Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Summary

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Abstract

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Introduction

The human retina located at the back of the eye functions as a light sensory tissue and plays a critical role in vision perception. Retinal cell dysfunction or cell death therefore causes vision problems or permanent blindness. Disorders involving degeneration or dysfunction of cells in different layers of the retina are known as degenerative retinal diseases, among which AMD is the most common type and the leading cause of irreversible blindness in the elderly in developed countries^1,2. The pathological process of AMD is associated with "drusen" accumulation between the RPE layer and the underlying Bruch's membrane, which in turn impairs RPE support of photoreceptor physiology, leading to neural retinal atrophy and vision loss^3,4,5. Thus far, there is no cure for advanced dry (non-neovascular) AMD. The emergence of stem cell therapy as a new paradigm in regenerative medicine brings the hope of replacing the dysfunctional or dead RPE cells with stem cell-derived healthy cells. Indeed, extensive preclinical studies of transplanting stem cells (e.g., human embryonic stem cell)-derived RPE cells into RPE-degenerative animal models have been performed^6,7, some of which have progressed to clinical trials^8,9 (NCT01344993, ClinicalTrials.gov). Recently, an alternative source of stem cells resident in the human RPE layer, the human RPE stem cells (hRPESCs), was identified by our lab and is currently being used in preclinical studies of hRPESC derived-RPE cell (hRPESC-RPE) transplantation therapy for AMD^10,11,12,13.

The subretinal injection technique is applied in the preclinical studies mentioned above by multiple groups, including our group. There are two general approaches for subretinal injection in animals: trans-vitreal and trans-scleral. The trans-vitreal approach has the advantage of the surgeon being able to directly observe the needle end as it penetrates the anterior eye, crosses the entire vitreal cavity adjacent to the lens, and penetrates the retina at the back to the eye to reach the subretinal space^14,15,16. However, it requires disrupting the retina in two locations (anterior and posterior), carries the risk of damaging the lens, and can result in backflow of cells into the vitreous when the needle is retracted. In contrast, the trans-sclera approach, in principle, avoids involvement of the retina and vitreous, and backflow exits the eye. In pigmented rodents, the surgeon can initially observe penetration of the sclera, but after passage into the pigmented choroid, the needle end is no longer visible. Without direct observation, breaching the retina is common and can result in retinal dissection and delivery of cells and/or blood into the vitreous. Moreover, because the eye surface is curved, it is very difficult to know which needle angles and depths are most effective for trans-scleral injections.

In this visualized article, we introduce a trans-scleral subretinal injection method informed by the use of post-surgical evaluations with Optical Coherence Tomography (OCT), which allows a detailed examination of the injection site. Our trans-scleral injection technique utilizes defined locations, angles, and depths for injection needles to produce very low surgical trauma and high reliability. Here, we specifically demonstrate the injection of hRPESC-RPE cells into the subretinal space of the RCS rat, a pre-clinical model of human AMD. With this injection method, we successfully and consistently delivered hRPESC-RPE cells into the subretinal space of RCS rat eyes with a very high success rate. Injection of cells was previously found to result in preservation of RCS photoreceptors at least 2 months after injection¹³. This procedure is performed under the dissecting microscope and is easy to learn. It requires two people (a surgeon and an assistant) to perform the injection and the average time of injection for each animal is less than 5 minutes. The defined angles and depths for injection needles make it possible for laboratories, where OCT is unavailable, to achieve successful subretinal injection. It allows for highly reproducible subretinal access and can be used not only for cell transplantation, but also for drug delivery and gene therapies.

Protocol

All procedures involving animals have been approved by the Institutional Animal Care and Use Committee (IACUC) at the State University of New York at Albany.

