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