CULTURE OF RECONSTRUCTED EPIDERMIS IN A DEFINED MEDIUM AT 33-DEGREES-C SHOWS A DELAYED EPIDERMAL MATURATION, PROLONGED LIFE-SPAN AND IMPROVED STRATUM-CORNEUM

In this study we compared human keratinocyte cultures grown at the air -liquid interface on de-epidermized dermis at 33 degrees C or at 37 de grees C in two different culture media: medium I - a fully defined ser um- and EGF-free medium; and medium II - a serum- and EGF-containing m edium, Cultures grown in medium II were initially hyperproliferative f ollowed rapidly by senescence, and had a high triglyceride content. Th e hyperproliferation was ascribed to the presence of EGF in the medium , In contrast, cultures grown in medium I at 33 degrees C showed a gre atly improved balance between cell proliferation and differentiation. They had a prolonged lifespan of at least 32 days without a significan t decrease in the number of living cell layers, a rate of proliferatio n similar to that of native epidermis and a low triglyceride content, Culturing at 37 degrees C increased the rate of differentiation withou t affecting the rate of proliferation, Furthermore, both at 33 degrees C and at 37 degrees C, keratin 6 was expressed only in the first supr abasal layer but was expressed in all suprabasal layers in cultures gr own in medium II, High keratin 6 expression was not directly linked to hyperproliferation but to deregulated terminal differentiation. Invol ucrin, transglutaminase and SPRR1 were abnormally expressed irrespecti ve of the culture conditions used, whereas SKALP expression was decrea sed in cultures grown in medium I, The epidermal lipid profile was bet ter in cultures grown in medium I; the relative amounts of ceramides, free fatty acids and cholesterol being comparable to native epidermis, Small-angle X-ray diffraction showed a slightly improved structural o rganization of stratum corneum lipids as demonstrated by the appearanc e of second-and third-order peaks of the 12-nm long phase and a marked reduction in the polycrystalline cholesterol peak.