Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

NOTE: All animal experiments were conducted in accordance with The Association of Research in Vision and Ophthalmology (ARVO) for the Use of Animals in Ophthalmic and Vision Research. Ethics approval was obtained from the Institutional Animal Care and Use Committee, SingHealth, Singapore. Animals were housed at the SingHealth Experimental Medicine Centre approved by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). This approval highlights that all animal experiments comply with the standards of the National Advisory Committee for Laboratory Animal Research guidelines set out by the Agri-Food and Veterinary Authority of Singapore. The following experimental protocol was established based on experiments conducted in 6 eyes of 6 Macaca fascicularis (4 male and 2 female, 4 to 6 years old, 2.8 to 4.0 kg) .

1. Achieving immunosuppression in the NHP model

1. Start the immunosuppression 7 days before surgery and continue immunosuppression throughout the follow-up period.
2. Weigh the NHP before the administration of systemic immunosuppression to ensure accurate medication dosage. The animal is weighed at baseline and weekly thereafter.
3. Use oral sirolimus, doxycycline, and minocycline to achieve systemic immunosuppression.
   1. Administer a loading dose of 2 mg of oral sirolimus followed by a daily maintenance dosing of 1 mg. Obtain baseline blood sirolimus level before administration and monitor this throughout the follow-up period. Ensure a concentration of at least 5 µg/L for adequate immunosuppression.
      NOTE: Sirolimus dose is not weight-adapted.
   2. Administer a dose of 7.5 mg/kg of oral doxycycline per day, two times per day.
   3. Administer a dose of 7.5 mg/kg of oral minocycline per day, two times per day.
4. During immunosuppression, monitor all NHPs for adverse systemic effects. Look for significant body weight loss (>10%), decreased appetite and water consumption, ungroomed hair loss, and abnormal behavior such as aggression and lethargy. Assessments will be done on Day 3, 14 and 1 month, followed by monthly assessments.

2. Instrument sterilization

1. Rinse the surgical instruments using distilled water.
2. Place the instruments in an ultrasonic bath filled with 500 mL of distilled water and 2 mL of instrument disinfectant. Clean the instruments using the sweep function of the ultrasonic bath for 15 min.
3. Remove the instruments from the ultrasonic bath. Rinse twice-thoroughly-with distilled water for 5 min each rinse. Air-dry the instruments after the rinse.
4. Place the instruments in an instrument box. Autoclave the box using the universal program setting (sterilization of instruments at 134 °C for 50 min: 30 min for autoclaving, 20 min for drying).

3. Preparation of preservative-free triamcinolone (40 mg/mL)

1. Using a 1 mL syringe, withdraw 1 mL of triamcinolone solution (10 mg/mL). Transfer it to a 15 mL conical tube and mix it with 4 mL of sterile balanced salt solution (BSS). 
2. Centrifuge the solution at 120 × g for 5 min. Ensure all the triamcinolone particles are at the bottom of the conical tube. Discard the supernatant (BSS) from the conical tube.
3. Re-suspend the triamcinolone particles with 5 mL of sterile BSS in the conical tube. Centrifuge the solution at 120 × g for 5 min. Discard the supernatant again.
4. Repeat step 3.3 to complete the washing of the triamcinolone particles with BSS (3x).
5. Re-suspend the triamcinolone particles with 0.25 mL of sterile BSS to achieve a concentration of 40 mg/mL.
6. Aspirate the re-suspended triamcinolone (40 mg/mL) with a new 1 mL syringe. Attach a 25 G blunt-tip flute needle, and keep the syringe with the triamcinolone solution ready for intraoperative use.

4. Pretreatment of NHP eyes with intravitreal plasminogen (0.25 µg/µL)

1. One week before the surgery, administer an intravitreal injection (20 µL) of monkey plasminogen (0.25 µg/µL).
2. Sedate the NHP before the procedure with an intramuscular injection of ketamine (10-20 mg/kg BW) and a subcutaneous injection of atropine (0.05 mg/kg BW). Administer tetracaine eyedrops for local anesthesia.
3. Before intravitreal injection, disinfect the periorbital region with 10% povidone-iodine. Disinfect the eye by administering 5% povidone-iodine to the conjunctival fornices of the NHP. Ensure the solution stays in the fornices for at least 1 min before rinsing thoroughly with sterile BSS.
4. Use a 250 µL syringe to aspirate the prediluted monkey plasminogen (0.25 µg/µL) from the vial. Attach a 30 G needle to the syringe, and keep the monkey plasminogen ready for intravitreal administration.
5. Use a pair of calipers to identify the injection site on the eye. Administer the intravitreal injection 3 mm away from the limbus.
6. Proceed with the injection with the needle directed towards the center of the globe. Upon removal of the needle from the globe, use a cotton applicator stick to tamponade the injection site and prevent the reflux of the intraocular contents.
7. Administer a lubricant gel or ointment to reduce immediate postoperative ocular surface irritation.

