This work was funded by the following funding: NEI R01EY033103 (MSS), Foundation Fighting Blindness (MSS),the Shulsky Foundation (MSS), the Joseph Albert Hekimian Fund (MSS), the Juliette RP Vision Foundation (YVL), Research to Prevent Blindness (unrestricted grant to the Wilmer Eye Institute at Johns Hopkins University and the Cullen Eye Institute at Baylor College of Medicine). We thank Dr. Malia Edwards (Johns Hopkins University School of Medicine) for kindly providing training on the microscope.

All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures were approved by the Johns Hopkins University Animal Care and Use Committee (approval M021M459).
1. Animals
<ol>
	<li>Use Rho::EGFP mice (aged P3-P6) as donors of retinal cell suspensions. 
	NOTE: This strain was a kind gift from Dr. T. Wensel, Baylor College of Medicine.</li>
	<li>Use OPN1LW-EGFP/NRL-/- mice14 (aged P3) as donors of retinal sheets.</li>
	<li>Use adult retinal degeneration Rd1/NS mice with immune deficiency (either sex) as recipients.</li>
	<li>House all mice in cages under a 12:12 h light-dark cycle with access to water and food as needed.</li>
</ol>
2. Collect neural retina (Figure 1)
NOTE: All the following steps were performed under sterile conditions. Supplier information of the research tools and products is provided in the Table of Materials.
<ol>
	<li>Donor mice preparation: Euthanize donor mice with an overdose of carbon dioxide and confirm euthanasia with cervical dislocation.</li>
	<li>Isolate mice eyeballs. To do so follow the steps below.
	<ol>
		<li>Carefully open pups&#39; eyelids using micro scissors to expose the eyeball.</li>
		<li>Use smooth forceps to grab the optic nerve and pull out the eyeball with a pair of forceps.</li>
	</ol>
	</li>
	<li>Isolate neural retina 
	NOTE: This step is performed under a dissection microscope.
	<ol>
		<li>Incise a hole in the center of the cornea using a 25 G sterile needle.</li>
		<li>Cut the cornea, through the hole, in half and enlarge the incision to the sclera and RPE, then remove the sclera and RPE.</li>
		<li>Use micro-toothed forceps to gently remove the lens and the vitreous to isolate the neural retina.</li>
		<li>Proceed with section 3 to prepare donor retinal suspension or section 4 to prepare donor retinal sheet, as required.</li>
	</ol>
	</li>
</ol>
3. Prepare donor retinal suspension (Figure 1)
<ol>
	<li>Incubate the neural retina in Papain solution at 37 &#176;C for 20-30 min until no cell clumps are detected. 
	NOTE: Gently pipette the cell mixture 15x every 10 min.</li>
	<li>Follow manufacturer&#39;s instructions of the Papain Dissociation Kit to collect single cells.</li>
</ol>
4. Prepare donor retinal sheets (Figure 1)
NOTE: This step follows section 2 and is performed under a dissection microscope.
<ol>
	<li>Put the isolated neural retina cup into a 35 mm Petri dish with prechilled sterile PBS.</li>
	<li>Use micro scissors to cut the neural retina cup into multiple retinal sheets (around 1 mm x 2 mm).</li>
</ol>
5. Prepare recipient mice
<ol>
	<li>Anesthetize recipient mice with intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine hydrochloride (20 mg/kg body weight).</li>
	<li>Confirm the surgical plane of anesthesia by ensuring the loss of blink and pain reflexes, regular breathing, and respiration.</li>
	<li>Assess the anesthetic depth by the lack of response to the tail pinch or pedal reflexes withdrawal.</li>
	<li>Always recheck the anesthetic depth during the operative procedure and adjust the anesthetic depth if the animal is responsive to pain. Keep the mice on the pre-warmed surgery table to prevent hypothermia.</li>
	<li>Dilate recipient pupils with 1% (wt/vol) tropicamide eye drops 5 min before the surgery. A well-dilated pupil can facilitate transpupillary visualization under the operating microscope.</li>
	<li>Disinfect the operating mouse eye and surrounding ocular tissues with povidone-iodine, wash with sterilized PBS.</li>
	<li>Apply 0.5% Proparacaine hydrochloride on the mouse eye for analgesia.</li>
</ol>
6. Subretinal transplantation of retinal grafts (Figure 2)
NOTE: All the following steps were performed under aseptic conditions. Surgical tools were autoclaved (use instrument trays to protect the tool tips and take the plunger out of the micro syringes to prevent them getting stuck in the lumen). Supplier information of the research tools and products is provided in the Table of Materials.
