Human Cell Culture Protocols , Third Edition

Human cell culture is not a new topic, but the development of new molecular techniques and reagents which can be used to investigate cell function and the responsible intracellular mechanisms make it a continuing requirement. This third edition of __Human Cell Culture Protocols__ expands upon the previous editions with current, detailed protocols for the isolation and culture of a range of primary cells from human tissues. With new chapters on pancreatic cells needed for basic studies on the pathogenesis of diabetes and for their application for islet transplantation, the book also delves into protocols for hepatocytes, skin cells, lung cells, parathyroid cells, gastric cells, renal cells, adipocytes, ovarian cells, bone cells, vascular smooth muscle cells, vascular endothelial cells, regulatory T cells, blood mononuclear cells, as well as new techniques being applied to human cell culture, particularly the use of biocompatible scaffolds to grow cells, the __in vitro__ use of laser microdissection to isolate cells from culture, and automated cell culture. Written in the highly successful __Methods in Molecular BiologyTM__ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, __Human Cell Culture Protocols, Third Edition__ makes it possible for a worker with basic cell culture training, whether in the fields of cell biology, gene therapy, and cell transplantation, to prepare cell cultures of the specific cell type necessary to forward their vital research. 001front-matter......Page 1 Human Cell Culture Protocols......Page 3 Dedication......Page 5 Preface......Page 7 Contents......Page 9 Contributors......Page 11 INDEX......Page 15 1. Background......Page 24 1.3. Biosafety......Page 25 2. Tissue Culture Laboratory: Design and Layout......Page 27 2.1.1. Laminar Flow Hood......Page 28 2.1.3. Centrifuge......Page 29 2.1.5. Autoclave......Page 30 3. Aseptic Technique......Page 31 4.1. Media......Page 32 4.2.3. Growth Curve......Page 33 4.2.5. Cryopreservation......Page 34 4.2.6. Cell Viability and Activity Assays......Page 35 References......Page 36 1. Introduction......Page 37 2.1. Reagent Setup......Page 38 2.3. Equipment......Page 40 3.1. MEF-Conditioned HES Medium......Page 41 3.2. L-Wnt3a-Conditioned Medium for Melanocyte Differentiation Medium (Mel-1)......Page 42 3.3. Dermal Stem Cell Culture......Page 43 3.4.2. Three-Dimensional Skin Reconstruct Culture......Page 44 3.4.3. Immunostaining......Page 46 3.5.2. Differentiated Melanocytes Express Melanocytic Markers......Page 47 4. Notes......Page 49 References......Page 50 1. Introduction......Page 52 2.1. Isolation and Purification......Page 54 2.2. Cultivation and Differentiation......Page 55 3. Methods......Page 56 3.1. Isolation and Purification......Page 57 3.2. Cultivation and Differentiation......Page 59 3.3. Application......Page 60 4. Notes......Page 61 References......Page 63 1. Introduction......Page 64 2.1. Reagents......Page 66 3.1. Parathyroid Cell Dispersion......Page 67 3.3. Long-Term Cultures......Page 68 3.4. Cell Line Establishment......Page 69 4. Notes......Page 71 References......Page 74 1. Introduction......Page 75 2.3.3. MTT Assay......Page 77 2.4. Measurements of Dynamic Hormone Secretion......Page 78 3. Methods......Page 79 3.2.1. Dithizone Staining of Islet b -Cells (See Note 6)......Page 80 3.2.2. Electron Microscopy......Page 81 3.3.2. ATP Synthesis......Page 82 3.4. Quantification of Islet Hormone Content (See Note 13)......Page 83 3.5. Measurements of Dynamic Hormone Secretion (See Note 25)......Page 84 3.6.1. Intact Islets......Page 85 4. Notes......Page 87 References......Page 91 1. Introduction......Page 92 2.1. Human Islet Isolation and Purification......Page 93 2.3. Culture of Human Pancreatic Non-endocrine Cells......Page 94 3.1. Human Islet Isolation from