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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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+agerelated+macular+degeneration.">Mechanisms of agerelated macular degeneration.</a> Neuron. 75, 26-39 (2012).</li><li>Abdelsalam, A., Del Priore, L. V., Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drusen+in+age-related+macular+degeneration:+Pathogenesis,+natural+course,+and+laser+photocoagulation-induced+regression.">Drusen in age-related macular degeneration: Pathogenesis, natural course, and laser photocoagulation-induced regression.</a> Surv Ophthalmol. 44 (1), 1-29 (1999).</li><li>Jager, R. D., Mieler, W. F., Miller, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration.">Age-related macular degeneration.</a> N Engl J Med. 358 (24), 2606-2617 (2008).</li><li>Lund, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+cells+rescue+visual+function+in+dystrophic+RCS+rats.">Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.</a> Cloning Stem Cells. 8 (3), 189-199 (2006).</li><li>Vugler, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+and+retinal+repair.">Embryonic stem cells and retinal repair.</a> Mech Dev. 124 (11-12), 807-829 (2007).</li><li>Schwartz, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+trials+for+macular+degeneration:+a+preliminary+report.">Embryonic stem cell trials for macular degeneration: a preliminary report.</a> Lancet. 379 (9817), 713-720 (2012).</li><li>Schwartz, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+retinal+pigment+epithelium+in+patients+with+age-related+macular+degeneration+and+Stargardt's+macular+dystrophy:+follow-up+of+two+open-label+phase+1/2+studies.">Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt's macular dystrophy: follow-up of two open-label phase 1/2 studies.</a> Lancet. 385 (9967), 509-516 (2015).</li><li>Stanzel, B. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+RPE+Stem+Cells+Grown+into+Polarized+RPE+Monolayers+on+a+Polyester+Matrix+Are+Maintained+after+Grafting+into+Rabbit+Subretinal+Space.">Human RPE Stem Cells Grown into Polarized RPE Monolayers on a Polyester Matrix Are Maintained after Grafting into Rabbit Subretinal Space.</a> Stem Cell Reports. 2 (1), 64-77 (2014).</li><li>Blenkinsop, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adult+retinal+pigment+epithelial+stem+cell-derived+RPE+monolayers+exhibit+key+physiological+characteristics+of+native+tissue.">Human adult retinal pigment epithelial stem cell-derived RPE monolayers exhibit key physiological characteristics of native tissue.</a> Invest Ophthalmol Vis Sci. 56 (12), 7085-7099 (2015).</li><li>Salero, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+RPE+can+be+activated+into+a+multipotent+stem+cell+that+produces+mesenchymal+derivatives.">Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives.</a> Cell Stem Cell. 10 (1), 88-95 (2012).</li><li>Davis, J. 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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.

