02348nas a2200229   4500000000100000000000100001008004100002260001500043653003500058653003700093653001300130653002300143653002900166100002100195700002200216700002000238245010400258856011300362490000600475520162300481022001402104       2020                            d  c2020-05-0810aalternatives to animal testing10aIn vitro skin irritation testing10aSubcutis10aTissue engineering10athree-layered skin model1 aFreia F. Schmidt1 aSophia Nowakowski1 aPetra J. Kluger00aImprovement of a Three-Layered in vitro Skin Model for Topical Application of Irritating Substances  uhttps://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2020.00388/full0 v83 a<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>  a2296-4185