Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

Abstract

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated.Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

Noninvasive evaluation of the skin barrier in reconstructed human epidermis using speckle analysis: Correlation with Raman microspectroscopy

“…In order to characterize the RHE models and evaluate the barrier function, a variety of invasive and noninvasive techniques were used. Protein levels and localization specific to different stages of keratinocyte differentiation were analyzed by immunofluorescence 6 . The assessment of lipid profiles was conducted by different separation techniques, specifically thin‐layer liquid chromatography and gas chromatography 7,8 .…”

“…Protein levels and localization specific to different stages of keratinocyte differentiation were analyzed by immunofluorescence. 6 The assessment of lipid profiles was conducted by different separation techniques, specifically thin‐layer liquid chromatography and gas chromatography. 7 , 8 With the advent of high‐performance liquid chromatography coupled with mass spectrometry (HPLC/MS), a more comprehensive evaluation of the lipid composition becomes possible.…”