Cultured keratinocytes (EpiCel™) (Aastrom)

Biopsies of human epidermis in a medium consisting of DMEM, supplemented with calf serum (5%), are trimmed of excess fat and cut into small pieces measuring approximately 2-5 mm per side. A maximum of 5 cm tissue is immersed in 20 ml of either USP lactated Ringers or Hepes-buffered saline containing 0.5 mg/ml thermolysin and refrigerated at 4°C for 16-24 h. Alternatively, the tissue is immediately incubated with thermolysin at 37°C for 2-3 h. After the thermolysin incubation, the epidermal layer is separated from the dermis with forceps and the keratinocytes isolated by incubating the tissue in a solution of EDTA (0.01%) and Trypsin (0.05%) at 37°C for 15 min, with mild agitation every 5 min. The trypsin is then inactivated with fetal bovine serum (FBS) and the keratinocytes plated in growth flasks previously seeded with lethally irradiated Swiss 3T3152 fibroblasts at a density of 40000 cells/cm. The keratinocytes are plated at 10000-20000 cells/cm in complete medium without EGF (cFAD-EGF: Ham’s F (25% v/v), DMEM (75% v/v)) including FBS (10% v/v), insulin (5 μg/ml), triiodothyronine (5 μg/ml), hydrocortisone (0.4 μg/ml) and cholera toxin 10^-10 M. Cell cultures are fed three times per week and epidermal growth factor (EGF 10 ng/ml), is added beginning with the second feeding. Following approximately 7 days of growth, the primary keratinocytes are isolated and suspended in cFAD-EGF medium.