Development of a Stable Chemically Defined Surface for the Culture of Human Keratinocytes under Serum-Free Conditions for Clinical Use

Abstract

Within the field of tissue engineering there is a need to develop new approaches to achieve effective wound closure in patients with extensive skin loss or chronic ulcers. This article exploits the well-known interdependency of epithelial keratinocytes and stromal fibroblasts in conjunction with plasma surface technology. The aim was to produce a chemically defined surface, which with the aid of a feeder layer of lethally irradiated dermal fibroblasts would improve the attachment and proliferation of the keratinocyte cell from which subconfluent cells can be transferred to wound bed models. Plasma copolymers of acrylic acid/octa-1,7-diene have been prepared and characterized by X-ray photoelectron spectroscopy. The fibroblasts and keratinocytes were cultured on plasma polymer-coated 24-well plates. Cell attachment and proliferation were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-eluted stain assay (MTT-ESTA) and DNA assay. Attachment and proliferation of both cell types on plasma polymer surfaces were compared with tissue culture plastic and collagen I, plus a negative control of a pure hydrocarbon layer. A pure acrylic acid surface, fabricated at a power of 10 W and containing 9.2% carboxylate groups, was found to promote both fibroblast and keratinocyte attachment and proliferation and permit the serum-free coculture of keratinocytes and irradiated fibroblasts.