Example 2
For embedding fibroblasts into the dermal layer (e.g. gel matrix), the protocol is as follows. First, the fibroblasts are detached using the trypsinization protocol described above. However, the pellet is re-suspended in complete E-medium low calcium (0.6 mM Ca++), supplemented with 0.5% (V/V) FBS (Invitrogen 16140071) and 2% penicillin/streptomycin (invitrogen 15140-122) and then added back to the flasks, where they are allowed to reach 50-60% confluence. Once again, the fibroblasts are detached according to the protocol described above. Once re-suspended, they are embedded into the dermal layer. From Day 0 to Day 1-2, the cells in the dermal layer are fed using complete E-medium low calcium (0.6 mM Ca++), supplemented with 0.5% (V/V) FBS (Invitrogen 16140071) and 100 μm ascorbic acid, RM/TI transglutaminase 50 μg/ml. From Day 1-2 to Day 3-4, the cells in the dermal layer are fed using complete E-medium low calcium (1.2 mM Ca++), supplemented with 0.5% (V/V) FBS (Invitrogen 16140071) and 100 μm ascorbic acid and RM/TI transglutaminase 50 μg/ml. From Day 14-18 on, the cells in the dermal layer are fed using complete cornification medium (1.8 mM Ca++), supplemented with 5% (V/V) FBS (Invitrogen 16140071) and 100 μm ascorbic acid and RM/TI transglutaminase 50 μg/ml.
