Isolating IPE and RPE Cells: A Technique for Obtaining Ocular Pigment Epithelial Cells from Rabbit Eye

Pigment epithelial cells are terminally differentiated cuboidal cells connected by tight junctions. These epithelial cells lining the iris are the iris pigment epithelial or IPE cells, and those near the retina are the retinal pigment epithelial or RPE cells.

To extract these cells, take an intact rabbit eye. Treat the eye with a disinfectant to remove any microorganisms from the eye's surface. Wash off the disinfectant with a buffer solution. Make an incision near the iris. Using this opening, make a complete circular cut along the iris boundary, dividing the eye into an anterior segment containing iris and a posterior part comprising retina and vitreous humor. 

Take the anterior segment and discard the lens to access the iris. Remove the iris and place it on a culture dish. Subsequently, take the posterior segment and discard the gelatinous vitreous humor and the retina from the eye bulb. 

Treat the iris and the eye bulb with trypsin and incubate. Trypsin degrades the tight junction and the extracellular matrix proteins, initiating dissociation of epithelial cells. Discard the trypsin and add a suitable culture medium. Gently scrape the iris and eye bulb surface to release the epithelial cells in the medium.

Finally, seed the IPE and RPE cells in separate wells. Cells are ready for further downstream assays. 

After euthanizing the animal, use curved scissors and Colibri forceps to enucleate the eyes, then clean the remaining muscle tissue and skin from the eyes. Collect the eyes in a 50-milliliter tube filled with non-sterile PBS, and transfer the tube to the laminar flow hood. Disinfect them by submerging in iodine-based solution for two minutes, then transfer the eyes to a 50-milliliter tube filled with sterile PBS.

Put one eye on a sterile gauze compress, and firmly hold the eye close to the optic nerve, then cut near the limit of the iris with a #11 scalpel. Using scissors, cut around the iris, and remove the anterior segment, and put it in a Petri dish leaving the bulb with the vitreous, until the retinal pigment epithelial, or RPE cells, are isolated.

To isolate iris pigment epithelial, or IPE cells, remove the lens with fine forceps, and delicately pull out the iris containing the IPE cells. Place the iris in a Petri dish, and wash it with sterile PBS. Add 500 microliters of 0.25% trypsin, and incubate for 10 minutes at 37 degrees Celsius. Remove the trypsin, add 500 microliters of complete medium, and scrape the IPE delicately with a flat fire-polished Pasteur pipette.

Collect the cell suspension, and put it in a 1.5-milliliter tube. Count the cells using the Neubauer chamber, and seed 0.2 million cells per well in a 24-well plate with 1 milliliter of complete medium. Place the plate in an incubator, and culture it at 37 degrees Celsius and 5% carbon dioxide.

To isolate RPE cells, remove the vitreous humor and retina from the posterior segment with thin forceps. Put the bulbs in a 24-well plate, and add 500 microliters of 0.25% trypsin per eye, and incubate for 10 minutes at 37 degrees Celsius. Remove trypsin, add 500 microliters of complete medium per globe, and scrape the RPE cells delicately with a curved fire-polished Pasteur pipette.

Collect the cell suspension, and put it in a 1.5-milliliter tube. Count the cells using the Neubauer chamber, and seed the cells as described previously. Place the plate in the incubator.