Characterization of the differentiated phenotype of an organotypic model of skin derived from human keratinocytes and dried porcine dermis.
A number of skin models have been developed, but a simple method for rapidly producing large quantities of differentiated epidermis has been missing. We show the differentiated phenotype of human keratinocytes in organotypic culture arising in vitro by air-exposure of keratinocytes cultured with feeders on dried pig dermis. Keratinocytes were seeded at low density on the dermis covered with irradiated NIH-3T3 feeder cells and after reaching confluence lifted to the air-medium interface. A well differentiated epidermis with distinct basal, spinous, granular and stratum corneum layers was formed within 1 week. In this way, 100 cm2 of the differentiated recombined human/pig skin (D-RHPS) can be obtained from 106 secondary keratinocytes in 14 days. The entire keratinocyte life cycle takes place on the dermal substrate - from single cells to stratified epidermis. The differentiation was characterized using a panel of monoclonal antibodies. Similarly as in the normal skin, keratin 14 was expressed in all cell layers, keratin 10 in suprabasal layers, beta1-integrin and epitopes to antibody LH8 in the basal layer, involucrin and transglutaminase in the granular and horny layer of the epidermis. Keratins 16 and 7/17, which are absent in the normal epidermis, but present suprabasally in the psoriatic one, were expressed strongly in all suprabasal layers and in a subpopulation of basal cells. The keratinocytes can be combined with two other cell types cultured either on the dermal side of the dermis and/or on the bottom of the dish. It appears that this simple skin model can be used in studies of epithelial/mesenchymal interactions and interactions between epidermal cells and infectious agents. It may be particularly useful for the study of human papilloma viruses.
Matousková
E
E
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague. matous@img.cas.cz
McKay
I
I
Povýsil
C
C
Königová
R
R
Chaloupková
A
A
Veselý
P
P
D016475:3T3 Cells; D000818:Animals; D015415:Biomarkers; D002454:Cell Differentiation; D018920:Coculture Techniques; D046508:Culture Techniques; D020405:Dermis / Q000033:anatomy & histology; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D007633:Keratins / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D010641:Phenotype; D012867:Skin / Q000033:anatomy & histology* / Q000166:cytology* / Q000378:metabolism; D013552:Swine
D015415:Biomarkers; D007633:Keratins