1. Pre-injection Preparation

1. Preparation of a hRPESC-RPE cell suspension
   NOTE: All the following steps are performed in sterile tissue culture hood and familiarity with basic sterile technique is required.
   1. Isolate primary hRPE cells from human donor eyes aged 50-90 years and culture cells in 24-well plates¹². Cryopreserve the cells at passage 1, thaw as needed and culture passage 2 (P2) cells for 4-5 weeks (Figure 1A) for injection.
   2. Remove the culture medium¹² and gently rinse wells twice with 500 µL pre-warmed 1X Dulbecco's Phosphate Buffered Saline without Calcium & Magnesium (1x DPBS-CMF) by adding 1X DPBS-CMF into wells using a 1,000 µL pipette and removing it using a vacuum.
   3. Add 300 µL trypsin/DNAse to each well. Incubate hRPESC-RPE cells in trypsin/DNAse (4 kU DNase per 1 mL of 0.25% trypsin-EDTA) for 4 min at 37 °C to dissociate the cells.
   4. Check the cells under the microscope to see if they have rounded up. Continue to incubate the cells in trypsin/DNase for an additional 2 min if they have not rounded up yet.
   5. Once the cells are rounded up, use a 1,000 µL pipette to triturate the detached cells from the well and transfer the trypsin/DNase containing detached cells into a 15-mL conical tube with equal volume of pre-warmed culture medium, to inactivate the trypsin/DNase.
   6. Rinse wells with pre-warmed 1X DPBS-CMF by gently pipetting up and down, especially around the edges of the well; then add these cells to the previous conical tube.
   7. Centrifuge the conical tube at 286 x g for 5 min at 4 °C to pellet the cells.
   8. Remove the supernatant and resuspend the cells with 1 mL culture medium.
   9. Count the cells using a hemocytometer.
   10. Centrifuge at 286 x g for 5 min at 4 °C to pellet the cells.
   11. Remove the supernatant and resuspend cells in sterile Balanced Salt Solution (BSS) at 50,000 cells/µL (to deliver 50,000 cells in 1 µL volume during subretinal injection).
   12. Transfer the final cell suspension (CS) into a 1.7-mL microcentrifuge tube and keep in an ice-water mixture until injection use.
2. Preparation of cell injector
   1. Insert a sterile 33-gauge beveled needle into the injection syringe and screw tightly to assemble the injector.
   2. Flush the injector with 100% ethanol 5-6 times.
   3. Flush the injector with 70% ethanol 5-6 times.
   4. Flush the injector with BSS 5-6 times.
   5. Mark the injector needle with a sterile black marker pen at a position of 600 µm away from the tip of the needle under the dissecting microscope (Figure 1B).
   6. Place the injector on a micromanipulator for injection.

