Human Cell Culture Protocols , Third Edition

Cover......Page 1
front-matter......Page 2
Human Cell Culture Protocols......Page 4
Dedication......Page 6
Preface......Page 8
Contents......Page 10
Contributors......Page 12
1. Background......Page 18
1.3. Biosafety......Page 19
2. Tissue Culture Laboratory: Design and Layout......Page 21
2.1.1. Laminar Flow Hood......Page 22
2.1.3. Centrifuge......Page 23
2.1.5. Autoclave......Page 24
3. Aseptic Technique......Page 25
4.1. Media......Page 26
4.2.3. Growth Curve......Page 27
4.2.5. Cryopreservation......Page 28
4.2.6. Cell Viability and Activity Assays......Page 29
References......Page 30
1. Introduction......Page 32
2.1. Reagent Setup......Page 33
2.3. Equipment......Page 35
3.1. MEF-Conditioned HES Medium......Page 36
3.2. L-Wnt3a-Conditioned Medium for Melanocyte Differentiation Medium (Mel-1)......Page 37
3.3. Dermal Stem Cell Culture......Page 38
3.4.2. Three-Dimensional Skin Reconstruct Culture......Page 39
3.4.3. Immunostaining......Page 41
3.5.2. Differentiated Melanocytes Express Melanocytic Markers......Page 42
4. Notes......Page 44
References......Page 45
1. Introduction......Page 48
2.1. Isolation and Purification......Page 50
2.2. Cultivation and Differentiation......Page 51
3. Methods......Page 52
3.1. Isolation and Purification......Page 53
3.2. Cultivation and Differentiation......Page 55
3.3. Application......Page 56
4. Notes......Page 57
References......Page 59
1. Introduction......Page 60
2.1. Reagents......Page 62
3.1. Parathyroid Cell Dispersion......Page 63
3.3. Long-Term Cultures......Page 64
3.4. Cell Line Establishment......Page 65
4. Notes......Page 67
References......Page 70
1. Introduction......Page 72
2.3.3. MTT Assay......Page 74
2.4. Measurements of Dynamic Hormone Secretion......Page 75
3. Methods......Page 76
3.2.1. Dithizone Staining of Islet b -Cells (See Note 6)......Page 77
3.2.2. Electron Microscopy......Page 78
3.3.2. ATP Synthesis......Page 79
3.4. Quantification of Islet Hormone Content (See Note 13)......Page 80
3.5. Measurements of Dynamic Hormone Secretion (See Note 25)......Page 81
3.6.1. Intact Islets......Page 82
4. Notes......Page 84
References......Page 88
1. Introduction......Page 90
2.1. Human Islet Isolation and Purification......Page 91
2.3. Culture of Human Pancreatic Non-endocrine Cells......Page 92
3.1. Human Islet Isolation from Donor Pancreas......Page 94
3.3. The Differentiation of These Cells into Insulin-Producing Cells In Vitro......Page 98
3.4. Further Maturation of the Partial Differentiated Non-endocrine Cells In Vivo......Page 100
References......Page 101
1. Introduction......Page 104
2.1. Hepatocyte Culture and Treatment......Page 105
2.2. P450 Substrate Cocktail......Page 106
3. Methods......Page 107
3.4. Treatment of Cultured Hepatocytes with Inducers......Page 108
3.5. Incubation of Hepatocytes with the P450 Substrate Cocktail......Page 109
3.8. HPLC/MS/MS Analysis......Page 110
4. Notes......Page 112
References......Page 113
1.1. Hepatocyte Isolation......Page 116
1.2. Functional Analysis......Page 118
1.3. Drug Metabolism......Page 119
2.1. Preparation of the Isolation Setup......Page 120
3.1. Isolation Procedure......Page 122
3.2. Percoll Purification......Page 124
3.3. Transaminase and Lactate-Dehydrogenase Measurement......Page 126
3.4. Urea Formation and NH4Cl Metabolism......Page 127
