Bovine enucleated eyes, obtained from the local abattoir, were
stored for 24 hours at 4°C to reduce adhesion between the neural
retina and the pigmented epithelium and were bisected posterior to the
ora serrata. After removal of the vitreous body and separation of the
retina, the eyecup was rinsed with RPMI-1640 (Gibco, Paisley, Scotland,
UK), and 1 ml trypsin (0.25%, Gibco) was introduced into the eyecup,
followed by incubation for 1 hour at 37°C. The retinal pigmented
epithelium (RPE) cells were pipetted out of the Bruch’s membrane,
washed with RPMI by centrifugation (10 minutes, 1500g), and
finally resuspended in RPMI 1640 supplemented with 5 mM glutamine, 100
U/ml penicillin, 100 mg/ml streptomycin, and 20% fetal bovine serum.
The cells were seeded at 5 × 104 cells/cm2 in tissue culture flasks and incubated
at 37°C in 5% CO2 atmosphere. The RPE cells
were used after the fourth passage.
Epithelial cells of the bovine lens were cultured according to Plouet
et al.
11 and were kindly provided by Yve Courtois,
INSERM, Paris. Normal human corneal fibroblasts were isolated from
peripheral cornea, as previously described,
12 and cultured
in RPMI-1640 medium supplemented with 10% fetal calf serum, 5 mM
glutamine, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C in a
5% CO
2 humidified incubator.