5. Surgical table and equipment setup

1. Establish a sterile field. When in the sterile field, wear surgical scrubs, mask, and hair cover at all times.
2. Prepare the preservative-free triamcinolone (40 mg/mL) for intraoperative visualization of vitreous (see section 3). Prepare the sterile BSS in a 10 mL syringe and lubricant in a 5 mL syringe. Place them on a drape.
3. Keep other instruments ready on a drape, including 3-0 silk, 7-0 vicryl sutures, cotton applicator sticks, wound closure strips, and chandelier endoillumination fiber wire.
4. Connect the vitrectomy set, including the high-speed vitrector, Venturi cassette, and the 25 G chandelier endoilluminator to the vitrectomy machine using sterile technique.
5. Open a 500 mL ophthalmic grade BSS bottle and connect it to the Venturi cassette according to the manufacturer's instructions. Proceed with priming the system.
6. Switch on the miOCT/surgical microscope. Select preset configurations of the surgical microscope for posterior segment surgery and illumination. Enter the details of the procedure, including ID, gender, laterality of the animal eye, and the name of the procedure.
7. Mount a non-contact, wide-angled, 128-degree fundus lens.
8. Attach sterile disposable hand-piece covers onto the surgical microscope/miOCT. Adjust the microscope position and focus using the foot pedal. Proceed with surgery.

6. Preparation of anesthesia and positioning of the animal (preferably performed by veterinarian team)

1. Ensure the NHP is fasted for at least 8 h before the induction of anesthesia to prevent regurgitation and vomiting. Sedate the NHP before the induction of anesthesia (see step 4.2 for sedation instructions).
2. Apply 1% tropicamide and 2.5% phenylephrine eyedrops at least 3x with 5 min intervals to achieve pupil dilation.
3. Administer an intramuscular injection of buprenorphine (0.005-0.03 mg/kg BW) 30 min before the surgery to achieve analgesia.
4. Intubate the NHP with an endotracheal tube, usually 3-5 mm in size. When attempting intubation, ensure that several sizes are available. Use the largest size that can be passed through the larynx without causing trauma. Measure end tidal CO₂ to ensure appropriate placement of endotracheal tube.
5. Deliver 2% isofluorane gas via the endotracheal tube to induce general anesthesia. Confirm the state of general anesthesia (lack of response to touch) by assessing the NHP's response to surrounding stimuli, including sounds and touch. Use 0.5-2% isofluorane gas to maintain the state of general anesthesia. 
6. Continuously monitor the NHP electrocardiogram, respiratory rate, blood pressure, and oxygen saturation during the entire surgery.
7. Position the NHP on the surgical table such that the eye is perpendicular to the surgical microscope. Administer a lubricant gel or ointment to the eye, which is not being operated on to reduce ocular surface irritation during anesthesia.
8. Cut the eyelashes using scissors to reduce the chance of infections.
9. Disinfect the periorbital region with 10% povidone-Iodine. Disinfect the eye by administering 5% povidone-iodine to the conjunctival fornices of the NHP. Ensure the solution stays in the fornices for at least 1 min before rinsing thoroughly with sterile BSS.
10. Position a sterile drape such that the pre-cut opening is centered over the eye to undergo surgery. Cover the eye with an adhesive surgical incision drape.
11. Perform a lateral canthotomy on the eye to undergo surgery.
12. Insert the Lieberman speculum to ensure adequate opening of the eyelids for the visualization of the eye.

7. Vitrectomy

NOTE: To access the subretinal space for the delivery of the PET-scaffold RPE graft, this protocol recommends a 4-port (valved) 25 G vitrectomy to be performed using a standard vitreoretinal surgical setup and a non-contact, wide-angled, 128° fundus lens. The protocol also recommends the use of a surgical microscope equipped with miOCT to guide several critical surgical steps, including the induction of foveal detachment, implantation of the RPE graft, and subretinal fluid drainage.