2. Subretinal Injection

1. Surgical area and animal preparation
   1. Weigh a 4-5 week old RCS rat (60-100 g) and anesthetize it using the isoflurane vapor delivery system.
      NOTE: To induce anesthesia keep the isoflurane flow rate at 5%. Confirm the depth of anesthesia by pressing the paws, and then reduce the flow rate to 2-3% for anesthesia maintenance during surgery.
   2. Place a sterile surgery drape, with a heating pad underneath, on the stage of the dissecting microscope to set up a sterile surgical area.
   3. Transfer the rat to the surgical area and place the rat in a nose cone connected to the isoflurane system to maintain anesthesia.
   4. Cover the rat's body with gauze. Pinch the rat's toe to confirm full anesthesia.
2. Trans-scleral subretinal injection under the microscope
   1. Apply a drop of eye lubricant on the rat's unoperated eye.
   2. Position the rat onto its right side with its left eye facing the ceiling for injection, its head towards the surgeon's right hand and its back towards the surgeon.
   3. Trim any whiskers that cover the eye with small scissors.
   4. Drip a small amount of eye wash from the temporal side of the left eye and collect the excess at the nasal side with a cotton applicator to rinse the eye.
   5. Dilate the pupil with 1% tropicamide and 2.5% phenylephrine (freshly made from 10% phenylephrine by diluting it in sterile 0.9% saline on the day of surgery) for a post-injection OCT exam by applying one drop of each.
   6. Gently pull the skin surrounding the eye 4-6 times to open the eyelid so that the eye is slightly proptosed for easier access to regions posterior to the limbus.
   7. Apply a drop of eye wash and keep the eye moist.
   8. Gently triturate the CS (prepared in step 1.1.12) and load the injector with 1.2 µL CS. The extra 0.2 µL is used to compensate for injection backflow.
      NOTE: Based on our measurements, about 5,000-8,000 cells are lost in the backflow with an injection of 50,000 cells/µL that equals to about 10-16% of cell loss and an extra of 0.2 µL CS was injected to compensate this cell loss.
   9. Place the injector filled with CS on a micromanipulator (or have an assistant hold it) vertically as RPE cells tend to sink easily in suspension.
   10. Apply a drop of 0.5% proparacaine (local topical anesthetic) on the eye and remove the excess with a cotton applicator.
       NOTE: This step should suppress the corneal reflex and prevent the eye from blinking during subsequent steps.
   11. Use forceps to grip the conjunctiva posterior to the limbus, rotate the eye nasally, and lift the conjunctiva to make a "tent".
   12. Use scissors to cut the top of the "tent" off to make a small opening in the conjunctiva and expose the underlying sclera.
   13. Use forceps to grip the edge of the remaining conjunctiva margin next to the limbus and rotate the eye nasally so that the pupillary axis is at an angle of about 30 degree relative to the table top (Figure 1C). Continued gripping of the conjunctiva margin is needed to provide a counter-force during needle insertions and to maintain the eye at an optimal angle.
   14. To make a pilot hole for cell injection, position the end of a sterile beveled 31-gauge insulin needle at 1,200-1,500 µm posterior to the limbus with the opening of the tip facing up.
   15. Adjust the angle of the insulin needle so that it is 10-15 degrees above the sclera (tangential to an imaginary plane at the intended injection site). Slowly penetrate the sclera-choroid complex to a needle depth of about 500 µm. In the pigmented rat, the needle end will 'disappear' beneath the pigmented choroid. For the brand of insulin needle used here, the distance from the needle tip to the bevel is 500 µm.
   16. Carefully withdraw the insulin needle (a very small effusion of blood may be seen).
   17. If excessive bleeding is noted, an eye spear can be applied to clear the hole, if necessary. Continued bleeding after the spear application indicates a vessel may have been damaged.
   18. Guide the RPE cell-loaded injector needle into the pilot hole, with the opening facing down, and at an angle of about 10-15 degrees relative to the local surface of the sclera.
   19. Gently insert the injector needle into the pilot hole to a depth of about 500 µm to access the subretinal space. There should be about a 100 µm margin between the edge of the black pen mark and the point where the needle is covered by the pigmented choroid (Figure 1C and 1D).
   20. Ask the assistant to gently depress the plunger of the injector syringe to inject the appropriate volume of cells (about 1.2 µL). Be ready to provide some counter-force as the assistant presses on the plunger.
       Note: Prior mock practice with the assistant can provide both the surgeon and assistant with the necessary experience with this step.
   21. While visually focusing on the pen mark edge, hold the injector in place for 25-30 s and then slowly retract the injector. A small amount of backflow is commonly observed.
       Note: If no backflow is observed, there may have been an intravitreal injection. If you see backflow through the seal or cells filling beneath the sclera, the injection was too shallow.
   22. Rinse the cell efflux from the injection site with the sterile eye wash 3 times and collect the excess with a cotton applicator.
   23. Apply a drop of eye lubricant on the operated eye and transfer the rat to the OCT station to examine the location of transplanted cells and the size of the subretinal bleb.

3. Post-injection Treatment

1. Anti-inflammatory and pain reliever treatment
   1. Inject buprenorphine at 0.1 mg/kg body weight in saline subcutaneously to decrease pain.
   2. Inject dexamethasone at 1.6 mg/kg body weight in saline by intraperitoneal injection (I.P.) for inflammation control.
2. Animal recovery
   1. Return the rat to the recovery cage under a heat lamp to maintain body temperature.
   2. Observe the operated eye for signs of hemorrhage.
   3. Observe the rat to ensure it comes out of anesthesia.
   4. Return the recovered animal to a fresh cage and flag the cage with a surgery card, and monitor daily for any signs of distress, ocular hemorrhage, or corneal opacities. Notify the veterinarian immediately if there are concerns.

