We engineer disease models to mimic aspects of retinal pathologies. With these models, we can better understand the role of RPE cells in diseases based on their response to physical changes, like when strain occurs on the monolayer or BREX membrane thickens with age. One of the biggest challenges is having a representative cell type.
Compared to the commonly used immortalized cell line, the primary cells isolated with this technique make our disease models more realistic to what happens in our eyes. This technique provides us with a method of obtaining high-quality cells without requiring extensive differentiation times. Furthermore, we use resources that are commonly available to research labs, which makes it more cost effective.
Moving forward, we want to look at how natural changes that happen in your eyes during aging may lead to the early stages of AMD. We want to better understand those changes so we can identify specific treatment targets. Once the enucleated porcine eyes from a USDA-inspected butcher shop arrive at the laboratory, proceed with exterior tissue removal.
Hold the muscle attached to the eye with one hand. Using a scalpel, gently cut away the tissue surrounding the sclera with the other hand. Use curved iris scissors to trim the optic nerve, then place the eye onto the ice with the cornea facing downwards.
For dissection, using tweezers, transfer the eye into the well containing 10%povidone-iodine solution and rotate it to ensure that it is fully coated. Place a sterile gauze onto the shallow inverted lid of the Petri dish. Place a cell strainer in a separate Petri dish.
Transfer the eye from the povidone-iodine solution into the well containing DPBS and wash it for roughly five seconds, then move it to the next DPBS-containing wells progressively to dilute the iodine solution. Afterwards, place the eye onto the sterile gauze. Using a scalpel, make a small incision below the ora serrata or roughly 1/3 down the globe from the iris edge.
Use iris scissors to cut evenly along the circumference of the globe parallel to the cornea. Use the tweezers to grab the optic nerve and gently lift the globe away from the gauze. Place the eye cup into the prepared cell strainer with the interior of the eye cup facing up.
Gently add cooled DPBS to the eye cup, ensuring the fluid level is just below the lowest point of the incision. To begin, sterilize and dissect the enucleated eyes obtained from a USDA-inspected butcher shop to prepare the eye cu. Gently place the eye cup with the interior facing up into the Petri dish containing the cell strainer.
Add cooled DPBS to the eye cup, ensuring the fluid level is just below the lowest point of the incision. Under a flashlight, find any areas where the neural retina is starting to detach from the retinal pigment epithelium. Then use blunt tweezers to gently grab the lifted neural retina and peel it away.
Gently collect the neural retina near the optic disc. Using a Pasteur pipette attached to a vacuum aspiration system, aspirate out the neural retina. Carefully add additional cooled DPBS to replace the aspirated volume.
Now, aspirate this added DPBS to remove any remaining neural retina. After removing the neural retina from all eye cups, gently add trypsin to each eye cup and replace the Petri dish lid. Incubate the eyes with trypsin at 37 degrees Celsius with 5%carbon dioxide for 30 minutes.
Under a flashlight, use 1, 000 microliter pipette to gently dislodge the retinal pigment epithelium cells from each eye cup. Transfer the dislodged cells to a 15 milliliter centrifuge tube. Wash the eye cup with cell culture media to collect any remaining cells as well as to neutralize the trypsin.
Mix these media containing cells with previously collected cells. Centrifuge the cell suspension at 250 G for five minutes at room temperature. Then remove the supernatant without disrupting the cell pellet.
Resuspend each cell pellet in three milliliters of DNase working solution. Incubate the cell suspension at 37 degrees Celsius with 5%carbon dioxide for 15 minutes, then centrifuge it at 150 G for five minutes at room temperature. Remove the supernatant and resuspend the cell pellet in three milliliters of fresh cell culture media.
Transfer cells obtained from two to three eyes in one well of a six-well plate and consider this as passage zero. Then carefully transfer the seeded six-well plate into a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. Primary retinal pigment epithelium cells were isolated from porcine eyes with characteristic pigmentation evident three days post isolation.
The cells achieved full confluence after one week, indicative of a healthy monolayer. Cultures exhibiting abnormal morphology and excessive pigmentation were recognized as contaminated and not used in the experiments.
