This article details the isolation of bovine iris pigment epithelial (IPE) and retinal pigment epithelial (RPE) cells from bovine eyes. The process involves careful dissection and enzymatic treatment to dissociate the cells for further study.
In the vertebrate eye, pigmented epithelial cells, PECs, are cuboidal cells containing melanin granules - a photoprotective pigment. They are lined by the extracellular matrix and bound together by tight junctions. The PEC monolayer adjacent to the retina are retinal pigment epithelial, RPE, cells; the PEC layer of the iris are called iris pigment epithelial, IPE, cells.
To isolate bovine IPE and RPE cells, begin with a bovine eye. Immerse the eye in appropriate disinfectant to eliminate any microbial contamination from its surface. Carefully incise the iris circumference to separate the anterior segment from the posterior segment.
From the anterior segment, remove the lens to reveal the iris. Further, separate the ciliary body from the iris. Meanwhile, excise the vitreous humor and retina from the posterior eye bulb. Treat the iris and eye bulb with trypsin.
Trypsin degrades components of both tight junctions between the pigmented epithelial cells and extracellular matrix, initiating cell dissociation. Now, replace trypsin from the iris and eye bulb with suitable media. Gently scrape the tissues to release the dissociating IPE and RPE cells into the media.
Transfer the cell suspensions into tubes and centrifuge. Resuspend the cell pellets in media and seed into multi-well plates. Incubate, allowing IPE and RPE cells to adhere and proliferate.
After cleaning, open the eye and cut around the iris to remove the anterior segment, then, put it in a Petri dish. Leave the bulb with the vitreous until RPE cells are isolated.
To isolate IPE cells, if the lens come with the anterior segment, remove the lens, and use fine forceps to delicately pull out the iris containing the IPE cells. Place the iris in a Petri dish and wash it with sterile PBS. Cut the ciliary body with the scalpel #10. After preparing the irises, incubate them with 2 milliliters of 0.25% trypsin at 37°C for 10 minutes.
Remove the trypsin and add 2 mL of complete medium. Then, isolate the cells by scratching with a flat fire-polished Pasteur pipette. Transfer the cell suspension into a 2-mL tube, and centrifuge the cells for 10 minutes at 120 x g.
Discard the supernatant, and resuspend the cells in 2 mL of complete medium. Seed 0.32 million cells per well in a 6-well plate in 3 mL of complete medium. Place the plate in an incubator, and culture it at 37°C and 5% carbon dioxide.
To isolate RPE cells, remove the vitreous humor and retina from the posterior segment with forceps. Avoid damaging the retinal pigment epithelium. Wash the bulb with PBS. After preparing both eyes, fill the bulb about three-fourths full with trypsin, and incubate for 25 minutes at 37°C, with the Petri dish lid on top of the bulb eye.
Remove the trypsin, and add 1 mL of complete medium. Using a curved fire-polished Pasteur pipette, remove the RPE cells. Collect the cell suspension within the bulb, and transfer it into a 1.5-mL tube for resuspension.
Centrifuge the cells for 10 minutes at 120 x g. Seed 0.32 million cells per well in a 6-well plate in 3 mL of complete medium. Place the plate in an incubator, and culture it at 37°C and 5% carbon dioxide.
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