Primary Mesenchymal Cells (eBook)

Contents 6
Introduction 7
Foreword to the Series 8
Articular Cartilage 9
1. INTRODUCTION 9
2. TISSUE PROCUREMENT AND PROCESSING 11
3. ASSAY TECHNIQUES 12
3.1 Chondrocyte re-differentiation in semi-solid media 13
3.2 Agarose differentiation assay 13
3.3 Alginate differentiation assay 13
3.4 Analysis of gene expression 14
3.5 RNA Analysis 14
3.6 RNase protection assay (RPA) 14
3.7 Immunohistochemical Analysis 14
4. CULTURE TECHNIQUES 15
4.1 Anchorage Dependent Expansion in Monolayer 15
4.2 Autologous human serum (AHS) versus fetal bovine serum (FBS) 15
4.3 Impact of Donor Age on Chondrocyte Re-differentiation 16
4.4 Development of a defined medium for articular chondrocyte culture 16
4.5 Re-differentiation of articular chondrocytes cultured in defined medium 19
5. UTILITY OF THE SYSTEM 19
6. CONCLUDING REMARKS 21
6.1 The Future Leads to Tissue Engineering 21
REFERENCES 22
Tendon and Ligaments 25
1. INTRODUCTION 25
2. TISSUE PROCUREMENT AND PROCESSING 27
3. ASSAY TECHNIQUES 29
4. CULTURE TECHNIQUES 30
5. UTILITY OF THE SYSTEM 31
6. CONCLUDING REMARKS 32
REFERENCES 32
Periodontal Ligaments 34
1. INTRODUCTION 34
2. TISSUE PROCUREMENT 36
3. ASSAY TECHNIQUES 40
4. CULTURE TECHNIQUES 41
5. UTILITY OF THE SYSTEM 45
6. CONCLUDING REMARKS 46
REFERENCES 46
Vascular Smooth Muscle 49
1. INTRODUCTION 49
1.1 Location of VSMCs in the blood vessel wall 50
1.2 VSMC phenotype and importance in disease 51
1.3 VSMC heterogeneity and tissue culture models 51
1.4 Importance of using human VSMC cultures 54
2. TISSUE PROCUREMENT AND PROCESSING 54
2.1 Obtaining human blood vessels 54
2.2 Processing tissue for VSMC isolation 55
2.3 Isolation of VSMCs from the micro-vasculature (pericytes) 57
2.4 Isolation of VSMCs from diseased vessels 57
3. CULTURE TECHNIQUES 58
3.1 Media and supplements 58
3.2 Culture vessels 59
3.3 Passaging of cultures 59
4. ASSAY TECHNIQUES 60
4.1 Identification of VSMCs 60
4.2 Cell markers 61
4.3 Growth 63
5. UTILITY OF SYSTEM 63
5.1 VSMC growth and migration 63
5.2 VSMC phenotypic diversity 64
5.3 VSMC contraction/differentiation 65
5.4 Human VSMCs and gene transfer 65
6. SUMMARY AND CONCLUDING REMARKS 66
REFERENCES 66
Skeletal Muscle 71
1. INTRODUCTION 71
1.1 Control of Muscle Development in Vertebrates 72
1.2 Regulation of Muscle Cell Determination and Migration 72
1.3 Regeneration of Skeletal Muscle 74
2. TISSUE PROCUREMENT AND PROCESSING 75
2.1 Tissue Procurement 75
2.2 Tissue Processing 76
2.3 Donor-to-Donor and Muscle-to-Muscle Variability 79
3. CULTURE TECHNIQUES 80
3.1 Choice of Culture Medium 80
3.2 Coating of Growth Surfaces 81
3.3 Enrichment of the Cell Population for Muscle Cells 83
3.4 Passaging and Conservation 84
3.5 Extending the Life Span of Muscle Cells 85
4. ASSAY TECHNIQUES 85
4.1 Morphology 86
4.2 Degree of Differentiation 88
4.3 Enhancement of Differentiation by Innervation 91
4.4 Gene Delivery to Cultured Muscle Cells 92
4.5 Making Your Own Muscle Cells 92
4.6 Human Skeletal Muscle Cell Lines 93
5. CLINICAL USE OF MYOBLASTS 94
5.1 Transplantation of Myoblasts 94
5.2 Myoblasts and Gene Therapy 94
5.3 Myoblasts and Treatment of Heart Failure 95
6. CONCLUDING REMARKS 96
REFERENCES 97