3.5. Glycolysis and Gluconeogenesis......Page 128
3.6. Sulforhodamine B Staining......Page 129
3.8. Periodic Acid Schiff Staining......Page 130
3.11. Preservation of Hepatocytes Polarity by 3D Extracellular Matrix......Page 132
4. Notes......Page 134
References......Page 136
1. Introduction......Page 138
2.3 SRB Assay......Page 139
2.5. Immunohisto chemistry and Confocal Analysis......Page 140
3.1.2. Infection of Human Hepatoma Cell Lines......Page 141
3.2.1 Quantification of Virus Effects via SRB Assay......Page 142
3.3.2. Quantification of LDH Leakage as a Cell Death Marker......Page 144
3.4.1. Generation of PCLS Using the Leica Vibrating Blade Microtome VT1200 S (Leica, Germany)......Page 146
3.4.2. Infection of PCLS......Page 148
3.4.4. Confocal Microscopy of Measles Vaccine Virus-Infected Tissue Slices......Page 149
4. Notes......Page 151
References......Page 152
1. Introduction......Page 154
2.3.1. Cell Growth......Page 156
2.3.5. Determination of Enzymatic Activities......Page 157
3. Methods......Page 159
3.1.2. Dissection and Dissociation......Page 160
3.2.2. Culture......Page 161
3.3.1. Cell Growth......Page 162
3.3.2. Dye Staining......Page 163
3.3.3. Immuno cytochemistry......Page 164
3.3.5. Determination of Enzymatic Activities......Page 165
3.4.1. EGF Supplementation......Page 166
3.4.2. Biological Matrix Coatings......Page 167
3.5.3. Quantification of Restitution......Page 169
4. Notes......Page 170
References......Page 171
1. Introduction......Page 174
2.2. Epithelial Cell Isolation......Page 175
2.4.3. SDS-PAGE and Western Blot......Page 176
2.4.4. Gene Profiling......Page 178
3.2. Epithelial Dissociation for the Generation of HIEC and Seeding......Page 179
3.4. Epithelial Dissociation for the Generation of PCDE and Seeding......Page 180
3.6.1. Cell Proliferation......Page 181
3.6.2. Indirect Immunofluorescence......Page 182
3.6.3. SDS-PAGE and Western Blot......Page 184
3.6.4. Gene Profiling......Page 186
4. Notes......Page 188
References......Page 189
1. Introduction......Page 192
2.4. Immunohisto­chemistry......Page 194
3. Methods......Page 195
3.1. Isolation of PTECs and Fibroblasts from Human Renal Tissue Using a Density Gradient......Page 196
3.2. Isolation of PTECs from Human Renal Tissue Using a Sieving Method......Page 198
3.4. Characterizing Cells: Immunohistochemistry......Page 199
3.5. Functional Assays: Brush Boarder Enzymes......Page 200
3.6. Functional Assays: Induction of cAMP......Page 201
References......Page 202
1. Introduction......Page 204
2. Materials......Page 206
3.1. Isolation of Whole Glomeruli......Page 208
3.2. Isolation of Glomerular Epithelial Cells......Page 209
3.3. Glomerular Mesangial Cell Culture......Page 211
3.5.1. Cell Morphology......Page 212
3.5.2. Envision Horseradish Peroxidase Immunohistochemistry Technique......Page 213
4. Notes......Page 215
References......Page 217
1.1. The Role of Adipose Tissue......Page 220
1.3. Regional Variations in Adipocyte Biology......Page 221
2.2. Adipocyte Isolation......Page 222
2.3.2. Isolated Mature Adipocyte Culture......Page 223
3.3. Adipocyte Isolation......Page 224
3.4.2. Adipocyte Culture......Page 226
4. Notes......Page 227
References......Page 229
1. Introduction......Page 232
2.1. Buffers and Media......Page 234
3. Methods......Page 235
3.2. Cell Isolation......Page 236
3.5. Cell Propagation......Page 238
