Evaluation of Cutaneous Irritancy Using Human Skin Recombinants

Kim, Bang Soon; Lee, Dong Youn; Han, Ji Hyun; Lee, Jong Hee; Cho, Kwang Hyun

Abstract

BACKGROUND: There is an increasing need for the development of in vitro models capable of substituting for animals in cutaneous irritancy studies. Until now, various culture models have been developed, including skin organ cultures, conventional and air-exposed cell cultures. The air-exposed 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 the concentrations and formulations which are applied in vivo. Recently, a new human skin recombinant, made of human keratinocytes cultured on de-epidermized dermis with fibroblast-populated collagen matrix, has been developed and appears to represent a better skin equivalent model than previous models.
OBJECTIVE
In the present study, monolayer-cultured human keratinocytes and the new human skin recombinants were evaluated for the test models of various skin irritants.
METHODS
The extent of skin irritancy induced after application of 3 different irritants (sodium lauryl sulfate, methyl paraben, and polyethylene glycol-400) was evaluated on the basis of (1) MTT assay, (2) neutral red uptake assay, (3) LDH release, and (4) release of IL-1 alpha. In the human skin recombinants, morphological perturbations and changes in the expression of differentiation-specific protein markers (keratin 1, involucrin, filaggrin, and loricrin) were also evaluated. To determine the difference between in vivo and in vitro models for the detection of irritancy, a patch test was performed on 11 normal human volunteers with various concentrations of the different irritants
RESULTS
The results of the present study show that irritant cytotoxicity correlates well with irritant concentration in both monolayer-cultured human keratinocytes and the new human skin recombinant. The new human skin recombinant is superior to monolayer culture as an in vitro model for skin irritancy screening in that the concentrations of test irritants are the same as in vivo. With the human skin recombinant, morphological changes were observed according to the irritant concentration.
CONCLUSION
The new human skin recombinant can be used as an alternative to animals for skin irritancy screening.