Reconstructed Human Epidermis: A Model to Study in vitro the Barrier Function of the Skin

Abstract

In previous studies, we have shown that architecture and differentiation of human epidermis reconstructed by culturing dissociated keratinocytes on deepidermized dermis at the air-liquid interface resembles very much those of epidermis in vivo. The various types of epidermal layers are present with the appropriate differentiation markers, such as the bullous pemphigoid antigen, suprabasal keratins, involucrin, membrane-bound transglutaminase and filaggrin, positioned almost like in normal epidermis. At the ultrastructural level, heterogeneous keratohyalin granules and intra- and extracellular membrane-coating granules are observed. After 7 days of culture, a compact stratum corneum covers the reconstructed epidermis. In the present work, the barrier function of the reconstructed epidermis was evaluated by measuring fluxes of ³H-water. Our results show that under the various culture conditions tested, the presence of the reconstructed epidermis reduces dramatically the permeability of the deepidermized dermis although the skin equivalent exhibits a higher permeability than normal skin. Conditions that favor terminal differentiation of the keratinocytes such as maintaining the cultures in delipidized serum improve the barrier function. Reduction of the relative humidity has a similar effect, whereas the age of the culture is of no influence. Experiments are in progress to reconstruct human epidermis with a barrier function as efficient as in normal skin.

Improvement of the Experimental Setup to Assess Cutaneous Bioavailability on Human Skin Models: Dynamic Protocol

“…Furthermore, particularly for human skin cultured on de-epidermized human dermis, vertical diffusion cells (e.g. modified Franz cells) have been used for determining percutaneous absorption in human skin models [9][10][11][12][13][14]. However, due to inappropriate cell and clamp design, the use of these cells might harm or even destroy the skin integrity of the model during the experiment.…”

Barrier Function of Reconstructed Epidermis at the Air-Liquid Interface: Influence of Dermal Cells and Extracellular Components

“…Recent studies suggest that the structural organization of stratum corneum lipids may be as important for barrier function as is the lipid composition itself [1][2][3][4][5][6][7][8][9][10], Cultured skin systems must thus ensure not only synthesis of both comeocytes and intercorneocyte lipid constituents, but also intercellular lipid organization. Recent progress in epithelial culture tech niques has led to the development of culture systems in which reconstructed epidermis ex hibits in vivo-like morphological and bio chemical differentiation [11][12][13][14][15][16][17][18][19][20][21], but barrier function remains subnormal [8,9,18,19,[22][23][24][25][26][27], Our laboratory has recently developed a culture model in which human kératinocytes proliferate and terminally differentiate on a synthetic porous membrane. Epidermal cells are exposed at the air-liquid interface (a pre requisite for terminal differentiation) and the reconstructed epidermis possesses the major structural characteristics of native epidermis [21].…”

Influence of Endothelial Cells on Structure, Biochemistry and Functionality of Epidermis Reconstructed on Synthetic Porous Membrane

“…Hydration as well as temperature can modify the lipid structural organization in the intercellular spaces. It is known that incubating fresh skin for 3 days induces a permeability coefficient 3 times higher than nonincubated fresh skin [7]. Since better results have been obtained for native and reconstructed epidermis when absorption study was performed at 32°C, further investigations could be done using a culture system where the medium under the equivalent would be at 37°C and the air over the reconstructed epidermis at 32°C, in order to mimic the native epidermis environment.…”

“…Until now, in all culture models developed with keratinocytes, mesenchymal cells and extracellular matrix, reconstructed epidermis displays morphological and biochemical characteristics of the native tissue from which it is derived. Skin models are interesting for pharmacological tests although they are more permeable compared to normal human skin [7,[9][10][11][12][13][14][15][16][17].…”