The current protocol shows in detail subretinal delivery of the whole retinal organoids in a large animal model, aiming at developing cell therapy approaches for retinal degeneration. The main advantage of this technique is that it allows for delivery of organoids directly to the target tissue, the retina. The technique aims to treat advanced blinding retinal degenerative conditions, such as age-related macular degeneration, retinitis pigmentosa, or leber congenital amaurosis.
Demonstrating the procedure will be Dr.Peterson Jones. For subretinal implantation of the organoids, use Stevens Tenotomy Scissors to perform a 0.5 to 1 centimeter lateral canthotomy in a four-month-old overnight-fasted anesthetized cat, and place an appropriately sized Barraquer eyelid speculum into the incision to keep the eyelids open with a surgical assistant regularly irrigating the cornea with BSS throughout the procedure. Use 0.5 Castroviejo corneal tying forceps and a small mosquito hemostat to gently grasp the bulbar conjunctiva next to the limbus.
Place two 6-0 silk sutures into the conjunctiva immediately adjacent to the limbus at the 4 and 8 o'clock positions to hold the eye in primary gaze and to retract the third eyelid. Clamp the ends of the sutures with small mosquito hemostats to help their manipulation, and place another suture at the 12 o'clock position at the limbus partially through the thickness of the limbus, taking care not to penetrate the eye. Loosely knot the suture, cutting the end short, and place a stay suture in the conjunctiva immediately adjacent to the limbus at the 4 o'clock position to hold the eye and primary gaze.
Reflect the bulbar conjunctiva between 10 and 2 o'clock, and use tenotomy scissors to incise the conjunctiva two to three millimeters from the limbus. Undermine the conjunctiva and clear the Tenon's capsule to expose the sclera at the two and 10 o'clock vitrectomy port sites three to five millimeters from the limbus. Use calipers to identify the sites for sclerotomy, avoiding the major scleral vessels that can be prominent in the cat.
Planning for a three millimeter region at the 10 o'clock instrument port for a right-handed surgeon, pre-place 6-0 or 7-0 polyglactin cruciate pattern sutures without tying at the sites of the proposed sclerotomies. Once the sutures are in place, have the assistant grasp the 12 o'clock stay sutures to help keep the globe in position, and use 0.12 millimeter Castroviejo forceps to hold the tissue next to the sclerotomy site. Uses a trocar to introduce a 23 gauge vitrectomy port through the sclera at both the 2 and 10 o'clock positions angled toward the optic nerve to avoid contacting the lens, and use tying forceps to gently push the irrigation port to determine whether the tips can be visualized within the vitreous.
Have the assistant place a Machemer magnifying irrigating vitrectomy lens onto the cornea to allow visualization of the posterior segment of the eye, and a 23 gauge vitrectomy probe cutter through the instrument port to perform a partial core vitrectomy. Next, introduce the needle of a one milliliter syringe containing one milliliter of triamcinolone solution into the instrument port and inject 250 to 500 microliters of the crystal suspension. When the solution has been delivered, Advance the vitrectomy probe through the instrument port to close to the optic nerve head with the port facing away from the retinal surface.
Use high vacuum to help detach the vitreal face from the retina, insert the injector through the instrument port toward the retinal surface, and extrude the cannula tip. Gently press the tip of the retinal surface and have the assistant to give a slight quick push on the syringe plunger to start the retinal detachment, reducing the injection pressure to permit a slow increase of the retinal detachment until the desired size is achieved. Use a 41 gauge cannula of the injector to slightly enlarge the detachment until retinal scissors can be introduced, and remove the 10 o'clock scleral port.
Then use a straight 2.850 millimeter slit knife oriented toward the optic nerve to avoid touching the lens to enlarge the sclerotomy at this site. Make the retinotomy on the bleb away from the optic nerve to avoid damaging retinal nerve fibers originating from the transplanted region, and avoid cutting major retinal vessels to reduce the risk of hemorrhage. To implant the organoids, insert a glass capillary loaded with organoids through the enlarged sclerotomy toward the retinotomy site, and use the tip of the capillary to slightly open the retinotomy to allow access to the subretinal bleb opening.
Have the assistant slowly press the plunger of the injector again to inject the organoids into the subretinal bleb. BSS should proceed organoids, the organs, and flush the retinotomy open. When all of the organoids have been delivered, leave the glass capillary in place for a few seconds before slowly removing the capillary from the eye, taking care to avoid any sudden release of fluid.
When the capillary has been removed, close the sclerotomy using the pre-placed suture in a cruciate pattern. Have the assistant remove the infusion port, and quickly tie the sclerotomy suture and close the peritomy with the polyglactin sutures in a simple continuous pattern. Close the lateral canthotomy with a 6-0 polyglactin suture and figure-eight skin sutures to perform the lateral canthus and use simple interrupted skin sutures to close the rest of the wound.
Then administer the appropriate post-surgical steroid and antibiotic cocktail. Human pluripotent stem cell-derived retinal organoid loading can be confirmed within the glass cannula of the injection device and during the surgery by direct visualization. The presence of the organoids within the subretinal space can be confirmed postoperatively by ophthalmic examination and fundus imaging.
Prior to euthanasia, confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography imaging can also be performed to assess the position of the organoids. These techniques can be used to demonstrate the persistence of retinal organoids in the subretinal space between the neural retina and the retinal pigment epithelium of the recipient eye. Histological and immunohistochemical analyses can be used to illustrate the survival of the xenogeneic graphs within the subretinal space of a large eye in an immunosuppressed animal.
Only those skilled in vitreoretinal surgery, such as trained veterinary ophthalmologists or vitreoretinal surgeons familiar with the species differences in the cat eye compared to human eye should undertake this procedure. The technique presented in this video should be applicable to other large animal models that are used for translating vitreoretinal surgical techniques to the clinics.
