In Vitro Approaches
The complexity of cell culture models varies from monolayers for adherent cells or suspensions for nonadherent cells, to cocultures or to epidermal reconstructs (3D model). Cocultures and epidermal reconstructs allow the study of paracrine or contact cell
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2.2.5
Muriel Cario-André and Maria Lucia Dell’Anna
Contents
Core Messages
2.2.5.1 Cell Isolation and Culture .................................... 219 Isolation of Skin Melanocytes and Keratinocytes ... Isolation and Culture of Skin Fibroblasts ............... Isolation and Culture of Hair Follicle Melanocytes and Keratinocytes .............................. Isolation and Freezing of Peripheral Blood Mononuclear cells (PBMC) .........................
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220 220 220 221
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2.2.5.2 In Vitro Reconstructed Epidermis ...................... 221 Preparation of Dead De-Epidermized Dermis ........ 221 Epidermal Reconstruction ....................................... 221
› 2.2.5.3 Functional Studies of NSV Cells Using Monolayers: Melanocytes, Keratinocytes, and Fibroblasts ...................................................... 222 2.2.5.4 Functional Studies Using Reconstructed Epidermis ............................................................... 223 2.2.5.5 New Analytic Techniques ..................................... 225 Fluorescence-Based Assays .................................... Proteomic ................................................................ Metabolomic/Lipidomic ......................................... Transcriptomic ........................................................
225 228 228 228
2.2.5.6 Concluding Remarks ............................................ 228 References ........................................................................... 228
M. Cario-André (*) Inserm U876, Centre de référence des maladies rares de la peau, Université V Segalen Bordeaux 2, Bordeaux, France e-mail: [email protected]
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The complexity of cell culture models varies from monolayers for adherent cells or suspensions for nonadherent cells, to cocultures or to epidermal reconstructs (3D model). Cocultures and epidermal reconstructs allow the study of paracrine or contact cell–cell effects. The tests on nonepidermal nonadherent cells, such as peripheral blood mononuclear cells (PBMC) expand the view, going beyond melanogenesis-associated metabolisms. The in vitro studies on hair follicle melanocytes help understand the maturation and differentiation of melanocytes and to unveil the defective steps of growth and migration in melanocyte precursors during the repigmentation. New analytic techniques are emerging which can be applied for investigating vitiligo.
2.2.5.1 Cell Isolation and Culture Melanocytes and keratinocytes can be cultured from both skin and hair follicle. Indeed, epidermal stem cells occur in the basal layer of the epidermis and within hair follicles. Follicular keratinocyte stem cells are situated in the bulge area, whereas those for melanocytes are found in the sub-bulge area.
M. Picardo and A. Taïeb (eds.), Vitiligo, DOI 10.1007/978-3-540-69361-1_2.2.5, © Springer-Verlag Berlin Heidelberg 2010
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Isolation of Skin Melanocytes and Keratinocytes Vitiligo cells are obtained from lesional or nonlesional skin of generalized vitiligo patients. The method for th
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