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

Abstract

To evaluate the epidermal barrier function of in vitro reconstructed epidermis, we measured the penetration of estradiol and water across human keratinocytes cultured in defined medium (DM), in the presence of proliferative fibroblasts (pF) or conditioned medium derived from pF, at the air-liquid interface on synthetic porous membrane, noncoated or coated with laminin, fibronectin, type I collagen or type IV collagen. Ultrastructural analysis showed a well-developed stratum corneum whatever the culture conditions. The permeability of reconstructed epidermis in DM on a noncoated porous membrane was 5- to 10-fold higher than human native epidermis, with both tracers. No significant change in barrier function was observed whatever the culture conditions.

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

“…Electron microscopy ( fig. 1a) revealed a well-organized multilayered epidermis showing basal, spinous, granular and horny layers after 7 days of emerged culture as shown previously [28]. Terminal epidermal differentiation appeared to be complete as anucleate cells of the stratum corneum exhibited thickened plasma membranes and tightly packed keratin filaments.…”

“…Figure 1 presents the electron microscopy for epidermis grown in DM which reached a terminal differentiation after 7 days of culture at the air-liquid interface. However, the reconstructed epidermis grown in this medium (control) is more permeable to water and estradiol than native human epidermis [28]. In vivo, epithelial differentiation is controlled by the supporting mesenchyme, and affected by different components of the dermis.…”

“…For histological and biochemical studies, thirdpassage keratinocytes were plated on 0.32-cm 2 cell culture inserts at a density of 7 ! 10 4 cells/insert in 1 ml of DMEM:Ham's F 12 medium (3:1) containing additives and supplemented with Ultroser G ® (2%) [28]. Inserts were placed in 24-well plates; each well contained 0.300 ml of that medium.…”

“…Percutaneous absorption was measured at 37 or 32°C directly in the cell culture insert as described previously [28]. As a control, native epidermis, derived from Caucasian abdominal skin, was separated from the dermis by immersion in hot water (60°C/60 s), and then introduced into the insert, after being placed in the glass cylinder.…”

“…To reach our goal, three culture conditions were compared: (1) keratinocytes cultured in defined medium (DM) [28], (2) keratinocytes cultured in medium previously conditioned by endothelial cells (CM), (3) keratinocytes cocultured with proliferative endothelial cells (pEC). After 5 days of culture under submerged conditions, cultures were lifted at the air-liquid interface for 9 days for biochemical analysis and 20 days for percutaneous absorption testing.…”