Simultaneous and separated culture of keratinocytes and fibroblasts on each side of a collagen membrane

Abstract We have developed a new cell culture system which is of benefit for the research of dermal-epidermal interaction. In this system, normal human keratinocytes are cultured on the upper surface of a permeable collagen membrane, on the undersurface of which human fibroblasts are simultaneously and separately cultured in a lifted condition. Both the keratinocytes and fibroblasts showed good proliferation and differentiation which were revealed by phase contrast microscopy as well as light and electron microscopies. A permeability test of the collagen membrane used in the present study showed that peptides of less than 30 kDa are able to penetrate through the membrane. Using this system, we estimated the total protein content and cornified envelope formation in the keratinocytes cultured with or without fibroblasts. We found that the total protein and cornified envelope formation were significantly increased when keratinocytes were cultured together with fibroblasts. When keratinocytes were cultured with fibroblasts in the condition of air-liquid interface, synthesis of the cornified envelope was further enhanced. These results indicate that fibroblasts really effect not only the proliferation but also the differentiation of keratinocytes, and the air-liquid interface enhances their effect on differentiation. This bi-phase separated cell culture system is a useful tool for the study of keratinocyte-fibroblast interaction.