Donor tissue was obtained from human fetal eyes, 16 to 20 weeks of
gestational age. Informed consent was obtained for the use of this
tissue before abortion and institutional approval was granted through
an agreement between Albert Einstein College of Medicine, the source of
the tissue, and Columbia University. The eye bulbs were washed
externally with 70% alcohol and then with phosphate-buffered saline
(PBS). The eyes were put into our standard RPE culture medium,
Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose supplemented
with 20% fetal calf serum (Hyclone, Logan, Utah), 2 mM l-glutamine and penicillin (50 unit/ml)/streptomycin (50
mg/ml) (Gibco, Grand Island, NY). The anterior segment with lens,
vitreous, and neural retina was removed. The posterior segment was
sliced into quadrants, and RPE patches were separated gently from
Bruch’s membrane and choroid, using fine forceps and microscopic
viewing. A distinct cleavage plane is identifiable between the taut
monolayer patch of RPE and the adjacent choroid so that an isolated
sheet of RPE can be pulled off. Each sheet was placed in a separate
culture plate. The edges of the sheet were pressed onto the surface of
the plate with the tip of a 26-gauge needle. The cultures were
maintained at 37°C in an incubator with a humidified atmosphere of
95% air/5% CO2, fed every 3 to 4 days, and
examined almost daily. To obtain cell suspensions, we washed the cells
with PBS three times and exposed them to 2.5% trypsin in Hank’s
solution with EDTA without Ca and Mg (Gibco) for 10 minutes at 37°C.
The monolayer was triturated into single cells or clusters of cells by
repeated pipetting. The concentration of cells in a suspension was
determined with a hemocytometer. The cells were either used for
transplantation or subcultured.