This article describes the isolation of retinal pigment epithelial (RPE) and iris pigment epithelial (IPE) cells from porcine eyes. The methodology involves careful dissection and culture techniques to obtain these specialized cells for further research.
The porcine eye contains a population of cuboidal epithelial cells that contain numerous melanosomes - organelles that help in light absorption - resulting in the pigmented appearance of these cells. Depending upon the location of these cells, epithelial cells present beneath the retina are called retinal pigment epithelial cells, or RPE cells, and the ones lining the iris are known as iris pigment epithelial cells, or IPE cells.
To isolate RPE and IPE cells, begin by taking a porcine eye. Submerge the eye in a suitable disinfectant solution to remove any microbes stuck to the eye's surface.
Remove the eye from the disinfectant solution and use a needle to make a small incision near the iris. Use this opening to make a complete circumferential cut along the iris boundary. This detaches the iris-containing anterior segment of the eye from the posterior eye bulb, which has the retina. Remove the excess portion and transfer the iris and the eye bulb into separate culture dishes.
Add a suitable culture media into the plate containing iris, and in the eye bulb. Scrape the tissue. The mechanical force loosens the epithelial cells, releasing them into the media. Aspirate the detached IPE and RPE cells. Seed them into separate culture plates and incubate till further use.
Clean remaining muscle tissue and skin from the eyes, and disinfect the eyes in iodine-based solution, and rinse them with PBS.
To isolate IPE cells, remove the lens and pull out the iris, as demonstrated. After preparing two irises, add 1 milliliter of complete medium and isolate the cells by scratching with a flat fire-polished Pasteur pipette, then count and seed the cells. Place the plate in the incubator.
To isolate RPE cells, remove the vitreous and retina from the posterior segment, and place the bulb in a Petri dish. Fill the bulb with 1 milliliter complete medium. Using a curved fire-polished Pasteur pipette, remove the RPE cells, collect the cell suspension within the bulb using a 1,000-microliter pipette, and transfer it into a 1.5-milliliter tube for resuspension. Then, count, and seed the cells, and incubate the plate as previously demonstrated.
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