HFRPE cells were obtained from three independent human fetal eyes at 18 to 22 weeks of gestational age (Advanced Bioscience Resources, Alameda, CA) as described before.
7 Microdissection was performed under sterile conditions aided by a dissecting microscope. The eyes were opened by a circumferential incision just above the ora serrata near the limbus, and the anterior segment and lens were separated. The posterior segment of the eye was cut into four quadrants and placed in a Petri dish containing Dulbecco’s minimum essential medium (DMEM; Sigma-Aldrich, St. Louis, MO). The neural retina and any remaining vitreous were removed. Sheets of RPE cells were separated from the choroid using fine forceps and immediately placed into a Petri-dish containing phosphate buffered saline (PBS) without Ca
2+/Mg
2+ (BioWhittaker, Walkersville, MD). After the separation of all four quadrants, the sheets were trypsinized (0.25% trypsin; Sigma-Aldrich) for 15 minutes. Growth medium consisting of DMEM (Sigma-Aldrich), 15% fetal bovine serum (Sigma-Aldrich), and a 1% solution of antibiotics and
l-glutamine (Sigma-Aldrich) was added, and the content was centrifuged at 2000 rpm. The supernatant was discarded, and the cells isolated from each eye were resuspended with growth medium into one well of a 24-well plate (BD Labware, Bedford, MA) and incubated for 1 week in 95% air and 5% CO
2 at 37°C. The cells were trypsinized and resuspended into a larger culture flask. The cultures were examined on a daily basis, and the growth medium was changed twice a week. At confluence, the cells were subcultured by trypsinization. Fourth- to sixth-passage HFRPE cells were used in the experiments.