Cell Preparation and Identification

1. All experiments are carried out on pigmented epithelial cells isolated from rabbit ciliary epithelium using a method modified from refs. 15,16. Figure 2 provides a schematic outline of the ciliary epithelial cell isolation, as described below.

2. Five- to 10-wk-old pigmented rabbits (Reimens, St. Agatha, Ontario, Canada) are anesthetized with 8 mg/100 g of sodium pentobarbital, and eyes are enucleated in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research.

3. Enucleated eyes are placed in sterile D-PBS, and a small incision is made at the corneo-scleral junction using a razor bade. The anterior segment of the eye is removed by cutting around the globe, starting at the incision, with blunt-ended scissors.

4. The ciliary body is then freed from the iris by making a small incision between the iridal and ciliary processes using iridectomy scissors.

5. The epithelium is then dissected from the underlying stroma under a dissecting microscope at 200x magnification, and treated for 30-40 min with 5 mL D-PBS containing collagenase (1.5 mg/mL) and pronase (1 mg/mL).

6. The enzyme reaction is terminated by the addition of 1.5 mL of 10% fetal calf serum (FCS), and the cell suspension centrifuged at 2000g for 5 min, washed with fresh sterile DMEM, and recentrifuged for 5 min.


Pigmented ^¿¡¿ii^SSL -T" I, w ' up to 14 days epithelial cell r4-BM

Fig. 2. Schematic diagram of rabbit PCE isolation and culture. See text for details.

7. The epithelial pieces are triturated gently with a sterile Pasteur pipet to yield single cells and small tissue explants.

8. Once isolated, cells are seeded onto glass cover slips at a density of 105 cells/mL in DMEM containing 5% FCS and antibiotics, and incubated in an atmosphere of 5% CO2/95% O2 at 37°C until use (up to a maximum of 14 d). PCE cells are clearly identified from NPCE cells by the presence of pigment granules. Furthermore, NPCE cells tend to have limited survival under the culture conditions used and, after 5, d ciliary epithelial cultures consisted primarily of PCE cells.

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