1. Perform a 360° conjunctival peritomy by incising the conjunctiva near the limbus using a pair of vannas scissors. Enlarge the peritomy by performing a blunt dissection.
2. Using a 25 G microvitreoretinal blade, perform a sclerotomy at 8 o'clock for the right eye or 4 o'clock for the left eye. Perform the sclerotomy 3 mm from the limbus of the eye.
3. Insert and suture a 25 G custom side port-infusion cannula using 7-0 vicryl suture. After confirming the intravitreal location, start the BSS infusion and set the system to maintain an IOP of 20 mmHg.
4. Using a 25 G flat head trochar, perform a sclerotomy at 2 o'clock for the right eye or 10 o'clock for the left eye, as in step 7.2.
5. Insert the 25 G chandelier light into the flathead trochar and secure it with sticky tape. Adjust the light source to approximately 60%.
6. Perform another sclerotomy, similar to step 7.2, at 10 o'clock for the right eye or 2 o'clock for the left eye. Place U-shaped vicryl 7-0 sutures around the sclerotomy without tying the knots. Insert the vitrectomy cutter tip through this sclerotomy.
7. Start the vitrectomy around the entry ports, followed by a short core vitrectomy with the following settings: maximum 5000 cuts per minute, maximum aspiration at 400 mmHg.
8. Inject 20-50 µL of triamcinolone (40 mg/mL) for better vitreous visualization.
9. Induce a posterior vitreous detachment (PVD) by separating the vitreous body from the retina.
   1. Position the vitrector above the optic disc to allow gentle induction of the PVD. Keep the vitrector only on aspiration at the maximum setting of 400 mmHg without any cutting involved.
   2. If required, use 25 G intraocular forceps to manipulate the vitreous at the disc margin to create a tear in the vitreous cortex to facilitate detachment.
      NOTE: PVD is considered successful if triamcinolone crystals glide unimpeded over the retinal surface.
10. Open the posterior hyaloid membrane with the cutter, and remove the detached vitreous skirt up to the vitreous base (at the retinal equator). Aspirate any remaining triamcinolone on the retinal surface.

8. miOCT-guided foveal detachment

1. Inject 1-2 mL of PFCL to cover the posterior pole up to the anterior, mid-peripheral retina.
2. Enter the eye with a subretinal injection cannula. Set the IOP to 0-4 mmHg on the vitrectomy machine (ensure perfectly watertight system; if needed, tie sutures around the ports).
3. Using either the 25/41 G customized dual-bore subretinal injection cannula or 25/38 G subretinal injection cannula connected to a 250 µL syringe, gently perform a subretinal injection of BSS to induce a localized retinal detachment. Once the bleb just crosses the fovea, stop the injection. Create a second bleb from a separate direction. Merge both the blebs to fully detach the fovea.
4. Enable the miOCT function to visualize bleb formation. Ensure the line and cube scans are in HD mode with the settings (512 x 128 pixels, scan width 4 mm) to acquire an image at the fovea. Observe the miOCT image for a complete detachment of the neural retina from the RPE layer at the fovea.
5. Enlarge the retinotomy to 1.5 mm with a pair of vertical 25 G vitreoretinal scissors to allow access to the subretinal space for transplantation.

9. Removal of native RPE

1. Set the IOP to 50 mmHg on the vitrectomy machine.
2. Remove the PFCL via active extrusion using a brushed silicone tip cannula.
3. Extend the sclerotomy with a 1.4 mm incision knife to allow the entrance of a 20 G instrument.
4. Using a custom 20 G extensible loop instrument, scrape the submacular host RPE for removal. Scrape an area that measures at least 2 x 3 mm.

10. Loading of the shooter for delivery of RPE cell monolayer transplant

1. For general instructions on the loading of a bullet-shaped graft cut from RPE cultures on PET cell carriers, refer to a previous publication²².

11. miOCT-guided graft implantation and position adjustment

1. Insert the tip of the shooter device through the sclerotomy at an IOP of 20 mmHg. Inject the implant towards the subretinal space via the retinotomy edge created from the retinal surface.
2. Inject the implant with the cell carrier side facing the Bruch's membrane and the RPE xenograft side facing the photoreceptors.
3. Enable the miOCT function to visualize the implant location. Ensure the implant is resting flat on the Bruch's membrane in the subretinal space, with an intact overlying retina. Ensure it is located a reasonable distance away from the created retinotomy and not impinged on the retinotomy site.
4. Adjust the implant position with the subretinal injection cannula or a 25 G curved intraocular scissor to ensure it is well-positioned under the macula.

