In vitro cultured human skin cells as alternatives to animals for skin irritancy screening

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Abstract
In the last few years a lot of attention has been paid to the development of the in vitro models which would substitute for animals in cutaneous irritancy studies. These models explore either organ or explant cultures using freshly excised skin or serial cultures of isolated skin cells (epidermal keratinocytes or dermal fibroblasts). The organ or explant models are suitable only for short exposures of skin samples to the compounds tested and the use of it will always be restricted by the limited availability of fresh human skin. The model that uses submerged cultures of keratinocytes or fibroblasts permits the production of a large number of cells, and permits large scale toxicity screening tests with many substances, that can be applied in a broad concentration range. Since the stratum corneum is absent in conventional (submerged) keratinocyte culture systems, this model is mainly suited for testing of water soluble compounds and it is less suitable for poorly soluble compounds and for topical products consisting of complex formulations which are made of active ingredients and their vehicles. This shortcoming can be overcome by using ‘organotypic cultures’in which keratinocytes are grown at the air-liquid interface on a suitable dermal substrate. Under these conditions, the culture forms a multilayered epidermis showing an overall structure which resembles that of a native epidermis. The presence of a coherent stratum corneum layer in these cultures permits the application of potential irritants at concentrations and in formulations as applied in vivo. For the evaluation of toxicity a number of tests have already been developed: assessment of cell viability, changes in cell morphology, modulation of cell proliferation and differentiation, monitoring of membrane damage, the measurements of the uptake or incorporation of radioactive precursors, establishment of the modulation of cell metabolism, determination of the release of inflammatory mediators, etc. All these in vitro techniques are still in a state of validation as far as their predictive value for in vivo skin irritancy is concerned.