Hbpital du Saint-saCrrment du centm hospitalier affilid, 1050 Chemin S a i n t e F q , Qu.t?bee, QC GlS 4L8, Canada he physico-chemical properties of a dermal-epidermal reconstructed skin provide numerous advantages for their use as permanent skin T replacement over full thickness skin injuries. The reconstructed skin elaborated in our laboratory by the new tissue engineering technique, the self-assembly approach, possesses a network of elastic fibers and collagen fibrils that confers suppleness and strength to the reconstructed tissue (Larouche et al., 2000). This tissue engineered skin has tremendous therapeutic values for autologous grafting of large burn wounds, giant nevi, and the healing of cutaneous ulcers.Our dermal-epidermal living human substitute is composed of skin fibroblasts that secrete their own extracellular matrix and skin keratinocytes that proliferate and differentiate into a cornified, stratified epithelium (Michel et al ., 1999). No synthetic or exogenous extracellular matrix, such as collagen or fibrin, has been added to the organotypic culture. The only extracellular matrix and basement membrane material found in our reconstructed skin has been secreted, and assembled by the autologous cells in culture and is thus fully immunocompatible with the donor. The cells that constitute this reconstructed skin retain the ability to proliferate, differentiate, and heal (Laplante et al ., 2000), which is of crucial importance in the long-term fonctionnality of the graft. The ability to secrete soluble factors enabling wound healing is a key function that is important for the use of our reconstructed skin as a woundhealing device.The keratinocytes are usually isolated from skin biopsy using an enzymatic digestion with thermolysin or dispase and/or trypsin (Cermain et al., 1993; Martinet et al., 1988; Rheinwald et al., 1975; Liu et al ., 1978). The major obstacle in the isolation of epithelial cells is the contamination with fibroblasts which can overgrow the keratinocytes. The fibroblasts can be cultured from explants, but this method is timeconsuming and not always successful. The mesenchymal cells can also be recuperated with a collagenase (enzymatic) treatment of the peeled dermis, and thus generating a more representative cell population. The simultaneous isolation of keratinocytes, fibroblasts, and endothelial cells using trypsin to digest the basal membrane, and explants to isolate fibroblasts has been documented (Normand et al., 1995). Their ability to reconstruct a tissue-engineered skin has not been investigated. ~ *Author to whom correspondence may be addressed. E-mail address: lude.germain8 chg. ulaval.coThe 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...