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