Donor Pancreas......Page 96 3.3. The Differentiation of These Cells into Insulin-Producing Cells In Vitro......Page 100 3.4. Further Maturation of the Partial Differentiated Non-endocrine Cells In Vivo......Page 102 References......Page 103 1. Introduction......Page 105 2.1. Hepatocyte Culture and Treatment......Page 106 2.2. P450 Substrate Cocktail......Page 107 3. Methods......Page 108 3.4. Treatment of Cultured Hepatocytes with Inducers......Page 109 3.5. Incubation of Hepatocytes with the P450 Substrate Cocktail......Page 110 3.8. HPLC/MS/MS Analysis......Page 111 4. Notes......Page 113 References......Page 114 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 173 2.2. Epithelial Cell Isolation......Page 174 2.4.3. SDS-PAGE and Western Blot......Page 175 2.4.4. Gene Profiling......Page 177 3.2. Epithelial Dissociation for the Generation of HIEC and Seeding......Page 178 3.4. Epithelial Dissociation for the Generation of PCDE and Seeding......Page 179 3.6.1. Cell Proliferation......Page 180 3.6.2. Indirect Immunofluorescence......Page 181 3.6.3. SDS-PAGE and Western Blot......Page 183 3.6.4. Gene Profiling......Page 185 4. Notes......Page 187 References......Page 188 1. Introduction......Page 190 2.4. Immunohisto­chemistry......Page 192 3. Methods......Page 193 3.1. Isolation of PTECs and Fibroblasts from Human Renal Tissue Using a Density Gradient......Page 194 3.2. Isolation of PTECs from Human Renal Tissue Using a Sieving Method......Page 196 3.4. Characterizing Cells: Immunohistochemistry......Page 197 3.5. Functional Assays: Brush Boarder Enzymes......Page 198 3.6. Functional Assays: Induction of cAMP......Page 199 References......Page 200 1. Introduction......Page 201 2. Materials......Page 203 3.1. Isolation of Whole Glomeruli......Page 205 3.2. Isolation of Glomerular Epithelial Cells......Page 206 3.3. Glomerular Mesangial Cell Culture......Page 208 3.5.1. Cell Morphology......Page 209 3.5.2. Envision Horseradish Peroxidase Immunohistochemistry Technique......Page 210 4. Notes......Page 212 References......Page 214 1.1. The Role of Adipose Tissue......Page 216 1.3. Regional Variations in Adipocyte Biology......Page 217 2.2. Adipocyte Isolation......Page 218 2.3.2. Isolated Mature Adipocyte Culture......Page 219 3.3. Adipocyte Isolation......Page 220 3.4.2. Adipocyte Culture......Page 222 4. Notes......Page 223 References......Page 225 1. Introduction......Page 228 2.1. Buffers and Media......Page 230 3. Methods......Page 231 3.2. Cell Isolation......Page 232 3.5. Cell Propagation......Page 234 3.7. Oil-Red-O-Staining......Page 235 4. Notes......Page 236 References......Page 239 1. Introduction......Page 240 2.3. Culture of Human GC......Page 245 2.7. Culture of Human Corpus Luteum Cells......Page 246 3.1. Culture of Porcine GC......Page 247 3.3. Culture of Human GC......Page 249 3.4. Primary Culture of Porcine ThCs......Page 250 3.6.1. Dispersion of Luteal Cells Without Enzyme Treatment......Page 251 3.7. Culture of Human Corpus Luteum Cells......Page 252 3.8.2. Histochemical Assay for Detection of Activity of 3-Beta-Hydroxysteroid Dehydrogenase......Page 253 3.8.4. Immunocyto­chemical Assay for Detection of Androgen Receptor (AR): Part B......Page 254 4. Notes......Page 255 References......Page 261 1.1. Location of VSMCs in the Blood Vessel Wall......Page 263 1.2. VSMC Phenotype and Importance in Disease......Page 264 1.3. VSMC Heterogeneity......Page 265 2.1. Media......Page 266 2.2. Culture Materials......Page 267 3.1.1. Explant Method......Page 268 3.1.3. Isolation of Microvascular VSMC......Page 269 3.1.5. Culture of VSMCs Within Intact Blood Vessels......Page 270 3.2.1. Cell Markers......Page 271 3.3. Passaging Cultures......Page 272 4. Notes......Page 273 References......Page 274 1. Introduction......Page 276 2.1. Culture Medium and Solutions for Isolating