The overall goal of this procedure is to isolate retinal stem cells from the ciliary epithelium of the eye. This is accomplished by first cleaning and cutting the eye in half, starting from the optic nerve, cutting through the cornea and meeting back at the optic nerve, and then removing the neural retina and lens. The second step of the procedure is to cut out the ciliary epithelium by cutting off the cornea iris and retinal pigmented epithelium.
The third step of the procedure is to enzymatically treat the ciliary epithelium, remove the sclera and mechanically dissociate the epithelium into single cells. The final step of the procedure is to resuspend the cells in serum free media and plate them in tissue culture plates containing serum free media with growth factors. Ultimately, results can be obtained that show prospectively the number of stem cells found in the ciliary epithelium of the eye through clonal sphere, formation in vitro.
Hi, my name is Brenda Coles. Our lab first came up with this method when we were researching papers on the regenerative capabilities of amphibians and fish in their eyes, and hypothesized that perhaps there was a cell in the mammalian eye with the same capabilities. Isolation of the retinal stem cells is performed in a specific sterile room dedicated to primary culture experiments.
Prior to starting this procedure, set up the dissecting microscope and cold light source and then lay out the sterile dissecting instruments. Each hood has a hot beat sterilizer for sterilizing the instruments between steps. We'll isolate retinal stem cells from eyes that have been placed immediately in a sterile Petri dish containing artificial cerebral spinal fluid After the removal from mice sacrificed according to approved animal ethics protocols, The most difficult aspect of this procedure is dissecting a round object under the microscope.
Great care must be taken so as not to destroy the tissue of interest Under the dissecting microscope, clean the eye with forceps. Get rid of the hair and the connected tissue that's attached to the cornea scleral border. Then transfer the eye to a new dish with A CSF while holding the eye stationary with serrated forceps.
Use angled micro dissecting scissors to remove the ocular muscles. Remove the optic nerve as well if it's still attached to the eye, try not to squish the eye during this process. Transfer the eyes to a new dish with a CSF.
Next used curved micro dissecting scissors to cut the eye in half. Begin at the optic nerve and cut through the middle of the cornea meeting back at the optic nerve where the cut was initiated. It is ideal if the two pieces of eye are roughly equivalent in size because this will make the next step easier.
Holding the corneas using two forceps, gently peel the two eye halves apart. Remove and discard the lenses viscera and the neural retina from the eye shells. Transfer the shells to a new dish with A CSF.
Now we are ready to isolate the ciliary epithelium. To begin orient the eye shell so that the cornea is on your right and the retinal pigmented epithelium is on your left. Gently pin the eye shell down with straight forceps on the RPE side.
Next, use the scalpel to cut the cornea and iris away from the ciliary epithelium of the eye by gently pushing down on the scalpel rather than using sawing motions. After that, cut the ciliary epithelium away from the RPE. Use non serrated forceps to transfer the strip of ciliary epithelium to a new 35 millimeter dish containing A CSF.
In the same way, isolate the ciliary epithelium from the other eye shell. Once the ciliary epithelium strips have been collected, transfer the strips into a 35 millimeter dish containing two milliliters of dis space. Place the dish with the strips in a 37 degree Celsius incubator for 10 minutes.
After 10 minutes, transfer the strips from the dis space into a dish containing two milliliters of filtered drips in Halle ur haase and kind reic acid. Place the dish at 37 degrees Celsius for 10 minutes. Now return to the dissecting microscope while holding the sclera down with straight forceps.
Use the bottom of the curve non-rated forceps to gently scrape the ciliary epithelium away from the sclera. Remove the sclera from the dish. All that should be left in the dish.
Now are the cells of interest and the enzyme solution. Using a fire polished cotton plugged pipette, transfer the solution into a 14 liter tube. Tritrate this solution 30 times to break apart the epithelial cells by gently forcing the solution in and out of the pipette centrifuge the tube for five minutes at 1500 RPM.
When the centrifugation is done, remove the tube from the centrifuge carefully. Since the cells are prone to coming off the bottom of the tube. At this stage, gently aspirate the majority of the SuperNet in using a fire polished pipette and then add one milliliter of trypsin inhibitor in serum free media to the cells.
Use a small borehole cotton plugged pipette to tri the sample approximately 50 times until it is a single cell suspension. Centrifuge the tube again for five minutes. At 1500 RPM, remove the supernatant and replace with one milliliter of your plating.
Medium refrigerate gently using a fire polished glass pipette to resuspend the cells. After counting the cells plate them at the desired density. In a 24 well plate, we usually plate 10 cells per microliter by filling each well first with medium, and then adding the cell suspension to get a final volume of 500 microliters of media.
Place the plate in a 37 degree Celsius carbon dioxide incubator where it will not be moved until you count the spheres that arise after seven days when this procedure is performed successfully. The dissected ciliary epithelial cells should look like this after being dissociated and plated at low density. After seven days in culture, the retinal stem cell spheres that arise are counted.
A sphere should be over 75 microns in diameter and free floating to be counted as a stem cell derived sphere. However, some cells will have limited proliferation and form PHE that do not meet size criterion and will not be counted as stem cell derived spheres. An example of such asteroid is shown here After its development.
This technique paved the way for researchers in the field of vision sciences to explore the possibility of using retinal stem cells in treating retinal disease.
