Immunocytochemistry of corneal endothelial cells proliferation uncoated, collagen and Matrigel-coated plates

A corneoscleral rim excision was made and the conjunctiva was dissected. Lens and aqueous humor were removed and the corneas were obtained.

Under sterile conditions, Descemet’s membrane were separated from corneal stroma and rinsed with basal stabilizer medium (SM) containing OptiMEM-I 8% fetal bovine serum (FBS) and 1% antibiotics. Corneal endothelia were peeled off from the Descemet’s membrane and a ~5 mm2 section was cultured in proliferative medium (PM) containing OptiMEM-I 8% FBS, 20 ng/mL of nerve growth factor (NGF), 5 ng/mL of epidermal growth factor (EGF), 200 µg/L of calcium chloride, 20 µg/mL of ascorbic acid, 0.08% chondroitin sulfate, and antibiotics over Matrigel, Collagen I or no coated plate until confluence (~90% of the plate showed adherent cells, ~15 days).