and Maintaining HUVEC Cultures......Page 277 2.3. Equipment......Page 279 3.2. Isolation and Primary Culture of Endothelial Cells......Page 280 3.3. Subculture of Endothelial Cells......Page 281 3.4. Characterisation of Endothelial Cells......Page 282 4. Notes......Page 283 Acknowledgements......Page 284 References......Page 285 1. Introduction......Page 286 2.1. General Equipment......Page 288 2.2. Tissue Culture Media, Supplements, and Other Reagents......Page 289 3.1. Human Peripheral Blood Mononuclear Cell Isolation......Page 290 3.2. Stimulation, Fixation, and Permeabilization Methodology......Page 291 3.3. Intracellular Phospho-Specific Flow Cytometry......Page 292 4. Notes......Page 293 References......Page 295 1. Introduction......Page 297 1.1.1. Phenotype......Page 298 1.1.2. Main Mechanism of Suppressive Function of CD4+CD25high Tregs......Page 300 2. Materials......Page 301 3.2. Isolation of CD4+CD25high Tregs by Dynal Magnetic Beads......Page 302 3.4. Culturing and Expansion of Human CD4+CD25high Tregs......Page 303 3.5.2. Proliferation Assay......Page 304 3.5.3. Intracellular Cytokine Staining......Page 305 4. Notes......Page 306 References......Page 307 1. Introduction......Page 310 2.2. Suspension and Three-Dimensional Culture Systems for Chondrocytes......Page 314 2.3.3. Proteoglycan Synthesis......Page 316 2.4. Analysis of mRNA......Page 317 2.5.3. Chromatin Immunoprecipitation (ChIP) Assays......Page 318 3.1. Isolation and Culture of Human Chondrocytes in Monolayer......Page 319 3.2.2. Suspension Culture Within Agarose......Page 322 3.2.4. Culture on 3D Scaffolds (See Note 11)......Page 323 3.2.5. Pellet Cultures......Page 324 3.3.1. Alcian Blue Staining......Page 325 3.3.3. Proteoglycan Synthesis......Page 326 3.3.4. Immuno cytochemistry......Page 327 3.3.5. Western Blotting......Page 328 3.4. Extraction and Analysis of RNA......Page 329 3.5. Analysis of Gene Transcription......Page 330 3.5.2. Cotransfections Using Plasmid Vectors for Expression of Recombinant Proteins......Page 331 3.5.4. Luciferase Assay......Page 332 3.5.6. Chromatin Immunoprecipitation Assay......Page 333 3.5.7. DNA Methylation Analysis......Page 335 4. Notes......Page 336 Acknowledgments......Page 341 References......Page 342 1. Introduction......Page 346 2.4. Phenotypic Characterisation......Page 348 3.1.1. Establishment of Primary Explant Cultures of Human Osteoblastic Cells......Page 349 3.1.2. Passaging Primary Cultures (E1–E1P1)......Page 352 3.1.3. Re-Plating Explant Cultures (E1–E2)......Page 353 3.1.5. Modifications to Basic Culture Sys tem: In Vitro Mineralisation......Page 354 3.2. Human Clonal Osteoblast Cell Lines......Page 355 3.3. Long-Term Storage of Cells......Page 356 3.4. Phenotypic Characteristics of Osteoblasts......Page 357 3.4.1. Alkaline Phosphatase Enzymatic Activity Assay......Page 358 3.4.2. Alkaline Phosphatase Histochemical Staining......Page 359 4. Notes......Page 360 References......Page 363 1. Introduction......Page 365 2.2. Tissue Culture......Page 366 2.4.1. Toluidine Blue Solution......Page 367 2.4.2. Tartrate-Resistant Acid Phosphatase Histochemistry......Page 368 3.1. Preparation of Dentine Discs and Coverslips......Page 369 3.2.1. Isolation of PBMC by Density Gradient......Page 370 3.2.2. Isolation of CD14+ Enriched PBMC......Page 371 3.3.1. Co-Culture......Page 372 3.3.2. Recombinant RANKL Cultures......Page 373 3.4. Characterization of Cells Produced in Human Osteoclast Cultures......Page 374 3.4.1. TRAP Staining......Page 376 3.4.2. Detection and Quantification of Resorption......Page 377 3.4.2.1. Toluidine Blue Staining......Page 378 3.4.2.3. Volume Measurements......Page 379 4. Notes......Page 380 References......Page 382 1. Introduction......Page 384 3.1. Osteoblast Isolation and Plating......Page 386 3.3. Exposure of Cells to Ho:YAG Laser Source......Page 