We wish to thank Patty Lederman for her assistance on the surgery and Susan Borden for RPE cell preparation. We also acknowledge NYSTEM C028504 for the funding for this project. Justine D. Miller is supported by NIH grant F32EY025931.

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
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Remove the culture medium12 and gently rinse wells twice with 500 &#181;L pre-warmed 1X Dulbecco&#39;s Phosphate Buffered Saline without Calcium &#38; Magnesium (1x DPBS-CMF) by adding 1X DPBS-CMF into wells using a 1,000 &#181;L pipette and removing it using a vacuum.</li>
		<li>Add 300 &#181;L 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 &#176;C to dissociate the cells.</li>
		<li>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.</li>
		<li>Once the cells are rounded up, use a 1,000 &#181;L 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.</li>
		<li>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.</li>
		<li>Centrifuge the conical tube at 286 x g for 5 min at 4 &#176;C to pellet the cells.</li>
		<li>Remove the supernatant and resuspend the cells with 1 mL culture medium.</li>
		<li>Count the cells using a hemocytometer.</li>
		<li>Centrifuge at 286 x g for 5 min at 4 &#176;C to pellet the cells.</li>
		<li>Remove the supernatant and resuspend cells in sterile Balanced Salt Solution (BSS) at 50,000 cells/&#181;L (to deliver 50,000 cells in 1 &#181;L volume during subretinal injection).</li>
		<li>Transfer the final cell suspension (CS) into a 1.7-mL microcentrifuge tube and keep in an ice-water mixture until injection use.</li>
	</ol>
	</li>
	<li>Preparation of cell injector
	<ol>
		<li>Insert a sterile 33-gauge beveled needle into the injection syringe and screw tightly to assemble the injector.</li>
		<li>Flush the injector with 100% ethanol 5-6 times.</li>
		<li>Flush the injector with 70% ethanol 5-6 times.</li>
		<li>Flush the injector with BSS 5-6 times.</li>
		<li>Mark the injector needle with a sterile black marker pen at a position of 600 &#181;m away from the tip of the needle under the dissecting microscope (Figure 1B).</li>
		<li>Place the injector on a micromanipulator for injection.</li>
	</ol>
	</li>
</ol>
2. Subretinal Injection
<ol>
	<li>Surgical area and animal preparation
	<ol>
		<li>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.</li>
		<li>Place a sterile surgery drape, with a heating pad underneath, on the stage of the dissecting microscope to set up a sterile surgical area.</li>
		<li>Transfer the rat to the surgical area and place the rat in a nose cone connected to the isoflurane system to maintain anesthesia.</li>
		<li>Cover the rat&#39;s body with gauze. Pinch the rat&#39;s toe to confirm full anesthesia.</li>
	</ol>
	</li>
	<li>Trans-scleral subretinal injection under the microscope
	<ol>
		<li>Apply a drop of eye lubricant on the rat&#39;s unoperated eye.</li>
		<li>Position the rat onto its right side with its left eye facing the ceiling for injection, its head towards the surgeon&#39;s right hand and its back towards the surgeon.</li>
		<li>Trim any whiskers that cover the eye with small scissors.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Apply a drop of eye wash and keep the eye moist.</li>
		<li>Gently triturate the CS (prepared in step 1.1.12) and load the injector with 1.2 &#181;L CS. The extra 0.2 &#181;L 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/&#181;L that equals to about 10-16% of cell loss and an extra of 0.2 &#181;L CS was injected to compensate this cell loss.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Use forceps to grip the conjunctiva posterior to the limbus, rotate the eye nasally, and lift the conjunctiva to make a &#34;tent&#34;.</li>
		<li>Use scissors to cut the top of the &#34;tent&#34; off to make a small opening in the conjunctiva and expose the underlying sclera.</li>
		<li>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.</li>
		<li>To make a pilot hole for cell injection, position the end of a sterile beveled 31-gauge insulin needle at 1,200-1,500 &#181;m posterior to the limbus with the opening of the tip facing up.</li>
		<li>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 &#181;m. In the pigmented rat, the needle end will &#39;disappear&#39; beneath the pigmented choroid. For the brand of insulin needle used here, the distance from the needle tip to the bevel is 500 &#181;m.</li>
		<li>Carefully withdraw the insulin needle (a very small effusion of blood may be seen).</li>
		<li>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.</li>
		<li>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.</li>
		<li>Gently insert the injector needle into the pilot hole to a depth of about 500 &#181;m to access the subretinal space. There should be about a 100 &#181;m 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).</li>
		<li>Ask the assistant to gently depress the plunger of the injector syringe to inject the appropriate volume of cells (about 1.2 &#181;L). 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.</li>
		<li>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.</li>
		<li>Rinse the cell efflux from the injection site with the sterile eye wash 3 times and collect the excess with a cotton applicator.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Post-injection Treatment
<ol>
	<li>Anti-inflammatory and pain reliever treatment
	<ol>
		<li>Inject buprenorphine at 0.1 mg/kg body weight in saline subcutaneously to decrease pain.</li>
		<li>Inject dexamethasone at 1.6 mg/kg body weight in saline by intraperitoneal injection (I.P.) for inflammation control.</li>
	</ol>
	</li>
	<li>Animal recovery
	<ol>
		<li>Return the rat to the recovery cage under a heat lamp to maintain body temperature.</li>
		<li>Observe the operated eye for signs of hemorrhage.</li>
		<li>Observe the rat to ensure it comes out of anesthesia.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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&#39; 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...

