separation of Peripheral Blood and Bone Marrow BLymphocytes 111
2.3 Separation of Splenic and Tonsillar B- Lymphocytes 112
2.4 Purification of B-Lymphocytes 113
3. ASSAY TECHNIQUES 115
3.1 B-Lymphocyte Antigens 115
3.2 Assays of B-Lineage Active Cytokines 116
4. CULTURE TECHNIQUES 117
4.1 Media and Conditions 117
4.2 Influence and Dependence on Growth Factors 118
4.3 Long - Term Culture Using Adherent Feeder Cell Layers 118
4.4 NIH3T3 CD40-Ligand Transfectants 123
5. UTILITY OF SYSTEM 125
5.1 B-Cell Differentiation 125
5.2 B- Cell Growth 125
5.3 B-Cell Signaling 126
5.4 B-Cell Survival and Apoptosis 126
6. CONCLUSION 126
LITERATURE CITED 127
Monocytes and Macrophages 132
1. INTRODUCTION 132
2. TISSUE PROCUREMENT AND PROCESSING 134
2.1 The Cell Source Limits the Type of Investigation 137
2.2 To Purify or Not to Purify 137
2.3 To Freeze or Not to Freeze 139
3. ASSAY TECHNIQUES 139
3.1 General Characteristics 139
3.2 Flow Cytometry 140
3.3 In Vitro Production of Macrophages and Detection of Monocyte-Macrophage Progenitor Cells Using the Colony-Forming Technique 140
4. CULTURE TECHNIQUES 145
4.1. Type of Growth Surface 145
4.2 Serum or Serum-Free 146
4.3 Other Culture Components 146
4.4 Growth Factors 146
4.5 Low Oxygen Tension 147
4.6 Production of Resident Macrophages on a Nonadherent Growth Surface 147
5. UTILITY OF THE SYSTEM 148
6. CONCLUDING REMARKS 149
ACKNOWLEGMENT 149
REFERENCES CITED 149
Isolation and Cultivation of Osteoclasts and Osteoclast-Like Cells 153
1. INTRODUCTION 153
2. TISSUE PROCUREMENT AND PROCESSING 156
2.1 Human Osteoclasts (In Vivo Formed) 156
2.2 Human Osteoclast-Like Cells (In Vitro Formed) 159
3. CULTURE TECHNIQUES 3.1 Preparation of Sterile Devitalized Bone or Ivory Discs for Resorption Studies 160
3.1 Preparation of Sterile Devitalized Bone or Ivory Discs for Resorption Studies 160
3.2 Culture of In Vivo Formed hOC 161
3.3 Culture of In Vitro Formed hOCL 162
4. ASSAY TECHNIQUES 4.1 Morphology and Ultrastructure 163
4.1 Morphology and Ultrastructure 163
4.2 Cytochemical Staining 164
4.3 Antigenic Profile 166
4.4 Molecular Profile 168
4.5 Bone Resorptive Function 168
5. UTILITY OF SYSTEM 170
6. CONCLUDING REMARKS 171
ACKNOWLEDGEMENTS 172
LITERATURE CITED 172
Isolation and Culture of Dendritic Cells 176
1. INTRODUCTION 176
2. ISOLATION OF DENDRITIC CELLS 178
2.1 Isolation of Specific Types of Dendritic Cells: Langerhans Cells 181
2.2 Other Methods to Increase the Number of Isolated Dendritic Cells 181
3. GENERATION OF DENDRITIC CELLS 182
3.1 Generation of DC from CD34+Cells 183
3.2 Generation of DC from CD14+ and Plastic-Adherent Cells 184
3.3 Choice of Medium 186
3.4 Methods for Loading DC with Antigen 188
4. ASSAYS FOR ASSESSING DENDRITIC CELLS 189
5. FUTURE DIRECTIONS 190
LITERATURE CITED 191
In Vitro Proliferation and Differentiation of CD34+ Cells to Neutrophils 197
1. INTRODUCTION 197
2. TISSUE PROCUREMENT AND PROCESSING 198
2.1 Obtaining CD34+ Cells 198
2.2 Preparation of Mat 199
2.3 Maintainance of Mature Cells In Vitro 199
3. ASSAY TECHNIQUES 200
3.1 CD34+ Cells 200
3.2 Measurement of Neutrophil Differentiation and Function 200
4. CULTURE TECHNIQUES 206
4.1 System Concepts 206
4.2 Static Systems: Gas Permeable Bags 206
4.3 Cell Culture Bag Materials 207
4.4 Optimal Volumes and Cell to Surface Area Ratio 207
4.5 Perfusion Systems 208
4.6 Growth Factors and Medium 209
4.7 Characterization of In Vitro Derived Neutrophils 210
5. UTILITY OF SYSTEM 212
6. CONCLUDING REMARKS 215
7. LITERATURE CITED 216
Isolation and Culture of Eosinophils 222
1. INTRODUCTION 222
2. ISOLATION OF EOSINOPHILS AND EOSINOPHIL PROGENITORS 224
2.1 Normal Peripheral Blood Eosinophils 224
2.2 Hypereosinophilic Donors 227
2.3 Umbilical Cord Blood Progenitors 227
2.4 Peripheral Blood and Bone Marrow Progenitors 229
3. IDENTIFICATION OF MATURE EOSINOPHILS 229
