Establishment of human skin organotypic cultures (skin-equivalents) as suitable bioassay models and investigation of dermal-epidermal interactions by using the culture system

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In vitro models of human skin utilizing keratinocytes grown upon fibroblast-contracted collagen gels have been known to demonstrate similar behavior to the cells in vivo. The purpose of our study is to provide suitable bioassay models for investigating the structure and function of epidermal or dermal tissue and to determine the dermal-epidermal interactions required for keratinocyte function, basement membrane formation and hair follicle formation utilizing the three-dimensional organotypic cultures such as skin-equivalents (SEs). Despite production of the epidermal basement membrane (BM) components, BM was rarely observed in SEs. Analyses through SEs have revealed that matrix metalloproteinases (MMPs) and plasmin are involved in BM formation. To elucidate the affect of these proteinases, we examined effects of inhibitors for MMPs and serine proteinase on the BM formation and the epidermal structure and differentiation in the SEs. By addition of both inhibitors the epidermis was well … More organized with multilayered keratinocytes consisting of basal, spinous, granular, and cornified layers. The epidermal differentiation markers were expressed in the upper layer of the epidermis and the sharply defined depositions specific for BM components were observed at the dermal-epidermal junction. Ultrastructurally, the extended and continuous lamina densa with approximately 30 nm thick was observed underneath the basal keratinocytes. These results suggest that the assembly of BM under the basal keratinocytes and the differentiation of epidermis are extensively promoted under the influence of inhibitors for MMPs and serine proteinases. In attempt on hair follicle formation by using this culture system, mouse dermal papilla cells were cultured at the dermal-epidermal interface in SEs. Cellular masses including the dermal papilla cells were frequently observed in the upper part of the dermis. Further studies are needed to determine whether the cellular masses are similar to primitive hair follicles. Less