Influence of fibroblasts on epidermization by keratinocytes cultured on synthetic porous membrane (insert) at the air-liquid interface

Abstract

Culture of keratinocytes on a noncoated porous synthetic membrane maintained at the air-liquid interface allows the establishment of a fibroblast/keratinocyte co-culture, without direct cell-cell contant between the two cellular layers. The influence of fibroblasts (proliferating, confluent or blocked by mitomycin C) on epidermization (i.e., expression of integrins and markers of epidermal differentiation) was studied by immunohistochemistry in two culture media. In the medium supplemented with VCS or Ultroser G and in the absence of fibroblasts, α2, α3, α5 and α6 subunits of integrins are expressed by the basal keratinocytes, except α5 which does not appear with the medium supplemented with Ultroser G. During stratification, the α3 subunit is the only one to persist on suprabasal cells and all the markers of epidermal differentiation studied (filaggrin, involucrin, transglutaminase, keratins K1/K10) are expressed at the 14th day of emerged culture. The presence of fibroblasts modifies the expression profile of integrins: when they are proliferative, the expression of α2 and α6 chains is delayed in the medium supplemented with FCS, and the α6 chain is absent in the medium supplemented with Ultroser G; when they are confluent or blocked by mitomycin C, greater changes are observed only in the medium supplemented with Ultroser G and lead to inhibition or delay of the expression of α2 and α6. In the presence of fibroblasts, only the expression of filaggrin (marker of terminal differentiation) is affected; it is delayed in the medium supplemented with FCS whatever the state of fibroblasts, and is inhibited in the medium supplemented with Ultroser G in the presence of proliferating and confluent fibroblasts.