RPE was removed from 64 donor eyes (obtained within 24 hours of death from the Eye Bank of the NORI, Amsterdam, The Netherlands) that were enucleated for the purpose of corneal transplantation, as described earlier, with minor modifications.
26 Briefly, the vitreous and neural retina were removed and the choroid together with the RPE was taken for incubation during 30 minutes at 37°C with 20 mg/mL Dispase (Roche Diagnostics, Mannheim, Germany). Then, the RPE was dissected and fragmented, and the purity of the RPE tissue was evaluated microscopically. RPE from eight eyes was brought into lysis buffer for the preparation of total lysates and for immunoprecipitation
(Fig. 1) . From the remaining 56 eyes, first-passage primary RPE cell cultures were established: RPE sheets from 22 eyes were seeded on 2.25-cm
2 glass coverslips and RPE sheets from 34 eyes into 75-cm
2 plastic culture vessels. The success rate was 13 (59%) of 22 eyes on glass coverslips and 15 (44%) of 34 eyes in plastic culture vessels. All RPE sheets were explanted in RPMI 1640 medium (Gibco, Merelbeke, Belgium), supplemented with 20% fetal calf serum (FCS, Gibco), 2 mM
l-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2.5 μg/mL amphotericin B. All 13 first-passage primary RPE cell cultures on glass coverslips were fixed in methanol after 2 to 3 days of culture and stored at −20°C. Eight of the former first-passage primary RPE cell cultures were used for immunocytochemistry. The 15 first-passage primary RPE cell cultures in 75-cm
2 tissue culture flasks were grown to confluence (BD Biosciences Labware, Franklin Lakes, NJ) and four of these were used for immunocytochemistry. Eleven RPE subcultures were isolated by trypsin treatment from the remaining first-passage primary RPE cell cultures and used for functional assays. RPE cell cultures were routinely examined by phase-contrast microscopy, immunocytochemistry, and Western blot to determine confluence and expression of cadherin and cytokeratin. The human neuroblastoma cell line SK-N-SH served as the positive control in molecular, immunocytochemical, and functional analyses of human N-cadherin-expressing cells.
27 The canine kidney epithelial cells MDCK/AZ
28 and the rat myofibroblasts DHD-FIB
29 were used respectively as noninvasive and invasive controls in the collagen type I assay. Canine kidney epithelial cells MDCKts.srcCl2
30 were used as a positive control for HGF-stimulated invasion. Conditioned medium (CM) of human colon cancer myofibroblasts CT5.3,
31 producing HGF, was used as a positive control for the induction of branching of MDCK/AZ cells expressing c-Met, inside collagen type I.
32 CM was harvested from two passage-4 primary RPE cell cultures (6–8 weeks old, covering 75 cm
2), from a passage-5 to -6 primary CT5.3 cell culture (passages 5–6, 1 week old, covering 75 cm
2) and an MRC-5 cell line (passage 15–16, 1 week old, covering 75 cm
2), expressing HGF, as follows. CM was collected over a total time of 72 hours, centrifuged for 10 minutes (1250
g at 4°C), and passed through a filter with a 0.2-μm pore size (Schleicher & Schuell, Dassel, Germany). CM was stored at −20°C until use for functional assays and for ELISA of HGF. To remove HGF from CM by immunoadsorption, 1 mL CM was incubated at 4°C for 3 hours with 2 μg anti-HGF antibody, followed by protein G Sepharose 4 beads (Amersham Pharmacia Biotech, Rainham, UK) for 1 hour. This procedure was repeated a second and third time to remove all HGF. As a control for specificity of the anti-HGF antibody precipitation, an isotype mouse IgG1 antibody was used.