Tissue engineering with HaCaT cells and a fibroblast cell line

Most skin models consist of primary cells. Our aim was to develop a highly reproducible skin model consisting only of cell lines to investigate irradi ation effects. The spontaneously immortalized human keratinocyte line HaCaT is known for its capacity for epidermal differentiation. As an organotypic coculture, HaCaT cells were grown air-exposed on top of a dermis equivalen t consisting of a murine fibroblast cell line (L929) in collagen. The techn ique for the preparation of this coculture system is described. After 3 wee ks a multilayered epithelium with signs of differentiation developed. The e xpression of several markers for differentiation and basal membrane formati on were compared with those of healthy human skin by immunohistochemical st aining, In the epithelium of the skin model several cytokeratins, especiall y keratin 10, and involucrin were expressed comparable to normal skin. Lami nin expression was found along the basal zone of the epithelium. BrdU label ing indicating proliferation was mainly found in the basal parts of the epi thelium. Differentiated cells showing DNA fragmentation were detected in th e upper parts of the epithelium by the TUNEL assay. Fluorescence in situ hy bridization was used to discriminate between HaCaT and L929 cells in the co culture, Some L929 cells growing on top of the epithelium could be detected . This might have been due to an invasion of highly proliferating L929 cell s and might be one of the limits of tissue engineering with cell lines. In conclusion, the organotypic coculture used as a skin model is a promising a dditional tool for addressing specific research questions.