Methods for the Preparation of an Autologous Serum-Free Cultured Epidermis and for Autografting Applications

: Cell culture techniques for producing a three-dimensional autologous epidermal autograft (cultured epidermal autograft) suitable for tissue grafting and wound healing procedures are described. This chapter commences with surgical biopsy of patient's skin tissue, further reduction of skin tissues to keratinocyte cells by enzymatic treatment, and recovery of viable adult keratinocytes in a new balanced buffered salt media supportive of the growth of clonally enriched isolated basal keratinocytes. Culture techniques required for the formation of a hole-free monolayer of undifferentiated basal keratinocytes without the use of an organotypic matrix substrate are accomplished with a specially designed nutrient basal media (HECK 109) that is a chemically defined and subsequent culture in this serum-free culture media supplemented with hormones and two human recombinant protein growth factors (EGF and IGF-1). Further culture techniques and media manipulations, including brief exposure to β-TGF to induce reversible G1-phase growth arrest, are followed by para-synchronous induction of a multilayered stratification and keratinizing epidermal differentiation, yielding a living three-dimensional epidermis formed entirely in cell culture. Protocols are listed for its enzymatic removal, floatation, and transfer for shipment to the clinic ready for surgical grafting to the self-same patient's debrided chronic leg ulcers. Recent clinical trial results have demonstrated the utility and efficacy of these grafts in forming durably healed chronic wounds.