Corneal Endothelial Cell Culture Protocol

The stripped corneal endothelium was placed in a culture plate coated with FNC Coating Mix^® (Athena Environmental Sciences, Baltimore, MD, USA). The pieces were left undisturbed overnight at 37 °C in a humidified 5% CO₂ chamber in proliferative growth medium, composed of Gibco^TM Opti-MEM-I (Thermo Fisher Scientific) supplemented with 8% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 5 ng/mL human recombinant EGF (PeproTech, Rocky Hill, NJ, USA), 100 μg/mL bovine pituitary extract (Biomedical Technologies, Stoughton, MA, USA), 200 mg/L calcium chloride (Invitrogen, Carlsbad, CA, USA), 0.08% chondroitin sulfate (Sigma-Aldrich), 50 μg/mL gentamicin, and 1× antibiotic/antimycotic solution (Thermo Fisher Scientific). The following day, the culture medium was replaced, and the cells were fed every other day. To minimize endothelial to mesenchymal transition, a previously published dual media was employed [26 (link)]. Here, passage 1 cells were grown to confluency in the described proliferative growth medium and then switched to stabilization medium, composed of Opti-MEM-I supplemented with 4% fetal bovine serum, 50 μg/mL gentamicin, and 1× antibiotic/antimycotic solution. Cells were maintained in stabilization medium for 1 week. Then, the cells were collected for biotinylation and protein extraction or for RNA isolation.