A simple reconstructed human epidermis: preparation of the culture model and utilization in in vitro studies

Abstract

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.

Type I Transglutaminase Accumulation in the Endoplasmic Reticulum May Be an Underlying Cause of Autosomal Recessive Congenital Ichthyosis

“…To demonstrate this, we compared the TG1 level in tAd5-EV and tAd5-TG1-FLAG virus-infected monolayer keratinocytes with the endogenous TG1 level in stratified keratinocyte cultures grown at the air-liquid interface. Air-liquid interface cultures mimic in vivo keratinocyte differentiation (41). The increase observed in raft cultures is consistent with a previous report from Steinert and colleagues (35) that TG1 levels increase more than 100-fold upon keratinocyte differentiation.…”

Protein Kinase C δ Increases Kruppel-like Factor 4 Protein, which Drives Involucrin Gene Transcription in Differentiating Keratinocytes

“…In this system, keratinocytes are plated onto a semipermeable membrane and grown at the airliquid interface. Under these conditions, keratinocytes assemble a highly differentiated multilayered epidermal equivalent that includes a basal proliferative layer, intermediate layers, and a cornified terminal layer (55). Our studies show that knockdown of KLF4 in these cultures severely compromises the differentiation process, resulting in formation of a disordered morphology and the absence of a cornified layer.…”

“…KLF4 Knockdown Suppresses Keratinocyte Differentiation and Reduces hINV Expression-To assess the impact of KLF4 knockdown on hINV expression during differentiation, we used an epidermal equivalent model that mimics in vivo epidermal differentiation (55). Keratinocytes were electroporated with control or KLF4 siRNA and then plated into Millicell chambers to test for the ability to form a stratified epidermal equivalent.…”

Alternative methods for the replacement of eye irritation testing

“…A similar approach employing RCE to predict eye irritation seems to be viable. In comparison to the reconstructed human epidermis, RCE has no cornified layer and shows a different morphology of viable cell layers (Katoh et al, 2013;Poumay et al, 2004).…”