HIGHLY DIFFERENTIATED SKIN EQUIVALENT MODEL FOR HIGH THROUGHPUT SCREENING OF TOPICAL THERAPEUTICS.
EpiDerm™ is an in vitro organotypic human skin model cultured at the air/liquid interface to produce a three-dimensional tissue with a stratum corneum possessing a barrier function similar to native epidermis. The model is suitable for topical application of creams or lotions, and has found use in applications including cytotoxicity, irritation, and/or efficacy screening of topical pharmaceutical or cosmetic products and ingredients. EpiDerm is presently produced on cell culture inserts, which can be handled and manipulated individually. The current abstract describes two experimental formats in which the tissues are cultured on one-piece 24-well or 96-well plates suitable for high-throughput applications. Histological evaluations show the experimental formats produce tissues which are indistinguishable from the standard format. To demonstrate the utility of the model, TNF-α secretion in response to topically applied phorbol ester (PMA) was measured by ELISA. PMA is a powerful tumor promoter which acts primarily via interaction with protein kinase C and has been shown to upregulate TNF-α expression. PMA treatment resulted in a 1.7-2.4 fold increase in TNF-α secretion but had no effect on tissue viability, as indicated by the MTT assay. In addition, high quality total RNA (absorbance ratios at 260/280 nm were 1.8 – 2.1; agarose gel electrophoresis ribosomal RNA bands were distinct and well defined) was extracted and purified from the tissues utilizing a procedure based on GSCN lysis and immobilization on glass-fiber filters. RNA yields averaged 46.2 ug/cm2 tissue. These data indicate that the new experimental EpiDerm formats may find utility in high-throughput screening applications such as in monitoring mRNA levels or the effects of PKC inhibitors and/or similar pharmaceutically important cellular targets.
EpiDerm, HTS, High throughput screening, Highly differentiated skin equivalent, MTT, MTT ET-50 tissue viability assay, MTT assay, TNF-a, Topical therapeutics
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