387 4. Notes......Page 389 References......Page 390 2.1. HepG2 Cell Line......Page 391 2.2. Chemicals and Solutions......Page 392 2.4. Laser Microdissection Microscope and Accessories......Page 393 3.2. Laser Microdissection......Page 394 3.3. Total RNA Extraction......Page 395 3.4. DNA Extraction......Page 396 3.6. Isolating Cells for Re-Culturing......Page 397 References......Page 398 1. Introduction......Page 399 2.4. Flow Cytometry and Alkaline Phosphatase Activity Analysis......Page 401 3.1. Isolation of Mononuclear Cells......Page 402 3.3. Cell Output Assays......Page 403 3.3.1. Osteogenic Induction and Alkaline Phosphatase Activity Assay......Page 404 3.3.2. Flow Cytometry......Page 405 4.1. Notes for Automated hMSC Culture......Page 406 4.2. Additional Notes for Developing Variant Automated Protocols......Page 408 Appendix (Automated Protocols)......Page 410 References......Page 412 1. Introduction......Page 413 2.1. Isolation of MSCs......Page 416 2.3. MSC Differentiation Cocktails......Page 417 2.4. Phenotyping Differentiated Adherent MSC, Including Tinctorial Stains......Page 418 2.5. Immunohisto­chemical Analysis......Page 419 2.6. Electron Microscopy......Page 420 3. Methods......Page 421 3.1.1. Bone Marrow-Derived MSC......Page 422 3.2. Culture of MSCs......Page 423 3.4. Adipocyte Differentiation Assay......Page 424 3.6. Osteocytic Differentiation......Page 425 3.9. Antibody Staining for Cell Type Characterisation......Page 426 4. Conclusions......Page 427 5. Notes......Page 428 References......Page 430 Additional Reading......Page 432 1. Introduction to cell culture / Christina Philippeos, Robin D. Hughes, Anil Dhawan and Ragai R. Mitry 2. Isolation and cultivation of dermal stem cells that differentiate into functional epidermal melanocytes / Ling Li, Mizubo Fukunaaga 3. Isolation, cultivation, and application of human alveolar epithelial cells / Nicole Daum, Anna Kuehn, Stephanie Hein, Ulrich F. Schaefer, Hanno Huwer, and Claus-Michael Lehr 4. Culture of parathyroid cells / Peyman Björklund and Per Hellman 5. Functional analysis of human islets of langerhans maintained in culture / Shanta J. Persaud, Bo Liu, and Peter M. Jones 6. Conversion of non-endocrine human pancreatic cells to insulin-producing cells for treatment of diabetes / Min Zhao and Guo Cai Huang 7. Evaluation of cytochrome P450 activities in human hepatocytes in vitro / María José Gómez-Lechón, Agustín Lahoz, José V. Castell, and María Teresa Donato 8. Human hepatocytes: isolation, culture, and quality procedures / Daniel Knobeloch, Sabrina Ehnert, Lilianna Schyschka, Peter Büchler, Michael Schoenberg, Jörg Kleeff, Wolfgang E. Thasler, Natascha C. Nussler, Patricio Godoy, Jan Hengstler, and Andrea K. Nussler 9. Preclinical testing ov virotherapeutics for primary and secondary tumors of the liver / Martina Zimmermann, Timo Weiland, Michael Bitzer, and Ulrich M. Lauer 10. Isolation and functional studies of human fetal gastric epithelium in primary culture / Pierre Chailler, Jean-Franc̦ois Beaulieu, and Daniel Ménard 11. Isolation, characterization, and culture of normal human intestinal crypt and villus cells / Jean-Franc̦ois Beaulieu and Daniel Ḿenard 12. Primary culture of human renal proximal tubule epithelial cells and interstitial fibroblasts / Claire C. Sharpe and Mark E.C. Dockrell 13. Glomerular epithelial and mesangial cell culture and characterization / Heather M. Wilson and Keith N. Stewart 14. Culture of isolated human adipocytes and isolated adipose tissue / Kirstin A. Carwswell, Mi-Jeong Lee, and Susan K. Fried 15. Primary culture of human adipocyte precursor cells: expansion and differentiation / Thomas Skurk and Hans Hauner 16. Primary culture of ovarian cells for research on cell interactions in the hormonal control of steroidogenesis / Jerzy F. Galas 17. Human vascular smooth muscle