The key step in the reconstruction of skin by the self-assembly approach is to use fibroblasts capable of secreting a mature extracellular matrix and keratinocytes that can associate with one another to form a stratified, differentiated epidermis. To determine the most efficient way to extract both cell types from a single cutaneous biopsy, five different enzymatic combinations were tested. The yield, viability and morphology of the recuperated cells were compared. The length and temperature of the enzymatic treatments influenced the number and the type of cells recuperated. The cells were then cultured in appropriate media in monolayers and reconstructed skin were produced using the self-assembly approach. No difference was observed between the different strains of cells.