Epidermal keratinocyte precursor cells are isolated from the follicular outer root sheath (ORS) of anagen phase plucked head hairs. ORS keratinocytes have a long life span and are highly proliferative, and can therefore produce a fully differentiated epidermis in vitro. Expanded keratinocytes are tissue-engineered in EpiDex® discs of 1 cm diameter, armed with a silicone membrane.
50-200 plucked hairs per patient are explanted onto microporous membranes of cell culture inserts carrying a feeder layer of growth-arrested human dermal fibroblasts on the undersurface. Within 14 days, ORS keratinocytes expand and reach confluency in a culture medium supplemented with human serum (10%) from type AB blood. Cells are detached with trypsin and cryopreserved in liquid nitrogen. Then, cells are plated on microporous membranes of cell culture inserts carrying the fibroblast feeder layer on their undersurface. After 36–48 hours, the cells are lifted to the air–liquid interface and grown for another 14–16 days, with three medium changes per week. After detachment from the microporous membrane, EpiDex® discs are attached to a silicone membrane, which is placed on culture medium fixed with 1% agarose in a shipment vessel.