Cardiomyocytes 108
1. INTRODUCTION 108
1.1 Cardiomyocyte Development and Differentiation 108
1.2 Cardiomyocyte Cultures 110
2. TISSUE PROCUREMENT AND PROCESSING 112
2.1 Human Myocardial Tissue 112
2.2 Cell Isolation 112
3. ASSAY TECHNIQUES 115
3.1 Morphology 115
3.2 Growth Characteristics 116
3.3 Myocardial Contractile Proteins 120
3.4 Cardiac Protein Gene Expression 120
3.5 Cellular Ultrastructure 121
3.6 Biochemical Identification 121
4. CULTURE TECHNIQUES 121
4.1 Cell Purification 121
4.2 Cell Culture 122
5. APPLICATIONS OF A CARDIOMYOCYTE CULTURE SYSTEM 124
5.1 Cardiac Development 124
5.2 Cardiac Physiology 124
5.3 Cardiac Pathology and Cellular Defenses 125
6. CONCLUSIONS 126
REFERENCES 126
Dermal Fibroblasts 130
1. INTRODUCTION 130
1.1 Brief history of fibroblast culture 130
1.2 Ease and scope of fibroblast culture 131
2. FIBROBLAST ISOLATION 132
2.1 Fibroblast populations and tissue types 133
3. CULTURE 133
3.1 Monolayer culture 133
3.2 Fibroblast Culture Lifespan 139
3.3 Three-dimensional culture 145
4. ANALYTICAL METHODS FOR THE STUDY OF FIBROBLAST CULTURES 150
4.1 Methods for Determining Cell Proliferation 150
4.2 Mathematical description of culture growth 153
4.3 Viability 154
4.4 Extracellular Matrix Synthesis 155
5. PRACTICAL APPLICATIONS OF DERMAL FIBROBLAST CULTURES 160
5.1 Studies of the properties of primary cultures 160
5.2 Fibroblast interactions with other cell types and feeder cultures 160
5.3 Tissue engineering of connective tissues other than dermis 161
5.4 Therapeutic and testing applications of threedimensional cultures 161
5.5 Extracellular matrix production 162
5.6 Gene therapy applications 162
6. CONCLUSION 163
ACKNOWLEDGEMENTS 163
REFERENCES 163
Adipose Tissue 178
1. INTRODUCTION 178
1.1 Organization of human adipose tissue 179
1.2 Adipose tissue function 180
2. TISSUE PROCUREMENT AND PROCESSING 181
2.1 General comments 181
2.2 Isolation of pre-adipocytes and adipocytes 181
3. CULTURE TECHNIQUES 182
3.1 Adipose tissue explants 182
3.2 Mature adipocytes 182
3.3 Pre-adipocytes 183
3.4 Pre-adipocyte differentiation 183
4. ASSAY TECHNIQUES 184
4.1 Morphological features of human pre-adipocyte differentiation 185
5. UTILITY OF THE SYSTEM 189
6. SUMMARY 189
REFERENCES 190
Mesenchymal Stem Cells 193
1. INTRODUCTION 193
2. TISSUE PROCUREMENT AND PROCESSING 195
2.1 Safety from Biohazards - Universal Precautions 195
2.2 Sources of hMSCs 196
2.3 Marrow Processing 196
3. CULTURE TECHNIQUES 198
3.1 hMSC Expansion in culture 198
3.2 Freezing cultured hMSCs 199
4. ASSAY TECHNIQUES 200
4.1 Flow Cytometry 200
4.2 Differentiation Assays 202
4.3 Differentiation Assays 206
5. UTILITY OF THE SYSTEM 206
6. CONCLUDING REMARKS 207
REFERENCES 208
Peripheral Blood Fibrocytes 212
1. INTRODUCTION 212
2. TISSUE PROCUREMENT AND PROCESSING 216
3. CULTURE TECHNIQUES 217
4. ASSAY TECHNIQUES 218
5. UTILITY OF SYSTEM 220
6. CONCLUDING REMARKS 221
REFERENCES 221
Osteoblasts 223
1. INTRODUCTION 223
1.1 Rationale for the use of isolated bone cells 223
2. TISSUE PROCUREMENT AND PROCESSING 227
3. CULTURE TECHNIQUES 227
4. ASSAY TECHNIQUES 232
4.1 HOB Characterization 232
5. UTILITY OF SYSTEM 236
6. CONCLUDING REMARKS 238
ACKNOWLEDGMENTS 238
REFERENCES 238