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Full-thickness skin substitutes are formed by plating NIKS® cells on top of a mixture of collagen and fibroblast cells. The cells differentiate into all the skin layers.
The NIKS® cells were derived from normal keratinocytes (BC-1-Ep) that were isolated from newborn human foreskin. They are not tumorogenic.
A collagen base is formed in specialized culture chambers, by mixing normal human neonatal fibroblasts, with type I collagen in Ham’s F-12 medium containing fetal bovine serum (FBS, 10%) and penicillin (100 units)/streptomycin (100 μg) (P/S). The collagen base is allowed to contract for 5 days. The NIKS keratinocytes are plated on the contracted collagen base at 3.5 × 105 cells per 50 μl of a mixture of Ham’s F-12/Dulbecco’s modified Eagle’s medium (DME, 3:1, final calcium concentration 1.88 mM) supplemented with FBS (0.2%), hydrocortisone (HC, 0.4 μg/ml), cholera toxin (CT,8.4 ng/ml), insulin (Ins, 5 μg/ml), adenine (Ade, 24 μg ml), and P/S. Cells are allowed to attach for 2 h before flooding culture chambers with medium (day 0). On days 1 and 2, cells are refed. On day 4, cells are lifted to the air interface with cotton pads and switched to cornification medium containing Ham’s F-12/DME (3:1, final calcium concentration 1.88 mM), supplemented with FBS (2%), HC (0.4 μg/ml), CT (8.4 ng/ml), Ins (5 μg/ml), Ade (24 μg/ml), and P/S. Cells are fed cornification medium every 3 d.