My research focuses on the development of stem cell-based therapy for advanced stages of retina diseases that cause photoreceptor degeneration. The goal of our research is to replace the loss of photoreceptors in patients with clinically safe and functional photoreceptor progenitors with the aim of improving their visual acuity or even restoring their vision. The main preclinical model is mice.
But their small size eyeball make it challenging to inject materials, especially under the retina. Developing the skill to perform successful subretinal injections requires patient and perseverance. We have successfully developed a laminin base for the receptor differentiation method that is able to generate photoreceptor progenitors in 32 days.
The subretinal transplantations of this photoreceptor progenitors cell suspensions in the rodent resulted in synaptic connectivity between the holes and graft in the eyes. This has led to partial visual restoration. Our approach is simple.
Direct visualization helps the surgeon maneuver the needle to the area of interest. Begin by placing PRDM in a water bath set at 37 degrees Celsius. Remove a cryovial containing day 32 HESC-derived photoreceptor progenitor cells from liquid nitrogen and keep it on dry ice.
Then thaw the cryovial in a water bath at 37 degrees Celsius for three to five minutes. Resuspend the cells in one milliliter of PRDM. Centrifuge them at 130g for four minutes.
After removing the supernatant, resuspend the pellet in one milliliter of PRDM. To count the cells, take 10 microliters of the cell suspension and mix with 0.2%Trypan Blue following the manufacturer's instructions. Pipette the cell mixture into the cell counting chamber slide.
And determine the cell number and viability using an automated cell counter. If cell viability is above 70%centrifuge the remaining cell suspension at 130g for four minutes. Remove the supernatant and resuspend the cell pellet in PRDM for transplantation.
Using a 10 microliter pipette tip, resuspend cell clumps in the microfuge tube until no clumps are visible. Load the suspension into a 33-gauge injection syringe and confirm the extrusion of cell solution through the needle. To begin, take an anesthetized mouse and apply one drop of 1%tropicamide and 2.5%phenylephrine into the eye to dilate the pupil.
Then apply an ophthalmic gel to the cornea to prevent dryness and anesthesia-induced cataracts. Next, set up an upright operating microscope with a direct light path in a sterile environment to perform the subretinal injection. To prepare the 10 microliter glass syringe, detach the needle hub and attach the 33-gauge blunt needle.
Take the metal hub cover and securely fasten the needle onto the syringe. Flush the needle with distilled water to test leakage and needle patency. Then empty the syringe and set it aside carefully.
Next, position the anesthetized mouse on a cushion and orient the treatment eye toward the microscope's objective. Apply 0.5%proparacaine hydrochloride and wait for 30 seconds. Then apply 150 microliters of ophthalmic gel to the eye and affix a round cover slip.
Perform a preliminary eye examination observing the cornea, iris, pupil, lens, and conjunctiva. Adjust the focal plane to visualize the fundus of the mouse eye through the pupil. Then align the head so that the optic head aligns with the center of the pupil.
Afterwards, tap the base of the tube containing HESC-derived photoreceptor progenitors to get a uniform cell suspension. Using a 10 microliter glass microliter syringe with a 33-gauge blunt needle, draw two microliters of cell mixture. Use a 30-gauge disposable needle to create a sclerectomy wound positioned two millimeters behind the limbus.
Maintain the needle's angle at approximately 45 degrees to prevent contact with the lens. Once the needle tip becomes visible within the eye, carefully retract the needle. Discard the used needle into a sharp bin to prevent needle prick injuries.
Now, take the glass syringe and insert the blunt needle into the sclerectomy wound. Advance the blunt needle without touching the lens until it reaches the opposite retina of the entry wound. Gently penetrate the retina until a pressure branch on the sclera is seen.
Then inject two microliters of the cell suspension, or PRDM media, into the subretinal space while maintaining gentle pressure on the syringe. The formation of a visible bleb indicates successful injection. Wait 10 seconds to allow the cells to settle down.
Gently retract the needle from the eye. To visualize the bleb, move the mouse head to position the eye under the microscope, and secure the position by gently holding the head. For intraoperative OCT, use the iOCT function on the operating microscope and select cube on the OCT screen.
Position the scanning area on the bleb by pressing the arrow buttons. To adjust the OCT, slide centering and focus for the best OCT quality. Press capture or scan to acquire the OCT scan of the bleb area.
And review the images to check the quality of the scans. Finally, remove the cover slip and gently clean the gel from the eye using gauze. Administer antibiotic ointment once after the injection to prevent infection.
The OCT scan revealed distinct HESC entities suspended in the cell treated eye. In contrast, the media treated eye displayed unclouded fluid, void of cells within the subretinal space.