Results

Using the technique described in this article, we consistently delivered hRPESC-RPE cells into the subretinal space of RCS rats by precisely controlling the location, angle, and depth of the injector needle inserting into the tissue (Figure 1B-D). Immediately following transplantation, an OCT examination was performed to observe the injection site and the subretinal bleb created by the transplanted cells. Post-surgical OCT evaluation serves as a screening...

Discussion

The subretinal injection technique depicted in this article is via the trans-scleral pathway, where the injector needle penetrates the outer layers (sclera-choroid-RPE complex) of the eye wall without harming the neural retina or disturbing the vitreous cavity. An alternative trans-vitreal approach has a potential risk of lens damage leading to cataract, since rodents' lens occupies the majority of the vitreous cavity. Compared to this method, our technique is less risky and causes minimal trauma as the injector need...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA (1x)	Life Technologies	25200-072
DNAse I	Sigma	DN-25
1xDulbecco’s Phosphate Buffered Saline without Calcium & Magnesium (1xDPBS-CMF)	Corning Cellgro	431219
Sterile Balanced Salt Solution (BSS)	Alcon	00065079550
Sterile eye wash	Moore Medical	75519
Sterile 0.9% saline	Hospira	488810
Proparacaine Hydrochloride Ophthalmic Solution (0.5%)	Akorn	17478026312
Tropicamide Ophthalmic Solution, USP (1%)	Bausch & Lomb	24208058559
Phenylepherine Ophtalmic Solution, USP (10%) stock	Bausch & Lomb	42702010305	This is used to make 2.5% Phenylepherine
Buprenex	Patterson	433502
Dexamethasone	APP Pharmaceuticals	63323051610
100% Ethanol	Thermo Scientific	615090040
70% Ethanol	Ricca Chemical Company	2546.70-5
Sterile GenTeal Lubricant Eye Gel	Novartis	78042947
Sterile Systane Ultra Lubricant Eye Drops	Alcon	00065143105
hRPESC-RPE cells	Not available commercially		Please refer to "Reference #12" for cell isolation and mainteinance.
24-well plates	Corning	3526
Conical tubes (15 ml)	Sarstedt	62554002
Microcentrifuge cap with o-ring	LPS inc	L233126
Capless Microcentrifuge tubes (1.7 ml)	LPS inc	L233041
Centrifuge	Eppendorf	5804R
Sterile alcohol wipe	McKesson	58-204
Sterile cotton tip applicators	McKesson	24-106-2S
Sterile Weck-Cel spears	Beaver-Visitec International	0008680
Sterile surgical drapes	McKesson	25-515
Gauze	McKesson	16-4242
Nanofil syringe (10 ul)	World Precision Instruments	Nanofil
Nanofil beveled 33-gauge needle	World Precision Instruments	NF33BV-2
Insulin syringe needles 31-gauge	Becton Dickinson	328418
Rat toothed forceps	World Precision Instruments	555041FT
Vannas Micro Dissecting Spring Scissors	Roboz	RS-5602
Circulating water T pump	Stryker	TP700
Heating pad	Kent Scientific	TPZ-814
Animal anesthesia system	World Precision Instruments	EZ-7000
Balance	Ohaus	PA1502
Stereo microscope	Zeiss	Stemi 2000
Microscope light source	Schott	ACE series
Bioptigen Envisu Spectral Domain Ophthalmic Imaging System	Bioptigen	R2210
Sterile black marker pen	Viscot Industries	1416S-100
Miniature measuring scale	Ted Pella Inc	13623
Infrared Basking Spot Lamp	EXO-TERRA	PT2144	This is used as a heating lamp for animals during the post-surgical recovery  phase