Isolating RPE and IPE Cells from Bovine Eye: A Procedure to Harvest and Culture Bovine Primary Pigment Epithelial Cells
Transcrição
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.