3.1 Morphology and Staining Characteristics 229
3.2 Cell Surface Markers 229
3.3 Specific Granule Proteins 230
4. METHODS FOR EOSINOPHIL CULTURE 230
4.1 Maintaining Viability of Eosinophil Isolated from Pheripheral Blood 230
4.2 Differentiation and Culture of Eosinophils from BM and CB Progenitors 231
4.3 Differentiation and Culture of Eosinophils from Peripheral Blood Progenitors 232
4.4 Quantitative Comparisons Between Natural and Cultured Eosinophils 232
5. APPLICATIONS 234
5.1 Characterization of Eosinophil-Mediated Functions In Vitro 234
5.2 Apoptosis and the Control of Eosinophilic Inflammation 234
5.3 Molecular Mechanisms of Differentiation and Transcriptional Control of Eosinophil- Specific Genes 235
5.4 Identification of Novel Enhancers and Inhibitors of Eosinophil Differentiation 235
REFERENCES 235
Isolation and Culture of Mast Cells and Basophils 243
1. INTRODUCTION 243
2. TISSUE PROCUREMENT AND PROCESSING 246
2.1 Progenitor Cells 246
2.2 Peripheral Blood Basophils 247
2.3 Tissue Mast Cells 248
2.4 Isolation of CD34+ Progenitor Cells 248
2.5 Isolation of Basophils from CML Blood 248
2.6 Isolation of Basophils from Normal Blood 249
2.7 Isolation and Purification of Mast Cells from Tissues 250
3. CULTURE TECHNIQUES 251
3.1 Culture of Basophils from Their Progenitor Cells 251
3.2 Culture of Mature Isolated Basophils (Normal And CML- Derived) 252
3.3 Culture of Mast Cells from Their Progenitors 252
3.4 Culture of Isolated Mature Tissue Mast Cells 253
4. UTILITY OF SYSTEMS 253
4.1 Basic Science and Cytokine Research 253
4.2 Clinical Immunology and Allergy Research 254
4.3 Pharmacology and Toxicology 254
5. CONCLUDING REMARKS 255
LITERATURE CITED 255
Purification and Culture of Erythroid Progenitor Cells 260
1. INTRODUCTION 260
2. PURIFICATION OF BFU-E 262
2.1 High Purity Method of BFU-E Purification (Method 1) [ 9,10] 262
2.2 High Yield Method of BFU-E Purification (Method II) [ 3,14] 269
2.3 Purification of BFU-E Using Magnetic Microbeads ( Method 111) 270
3. IN VITRO GENERATION OF CFU-E FROM PURIFIED BFU-E 273
3.1 Methylcellulose Cell Culture for Generation of CFUE [8] 273
3.2 CFU- E Generation from BFU- E in Liquid Suspension Culture 274
4. ERYTHROID PROGENITOR CELL ASSAYS 275
4.1 Semi-Solid Culture of BFU-E and CFU-E 275
4.2 Determination of Cell Purity in Liquid Culture 277
4.3 Morphology of BFU-E and CFU-E 277
5. UTILITY OF PURIFICATION PROCEDURES 278
5.1 Transitional Change of Growth Factor Requirements and Receptors 278
5.2 Studies of Surface Receptors 281
5.3 Studies on Apoptosis 282
5.4 Studies on Signal Transduction and Protein Phosphorylation 283
6. CONCLUSIONS 285
ACKNOWLEDGMENTS 285
REFERENCES 286
In Vitro Development of Megakaryocytes and Platelets 288
1. INTRODUCTION 288
2. TISSUE PROCUREMENT AND PROCESSING 292
2.1 Isolation of Lineage-Light-Density CB Cells 292
2.2 Isolation of Lineage-Light-Density Fetal Liver Cells 294
2.3 Isolation of Lineage-Light-Density FBM Cells 297
2.4 Preparation of Cells for FACS 298
3. ASSAY TECHNIQUES 301
3.1 Flow Cytometric Analysis of Cell Surface Markers 301
3.2 Flow Cytometric Analysis of DNA Content 302
4. CULTURE TECHNIQUES 304
4.1 Serum- Deprived Media 305
4.2 Colony-Forming Unit Assay 306
5. UTILITY OF SYSTEMS 308
6. CONCLUDING REMARKS 310
LITERATURE CITED 311
Perspectives, Ethics and Clinical Issues in the Use of Primary Human Cells 317
1. INTRODUCTION 317
2. HISTORICAL AND LEGAL PERSPECTIVES 318
2.1 Whole Organ Transplantation 318
2.2 Tissue, Cell and Gene Transplantation 323
3. ETHICAL ISSUES AND RESPONSIBILITIES 327
3.1 Acquisition and Distribution 327
3.2 Selection of Donors and Recipients 330
3.3 Genetic Manipulation 333
4. HARVESTING, PREPARATION AND BANKING ISSUES 334
4.1 Safety and Efficacy of the Products 334
4.2 Ownership and Cells, Tissues and Organs as Commodities 336
5. SUMMARY 337
REFERENCES 338