The preparation of a reconstructed human epidermis is described with examples of its utilization in in vitro studies. The model was obtained by culturing normal human keratinocytes at high cell density for 14 days in serum-free and high calcium (1.5 m M) medium on an inert polycarbonate filter at the air-liquid interface. These stratified cultures showed histological features similar to those observed in vivo in the epidermis: a proliferating basal layer and differentiating spinous, granular, and cornified layers. Electron microscopy illustrated lamellar bodies, junctions and keratohyalin granules. Immunofluorescent localization of epidermal markers (keratins 14 and 10, involucrin and filaggrin) revealed typical differentiation. This in vitro reconstructed tissue was used in studies of toxic effects of chemicals. The modelled tissue showed progressive cytotoxicity of a skin irritant (benzalkonium chloride) and a sensitizer (dinitrochlorobenzene) as assessed by MTT assay. Moreover, differential release of interleukin-1alpha and interleukin-8 were measured after 20 h of incubation allowing the irritant to be distinguished from the sensitizer. Permeation studies indicated efficient barrier function of the reconstructed epidermis, as well as metabolizing properties towards hormones. This model can be custom-made and is potentially useful for studies involving keratinocytes in the epidermis, in basic science, dermatology or toxicology.

ABSTRACT:This study was designed to investigate the human absorption, disposition, and mass balance of 14

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Culture models of tissues and organs are valuable tools developed by basic research that help investigation of the body functions. Modelling is aimed at simplifying experimental procedures in order to better understand biological phenomena, and consequently, when suYciently characterized, culture models can also be utilized with high potential in applied research. In skin biology and pathology, the development of cultures of keratinocytes as monolayers has allowed the elucidation of most functional and structural characteristics of the cell type. Beside the multiple great successes that have been obtained with this type of culture, this review draws attention on several neglected characteristics of monolayer cultures. The more sophisticated models created in order to reconstruct the fully diVerentiated epidermis have followed the monolayers. The epidermal reconstruction produces all typical layers found in vivo and thus makes the model much less simple, but only this kind of model allows the study of full diVerentiation in keratinocyte and production of the corniWed barrier. In addition to its interest in basic research, the reconstructed epidermis is currently gaining a lot of interest for applied research, particularly as an alternative to laboratory animals in the chemical and cosmetic industry. Today several commercial providers propose reconstructed skin or epidermis, but in vitro assays on these materials are still under development. In order to be beneWcial at long term, the validation of assays must be performed on a material whose availability will not be interrupted. We warn here providers and customers that the longevity of in vitro assays will be guaranteed only if these assays are done with welldescribed models, prepared according to published procedures, and must consider having a minimum of two independent simultaneous producers of similar material.

The European Centre for the Validation of Alternative Methods (ECVAM) Skin Irritation Task Force was established in 1996, to review the status of the development and validation of alternative tests for skin irritation and corrosion, and to identify appropriate non-animal tests for predicting human skin irritation that were sufficiently well-developed to be prevalidated and validated by ECVAM. The EpiDerm™ method, based on a reconstituted human skin model, was proposed as being sufficiently well advanced to enter a prevalidation (PV) study. Based on a review of test protocols, prediction models (PMs), and data submitted by test developers on ten specified chemicals, with 20% sodium lauryl sulphate as a reference standard, the task force recommended the inclusion of four other tests: EPISKIN™ and PREDISKIN™, based on reconstituted human epidermis or on human skin; the non-perfused pig-ear test, based on pig skin; and the skin integrity function test (SIFT), with ex vivo mouse skin. The prevalidation study on these methods was funded by ECVAM, and took place during 1999–2000. The outcome of the PV study was that none of the methods was ready to enter a formal validation study, and that the protocols and PMs of the methods had to be improved in order to increase their predictive abilities. Improved protocols and PMs for the EpiDerm and EPISKIN methods, the pig ear test, and the SIFT were presented at an extended Task Force meeting held in May 2001. It was agreed that, in the short term, the performance of the revised and harmonised EpiDerm and EPISKIN methods, as well as the modified SIFT, should be evaluated in a further study with a new set of 20 test chemicals. In addition, it was decided that the SIFT and the pig-ear test would be compared to see if common endpoints (transepidermal water loss, methyl green-pyronine stain) could be identified.