Development of a stratum corneum and barrier function in an organotypic skin culture

Abstract

The stratum corneum of human skin is responsible for maintaining the epidermal permeability barrier. We have developed a bilayered skin culture (SC) which forms a corneum 35 +/- 1 cell layers thick 21 days after being raised to the air-liquid (A/L) interface. By the 7th day after raising to the A/L interface the corneocytes were irregularly shaped and had cross-sectional areas (CSA) of > or = 300 microns 2. By the 21st day the corneocytes had assumed polygonal shapes and had a CSA (100-250 microns 2) similar to that of human foreskin. The total lipid (TL) content of the corneum averaged 5-7% of the lyophilized weight. Ceramide content increased from 20% of TL at day 7 of A/L interface culture to 30% at day 21. Triglycerides decreased from 43% to 17% of TL during the same period. Free fatty acids comprised 5.5% of TL at day 21 of A/L interface culture. The intercorneocyte spaces contained stacks of lipid lamellae. However, the stacks lacked the Landmann unit repeat. Abnormal lamellar structures were observed in both the intra- and extracorneocyte spaces. Transepidermal water loss (TEWL) was > 4 mg/cm2 per h throughout the culture period. Lipid supplementation of the culture medium and culturing in a low humidity environment improved barrier function by 50%. However, the effects were not additive. The SC developed a near-normal corneum, but did not achieve barrier competence, due at least partially to abnormalities in lipid composition and organization.(ABSTRACT TRUNCATED AT 250 WORDS)

Herpes simplex virus type 1 exhibits a tropism for basal entry in polarized epithelial cells

“…These results partly differ from those obtained in organotypic, stratifying cultures in vitro (Syrjänen et al, 1996;Hukkanen et al, 1999). It is well known, however, that such cultures do not represent the full barrier competence of epidermis in vivo (Nolte et al, 1993). In our view, explant cultures as used in this study are closer to the in vivo situation and are, therefore, more suitable to study HSV entry into epithelia.…”

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

“…No correlation between permeability and the number of cell layers in the stratum corneum was found, confirming data from Elias et al [2], However, Mak et al [18] showed that improved barrier function to water in cul tured epidermis correlated with an increase in the number of horny layers. This suggests that, while a minimal thickness is probably required for barrier function [8], there is a point at which stratum comeum thickness no longer affects this function. Changes in cul ture conditions might lead to changes in lipid organization within the stratum comeum, in keratin synthesis and in the formation of hor ny envelopes, probably due to prematuration of keratinocytes in vitro [2.…”

“…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].…”

Generation of Human Epidermal Constructs on a Collagen Layer Alone

“…Within synthetic constructs, it was possible to identify cell layers comparable with those observed in natural skin; nevertheless, the number of the cell layers (4-6 layers, including basal cells) was lower than in natural skin and other models of bilayered skin equivalents. 43,45,51,52 The ultrastructure of the synthetic construct showed a typical structure in the basal stratum with the natural epidermis. 53 However, electron microscopy analysis revealed that the overall organization of these layers, as well as keratinocyte differentiation, was poorer than with natural epidermis, including the formation of junctional complexes, basement membrane, and keratinization.…”