Keratinocytes are harvested from a skin biopsy, expanded and seeded on a flexible medical-grade silicone coated with a chemically controlled plasma polymer film which supports cell growth.
A skin biopsy of approximately 4 cm2 surface area and 0.4-0.6 mm thickness was taken from a patient, under a local anesthesia. The tissue was then cut, in a clean-room, into small pieces and kept in trypsin at 4°C, overnight. Harvested keratinocytes were seeded on a feeder layer of lethally irradiated 3T3 fibroblasts in Green’s medium. Cells were cultured in a DMEM and Ham’s F12 medium (3:1 ratio) supplemented with fetal calf serum (FCS) (10%), EGF (10 ng/ml), hydrocortisone (0.4 µg/ml), cholera toxin (10-7 M), adenine (1.8×10-4 M), insulin (5 µg/ml), transferrin (5 µg/ml), glutamine (2×10-3 M), tri-iodothyronine (2×10-7 M), amphotericin B (0.625 µg/ml), penicillin (100 IU/ml) and streptomycin (100 µg/ml). When reaching 80% confluency of passage 3, cells were detached using trypsin, frozen in a freezing medium containing FCS (90%) and DMSO (10%) and stored in liquid nitrogen until use. Thawed cells were seeded on a Myskin® dressing at 1–2×106 cells/28 cm2 surface area, in complete Green’s medium, for 24 h.