Cellular artificial skin substitute produced by short period simultaneous culture of fibroblasts and keratinocytes

Abstract

We developed a novel artificial skin substitute consisting of two collagen sponge layers with different pore sizes and cross-link densities. Fibroblasts suspended in 0.5 ml Dulbecco-modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) were seeded on the lower dermal sponge layer, then epidermal collagen sponge and 0.1 ml suspension of keratinocytes in KGM were layered in this order. After a few hours, the medium was changed to DMEM + 5% FBS. These processes were carried out in one day, and the composite layers were then cultured by the air-liquid interface culture method. Three to five days after seeding, keratinocytes had grown to about ten layers, and fibroblasts had grown three-dimensionally into the lower dermal sponge layer. This novel cellular artificial skin substitute was grafted onto nude mice and took in 4 weeks. This skin substitute has the advantage of a shorter culturing period than previously cultured skins, and may be clinically useful for grafting that is urgently required in patients with severe generalised burns.