Fluorescence imaging of reepithelialization from skin explant cultures on acellular dermis

Reconstituted skin models are valuable tools in studies of cutaneous biology although they are not generally devised to visualize and quantify the time course of reepithelialization. We describe a skin explant culture technique coupled with vital microscopy allowing sequential imaging of epithelial outgrowth while maintaining the tissue in culture. Radial expansion of neo-epidermis was initiated from a 2-mm skin punch biopsy explanted onto acellular human dermis and maintained at the air-liquid interface in serum-containing keratinocyte medium. Microscopic viewings and surface area measurements of newly formed epidermis were performed repeatedly using fluorescein-based cell staining and subsequent image analysis. The labeling and visualization procedures did not interfere with neo-epidermal growth or tissue architecture. In order to appraise the versatility of the model, we studied the effect of epidermal growth factor supplementation on the course of skin resurfacing as related to tissue morphology and proliferative activity over a 10-day cultivation period. Exogenous epidermal growth factor at 10 ng/ml increased the radial outgrowth rate (mean, + 13.3 percent), papillomatosis index (+ 19.2 percent), epithelial thickness (+ 49.8 percent), and fraction of Ki-67 positive basal cells (+ 18.4 percent) in neo-epidermis produced from a biopsy of normal human skin. The contribution of this miniaturized and convenient format for concurrent studies of dynamic and qualitative features of neo-epidermis should be a useful complement to existing assays of epidermalization.