Improvement of human keratinocyte isolation and culture using thermolysin

We propose a modification of the conventional keratinocyte isolation method which has shown a significant improvement in the purity, colony forming efficiency (c.f.e.) and growth capacity of the isolated epidermal cell population. This method utilized thermolysin since it selectively digests the dermo-epidermal junction. Following separation from the dermis, the epidermis was digested with trypsin to obtain a single cell suspension. Compared with the conventional procedure, this isolation method was shorter and resulted in (i) cells displaying a higher colony forming efficiency, (ii) cells reaching confluence 1-3 days earlier, (iii) cells not contaminated by fibroblasts, (iv) a cell population containing all the basal layer keratinocytes. These cells were suitable for the establishment of primary cultures and could be subcultured. Such cell populations should be advantageous in studies of epithelial-mesenchymal interactions in which keratinocyte populations, free of fibroblasts, are desirable. In the treatment of extensively burned patients using cultured epidermal sheets, the main problem remains the time required for their production. Thus, the absence of fibroblast overgrowth of the keratinocyte cultures and the significantly reduced time to obtain confluent cultures and epidermal sheets with our method have very important implications for the treatment of large burn wounds.