cell culture / Diane Proudfoot and Catherine Shanahan 18. Culture of human endothelial cells from umbilical veins / Richard C.M. Siow 19. Human peripheral blood mononuclear cell culture for flow cytometric analysis of phosphorylated mitogen-activated protein kinases / Athanasios Mavropoulos, Daniel Smyk, Eirini I. Rigopoulou, and Dimitrios P. Bogdanos 20. Human CD4+CD25(high [superscript]) CD127 (low/neg [superscript]) regulatory T cells / Haibin Su, Maria Serena Longhi, Pengyun Wang, Diego Vergani, and Yun Ma 21. Human chondrocyte cultures as models of cartilage-specific gene regulation / Miguel Otero, Marta Favero, Cecilia Dragomir, Karim El Hachem, Ko Hashimoto, Darren A. Plumb, and Mary B. Goldring 22. Isolation and culture of human osteoblasts / Alison Gartland, Robin M.H. Rumney, Jane P. Dillon, and James A. Gallagher 23. Human osteoclast culture and phenotypic characterization / Ankita Agrawal, James A. Gallagher, and Alison Gartland 24. Effects of temperature generated from the holmium: Yag laser on human osteoblasts in monolayer tissue culture / Moustafa I. Hafez, Anne Sandison, Richard R. H. Coombs, Ian D. McCarthy, and Al-Shymaa M. Hafez 25. Laser microdissection microscopy: application to cell culture / Ahlam Mustafa, Cathy Cenayko, Ragai R. Mitry, and Alberto Quaglia 26. Automated adherent human cell culture (mesenchymal stem cells) / Robert Thomas and Elizabeth Ratcliffe 27. Culturing and differentiating human mesenchymal stem cells for biocompatible scaffolds in regenerative medicine / William R. Otto and Catherine E. Sarraf. Introduction To Cell Culture -- Isolation And Cultivation Of Dermal Stem Cells That Differentiate Into Functional Epidermal Melanocytes -- Isolation, Cultivation, And Application Of Human Alveolar Epithelial Cells -- Culture Of Parathyroid Cells -- Functional Analysis Of Human Islets Of Langerhans Maintained In Culture -- Conversion Of Non-endocrine Human Pancreatic Cells To Insulin-producing Cells For Treatment Of Diabetes -- Evaluation Of Cytochrome P450 Activities In Human Hepatocytes In Vitro -- Human Hepatocytes: Isolation, Culture, And Quality Procedures Hepatocytes In Vitro -- Human Hepatocytes: Isolation, Culture, And Quality Procedures -- Preclinical Testing Of Virotherapeutics For Primary And Secondary Tumors Of The Liver -- Isolation And Functional Studies Of Human Fetal Gastric Epithelium In Primary Culture. Isolation, Characterization, And Culture Of Normal Human Intestinal Crypt And Villus Cells -- Primary Culture Of Human Renal Proximal Tubule Epithelial Cells And Interstitial Fibroblasts -- Glomerular Epithelial And Mesangial Cell Culture And Characterization -- Culture Of Isolated Human Adipocytes And Isolated Adipose Tissue -- Primary Culture Of Human Adipocyte Precursor Cells: Expansion And Differentiation -- Primary Culture Of Ovarian Cells For Research On Cell Interactions In The Hormonal Control Of Steroidogenesis -- Human Vascular Smooth Muscle Cell Culture -- Culture Of Human Endothelial Cells From Umbilical Veins -- Human Peripheral Blood Mononuclear Cell Culture For Flow Cytometric Analysis Of Phosphorylated Mitogen-activated Protein Kinases -- Human Cd4+cd25(high) Cd127 (low/neg) Regulatory T Cells. Human Chondrocyte Cultures As Models Of Cartilage-specific Gene Regulation -- Isolation And Culture Of Human Osteoblasts -- Human Osteoclast Culture And Phenotypic Characterization -- Effects Of Temperature Generated From The Holmium: Yag Laser On Human Osteoblasts In Monolayer Tissue Culture -- Laser Microdissection Microscopy: Application To Cell Culture -- Automated Adherent Human Cell Culture (mesenchymal Stem Cells) -- Culturing And Differentiating Human Mesenchymal Stem Cells For Biocompatible Scaffolds In Regenerative Medicine. Edited By Ragai R. Mitry, Robin D. Hughes. Includes Bibliographical References And Index.