This protocol will outline an easy and efficient way to successfully isolate primary mouse retinal pigmented epithelium cells. Retinal pigmented epithelium cells play a very important role to maintain the photoreceptor cells and to ensure the normal function of the retina. Our lab has been isolating primary mouse pigmented epithelium cells as an important tool to study the outer blood-retinal barrier and related diseases such as age-related macular degeneration.
To extract the mouse eyes, ethically euthanize the mouse according to your institution's approved methods, then place the mouse on a sterile underpad and nucleate the eye by pressing a 5/45-style tweezer on the orbital area to induce proptosis, then move the tweezer to the posterior of the eye and gently pull to remove the eye ensuring that the optic nerve is still intact. While using forceps, submerge the eyes in a 2-milliliter microcentrifuge tube containing 5%povidone iodine. Incubate the eyes at room temperature for 2 to 3 minutes.
Remove the eyes from the povidone iodine and place them in a Petri dish. While using a sterile transfer pipette, wash the eyes with Hanks'Balanced Saline Solution until the orange color of the povidone iodine is gone. This takes approximately 2 to 3 washes.
Place the eyes in a new Petri dish and submerge them in cold complete RPE cell growth media. From there, you can begin dissecting under a dissecting microscope. Under the microscope, use tweezers and/or Vannas scissors to pull or cut away connective tissue in extraocular muscles.
Eyes can then be held for an hour in cold RPE cell culture media. However, it is preferred to proceed directly to the next step after cleaning the eyeball. Transfer the eyes to a 2-milliliter microcentrifuge tube containing the enzyme A solution, then incubate the eyes inside an incubator at 37 degrees Celsius for 40 minutes.
Remove the eyes from the enzyme A solution and place them in a new 2-milliliter microcentrifuge tube containing the enzyme B solution, then place them inside an incubator and incubate at 37 degrees Celsius for 50 minutes. Remove the eyes from enzyme B and place them in a 60-millimeter Petri dish and submerge them and complete RPE cell growth media, then incubate the eyes at 4 degrees Celsius for 30 minutes. Place the dish under the dissecting microscope and begin dissecting.
Use M5S forceps and a needle of a syringe to make an incision in the ora seratta section. Now with two forceps, grasp the inner part of the incision with one forcep and grasp the cornea with the other forcep. Begin to slowly tear the cornea from the retina by pulling the cornea.
Be sure to tear in small increments and move down the tear as you remove the cornea. This will ensure that the RPE will remain mostly intact and you will get a cleaner tear. Once the cornea is completely detached, you should be able to see the iris and lens.
Remove both the iris and lens with forceps to maintain the purity of the isolation. To remove the neural retina from the RPE layer, grasp the outer layer of the eyecup with one tweezer and position an arm of a second tweezer between both the RPE and neural layers. From there, gently pull or scoop the neural retina away from the RPE layer, then discard the neural retina.
Move the RPE layer to a different place on the dish free of debris. To separate the RPE from the choroid and sclera, gently scrape the inside of the eyecup with a 5/45-style tweezer to ensure the separation of the RPE cells. The RPE cells appear cloudy when suspended and complete RPE cell growth media.
Aspirate the cells using a 3.2-milliliter sterile transfer pipette and transfer to a new 15-milliliter centrifuge tube containing complete RPE cell growth media. The cells can be centrifuged at 300g for 10 minutes at room temperature. You can then aspirate out the supernatant and resuspend the pellet in warmed RPE cell growth media.
Plate the cells on a 100-millimeter Petri dish and incubate at 37 degrees Celsius and 5%CO2. These images show a successful isolation at passage 0 and passage 1, which is evident by the pigmentation of the RPE cells. At passage 4, the cells become less characteristic and exhibit less pigment, show a spindle-like appearance or become more robust with a larger surface area.
We validated the RPE cells with an RPE-specific marker, RPE65 and a cytoskeletal protein F-actin. We also checked for potential contamination with Muller cells by staining with a Muller cell-specific marker, vimentin. This protocol is a simplified adaptation of existing protocols.
The protocol uses materials and associated equipment that are available in most research labs, which will help isolating primary mouse RPE cells in an effective way.
