Noll, M., M-L merkle, M. Kandsberger, T. Matthes, H. Fuchs, and T. Graeve. Reconstructed Human Skin (AST-2000) as a Tool for Pharmacotoxicology. ATLA 27: 302. 1999.

Specific cells have been isolated from organs and cultivated in culture, as well as applied in biological, medical and pharmaceutical research and technology. However, such in vitro processes have mainly been performed with single cells. A further development is the reconstruction of three-dimensional cell structures such as the living human skin model presented here. We have developed a model of reconstructed, bilayered human skin (AST-2000). Like natural human skin, AST-2000 consists of a lower dermal layer composed of dermal fibroblasts embedded in a biomatrix, and an overlying cornified epidermal layer of living human keratinocytes. For standardization purposes, dermal fibroblasts were isolated from human bioptic material, serially subcultured, and cryopreserved for further use. Both commercially available keratinocytes and primary keratinocyte cultures were used in the construction of epidermis. Fibroblasts were embedded in the biomatrix in culture insets. Keratinocytes were seeded on this biomatrix and were cultured in a well-defined medium first submersed, followed by culture at the gas/medium interface. Histologic sections were prepared and specific staining techniques were used to analyse the tissue structure, the microstructure, and the growth and differentiation behaviour of the skin model. The model showed very good correlation with the in vivo skin. Skin damage or cytotoxicity was measured as decreased tetrazolium vital dye metabolism. Test substances were applied topically to the stratum corneum of the reconstructed skin. In time-course, or concentration course, experiments the irritation potential of several substances was evaluated. The results show a good correlation to the human patch test and rabbit skin test. We conclude that the in vitro assay using the AST-2000 skin model is a promising alternative to in vivo animal testing for pharmacological, immunological and molecular biological investigations.