<ol>
	<li>Anterior chamber paracentesis: Penetrate peripheral cornea into anterior chamber with an insulin needle to allow aqueous humor to passively egress, to prevent the intraocular pressure (IOP) spikes during transplantation. 
	NOTE: A successful paracentesis is indicated by the efflux of aqueous humor through the cornea hole.</li>
	<li>Put a drop of sodium hyaluronate and a glass coverslip (5 mm diameter) on top of the cornea to enable transpupillary visualization of the fundus under the surgical scope.</li>
	<li>Use toothed forceps to expose the injection locus (1 mm post-corneal limbus) by pressing either the superior or inferior eye wall, as required by the experiment, toward the center of transpupillary vision.</li>
	<li>Use a microinjection needle to perpendicularly penetrate the sclera.</li>
	<li>Carefully rotate the needle direction tangentially to allow complete penetration of the sclera-choroid-RPE complex into the subretinal space.</li>
	<li>Insert the needle tangentially to access the terminal injection locus. 
	NOTE: Verify the complete positioning of the needle&#39;s bevel by transpupillary vision guidance. Use the superficial retinal vessels as an anatomical reference for accurate needle positioning within the subretinal space.</li>
	<li>Inject retinal grafts 
	NOTE: The presence of small bubbles preloaded in the syringe can facilitate validation of the accurate subretinal positioning of donor cells. Elevated IOP resulting from the subretinal injection increases the risk of donor cell reflux.
	<ol>
		<li>If the cornea becomes cloudy24,25, an indicator of high IOP, keep the needle in the subretinal space for at least 3 min until the cornea clears, an indicator that the IOP has reduced sufficiently. 
		NOTE: In rare cases, the IOP may take longer to normalize, and it is recommended keeping the needle in place until the clarity of the cornea is restored even if this would take 8-10 min. Observe under a surgical microscope to prevent damage to retinal structures.</li>
	</ol>
	</li>
	<li>Grasp the injection hole edge with micro-toothed forceps and quickly withdraw the needle. 
	NOTE: The criteria for successful subretinal delivery of a retinal cell suspension and sheet are a constant bleb and a visible white sheet in the subretinal space, respectively.</li>
	<li>Clean the surgical area of the eye with artificial tears and apply ointment if necessary.</li>
	<li>Post-surgery administration
	<ol>
		<li>Place transplanted mice in a pre-warmed (37 &#176;C) clean recovery cage and carefully monitor for distress.</li>
		<li>Apply a drop of topical 0.5% Proparacaine Hydrochloride on the surgical eye 2-3 times if distress occurs.</li>
		<li>Return mice to the house cage after the mice are completely alert and mobile. Place a small number of food pellets and a gel cup in the cage if the mice have trouble reaching the food hopper. 
		NOTE: Keep the eye surface humid until anesthesia wears off to prevent cataract formation.</li>
	</ol>
	</li>
</ol>
7. Multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging
<ol>
	<li>Two months post-transplantation, anesthetize recipient Rd1/NS mice (n=5) by intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine hydrochloride (20 mg/kg body weight) for imaging.</li>
	<li>Dilate pupils with 1% (wt/vol) tropicamide eye drops.</li>
	<li>Perform standard 55&#176; multimodal imaging using the confocal scanning laser ophthalmoscope cSLO system22.</li>
	<li>Obtain MR and SD-OCT images using 30 frames of ART averaging.</li>
</ol>
8. Histological staining
<ol>
	<li>Prepare frozen slides of transplanted Rd1/NS mice eyes as previously reported 14.</li>
	<li>Block and penetrate the frozen slides of transplanted retinae using a mixture of 5% goat serum and 1% Triton X100 in PBS.</li>
	<li>Incubate samples with primary antibodies over night at 4 &#176;C and rinse in PBS (5 min, 3x). The primary antibodies include goat anti-GFP-FITC (1:200), rabbit anti-recoverin (1:1000), and rabbit anti-S-opsin (1:500).</li>
	<li>Incubate the samples with secondary antibody (goat anti-rabbit Cy3, 1:500) and counterstained with DAPI. 
	NOTE: The suppliers of primary and secondary antibodies are listed in Table of Materials.</li>
</ol>

