The overall goal of the following experiment is to transplant enriched photoreceptor precursor cells into a recipient mouse retina. This is achieved by first isolating the retina eye of young donor animals. In the second step, the postnatal photoreceptor precursor cells are isolated from the retinal cell population by CD 73 based magnetic associated cell sorting.
Next, the photoreceptor precursors are transplanted into the subretinal space of recipient animals to facilitate their integration into the recipient retina. Ultimately, the integration potential and behavior of the transplanted photoreceptor precursor cells can be analyzed by immunofluorescence microscopy. This method can help to answer key questions in the field of retinal regeneration, such as what is the capacity of transplanted photoreceptors to integrate in the neural circuitry of the host for restoration of vision.
The implications of this technique extend beyond the treatment of retinal degenerative diseases. Once the installation and enrichment of photoreceptor precursors by max can easily adapted to GMP conditions, Visual demonstration of this method is critical As the transplantation steps are difficult to learn, the subtle handling and placement of the needle in the subretinal space is crucial to ensure maximum success and reproducibility To dissociate the retinas. Begin by briefly rinsing the heads of postnatal day four pups in 70%ethanol, followed by A PBS wash.
Then transfer the heads to cold HBSS to nucleate the eyes for each eye. First, open the eyelid. Then using a pair of curved forceps.
Grasp the organ at the optic nerve region and pull the eye carefully out of the orbit. Next to isolate the retina, introduce the tip of a pair of closed scissors into the optic nerve and open the blades. Peel the retinal pigment, epithelium, choroid out, and use curved forceps to remove the lens and flood vessels.
Incubate the isolated retinas in a 1.5 milliliter reaction tube containing papain solution for 30 to 60 minutes in a shaker at 400 RPM and 37 degrees Celsius. While the retinas are incubating, dispense one milliliter of OVO moid solution into a 15 milliliter reaction tube, labeled one then to a two milliliter reaction tube labeled two at 60 microliters of DNAs one 60 microliters of OVO MOID solution and 520 microliters of EBSS. After the incubation transfer the papain solution containing the partially digested retinas to reaction tube two, and then iterate the retinas 10 times with a fire polished pipette.
Gently layer the single cell suspension on top of the OVO MOID solution in reaction tube one and centrifuge the cell solution for five minutes at 300 Gs and room temperature. Then discard the supernatant and resuspend the cells in 500 microliters of max buffer. To sort by max first incubate the cells in 10 micrograms per milliliter of rat anti CD 73 antibody on ice.
After five minutes, wash the cells in 10 milliliters of max buffer. Then resuspend the pellet in 480 microliters of max buffer, and incubate the cells in 120 microliters of go anti RET IgG microbeads on ice without agitation, shaking or mixing. After 15 minutes, wash the cells in five milliliters of max buffer during the wash, place an LS column onto a max magnet and put a pres separation filter on top of the column.
Next, hydrate the filter and the column with three milliliters of max buffer. Collecting the buffer in a 15 milliliter tube labeled W for wash. When the cells have finished spinning down, re suspend the pellet in 500 microliters of max buffer and unload the cells into the filter.
Add one milliliter of max buffer on top of the cells, and then collect the negative fraction in a 15 milliliter reaction tube labeled minus for negative. Next, add three milliliters of max buffer to the column three times to wash it. Now remove the column from the magnetic stand.
Install it on top of a 15 milliliter reaction tube labeled plus for positive. And quickly load five milliliters of max buffer into the column. Plunge all of the buffer through the column, and then after spinning down the positive fraction, re suspend the pellet in 500 microliters of max buffer.
Place the cells at four degrees Celsius and count them. Then adjust the concentration to two times 10 to the fifth cells per microliter in max buffer, keeping the cells at four degrees Celsius for transplantation of the isolated photoreceptor precursor cells dilate the pupils of a sedated adult mouse with a drop of phenyl left vent tropic chaid while the pupils dilate. Use a diamond pen to cut a cover slide into approximately five by five millimeter pieces.
Place the mouse under a stereo microscope, restrain the animal in a mouse head holder, and then apply a drop of ICIC gel to each eye to prevent drying. Now use a 30 gauge half inch needle to make a small hole at the border between the sclera and the cornea. Then place one of the five by five millimeter covers slide pieces on top of the cornea, allowing direct visualization of the retina.
Next, flush a pre sterilized microliter syringe several times with sterile deionized water, and then load the syringe with one of the sorted cell suspension. Direct the needle diagonally through the conjunctiva and sclera placing it in the nasal half of the retina. Then gently punch a hole through the retina to the subretinal space and inject the cells.
Confirm the bullous uniformed detachment of the retina, which should cover approximately a quarter of the retinal space without any bleeding. Then withdraw the syringe gently and flush it several more times with the ionized water. Finally, release the mouse from the restrainer.
Inject the animal with aamaz oil hydrochloride to reverse the effects of the anesthesia and allow it to recover in a dark 25 degrees Celsius chamber in order to assess the ability of rod photoreceptors to integrate into the mouse retina. A mouse reporter line was used in which GFP is driven by the neural retina losing zipper or NRL promoter as demonstrated in these histograms. The initial retinal cell suspension contained 30.4%GFP positive rod photoreceptors.
Following CD 73 based max sorting and enrichment of up to 86.9%of the GFP positive photoreceptor precursor cells was observed in the CD 73 positive fraction in the CD 73 negative fraction. Only 9.9%of the cells were positive for GFP. The enrichment of N-R-L-G-F-P positive cells following CD 73 based max is also observed.
After plating in vitro, the donor cells remain at the site of injection as defined by the bullous detached host retina and integrate into the outer nuclear layer of the host retina acquiring the morphology of mature photoreceptors. Once master magnetic associated cell sortin can be performed within two hours if correctly.Done. This procedure can be also adapted for the enrichment of stem cell derived photoreceptors or even other retinal stem cells.
Given the availability of specific antibodies to answer additional questions in regard to restorative or rescue by cell transmutation for the retina. After watching this video, you should have a good understanding of how to transplant. Photo max enriched photoreceptor precursor cells to this subretinal space of recipient mice.
