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

We developed a novel artificial skin substitute consisting of two collagen sponge layers with different pore sizes and cross-link densities. Fibroblas ts 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 epider mal collagen sponge and 0.1 ml suspension of keratinocytes in KGM were laye red in this order. After a few hours, the medium was changed to DMEM + 5% F BS. These processes were carried out in one day, and the composite layers w ere then cultured by the air-liquid interface culture method. Three to five days after seeding, keratinocytes had grown to about ten layers, and fibro blasts had grown three-dimensionally into the lower dermal sponge layer. Th is novel cellular artificial skin substitute was grafted onto nude mice and took in 4 weeks. This skin substitute has the advantage of a shorter cultu ring period than previously cultured skins, and may be clinically useful fo r grafting that is urgently required in patients with severe generalised bu rns. (C) 1999 The British Association of plastic Surgeons.