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MSS is/was a paid advisor to Revision Therapeutics, Johnson &#38; Johnson, Third Rock Ventures, Bayer Healthcare, Novartis Pharmaceuticals, W. L. Gore &#38; Associates, Deerfield, Trinity Partners, Kala Pharmaceuticals, and Acucela. MSS receives sponsored research support from Bayer. These arrangements have been reviewed and approved by the Johns Hopkins University in accordance with its conflict-of-interest policies. KVL is named as inventors on patent applications at University of Colorado. MSS and YVL are named as inventors on patent applications at Johns Hopkins University.

Subretinal transplantation in mice is technically challenging due to the small size of mouse eyes. In this study, we developed a simple and reproducible platform for subretinal transplantation in mouse recipients. The platform allows consistency in donor viability protection, successful subretinal delivery, and ensures a low complication rate.
The subretinal transplantation technique depicted here was developed based on the trans-scleral route, where the injection needle penetrates the outer layers (sclera-choroid-RPE complex) of the eyewall. Compared to the trans-corneal19,20 and trans-vitreous21,26,27&#160;approach, the trans-scleral approach directly enters the subretinal space without penetrating the anterior segment structures and neural retina, thus enabling a harmless transplantation and reducing the risk of donor cell reflux through the penetrating hole. A challenge of the trans-scleral approach is to ensure the needle tip propagates along the subretinal space before arriving at the terminal delivery site, while avoiding potential disturbance of the adjacent RPE and choroid layers. It is challenging to achieve these goals through a traditional trans-scleral approach28,29 without direct visual guidance. In this study, we developed a transpupillary vision-guided platform using a surgical microscope and external illumination of the retina to facilitate subretinal transplantation in mouse models. The platform allows the operator to track the surgical process and the recipients&#39; retinal condition by providing real-time visualization of the recipient fundus under the surgical microscope. For example, successful penetration of the sclera-choroid-RPE complex can be simply validated by a relatively bright reflectance of the needle tip via transpupillary visualization. Importantly, when guided by transpupillary visualization, the operator can accurately modulate the angle and the depth of injection according to the relative position between the needle and anatomic structures in the recipient retina (e.g., retinal vessels and optic nerve head). Moreover, accurate administration of materials using this technique can minimize RPE, choroid trauma, and other surgical complications. Indeed, we found hemorrhages can be minimized by avoiding maneuvers in close proximity to retinal blood vessels with the help of transpupillary visualization.
Reflux of donor cells is a major cause of surgical failure post-injection29,30. There are multiple factors involved in promoting donor cell reflux. The high intraocular pressure (IOP) of the recipient eyes caused by the injected donor cell mixtures is an important factor that promotes the reflux of cells out of the eye. Our protocol reduces the recipients&#39; IOP by anterior chamber penetration at the beginning of the surgery and enables the IOP to lower spontaneously by aqueous fluid egress before pulling out the vitreous needle from the vitreous cavity. A second factor is the unsealed injection tunnel in the recipients&#39; eyes. To prevent graft reflux back through the injection tunnel, we use a small size micro-injection needle (34G) to create a self-sealing tunnel when penetrating the eyewall and toothed micro-forceps to hold the edges of the external tunnel opening when pulling out the needle. In conventional transplantation, refluxed grafts are hardly detected because they may occur rapidly or dissipate quickly. The sodium hyaluronate, upon application of the coverslip, almost invariably slides off the cornea to cover most of the limbus and sclera including the sclerotomy site. Following cell injection, the sodium hyaluronate at the sclerotomy site can help to slow down the bulk flow of refluxed contents, if any, and help to make them visually detectable under the surgical microscope. Moreover, the absence of reflux can also be verified by tracing the constancy of the subretinal bleb for cell suspensions and the intraocular location of the retinal sheet using our protocol, which could be useful for laboratories where optical coherence tomography (OCT) is unavailable.
While a substantial number of photoreceptor cells survived in the transplanted retinal sheets, no discernible cellular orientation, or sheet orientation, could be detected. Although delivery of 3D retinal sheets to large animals has been established26,31,32, the subretinal space in mice, coupled with the lack of intraoperative retinal imaging capability, makes it very challenging to ensure the maintenance of sheet orientation intraoperatively as well as in the immediate postoperative period during which the transplanted cells or sheets may become unstable as the mouse regains ambulation.
We describe a trans-scleral surgical platform with direct transpupillary vision guidance for the subretinal transplantation in mouse recipients. This platform enables high accuracy of injection and a low rate of surgical complications. This platform enables precise delivery of known doses of cells, is relatively easy to learn, and facilitates subretinal delivery in addition to intra-retinal or intravitreal injections for different types of therapeutic agents including gene therapy.

