Isolation and Culture of Human RPE Cells

The use of human material was approved by the Ethics Committee of the University of Leipzig and was performed according to the Declaration of Helsinki. Human RPE cells were obtained from several donors within 48 h of death, and were prepared and cultured as follows. After the vitreous and the retina were removed, the RPE cells were mechanically harvested, separated by digestion with 0.05% trypsin and 0.02% EDTA, and washed two times with PBS pH 7.2 (1.54 mM KH₂PO₄; 155.17 mM NaCl; 2.71 mM Na₂HPO₄x7H₂O, Invitrogen, Paisley, UK). The cells were suspended in complete Ham F-10 medium containing 10% fetal bovine serum, GlutaMAX II, and penicillin/streptomycin, and were cultured in tissue culture flasks (Greiner, Nürtingen, Germany) in 95% air/5% CO₂ at 37 °C. Cells of passages 3 to 5 were used. The epithelial nature of the RPE cells was routinely identified with immunocytochemistry using the monoclonal antibodies AE1 (recognizing most of the acidic type I keratins) and AE3 (recognizing most of the basic type II keratins), both from Chemicon. To test the substances, cultures that reached approximately 90% confluency were growth arrested in medium without serum for 5 h. Subsequently, media containing 0.5% serum with and without test substances were added.