This protocol allows for the efficient isolation and culture of mouse RPE cells. Healthy primary mouse RPE cultures are valuable models to understand the mechanisms of underlying eye diseases. Confluent and viable RPE cultures obtain within three days and can be grown for weeks.
This protocol has been successfully used to understand the mechanisms underlying early age-related macular degeneration, which is the leading cause of blindness among the elderly in developed countries. It is important to visualize the dissection steps. Details are critical for the success of the protocol, for instance, how to handle the eyeballs so that the sclera isn't punched, where exactly cuts should be performed, and how to embed the eye cups in trypsin so that they stay open.
Begin with preparing all the necessary reagents for the protocol. Then, start cleaning the eyeball by carefully removing all connective tissue, blood, and muscles, using Dumont number five forceps and angled scissors, without making any cuts in the sclera. Transfer the eyeball to a fresh dish containing three milliliters of HEPES-free HBSS buffer regularly to keep the eye fresh and clean and avoid any contamination.
Use the optic nerve as a handle to hold the eyeball and make a hole in the center of the cornea with a sharp carbon steel number 11 blade. Make three incisions in the cornea through the hole with Dumont number five scissors, ensuring that there is sufficient space to remove the lens. Hold the optic nerve and apply slight pressure to the ora serrata with the base of the angled scissors, until the lens comes out completely.
Keep the iris epithelium intact to prevent the detachment of the neural retina and RPE during incubation. Place the eye in HEPES-free HBSS buffer. Incubate the eyes without lenses in hyaluronidase solution at 37 degrees Celsius for 45 minutes in a 5%carbon dioxide aerated incubator to detach the neural retina from the RPE.
Place each eye in a new well with 1.5 milliliters of cold HBSS HEPES buffer per well and incubate it on ice for 30 minutes. After washing, place each eye into a 35 millimeter culture dish with fresh HBSS HEPES buffer. Cut the cornea through the original incisions with eight centimeter Vannas scissors until the ora serrata is reached, then cut below the ora serrata to remove the iris epithelium and cornea.
Hold the ora serrata with curved tweezers and using angled micro forceps, pull away the neural retina, making sure that the RPE layer is not cut. Then, cut the internal attachment to the optic nerve. Cut the optic nerve and transfer each eye cup to a different 12 well plate containing 1.5 milliliters of fresh trypsin-EDTA per well.
Ensure that the eye cups remain opened and completely submerged in the trypsin. Incubate the eye cups at 37 degrees Celsius for 45 minutes in a 5%carbon dioxide incubator. Collect each eye cup together with any RPE sheets detached during the trypsin incubation and transfer them into a 12 well plate containing 1.5 milliliters of FBS solution per well.
If RPE sheets remain in the trypsin solution, use a micropipette to transfer them to the well with FBS solution. Hold each eye cup by the optic nerve and shake it face down into the 12 well plate containing 1.5 milliliters of 20%FBS in HBSS HEPES buffer until complete detachment of the RPE sheets is achieved. Collect any RPE sheets and RPE clusters with a micropipette, avoiding any white pieces of sclera or choroid, which could contaminate the cultures, and place them in a 15 milliliter tube.
Pull two eyes from the same mouse in one tube. Then, centrifuge the RPE mixture at 340 x g for two minutes at room temperature and discard the supernatant. Gently resuspend the RPE pellet in a one milliliter mixture of 0.25%trypsin-EDTA and incubate it for one minute in a water bath at 37 degrees Celsius to disaggregate the RPE sheets into single cells.
Then gently pipette up and down 10 times, avoiding bubble formation. Add nine milliliters of freshly prepared RPE medium to dilute and inactivate the trypsin and centrifuge the mixture at 340 x g for two minutes at room temperature. Aspirate the supernatant and carefully resuspend the cell pellet in 150 microliters of RPE medium with 5%FBS.
Next, remove PBS from the bottom chamber of the previously prepared laminin coated membrane insert and add 700 microliters of RPE medium to it. Remove the laminin from the upper chamber of the membrane insert and distribute the RPE cell suspension drop-wise and uniformly to the center of the chamber. Place the membrane insert undisturbed in a 5%carbon dioxide incubator at 37 degrees Celsius for at least 24 hours.
Then check the membrane insert under the microscope to make sure that most RPE cells are attached to the insert and the confluence is at least 50%Do not change the media during the first 72 hours. RPE cells isolated using this protocol showed the typical RPE size, morphology, and pigmentation. A highly-polarized RPE monolayer with two nuclei joined by tight junctions, along with the presence of apical microvilli and basal infoldings, was observed after one week.
Polarization of the RPE monolayer was confirmed by TER values, with the average higher than 200 ohms centimeter squared, which remained stable over time. In phagocytosis assays performed on FITC labeled bovine POS, engulfment and digestion of POS was observed, indicating formation of a functional confluent monolayer of RPE cells. Viable cultures require short dissection time, which is achieved with experience.
Some RPE cells may stay attached to the neural retina, but if several RPE sheets remain attached, the protocol will not succeed. Fresh hyaluronidase must be used every time to prevent this problem. These functional primary mouse RPE cell cultures are a valuable tool to study the pathobiology of the RPE in many eye diseases.
For example, this protocol made it possible to study the pathobiology of the RPE in a mouse model of macular degeneration.
