RT-PCR Corneal endothelial cells proliferation uncoated, collagen and Matrigel-coated plates

Six White New Zealand rabbits 3-month-old were sacrificed under general anesthesia and a lethal intracardiac injection of sodic pentobarbital. 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). The plates were tripzinised and cultured in SM. Morphological changes were photodocumented. RNA was isolated from CEC cultured in PM and SM. Final point RT-PCRs were made to analyze the expression of the specific CECs markers: glypican-4 (GPC4), tight junction protein 1 (TJP1), and CD200; housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) was used. Electrophoresis of PCR products was performed on a 2% agarose gel and the bands were photodocumented. Immunocytochemistry was performed to analyze the presence of GPC4 and Na/K-ATPase in CEC cultured in each condition. Images were obtained with a fluorescence inverted microscope.