12. miOCT-guided drainage of subretinal fluid

1. Using a brushed silicone tip cannula, perform a fluid-air exchange and careful subretinal fluid drainage. Attempt gentle subretinal fluid aspiration from the bleb retinal detachment and retinotomy edge apposition.
2. Enable the miOCT function for real-time visualization of adequate subretinal fluid drainage until the retina is reattached over the implant.

13. Ending the operation

1. Close the working port sclerotomy using the preplaced 7-0 vicryl suture. Remove the 25 G chandelier and 25 G infusion cannula. Close these sclerotomies with 7-0 vicryl sutures.
2. Administer 2 mg in 0.05 ml of intravitreal preservative free triamcinolone (40 mg/ml) at the 8 o’clock sclerotomy prior to suturing.
3. Palpate the eye to ensure that the IOP is within the acceptable range. Inject filtered air (or BSS) via a 30 G needle if required.
4. Suture the conjunctiva with 7-0 vicryl sutures and canthotomy with 5-0 prolene (remove after 10-14 days).

14. Postoperative animal care

1. Position the NHP face down for 1 h post-surgery. Do not leave the animal unattended until consciousness has been regained. Ensure that a veterinarian and animal care technician are available for observation and support during the postoperative process.
2. Apply topical antibiotic (tobramycin), steroid (dexamethasone) ointment, and homatropine eye drops twice a day for 5 days postoperatively.
3. Administer another subcutaneous buprenorphine (0.005-0.03 mg/kg BW) injection 6 h after surgery for adequate pain control.
4. Return the NHP to the company of other animals only when it has fully regained consciousness.
5. Conduct multimodal imaging follow-ups on Day 3, 14 and Month 1 after the procedure, followed by monthly checkups. Perform ERGs every month after the procedure. Remove the 5-0 prolene sutures for the canthotomy at Day 14 concurrently with the sedation period used for multimodal imaging. The remaining sutures are resorbable, 7-0 vicryl sutures, which do not require removal. 

15. Postoperative monitoring methods for multimodal imaging

1. Fast the NHP overnight. Sedate the NHP just before imaging (see step 4.2 for drug and concentration for sedation). If sedation is insufficient to stop eye movement, consider the use of general anesthesia.
2. Apply 1% tropicamide and 2.5% phenylephrine eyedrops to achieve pupil dilation before imaging (see step 6.2).
3. Perform autofluorescence (AF), fundus fluorescein angiography (FFA), and optical coherence tomography (OCT) using a high-resolution OCT machine with a 55° field lens and 30° field lens.
   1. Administer intravenous 10% fluorescein (0.1 mL/kg BW) for FFA. To obtain an early phase image, capture an image within 30 s of injection. For a late phase image, capture an image 5-10 min after the injection.
4. Perform fundus photography using a fundus camera between the early and late phases of FFA.

16. Postoperative monitoring methods for full-field electroretinogram (ERG) studies

1. Fast the NHPs overnight. Sedate the NHP before ERG studies (see step 4.2 for drug and concentration for sedation). Throughout the ERG recordings, re-administer sedation when appropriate.
2. Separate multimodal imaging and ERG recordings with an interval of at least 2-3 days.
3. Once sedated, ensure the NHP is dark-adapted for 30 min before the ERG recording.
4. Position the stainless-steel subdermal needle electrodes at the left and right lateral canthi (reference electrodes) and the back of the NHP body (ground electrode). Place the ERG contact lens electrodes on the NHP cornea using vidisic gel to aid contact and adhesion.
5. Base all ERG testing on the human protocols recommended by the International Society for Clinical Electrophysiology of Vision (ISCEV)¹⁴. Commence ERG recording under scotopic conditions and start with the dimmer flashes. Follow the ISCEV recommendations for recommended interstimulus intervals.
6. Ensure the NHP is light-adapted for 10 min before photopic testing, again using standard ISCEV recommendations for background strength.

17. Euthanasia of NHP

1. To euthanize the NHP for enucleation, administer intravenous sodium pentobarbital (75 mg/kg), as recommended by the Panel on Euthanasia of the American Veterinary Medical Association.