In vitro reconstitution of differentiated human epithelia

By Pellegrini, G. - Gherzi, R. - Franzi, A.T. - D'Anna, F. - De Luca, M. - Cancedda, R.

Human keratinocytes obtained from skin biopsies can be serially cultured in vitro. When plated on lethally irradiated 3T3 fibroblasts, keratinocyte colonies reconstitute a stratified squamous epithelium devoid of stratum corneum. The expression of a mature cornified epidermis, expressing all the morphological and biochemical markers of the in vivo epidermis, can be obtained by the "emerged dermal equivalent" culture system. Melanocytes grown under the same culture conditions, maintain a physiological melanocyte/keratinocyte ratio, are organised in the basal layer of the cultured epidermis, and maintain differentiated functions such as dendritic arborisation, melanin synthesis and melanosome transfer. This allows the reconstitution of an epidermis physiologically populated by functionally active melanocytes. Epithelial cells from different mucosal body sites, namely palate, urethra, conjunctiva, cornea and vagina, can also be cultured and maintain the characteristics of the original donor sites. The in vitro reconstituted human epithelia, permanently transplanted onto patients presenting large epidermal or mucosal defects, retain the characteristics of the original donor site, suggesting an intrinsic site-specific differentiation programme. These three-dimensional human epithelium models could prove useful in standard cytotoxicity assays and could be used as a tool to study the effects of a variety of compounds on normal human epithelia in vitro.