地図状委縮の目の大きいモデルにヒト胚性幹細胞由来網膜色素上皮細胞の網膜下移植

The overall goal of this procedure is to create a large-eyed animal model of geographic atrophy to transplant human embryonic derived-retinal pigment epithelial cells into the eye and assess donor cell integration using optical coherence tomography. This method can help answering key questions in the field of regenerative medicine, such as, stem cell based treatment for degenerative diseases. The main advantage of this surgical setting is the unlimited source of healthy cells and the surgical setting for subretinal injection, which has many similarity with a procedure intended for patient.

The implications of this technique extends towards therapy of age-related macular degeneration through the ostentation of retinal pigment epithelial cells derived from human embryonic stem cells. Our animal technician, Monica Aronsson, is holding in position the rabbit so that we can visualize and image the retina with optical coherence tomography. After anesthetizing a rabbit according to the text protocol, place the rabbit under the surgical microscope with the head facing the surgeon.

Then, use a lid retractor to remove the eyelids and a sterile cloth to remove the nictitans membrane. For microsurgery, use a two-port or optional three-port 25-gauge transvitreal pars plana technique with non-valved trocars for the insertion of microsurgical instruments. The multi-function vitrectomy machine has ports to connect an infusion canula, endoillumination, a vitrector, and an endolaser.

For subretinal injections, only the endoillumination is mandatory which makes a two-port setup sufficient. Using clot forceps to grab and displace the conjunctiva, overline the insertion site. Transsclerally insert one of the two upper trocars, one to two millimeters from the limbus, at a thirty to forty-five degree limbus parallel angle.

Proceed to insert the trocar to the middle of the tip. Then, turn the trocar ninety degrees and advance it into the eye, aiming at the posterior pole. This will prevent the postsurgical leakage from this sclerotomy and also decrease the risk of endophthalmitis.

Repeat with the second upper trocar. When inserting the trocar, it is important to be one to two millimeters from the limbus to tunnel the insertion and aim at the posterior pole to minimize the risk of infection, vitreous bleeding, retinal detachment, and our lens touch. Apply synthetic tears as a contract gel to the cornea then position a single-use flat contact lens on the cornea to visualize the retina.

Next, drop five hundred microliters of Sodium iodate into a one milliliter syringe connected to an extension tube operated by an assistant and a 38-gauge poly-tipped canula operated by the surgeon. Insert the endoillumination probe through the upper left trocar. Then, insert the injection canula through the upper right trocar and advance the canula through the vitreous space towards the retina, aiming for the area just below the optic nerve head.

Allow the tip of the canula to slowly touch the retina until a focal whitening is visible. Do not let the canula penetrate the retina since this may cause hemorrhaging. Now, penetrating the retina with the needle, inject fifty microliters of Sodium iodate subretinally over a five second period.

Since there is a natural cleavage plane between the retina and the underlying choroid, a clearly visible semi-transparent bleb should gradually form during the injection. While injecting, slowly retract the needle but make sure the tip is maintained within the bleb to minimize reflux. Following removal of the endoillumination and injection canula, use clot forceps to remove the trocars.

Then use the tip, or the blunt end of the forceps, to apply light pressure for thirty seconds to the soft-sealing sutureless sclerotomy. During recovery, attend to the animal according to the text protocol and wait seven days for transplantation. Seven days after injection of Sodium iodate and following administration of intravitreal Triamcinolone, according to the text protocol, remove the cell differentiation medium from a twenty-four well confluent hESC-RPE monolayer and use five hundred microliters of PBS without calcium and magnesium to wash each well.

After trypsinizing the cells, tilt the plate and carefully remove the trypsin to allow the cells to remain attached to the plate. Collect the cells in eight hundred microliters of fresh thirty-seven degrees Celsius pre-warmed differentiation medium by gently pipetting, or even scraping if needed, to obtain a single cell suspension. Using zero point two percent trypan blue, count the cells in a hemocytometer chamber according to the manufacturer's instructions.

Then add five milliliters of differentiation medium to the cell suspension and centrifuge the cells at three hundred times g, at room temperature for five minutes. Discard the supernatant and re-suspend the pellet in freshly filter sterilized PBS at a final concentration of one thousand cells per microliter. Aliquot the previous cell suspension into six hundred microliter aliquots and keep them on ice until and during surgery.

After anesthetizing the rabbit and dilating the pupils according to the text protocol, place the rabbit with the head facing the surgeon. Use the lid retractor to remove the eyelids and a sterile cloth to remove the nictitans membrane. For microsurgery, use a two-port or optional three-port 25-gauge transvitreal pars plana technique with trocars placed in the same positions as demonstrated earlier in this video.

If the previous sclerotomies are visible perform trocar insertion just adjacent to but not through these insertion sites to minimize the risk of postoperative leakage. Apply synthetic tears to the eye as before and place a single-use flat contact lens over the solution on the cornea to visualize the retina. After properly positioning the tip and aiming for the center of the Sodium iodate pre-treated area, distinguished by a characteristic metallic endoillumination reflex, subretinally inject fifty microliters of a gently mixed hESC-RPE suspension.

The neurosensory retina should separate easily, creating a clearly visible bleb. During the injection, slowly retract the needle but make sure the tip is maintained within the bleb to minimize reflux. After removal of the endoillumination and injection canula, use clot forceps to remove the trocars and use the blunt-end of the forceps to apply light pressure for thirty seconds as before.

Use a spectral domain optical coherence tomography, or SD-OCT device, with the accompanying software to obtain cross-sectional B-scans of the treated animals, according to manufacturer's instructions. Place the anesthetized and pupil-dilated animal in an adjustable mount to obtain an unobstructed path from the instrument light source to the rabbit retina. To avoid image blurring, keep the cornea moist by flushing with topical saline every thirty to sixty seconds.

Obtain at least three OCT scans with simultaneous infrared confocal scanning laser ophthalmoscopy or confocal SLO reflectance reference images representing the upper, central, and lower portion of the injected area. Obtain multiple simultaneous laser colors of en-face fundus images with confocal SLO blue, green, infrared, and multicolored laser reflectance. Capture blue light autofluorescence images using the blue light laser capability of the SD-OCT device.

Representative in vivo images of BAF, IR-cSLO, and SD-OCT of a normal albino rabbit retina are shown here. A blow up of the SD-OCT image illustrates the different retinal layers with their distinctive levels of light reflection captured by the SD-OCT instrument. As shown in this SD-OCT image, taken three months after injection of fifty microliters of one millimolar Sodium iodate solution, a bleb is created that resolved and progressively degenerated the outer retina.

Thinning of the outermost neuroretinal layers in the SD-OCT and the hypo-BAF areas can be observed, corresponding to RPE loss, thus recreating a GA-like phenotype. Upon identification of the area of damage, fifty thousand hESC-RPE cells were re-injected for transplantation, creating a second bleb that resolved. Mild to moderate hyper-BAF areas are present with a focally increased hyper-BAF on the borderline of the area of damage, indicative of chronic stress of the native RPE.

The bleb corresponding to the injection of hESC-RPE's in non pre-treated eyes showing patches of pigmented cells in the MC image and well preserved retinal structures in the SD-OCT scan is shown for comparison. Mastering this pretty clinical method for subretinal injection will facilitate clinical transplantation of cell-based treatment for conditions, such as, age-related macular degeneration. After watching this video, you should have a good understanding on how to set up subretinal injections in a life type model, how to prepare a human embryonic stem cell derived cells for subretinal injections, and how to visualize integration of donor cells using optical coherence tomography.