Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

The protocol is significant since it shows the isolation and transfection of pigment epithelial cells from five mammals as an optimal system to study ocular gene therapy in various setups. The main advantage of this method is its flexibility, transferability, high cell yield, viability and transfection rate due to the five species and the non-viral SB100X transposon system used. These techniques have been developed to study gene therapy approaches targeting retinal and iris pigment epithelial cells, which are isolated and transfected in the present protocol, but are usable for any ocular research.

Demonstrating the procedure will be Gregg Sealy, a technician from the laboratory. 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 number 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 one 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 the 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. Clean remaining muscle tissue and skin from the eyes and disinfect the eyes in iodine-based solution and rinse them with PBS.

To isolate IPE cells, remove the lens and pull out the iris as described previously. After preparing two irises, add one milliliter of complete medium and isolate the cells by scratching with a flat, fire-polished Pasteur pipette, then count and seed the cells. Place the plate in the incubator.

To isolate RPE cells, remove the vitreous and retina from the posterior segment and place the bulb in a Petri dish. Fill the bulb with one milliliter complete medium. Using a curved, fire-polished Pasteur pipette, remove the RPE cells.

Collect the cell suspension within the bulb using a 1, 000 microliter pipette, and transfer it into a 1.5 milliliter tube for resuspension. Then count and seed the cells and incubate the plate as previously demonstrated. After cleaning, open the eye and cut around the iris to remove the anterior segment, then put in a Petri dish.

Leave the bulb with the vitreous until RPE cells are isolated. To isolate IPE cells, if the lens come with the anterior segment, remove the lens and use fine forceps to delicately pull out the iris containing the IPE cells. Place the iris in a Petri dish and wash it with sterile PBS.

Cut the ciliary body with a scalpel number 10. After preparing the irises, incubate them with two milliliters of 0.25%trypsin at 37 degrees Celsius for 10 minutes. Remove the trypsin and add two milliliters of complete medium, then isolate the cells by scratching with a flat, fire-polished Pasteur pipette.

Transfer the cell suspension into a two milliliter tube and centrifuge the cells for 10 minutes at 120 times G.Discard the supernatant and resuspend the cells in two milliliters of complete medium. Seed 0.32 million cells per well in a six well plate in three milliliters 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 forceps. Avoid damaging the retinal pigment epithelium. Wash the bulb with PBS.

After preparing both eyes, fill the bulb about about three-fourths full with trypsin, and incubate for 25 minutes at 37 degrees Celsius with the Petri dish lid on top of the bulb eye. Remove the trypsin and add one milliliter of complete medium. Using a curved, fire-polished Pasteur pipette, remove the RPE cells.

Collect the cell suspension within the bulb and transfer it into a 1.5 milliliter tube for resuspension. Centrifuge the cells for 10 minutes at 120 times G.Seed 0.32 million cells per well in a six well plate in three milliliters of complete medium. Place the plate in an incubator and culture it at 37 degrees Celsius and 5%carbon dioxide.

Culture the cells at 37 degrees Celsius and 5%carbon dioxide in a humidified incubator. After three to four days, pipette up and down to collect nonadherent cells and put half of the volume in another well. Fill the well to one milliliter with complete medium.

After another three to four days, repeat the cell collection, this time pooling the nonadherent cells from two wells into one. Add medium to all wells. Observe the cells and change the medium two times per week.

When cells reach confluence, switch to complete medium with 1%FBS. Using the aforementioned protocols, IPE and RPE cells were successfully isolated and cultured from five different species. The RPE expression pattern was confirmed in primary bovine IPE cells by immunofluorescence for RPE65.

Cell viability was studied to exclude a potential toxicity of the electroporation buffer using a commercially available cytotoxicity assay kit and a knot electroporated control. No impact on cell viability was observed. Precultured pigment epithelial cells from different species were transfected with the yellow fluorescent Venus protein.

Transfected RPE19 cells were used as a positive control. The quantification of the transfection efficiency in precultured pig RPE is shown here. Freshly isolated rabbit pigment epithelial cells were transfected with the yellow fluorescent protein Venus and were monitored by microscopy.

Pigment epithelial cells were transfected with pigment epithelium derived factor and protein secretion was monitored by Western blot. The signal for transfected cells was higher compared with non-transfected cells for all species and days studied, and no decrease in protein secretion was observed within this time. It is crucial to avoid scraping the cells with sharp tools.

Here, fire-polished Pasteur pipettes are used, except in small mouse eyes, where a round scalpel must be used. Pigment epithelial cells can be used to develop gene therapy approaches and to simulate pathological conditions, for example, oxidative stress damage. These models can also be used to test therapeutic drugs.