Improvement of a Three-Layered in vitro Skin Model for Topical Application of Irritating Substances

<p>In the field of skin tissue engineering, the development of physiologically relevant <italic>in vitro</italic> skin models comprising all skin layers, namely epidermis, dermis, and subcutis, is a great challenge. Increasing regulatory requirements and the ban on animal experiments for substance testing demand the development of reliable and <italic>in vivo</italic>-like test systems, which enable high-throughput screening of substances. However, the reproducibility and applicability of <italic>in vitro</italic> testing has so far been insufficient due to fibroblast-mediated contraction. To overcome this pitfall, an advanced 3-layered skin model was developed. While the epidermis of standard skin models showed an 80% contraction, the initial epidermal area of our advanced skin models was maintained. The improved barrier function of the advanced models was quantified by an indirect barrier function test and a permeability assay. Histochemical and immunofluorescence staining of the advanced model showed well-defined epidermal layers, a dermal part with distributed human dermal fibroblasts and a subcutis with round-shaped adipocytes. The successful response of these advanced 3-layered models for skin irritation testing demonstrated the suitability as an <italic>in vitro</italic> model for these clinical tests: only the advanced model classified irritative and non-irritative substances correctly. These results indicate that the advanced set up of the 3-layered <italic>in vitro</italic> skin model maintains skin barrier function and therefore makes them more suitable for irritation testing.</p>