Primary AECs were isolated from TNIL amnion (about 10 g), peeled from the chorion layer, and dispersed by successive treatments with 0.125% collagenase and 1.2% trypsin (17, 42). The dispersed cells were plated in a 1:1 mixture of Ham’s F12/DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), EGF (10 ng/ml), 2 mM l-glutamine, penicillin G (100 U/ml), and streptomycin (100 mg/ml) at a density of 3 million to 5 million cells per T75 and incubated at 37°C with 5% CO2 until they were 80 to 90% confluent.

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