The living skin equivalent as a model in vitro for ranking the toxic potential of dermal irritants

Abstract

The living skin equivalent (LSE) is an organotypic co-culture composed of human dermal fibroblasts in a collagen-containing matrix overlaid with human keratinocytes that have formed a stratified epidermis. This model system was used as a dermatotoxicity model in vitro for studying the effects of test samples topically applied to the air-exposed epidermis. Using the colorimetric thiazolyl blue (MTT) conversion assay as a measure of mitochondrial function, the extent of cytotoxicity induced by several well-characterized chemical irritants was evaluated in the LSE. For the seven chemical irritants tested, the concentrations that inhibited MTT conversion by 50% were approximately those threshold concentrations at which irritation was seen in human skin. In addition, nine chemicals that were classified as non-irritating to human skin, including solids and water-insoluble substances, exhibited minimal or no inhibition of MTT conversion when tested at full strength. Time- and dose-dependent release of the pro-inflammatory mediators, prostaglandin E2, prostacyclin and interleukin-1-alpha (IL-1α) was seen in LSE exposed to two known chemical irritants, morpholine and hydroxylamine sulphate, but not in LSE exposed to a minimally irritating test sample. The permeability constants ( K p values) for water, caffeine, hydrocortisone, oestradiol and benzoic acid were determined in the LSE. In all cases the LSE was more permeable than human skin with K p values ranging from 0.56 ± 0.10 × 10 −3 cm/hr for hydrocortisone to 29 ± 5 × 10 −3 cm/hr for water penetration. The data suggest that organotypic skin cultures can be used as model systems for studying certain aspects of chemically induced dermal irritation. Using rates of water penetration as the most meaningful assessment of barrier competence, the LSE is approximately 30-fold more permeable than human skin. Although incomplete as a percutaneous absorption model, the presence of this partial barrier does influence the responses of cells in the LSE to topically applied chemicals.