3.7. Oil-Red-O-Staining......Page 239
4. Notes......Page 240
References......Page 243
1. Introduction......Page 244
2.3. Culture of Human GC......Page 249
2.7. Culture of Human Corpus Luteum Cells......Page 250
3.1. Culture of Porcine GC......Page 251
3.3. Culture of Human GC......Page 253
3.4. Primary Culture of Porcine ThCs......Page 254
3.6.1. Dispersion of Luteal Cells Without Enzyme Treatment......Page 255
3.7. Culture of Human Corpus Luteum Cells......Page 256
3.8.2. Histochemical Assay for Detection of Activity of 3-Beta-Hydroxysteroid Dehydrogenase......Page 257
3.8.4. Immunocyto­chemical Assay for Detection of Androgen Receptor (AR): Part B......Page 258
4. Notes......Page 259
References......Page 265
1.1. Location of VSMCs in the Blood Vessel Wall......Page 268
1.2. VSMC Phenotype and Importance in Disease......Page 269
1.3. VSMC Heterogeneity......Page 270
2.1. Media......Page 271
2.2. Culture Materials......Page 272
3.1.1. Explant Method......Page 273
3.1.3. Isolation of Microvascular VSMC......Page 274
3.1.5. Culture of VSMCs Within Intact Blood Vessels......Page 275
3.2.1. Cell Markers......Page 276
3.3. Passaging Cultures......Page 277
4. Notes......Page 278
References......Page 279
1. Introduction......Page 281
2.1. Culture Medium and Solutions for Isolating and Maintaining HUVEC Cultures......Page 282
2.3. Equipment......Page 284
3.2. Isolation and Primary Culture of Endothelial Cells......Page 285
3.3. Subculture of Endothelial Cells......Page 286
3.4. Characterisation of Endothelial Cells......Page 287
4. Notes......Page 288
Acknowledgements......Page 289
References......Page 290
1. Introduction......Page 292
2.1. General Equipment......Page 294
2.2. Tissue Culture Media, Supplements, and Other Reagents......Page 295
3.1. Human Peripheral Blood Mononuclear Cell Isolation......Page 296
3.2. Stimulation, Fixation, and Permeabilization Methodology......Page 297
3.3. Intracellular Phospho-Specific Flow Cytometry......Page 298
4. Notes......Page 299
References......Page 301
1. Introduction......Page 304
1.1.1. Phenotype......Page 305
1.1.2. Main Mechanism of Suppressive Function of CD4+CD25high Tregs......Page 307
2. Materials......Page 308
3.2. Isolation of CD4+CD25high Tregs by Dynal Magnetic Beads......Page 309
3.4. Culturing and Expansion of Human CD4+CD25high Tregs......Page 310
3.5.2. Proliferation Assay......Page 311
3.5.3. Intracellular Cytokine Staining......Page 312
4. Notes......Page 313
References......Page 314
1. Introduction......Page 318
2.2. Suspension and Three-Dimensional Culture Systems for Chondrocytes......Page 322
2.3.3. Proteoglycan Synthesis......Page 324
2.4. Analysis of mRNA......Page 325
2.5.3. Chromatin Immunoprecipitation (ChIP) Assays......Page 326
3.1. Isolation and Culture of Human Chondrocytes in Monolayer......Page 327
3.2.2. Suspension Culture Within Agarose......Page 330
3.2.4. Culture on 3D Scaffolds (See Note 11)......Page 331
3.2.5. Pellet Cultures......Page 332
3.3.1. Alcian Blue Staining......Page 333
3.3.3. Proteoglycan Synthesis......Page 334
3.3.4. Immuno cytochemistry......Page 335
3.3.5. Western Blotting......Page 336
3.4. Extraction and Analysis of RNA......Page 337
3.5. Analysis of Gene Transcription......Page 338
3.5.2. Cotransfections Using Plasmid Vectors for Expression of Recombinant Proteins......Page 339
3.5.4. Luciferase Assay......Page 340
