In previous studies, we have shown that architecture and differentiation of human epidermis reconstructed by culturing dissociated keratinocytes on deepidermized dermis at the air-liquid interface resembles very much those of epidermis in vivo. The various types of epidermal layers are present with the appropriate differentiation markers, such as the bullous pemphigoid antigen, suprabasal keratins, involucrin, membrane-bound transglutaminase and filaggrin, positioned almost like in normal epidermis. At the ultrastructural level, heterogeneous keratohyalin granules and intra- and extracellular membrane-coating granules are observed. After 7 days of culture, a compact stratum corneum covers the reconstructed epidermis. In the present work, the barrier function of the reconstructed epidermis was evaluated by measuring fluxes of 3H-water. Our results show that under the various culture conditions tested, the presence of the reconstructed epidermis reduces dramatically the permeability of the deepidermized dermis although the skin equivalent exhibits a higher permeability than normal skin. Conditions that favor terminal differentiation of the keratinocytes such as maintaining the cultures in delipidized serum improve the barrier function. Reduction of the relative humidity has a similar effect, whereas the age of the culture is of no influence. Experiments are in progress to reconstruct human epidermis with a barrier function as efficient as in normal skin.

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