These cells are isolated from donated corneas. Confluent endothelial cultures exhibit polygonal morphology.
For endothelial cell isolation, each cornea is placed in a Petri dish containing Medium 199 and gentamicin (50 ug/mL). Under a dissecting microscope, the Descemet’s membrane, with the attached endothelium, is stripped from the stroma and placed in a 15-mL centrifuge tube containing EDTA (0.2 mg/mL) in Hanks’ balanced salt solution, pH 7.4. After incubation for 1 hour at 37°C, cells are detached by vigorous disruption with a glass pipette. Cells are then pelleted and resuspended in culture medium consisting of OPTIMEM-1, supplemented with fetal bovine serum (8%), FGF (40 ng/mL), EGF (5 ng/mL ), NGF (20 ng/mL ), ascorbic acid (20 ug/mL), human lipids (0.005%), calcium chloride (200 mg/L ), chondroitin sulfate (0.08%), RPMI-1640 multiple vitamin solution (1% ), gentamicin (50 ug/mL), and antibiotic/antimycotic solution (1:100). Medium is changed every other day until cells reach confluency (within 10 to 14 days).
EMBRYONIC DEVELOPMENT & STEM CELL COMPENDIUM
