Primary cultures of porcine retinal pigment epithelial (pRPE) cells were isolated from freshly enucleated eyes of specific pathogen-free (SPF) Danish Landrace pigs (3 months old, 30 kg). Eyes were sterilized in 70% ethanol for 25 seconds. The sclera was carefully removed from the eyes, first by sharp incision 3 mm behind the limbus and thereafter by blunt dissection until only the optic nerve was adherent, leaving the uvea intact. Thereafter the eyes were incubated for 60 minutes at 37°C with 2 mg/mL collagenase (Sigma-Aldrich) dissolved in culture medium (DME-H16; Invitrogen-Life Technologies, Taastrup, Denmark). The eyes were place with the cornea downward. The optic nerve head was removed and the uvea-Bruch’s membrane-RPE complex was then isolated in three concentric zones numbered 3, 2 and 1, centered around the putative lesion area. The zones were selected to match the areas in
Figure 1(zone 1 corresponds to areas I and IV, zone 2 corresponds to areas II and V, and zone 3 corresponds to areas III and IV). It was then possible to remove the RPE in small sheets using gentle manipulation by the water-jet method. Contamination of choroidal cells was avoided by washing the cells in calcium-magnesium–free phosphate-buffered saline (CMF-PBS). The isolated sheets were seeded directly into 35-mm tissue culture dishes (Invitrogen-Life Technologies). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal calf serum, 300 mg/mL glutamine, 50 mg/mL gentamicin, and 2.5 mg/mL amphotericin B and maintained in humidified 10% CO
2 at 37°C.
12 The medium was changed every second day.
Figure 2shows phase-contrast micrographs of the cultured porcine RPE cells derived from the three anatomic regions.
The primary pRPE cultures were harvested on day 7 with 1× trypsin-EDTA (Sigma-Aldrich). These cells were maintained and propagated in three passages. Cells in each of these passages were seeded in 96-well, clear, flat-bottomed, treated tissue culture microplates (model 3595; Corning, Inc., Corning, NY) and assayed for proliferation. We seeded 2000 cells/well, approximately 60 cells/mm2. Uniform seeding density was obtained by measuring the cell density in the stock solution of cells by triplicate counts of a 10 μL sample in a standard hemocytometer chamber. The stock solution was then diluted to a concentration of 20,000 cells/mL, and 100 μL of this solution was transferred to each of the wells in the 96-well plate by an automated pipette (Response 4850 10–500 μL; Eppendorf, Horsholm, Denmark). This procedure was repeated for each primary isolate each time the 96-well plates were seeded. For both proliferation assays, all 96-well plates were seeded at the same day, and each plate was seeded with isolates from all the three anatomic region in random order, to avoid systematic seeding artifacts.
A colorimetric method was used to estimate the number of viable cells. The assay is based on mitochondrial enzymatic conversion of the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] to a formazan salt (CellTiter 96 AQueous; Promega Corp., Madison WI). We incubated the RPE cells for 2 hours with 20 μL of the tetrazolium compound and then measured the quantity of the formazan reaction product spectrophotometrically as the absorbance at 490 nm. Quadruplicates of each zone from two different pig eyes were obtained at 2, 4, 8, 10, 14, and 21 days for three passages. For each of the investigated anatomic origins, we verified that the measured absorbance was directly proportional to the number of RPE cells. Primary cultures of confluent porcine RPE cells from zones I, II, and III were trypsinized, and the total number of cells was determined as described earlier. Thereafter dilution curves of 100,000, 50,000, 25,000, 12,500, 6,250 and 3,125 cells/well were prepared in triplicate and incubated with the tetrazolium compound, and the absorbance measured (data not shown).
DNA synthesis was measured by incorporation of [3H]thymidine. Cells were incubated with 2 μCi/mL [3H]thymidine for 24 hours, and then the medium was removed and the cells were washed three times with CMF-PBS. Finally, the cells were frozen at −20°C, and subsequently, the lysed with sodium citrate and the [3H]thymidine incorporation was measured in a scintillation counter. Results are expressed as counts per minute (cpm) per well from quadruplicate samples.