Cultured porcine epithelial grafts: An improved method

An improved method of in vitro cultivation of porcine keratinocytes by which keratinocyte sheets suitable for grafting can be generated rapidly is described. Epidermis from splitthickness porcine skin is enzymatically separated from dermis with 0.25% Dispase solution (37°C) within 3 h, and trypsinized to a single cell suspension. Keratinocytes are grown in Dulbecco-Vogt modified Eagle medium supplemented with 20 ng/ml hydrocortisone, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 20% (cells from six-month-old pigs) or 10% fetal calf serum (cells from two-month-old pigs). Freshly isolated keratinocytes are plated at a density of 1.25 × 10⁶ cells/ml since their plating efficiency is about 15 times lower than that of human keratinocytes grown under comparable conditions. Primary keratinocytes plated on plastic grow to confluence faster than those plated on lethally irradiated 3T3-J2 feeder layer cells. Porcine keratinocytes grown on plastic reach senescence in the third passage but, when subsequently cultivated on a lethally irradiated 3T3- J2 feeder layer, can be passaged up to seven times. Nevertheless, plating efficiency of second-passage porcine keratinocytes is only about 5% - 7%, whereas that of human newborn foreskin keratinocytes is 20%-30%. Confluent stratified primary cultures grown on plastic, or secondary cultures grown on feeder layers, are used for grafting. The sheets are detached with Dispase solution and stapled to vaseline gauze to facilitate handling. Epidermal regeneration from porcine grafts produced by this method has been demonstrated after transplantation to full-thickness wounds excised to muscle fascia in donor animals.