Separation of the epidermal sheet by dispase.

Dispase is a bacterial neutral protease which is obtained from the culture filtrate of Bacillus polymyxa. After 24-h treatment of the human skin with 500 and 1000 U/ml dispase, the epidermal sheet was easily peeled from the dermis, and its undersurface retained rete ridges. Electron microscopic observation showed that the basal surface was composed of cells with numerous slender villi and cytoplasmic projections. Although the intercellular spaces of the spinous as well as the basal layers were wide, all desmosomes were intact with their accompanying tonofilaments. An epidermal cell suspension was obtained by incubating the dispase-separated epidermal sheet in trypsin solution for 15 min and the viability of the cells was more than 95% as measured by the trypan blue dye exclusion test. A culture of keratinocytes was established by seeding the dissociated cells in the usual way.