Transplantation of photoreceptor and retinal pigmented epithelial (RPE) cells provides potential therapy for retinal degenerative diseases such as age-related macular degeneration (AMD), Stargardt disease, and retinitis pigmentosa (RP)1,2,3,4,5,6,7. To replenish or replace the diseased photoreceptors and RPE cells in degenerated retinae, the subretinal space is particularly well suited as a transplantation target given the laminar anatomy of host photoreceptors and RPE cells. While surgical procedures of sub-retinal transplantation of RPE cells are well-established in large animals8,9,10 and clinical trials11,12,13, challenges facing photoreceptor transplantation research include the scarcity of transgenic large animal models and the limited understanding of in-depth synaptogenesis mechanisms involved in neuronal transplantation, among other concerns. Genetically modified murine models, with various types of retinal degeneration mutants, provide useful tools for studying molecular mechanisms in the context of transplantation and steering the development of effective cell replacement therapies at the preclinical stage14,15,16,17,18.
Unlike the relatively large eye and small crystalline lens in large animals (e.g., pig, monkey), the small size and large crystalline lens of mouse eyes make them difficult surgical targets, especially for subretinal transplantation in which physical space constraints and limited direct visualization are the core challenges.
Current approaches can be classified into three major types based on the injection route. First, in the case of the trans-corneal approach, the needle is passed through the cornea into the vitreous cavity and then into the subretinal space19,20. Successful subretinal delivery can be achieved using this method but the damage to anterior segment structures (i.e., cornea, iris, lens) is a major risk that may severely impede downstream in vivo analysis. Second, in the case of the trans-vitreous approach, the needle enters the vitreous cavity through the pars plana and then into the subretinal space21. This approach is widely used in humans and large animals. However, there is a potential risk of lens damage in rodents as the lens occupies a larger relative volume of the vitreous cavity. Notably, both trans-corneal and trans-vitreous protocols require penetration of the neural retina to arrive at the subretinal space, which causes damage to the host retina and increases the risk of donor cell reflux through the penetration hole. Third, in the case of the trans-scleral approach22,23, the needle penetrates through the scleral-choroid-RPE complex and directly enters the subretinal space. This approach reduces the potential trauma of anterior segment structures and the vitreous cavity. However, without direct visualization of the host fundus, surgical failures caused by trans-retinal punctures, RPE detachment, and choroid hemorrhage are commonly detected.
Here, we developed a trans-scleral surgical platform with direct transpupillary vision guidance for subretinal transplantation in mouse recipients. Validation of the viability of donor retinal sheets and cell suspensions before transplantation was performed. Successful delivery of the donor cells into the subretinal space of a retina degeneration mouse model was confirmed. Only rare surgical complications were detected. In addition, transplanted photoreceptors in the retinal grafts survived and showed evidence of advanced maturation into &#34;adult&#34; rods and cones two months post-transplantation.