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 countries1,2. The pathological process of AMD is associated with &#34;drusen&#34; accumulation between the RPE layer and the underlying Bruch&#39;s membrane, which in turn impairs RPE support of photoreceptor physiology, leading to neural retinal atrophy and vision loss3,4,5. 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 performed6,7, some of which have progressed to clinical trials8,9 (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 AMD10,11,12,13.
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 space14,15,16. 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.

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

development of a refined protocol for trans-scleral subretinal transplantation of human retinal pigment epithelial cells into rat eyes

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.

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...

Watch this Scientific Journal Video about Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes at JoVE.com

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

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.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

development-refined-protocol-for-trans-scleral-subretinal

Research

JoVE Journal

Neuroscience

9.4K Views.  Neural Stem Cell Institute. 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. 

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

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Developmental Biology

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Medicine

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

The subretinal implantation of retinal pigmented epithelium (RPE) is one of the most promising approaches for the treatment of degenerative retinal diseases. However, the performance of preclinical studies on large-eye animal models remains challenging. This report presents guidelines for the subretinal transplantation of RPE on a cell carrier into minipigs.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial ...

Bioengineering

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

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

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

Development of a Surgical Technique for Subretinal Implants in Rats

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been undertaken to develop regenerative medicine-based therapies for AMD, including stem cell-based therapies. Rodents as animal models for retinal degeneration are a foundation for translational research, due to the broad spectrum of strains that develop retinal degeneration diseases at different stages. However, mimicking human therapeutic delivery of subretinal implants in rodents is challenging, due to anatomical differences such as lens size and vitreous volume. This surgical protocol aims to provide a guided method for transplanting implants into the subretinal space in rats. A user-friendly comprehensive description of the critical steps has been included. This protocol has been developed as a cost-efficient surgical procedure for reproducibility across different preclinical studies in rats. Proper miniaturization of a human-sized implant is required prior to conducting the surgical experiment, which includes adjustments to the dimensions of the implant. An external approach is used instead of an intravitreal procedure to deliver the implant to the subretinal space. Using a small sharp needle, a scleral incision is performed in the temporal superior quadrant, followed by paracentesis to reduce intraocular pressure, thereby minimizing resistance during the surgical implantation. Next, a balanced salt solution (BSS) injection through the incision is carried out to achieve focal retinal detachment (RD). Lastly, insertion and visualization of the implant into the subretinal space are conducted. Post-operative assessment of the subretinal placement of the implant includes imaging by spectral domain optical coherence tomography (SD-OCT). Imaging follow-ups ascertain the subretinal stability of the implant, before the eyes are harvested and fixated for histological analysis.

The present protocol describes the scleral approach for subretinal device implantation, a feasible surgical technique for implementation in animal models of retinal diseases in research.

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...