Industry experience with alternative methods. Chemical safety for the 21st century

Reconstruction ofa skin equivalent using an immortalized human keratinocyte line, HaCaT, was investigated in an attempt to generate an in vitro system representative for human skin. Three different substrates were used to establish air-exposed cultures of HaCaT cells: de-epidermized dermis, collagen gels, and filter inserts. Effects of variations in culture conditions on tissue morphology, on the expression of proliferation-specific and differentiation-specific protein markers, and on lipid profiles were investigated. When grown at the air-liquid interface HaCaT cells initially developed a multilayered epithelium, but during the course of culture marked alterations in tissue architecture were observed. Ultrastructurally, a disordered tissue organization was evident as judged from the presence of rounded cells with abnormally shaped nuclei. Keratins K1 and K10 were irregularly expressed in all cell layers, including stratum basale. Staining of K6/K16 was evident in all cell layers. Locally, basal and suprabasal cells were positive for K4 and additionally expressed K13 and K19. The cornified envelope precursors were expressed only in older cultures (>2 wk after air exposure), except for transglutaminase and small proline rich protein 1, which were irregularly expressed in both early and older cultures. In addition, HaCaT cells showed an impaired capacity to synthesize lipids that are necessary for a proper barrier formation as indicated by the absence of free fatty acids and a very low content and incomplete profile of ceramides. Our data demonstrate that the ultimate steps of terminal differentiation in HaCaT cells do not occur irrespective of the type of substrate or the culture conditions.