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 irradiation 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 equivalent consisting of a murine fibroblast cell line (L929) in collagen. The technique for the preparation of this coculture system is described. After 3 weeks a multilayered epithelium with signs of differentiation developed. The expression of several markers for differentiation and basal membrane formation were compared with those of healthy human skin by immunohistochemical staining. In the epithelium of the skin model several cytokeratins, especially keratin 10, and involucrin were expressed comparable to normal skin. Laminin expression was found along the basal zone of the epithelium. BrdU labeling indicating proliferation was mainly found in the basal parts of the epithelium. Differentiated cells showing DNA fragmentation were detected in the upper parts of the epithelium by the TUNEL assay. Fluorescence in situ hybridization was used to discriminate between HaCaT and L929 cells in the coculture. Some L929 cells growing on top of the epithelium could be detected. This might have been due to an invasion of highly proliferating L929 cells 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 additional tool for addressing specific research questions.