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			<td height="21" style="height:21px;width:259px;">0.25% Trypsin-EDTA (1x)</td>
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			<td style="width:256px;">Sigma</td>
			<td style="width:119px;">DN-25</td>
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			<td height="63" style="height:63px;width:259px;">1xDulbecco&#8217;s Phosphate Buffered Saline without Calcium &#38; Magnesium (1xDPBS-CMF)</td>
			<td style="width:256px;">Corning Cellgro</td>
			<td style="width:119px;">431219</td>
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			<td style="width:256px;">Alcon</td>
			<td style="width:119px;">00065079550</td>
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			<td height="21" style="height:21px;width:259px;">Sterile 0.9% saline</td>
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			<td style="width:119px;">488810</td>
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			<td height="42" style="height:42px;width:259px;">Proparacaine Hydrochloride Ophthalmic Solution (0.5%)</td>
			<td style="width:256px;">Akorn</td>
			<td style="width:119px;">17478026312</td>
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			<td height="42" style="height:42px;width:259px;">Tropicamide Ophthalmic Solution, USP (1%)</td>
			<td style="width:256px;">Bausch &#38; Lomb</td>
			<td style="width:119px;">24208058559</td>
			<td style="width:231px;"></td>
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			<td height="42" style="height:42px;width:259px;">Phenylepherine Ophtalmic Solution, USP (10%) stock</td>
			<td style="width:256px;">Bausch &#38; Lomb</td>
			<td style="width:119px;">42702010305</td>
			<td style="width:231px;">This is used to make 2.5% Phenylepherine</td>
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			<td style="width:256px;">Patterson</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">100% Ethanol</td>
			<td style="width:256px;">Thermo Scientific</td>
			<td style="width:119px;">615090040</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">70% Ethanol</td>
			<td style="width:256px;">Ricca Chemical Company</td>
			<td style="width:119px;">2546.70-5</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:259px;">Sterile GenTeal Lubricant Eye Gel</td>
			<td style="width:256px;">Novartis</td>
			<td style="width:119px;">78042947</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Sterile Systane Ultra Lubricant Eye Drops</td>
			<td style="width:256px;">Alcon</td>
			<td style="width:119px;">00065143105</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:259px;">hRPESC-RPE cells</td>
			<td style="width:256px;">Not available commercially</td>
			<td style="width:119px;"></td>
			<td style="width:231px;">Please refer to &#34;Reference #12&#34; for cell isolation and mainteinance.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">24-well plates</td>
			<td style="width:256px;">Corning</td>
			<td style="width:119px;">3526</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Conical tubes (15 ml)</td>
			<td style="width:256px;">Sarstedt</td>
			<td style="width:119px;">62554002</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Microcentrifuge cap with o-ring</td>
			<td style="width:256px;">LPS inc</td>
			<td style="width:119px;">L233126</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Capless Microcentrifuge tubes (1.7 ml)</td>
			<td style="width:256px;">LPS inc</td>
			<td style="width:119px;">L233041</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Centrifuge</td>
			<td style="width:256px;">Eppendorf</td>
			<td style="width:119px;">5804R</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile alcohol wipe</td>
			<td style="width:256px;">McKesson</td>
			<td style="width:119px;">58-204</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile cotton tip applicators</td>
			<td style="width:256px;">McKesson</td>
			<td style="width:119px;">24-106-2S</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile Weck-Cel spears</td>
			<td style="width:256px;">Beaver-Visitec International&#160;</td>
			<td style="width:119px;">0008680</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile surgical drapes&#160;</td>
			<td style="width:256px;">McKesson</td>
			<td style="width:119px;">25-515</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Gauze</td>
			<td style="width:256px;">McKesson</td>
			<td style="width:119px;">16-4242</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Nanofil syringe (10 ul)</td>
			<td style="width:256px;">World Precision Instruments</td>
			<td style="width:119px;">Nanofil</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Nanofil beveled 33-gauge needle</td>
			<td style="width:256px;">World Precision Instruments</td>
			<td style="width:119px;">NF33BV-2</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Insulin syringe needles 31-gauge</td>
			<td style="width:256px;">Becton Dickinson</td>
			<td style="width:119px;">328418</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Rat toothed forceps</td>
			<td style="width:256px;">World Precision Instruments</td>
			<td style="width:119px;">555041FT</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:259px;">Vannas Micro Dissecting Spring Scissors</td>
			<td style="width:256px;">Roboz</td>
			<td style="width:119px;">RS-5602</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Circulating water T pump&#160;</td>
			<td style="width:256px;">Stryker</td>
			<td style="width:119px;">TP700</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Heating pad</td>
			<td style="width:256px;">Kent Scientific</td>
			<td style="width:119px;">TPZ-814</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Animal anesthesia system</td>
			<td style="width:256px;">World Precision Instruments</td>
			<td style="width:119px;">EZ-7000</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Balance</td>
			<td style="width:256px;">Ohaus</td>
			<td style="width:119px;">PA1502</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Stereo microscope</td>
			<td style="width:256px;">Zeiss</td>
			<td style="width:119px;">Stemi 2000</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Microscope light source</td>
			<td style="width:256px;">Schott</td>
			<td style="width:119px;">ACE series</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:259px;">Bioptigen Envisu Spectral Domain Ophthalmic Imaging System</td>
			<td style="width:256px;">Bioptigen</td>
			<td style="width:119px;">R2210</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile black marker pen</td>
			<td style="width:256px;">Viscot Industries</td>
			<td style="width:119px;">1416S-100</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Miniature measuring scale</td>
			<td style="width:256px;">Ted Pella Inc</td>
			<td style="width:119px;">13623</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">Infrared Basking Spot Lamp&#160;</td>
			<td style="width:256px;">EXO-TERRA</td>
			<td style="width:119px;">PT2144</td>
			<td style="width:231px;">This is used as a heating lamp for animals during the post-surgical recovery&#160; 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

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Enrichment of Bruch's Membrane from Human Donor Eyes

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of the center&#160;of the retina, called the macula, resulting in the loss of central vision. Early AMD is characterised by the presence of small, yellowish lesions called soft drusen that can progress onto late AMD such as geographic atrophy (dry AMD) or neovascularisation (wet AMD). Although the clinical changes are well described, and the understanding of genetic influences on conferring AMD risk are getting ever more detailed, one area lacking major progress is an understanding of the biochemical consequences of genetic risk. This is partly due to difficulties in understanding the biochemistry of Bruch&#8217;s membrane, a very thin extracellular matrix that acts as a biological filter of material from the blood supply and a scaffold on which the retinal pigment epithelial (RPE) cell monolayer resides. Drusen form within Bruch&#8217;s membrane and their presence disrupts nutrient flow to the RPE cells. Only by investigating the protein composition of Bruch&#8217;s membrane, and indeed how other proteins interact with it, can researchers hope to unravel the biochemical mechanisms underpinning drusen formation, development of AMD and subsequent vision loss. This paper details methodologies for enriching either whole Bruch&#8217;s membrane, or just from the macula region, so that it can be used for downstream biochemical analysis, and provide examples of how this is already changing the understanding of Bruch&#8217;s membrane biochemistry.

Identifying proteins specifically associated with Bruch&#8217;s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

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 ...