<table><tbody><tr><td>Artificial tears</td><td>CareAll</td><td>P31447-04</td><td></td></tr><tr><td>Coverslips (5mm in diameter)</td><td>Deckglaser</td><td>N/A</td><td></td></tr><tr><td>Goat anti-GFP (FITC)</td><td>Abcam</td><td>Ab6662</td><td></td></tr><tr><td>Goat-anti-rabbit Cy3&amp;nbsp;</td><td>Invitrogen</td><td>A10520</td><td></td></tr><tr><td>Insulin syringe (30G)</td><td>Easy Touch</td><td>08496-3015-11</td><td></td></tr><tr><td>Ketamine</td><td>VETone Zetamine</td><td>AH2017J</td><td></td></tr><tr><td>Live Dead Viability Kit</td><td>Thermo Fisher Scientific</td><td>L3224</td><td></td></tr><tr><td>Micro scissor</td><td>Harvard Apparatus</td><td>72-8503</td><td></td></tr><tr><td>Micro smooth forceps</td><td>ASICO</td><td>AE-4360</td><td></td></tr><tr><td>Micro toothed forceps</td><td>World Precision Instruments</td><td>555041FT</td><td></td></tr><tr><td>Microinjection needle (26G)</td><td>Hamilton</td><td>7804-03</td><td></td></tr><tr><td>Microinjection needle (34G)</td><td>Hamilton</td><td>207434</td><td></td></tr><tr><td>Microinjection syringe</td><td>Hamilton</td><td>7633-01</td><td></td></tr><tr><td>Papain dissociation kit</td><td>Worthington Biochemical</td><td>LK003150</td><td></td></tr><tr><td>Petri-dish (35 mm)</td><td>Thermo Fisher Scientific</td><td>FB012920</td><td></td></tr><tr><td>Povidone-iodine (10%)</td><td>Betadine Solution</td><td>N/A</td><td></td></tr><tr><td>Proparacaine Hydrochloride (0.5%)</td><td>Keeler</td><td>AX0500</td><td></td></tr><tr><td>Rabbit anti-recoverin</td><td>Millipore</td><td>Ab5585</td><td></td></tr><tr><td>Rabbit anti-S-opsin</td><td>Millipore</td><td>Ab5407</td><td></td></tr><tr><td>Sodium hyaluronate</td><td>Johnson &amp;amp; Johnson Vision&amp;nbsp;</td><td>10-2400-11</td><td></td></tr><tr><td>Sterile cotton swabs</td><td>Puritan</td><td>25-806 2PC</td><td></td></tr><tr><td>Sterile needle (25G)</td><td>BD PrecisionGlide Needle</td><td>305122</td><td></td></tr><tr><td>Sterile towel drapes</td><td>Dynarex</td><td>4410</td><td></td></tr><tr><td>Surgical materials/reagents</td><td></td><td></td><td></td></tr><tr><td>Tropicamide ophthalmic solution</td><td>Henry Schein</td><td>112-7192</td><td></td></tr><tr><td>Xylazine</td><td>AnaSed Injection</td><td>N/A</td><td></td></tr></tbody></table>

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.