3.5.6. Chromatin Immunoprecipitation Assay......Page 341
3.5.7. DNA Methylation Analysis......Page 343
4. Notes......Page 344
Acknowledgments......Page 349
References......Page 350
1. Introduction......Page 354
2.4. Phenotypic Characterisation......Page 356
3.1.1. Establishment of Primary Explant Cultures of Human Osteoblastic Cells......Page 357
3.1.2. Passaging Primary Cultures (E1–E1P1)......Page 360
3.1.3. Re-Plating Explant Cultures (E1–E2)......Page 361
3.1.5. Modifications to Basic Culture Sys tem: In Vitro Mineralisation......Page 362
3.2. Human Clonal Osteoblast Cell Lines......Page 363
3.3. Long-Term Storage of Cells......Page 364
3.4. Phenotypic Characteristics of Osteoblasts......Page 365
3.4.1. Alkaline Phosphatase Enzymatic Activity Assay......Page 366
3.4.2. Alkaline Phosphatase Histochemical Staining......Page 367
4. Notes......Page 368
References......Page 371
1. Introduction......Page 374
2.2. Tissue Culture......Page 375
2.4.1. Toluidine Blue Solution......Page 376
2.4.2. Tartrate-Resistant Acid Phosphatase Histochemistry......Page 377
3.1. Preparation of Dentine Discs and Coverslips......Page 378
3.2.1. Isolation of PBMC by Density Gradient......Page 379
3.2.2. Isolation of CD14+ Enriched PBMC......Page 380
3.3.1. Co-Culture......Page 381
3.3.2. Recombinant RANKL Cultures......Page 382
3.4. Characterization of Cells Produced in Human Osteoclast Cultures......Page 383
3.4.1. TRAP Staining......Page 385
3.4.2. Detection and Quantification of Resorption......Page 386
3.4.2.1. Toluidine Blue Staining......Page 387
3.4.2.3. Volume Measurements......Page 388
4. Notes......Page 389
References......Page 391
1. Introduction......Page 394
3.1. Osteoblast Isolation and Plating......Page 396
3.3. Exposure of Cells to Ho:YAG Laser Source......Page 397
4. Notes......Page 399
References......Page 400
2.1. HepG2 Cell Line......Page 402
2.2. Chemicals and Solutions......Page 403
2.4. Laser Microdissection Microscope and Accessories......Page 404
3.2. Laser Microdissection......Page 405
3.3. Total RNA Extraction......Page 406
3.4. DNA Extraction......Page 407
3.6. Isolating Cells for Re-Culturing......Page 408
References......Page 409
1. Introduction......Page 410
2.4. Flow Cytometry and Alkaline Phosphatase Activity Analysis......Page 412
3.1. Isolation of Mononuclear Cells......Page 413
3.3. Cell Output Assays......Page 414
3.3.1. Osteogenic Induction and Alkaline Phosphatase Activity Assay......Page 415
3.3.2. Flow Cytometry......Page 416
4.1. Notes for Automated hMSC Culture......Page 417
4.2. Additional Notes for Developing Variant Automated Protocols......Page 419
Appendix (Automated Protocols)......Page 421
References......Page 423
1. Introduction......Page 424
2.1. Isolation of MSCs......Page 427
2.3. MSC Differentiation Cocktails......Page 428
2.4. Phenotyping Differentiated Adherent MSC, Including Tinctorial Stains......Page 429
2.5. Immunohisto­chemical Analysis......Page 430
2.6. Electron Microscopy......Page 431
3. Methods......Page 432
3.1.1. Bone Marrow-Derived MSC......Page 433
3.2. Culture of MSCs......Page 434
3.4. Adipocyte Differentiation Assay......Page 435
3.6. Osteocytic Differentiation......Page 436
3.9. Antibody Staining for Cell Type Characterisation......Page 437
4. Conclusions......Page 438
5. Notes......Page 439
References......Page 441
Additional Reading......Page 443
back-matter_INDEX......Page 444