Retinal grafts are successfully delivered into the subretinal space and survived in vivo. 
The performance of our subretinal transplantation platform was assessed in Rd1/NS recipient&#160;mice aged 6-8 weeks when their residual retinae were severely thinned due to the almost complete outer nuclear layer (ONL) degeneration. Given the fragility of the retina, the scarcity of cone photoreceptors, and the relatively poor viability of cells in suspension compared to sheet donors, cone-rich mice (OPN1LW-EGFP;NRL-/-)14 were used as the sources of donor retinal sheets and rod-rich mice (Rho::EGFP) as the donor for cell suspensions. Cone-rich retinal sheet and rod-rich cell suspensions were successfully delivered into the subretinal space of all recipient mice using our surgical platform, as confirmed by the subretinal bleb and white sheet seen using transpupillary visualization immediately following injection. Two months post-transplantation, multimodal cSLO imaging was performed to track the states of retinal grafts in vivo. Cross-sectional OCT scanning showed that retinal grafts survived in the subretinal space and reconstituted the ONL in all recipient mice (Figure 3A). Infrared imaging detected no obvious cataracts in all transplanted eyes (Figure 3B). Other surgical complications including hemorrhage were rarely detected in transplanted retinae (n = 1/10 eyes) by multicolor reflectance imaging at two months post-transplantation (Figure 3C). We performed histological staining to further assess the survival and extent of maturation of photoreceptors in retinal grafts. Abundant cone photoreceptors expressing OPN1LW:EGFP and S-opsin in transplanted retinal sheets were observed. Likewise, transplanted retinal cell suspensions showed a large proportion of Rec+ photoreceptors in vivo, including numerous Rho::EGFP+ rods (Figure 3D). The non-transplanted control mice showed severe ONL degeneration with sparse residual cone photoreceptors (Rec+). No EGFP signal was detected in non-transplanted Rd1/NS retina (Figure 3D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65448/65448fig01.jpg" /> 
Figure 1: Schematic of collecting donor retinal sheet and cell suspensions. Schematics showing the key steps of collecting retinal cell suspensions and sheets from donor Rho::EGFP mice. Bright-field and fluorescent imaging showed representative images of dissociated cells and a dissected sheet isolated from Rho::EGFP mice. Yellow dotted line: incisional margin. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65448/65448fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65448/65448fig02v3.jpg" /> 
Figure 2: Procedures of subretinal transplantation of retinal sheets and cell suspension in Rd1/NS mice. (A)&#160;Schematic images of&#160; recipient mice preparation. (B) Key surgical procedures and corresponding diagrams showing subretinal transplantation of retinal cell suspensions and sheets. Surgical steps include: 1. penetrate the anterior chamber; 2. drop the sodium hyaluronate on the cornea, then mount the coverslip on top of the sodium hyaluronate to facilitate the transpupillary visualization; 3. penetrate outer layers of the eye wall (sclera-choroid-RPE complex). Penetration angles of the needle are on the top right panel of the illustration; 4. insert injection needle; 5. inject grafts. Representative images show the successful delivery of retinal cell suspensions and sheet in two individual recipients, respectively. Asterisk: optic nerve head; Red arrowheads: retinal blood vessel; White arrowhead: penetrated needle tip; White arrows: grafted retinal suspensions or sheet. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65448/65448fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65448/65448fig03.jpg" /> 
Figure 3: Successful delivery of retinal grafts into the subretinal space and survival in vivo. (A)&#160;Representative SD-OCT images showed the subretinal distribution of transplanted retinal sheets and cell suspensions in two individual Rd1/NS mice, respectively. Non-transplanted Rd1/NS mice were collected as a control. Region of Interest (ROI) is indicated by yellow-dotted boxes on infrared (IR) fundus images. Inner retina is designated as laminae of retinal ganglion cell layer (RGC), inner plexiform layer (IPL), and inner nuclear layer (INL). (B) Representative infrared (IR) image of a transplanted Rd1/NS mouse showed no cataract through the dilated pupil. (C) Representative multicolor reflectance (MR) images of transplanted and non-transplanted Rd1/NS eyes. The transplanted eyes presented no obvious surgical complications including hemorrhage. (D) Immunohistochemistry (IHC) staining of transplanted- (sheet and suspension) and non-transplanted (control) Rd1/NS mice. The data showed numerous grafted photoreceptors expressing specific markers of photoreceptors (Rec), rods (Rho::EGFP), L/M cones (OPN1LW:EGFP), and S-cones (S-opsin) at two months post-transplantation. Recipient retinal laminae were identified by DAPI staining (blue). Retinal grafts were identified by EGFP reporter (green). The non-transplanted mice retina showed severe ONL degeneration with sparse residual cone photoreceptors (Rec+). No EGFP signal was detected in non-transplanted Rd1/NS retinae. Magnified images were presented on the right two panels. Abbreviations: RGC: retinal ganglion cell layer; INL: inner nuclear layer. ONL: outer nuclear layer. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65448/65448fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model at JoVE.com

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

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. ...

transpupillary-guided-trans-scleral-transplantation-subretinal-grafts

Universidad San Sebastian

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Research

JoVE Journal

Neuroscience

1.1K Views.  Johns Hopkins University School of Medicine. 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.

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

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Medicine

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

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 ...

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.

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 ...

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 ...

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, ...

Biology

Revitalizing Sight: Stem Cell Therapy for Retinal Degeneration

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal diseases at advanced stages. We have shown human recombinant retina-specific laminin isoform matrix is able to support the differentiation of human embryonic stem cells (hESCs) to photoreceptor progenitors. In addition, sub-retinal injection of these cells has also shown partial restoration in the rd10 rodent and rabbit models. Sub-retinal injection is known to be an established method that has been used to deliver pharmaceutical compounds to the photoreceptor cells and retinal pigmented epithelial (RPE) layer of the eye due to its proximity to the target space. It has also been used to deliver adeno-associated viral vectors into the sub-retinal space to treat retinal diseases. The sub-retinal delivery of pharmaceutical compounds and cells in the murine model is challenging due to the constraint in the size of the murine eyeball. This protocol describes the detailed procedure for the preparation of hESC-derived photoreceptor progenitor cells for injection and the sub-retinal delivery technique of these cells in genetic retinitis pigmentosa mutant, rd10 mice. This approach allows cell therapy to the targeted area, in particular the outer nuclear layer of the retina, where diseases leading to photoreceptor degeneration occur.

Author Spotlight: Advancing Vision Restoration - Stem Cell-Based Therapy for Retinal Diseases 

We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice.

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal ...