The authors have no acknowledgements.

The study protocol was approved by the Ethics Committee of the Low Vision Academy and all subjects signed a written consent in accordance with the Helsinki Declaration. This research study has received ethical approval from both Loughborough and Sheffield Universities.
NOTE: The inclusion and exclusion criteria of dry age-related macular degeneration patients to receive suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) is described in Table 1.
1. Diagnosis of Dry Age-related Macular Degeneration Patients
<ol>
	<li>Ascertain the diagnosis with confocal scanning laser ophthalmoscope, SD-OCT, and MY.</li>
	<li>Evaluate each group&#39;s BCVA for far and near distance. Measure VA for near vision (close-up) in points (Pts). Measure BCVA at time 0 (T0), 90 (T90), and 180 days (T180) compared to early treatment diabetic retinopathy study (EDTRS) charts at 4 meters in logMAR.</li>
	<li>Record electrical scotopic, mesopic, and photopic cell activity, or flash ERG, according to the standards set in 2009 by the International Society for Clinical Electrophysiology of Vision (ISCEV)11.</li>
</ol>
2. Anesthetization
NOTE: The gold standard in anesthesia during LRRT is topical anesthesia, reinforced by sub-tenon&#39;s infiltration of anesthetic and sedation. In specific cases, general anesthesia is preferred.
<ol>
	<li>Obtain corneal and conjunctival anesthesia by applying topical local anesthetics instilled dropwise 15&#160;- 20 min before the surgery with lidocaine at 4% and ropivacaine at 1%.</li>
	<li>Inject anesthesia by infiltration directly into sub-conjunctival and subtenon&#39;s spaces.</li>
	<li>Use local infiltration both in the abdominal region, before the adipose tissue is extracted, and in the sub-conjunctival and sub-tenon&#39;s spaces, 12 mm from the limbus. Adopt local anesthetic of carbocaine or marcain mixed with 1,200 IU epinephrine.</li>
	<li>Provide intraoperative sedation through the anesthetic, which can be performed properly by using fentanyl as a narcotic analgesic through repeated small boli. The dosage is generally 0.025 mg of fentanyl with 1 mg of midazolam per bolus.</li>
</ol>
3. Limoli Retinal Restoration Technique Preparation
NOTE: This technique represents a variant of Pelaez&#39;s intervention by which orbital autologous fat is transplanted in the subscleral space1,6,7,12. Surgically grafted cells can produce many GFs with neurotrophic and angiotrophic properties in the surrounding tissue, choroid, and retina18,19,20,21,22,23,24,25. In LRRT, the distance between grafted autologous cells and choroid is reduced by means of deep sclerectomy, and the contact area between the stalk and choroid is expanded to promote the paracrine autologous cell secretion into the choroidal flow9,10,14.
<ol>
	<li>Perform proper disinfection of each eye before surgery with cellular grafting between the choroid and sclera, a procedure called Limoli Retinal Restoration Technique (LRRT)15,16,17.</li>
	<li>Graft the ADSCs, obtained by Coleman et al. and Lawrence&#39;s technique (Figure 1) from abdominal fat, in the SVF in the sovrachoroidal space15,16,17.</li>
	<li>Infiltrate adipose pedicle with platelets derived from PRP gel obtained through the following steps.</li>
	<li>Centrifuge blood6,12 and collect platelet-rich plasma (PRP). The stimulus to platelet degranulation causes GF release in the adipose pedicle6,12.</li>
</ol>
4. Technical Specifications and Strategy
Note: Fat tissue is collected and purified from the abdominal subcutaneous layer of patients, according to the Lawrence and Coleman technique17(Table of Materials).
<ol>
	<li>Manually harvest 10 mL of fat tissue from the abdominal subcutaneous layer of each patient, using a 3-mm blunt cannula connected to a locking syringe, according to the Lawrence and Coleman technique17 (Figures 2A/2B).</li>
	<li>Separate pure SVF of fat tissue from blood, fat, oil, and liquid by centrifugation for 5 min at 1,500 x g at 20 &#176;C (Figure 2C). The SVF is very rich of ADSCs17.</li>
	<li>Collect 8 mL of human peripheral blood with a 22 G needle and in a separate tube for PRP preparation.</li>
	<li>Centrifuge the collected blood for 5 min at 1,500 x g at 20 &#176;C (Figure 2D). In LRRT, the ensuing changes result in better survival of the autologous fat graft, ADSC proliferation, which favors increased choroidal perfusion, and a more comprehensive modulation of the action of those factors that are secreted only by fat7,11,17.</li>
	<li>Build the suprachoroidal pocket (more details in step 4, in particular 4.4 and 4.5) to accommodate the graft obtained from orbital fat and saturate the residual volume of this pocket with a mixture of ADSCs from SVF and PRP, obtained according to the Lawrence and Coleman technique17.</li>
</ol>
5. Suprachoroidal Autograft by LRRT (Limoli Retinal Restoration Technique): Surgical Procedure and Technical Details
<ol>
	<li>Anchor the sclera with 6-0 silk suture, near the inferior-temporal limbus.</li>
	<li>Open the subconjunctival and subtenonian space at 11 mm from the inferior-temporal limbus, using 5.5&#34; Westcott Tenetomy curved scissors.</li>
	<li>Insert the Limoli-Basile conjunctival retractor in this space to make a scleral surgical field.</li>
	<li>Using a 5-mm crescent knife angled bevel up, pre-cut a flap on the side in the sclera at 8 mm, from the limbus. The flap hinge is always radial and to the left of the surgeon.</li>
	<li>In the inferior-temporal quadrant, at 8 mm from the limbus, open a deep scleral door of about 5 mm on the side by radial hinge by using a crescent knife, angled bevel up. Carry out sclerectomy at an adequate depth to view the slate color of the choroid.</li>
	<li>Create a gap by removing a little operculum in the distal part of the flap, in order to facilitate blood circulation in the subsequent suprachoroidal autograft.</li>
	<li>Extract with ophthalmological forceps the orbital fat from a gap above the inferior oblique muscle. Make sure the extracted fat is sufficiently vascularized to allow it to survive after its implantation.</li>
	<li>Gently place the autologous fat flap on the choroidal bed and suture with choroidal 6/0 polyglactin fiber at the proximal edge of the door.</li>
	<li>Suture the scleral flap to avoid compression on the fat pedicle or on its nutrient vessels.</li>
	<li>Infiltrate the stroma of the fat pedicle with 1 mL of PRP gel (obtained by centrifugation of the blood material, separation of the component, and platelet degranulation26) using a 30 G angled (30&#176;) cannula.</li>
	<li>Prepare the sides of the conjunctiva for the suture. Then, remove the conjunctival retractor.</li>
	<li>Suture the conjunctiva, using 6/0 polyglactin fiber.</li>
	<li>Before closing, leave a space to insert into the subscleral space, between the flap, the choroid, and the choroidal autograft, a small flexible plastic tube with the autologous fat graft.</li>
	<li>Saturate the residual space between the autologous fat graft, choroid, and scleral flaps with 0.5 cc of SVF (rich of ADSCs), previously prepared in step 3.2, by a small flexible plastic tube, inserted into the scleral pocket.</li>
	<li>After saturating the residual space, close the suture.</li>
	<li>After surgery, administer three days of antibiotic therapy with 500 mg azithromycin. Also, provide eye-drop therapy with an antibiotic and steroid combination, such as Chloramphenicol and Betamethasone, for about 15 - 20 days. 
	NOTE: An autograft made up of fat cells, ADSCs from SVF, and PRP has now been obtained26. Reduce the distance between the grafted autologous cells and choroid by deep sclerectomy to stimulate paracrine secretion of autologous cells into the choroidal flow. For the same purpose, expand the area of contact between the stalk and choroid.</li>
</ol>

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Presented at ARVO 2015, May 3-7 - Denver, CO - USA.

The primary purpose of this study was to evaluate whether the suprachoroidal graft of adipocytes, ADSCs in SVF, and PRP could improve VA and retinal sensitivity in dry AMD-affected eyes over time. Another main objective was to demonstrate possible therapeutic effects of these cells, based on the recent literature, since several preclinical studies have suggested that GF-based therapy could be useful for patient care in several diseases.
In fact, some studies have shown that autologous human induced pluripotent stem cells (iPSC) could represent a cellular source for the graft, aimed at retinal pigment epithelium (RPE) regeneration in tissue replacement therapy for AMD18,19. These cell sheets are generated as a monolayer which could express typical RPE markers and exhibit polarized GF secretion, showing phagocytotic ability, as well as gene-expression patterns similar to those of native RPE18,19. Upon transplantation, autologous non-human primate iPSC-RPE cell sheets showed neither immune rejection nor tumor formation18,19.
The present study presents some different characteristics. We analyzed directly in dry AMD-affected human eyes whether the suprachoroidal autograft cell can improve visual performance.
Besides, sovrachoroidal graft of autologous cells according to LRRT has always proven to be safe. We have never registered sub-retinal neovascularization, macular edema, retinal detachment, or other retinal problems in the first-year post intervention. On the other hand, inappropriate surgical procedures can theoretically lead to perforation of the choroid with subsequent bleeding, but in our research no eye was damaged. However, it is possible to have sub-conjunctival hemorrhage that is usually reabsorbed within a few days and does not present again as a complication.
Recent studies have provided ample evidence of a significant increase in scotopic ERG values, BCVA and MY, at 90 and 180 days post autologous graft. However, the increase was greater if the retinal thickness average (RTA) recorded by SD-OCT was higher11,26. It is believed that the surgically grafted cells can produce GFs in the surrounding tissue, choroid, and retina, and that they have neurotrophic and angiotrophic properties, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), pigment-epithelium-derived factor (PEDF), interleukin (IL), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and placental growth factor (PlGF), while platelets produce platelet-derived grow factor (PDGF), platelet-derived angiogenesis factor (PDAF), etc.6,7,12,13,21
Graft placement near the choroid is believed to allow the produced GFs to enter the choroidal flow, to reach the endothelial cell receptors, RPEs, Muller cells, photoreceptors, and finally to interact with them. In LRRT, the autologous grafted elements are useful, each in their own way, for regeneration. The fat cells produce bFGF, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), IL, transforming growth factor &#946; (TGF-&#946;), PEDF, and adiponectin21. The ADSCs produce bFGF, VEGF, M-CSF, GM-CSF, PlGF, TGF-&#946;, hepatocyte growth factor (HGF), IGF-1, IL, and angiogenin6,7. The platelets produce PDGF, IGF-1, TGF-&#946;, VEGF, bFGF, EGF, PDAF, and thrombospondin (TSP)6,12.
Some factors promote endothelial regeneration, and some stimulate ADSC proliferation, thus favoring both autologous fat and adipocyte survival, while others inhibit neovascular processes22,23,24. PEDF and bFGF favor photoreceptor survival, while EGF exerts its action on M&#252;ller cells by triggering endogenous bFGF transcription and stimulating ADSCs to increase their secretory activity25,27. Though GFs are normally secreted by RPEs, this does not occur in atrophic maculopathy as a result of the RPE/choriocapillaris complex. Paracrine GF secretion by graft cells contributes to favoring photoreceptor and choriocapillaris survival28. Moreover, M-CSF, GM-CSF, and IL have anti-inflammatory and chemotactic effects on macrophages, which are involved in the elimination of intraretinal cell debris, a function that is physiologically carried out by RPEs29,30.
The cell types grafted behind the choroid can ensure constant GF secretion in the choroidal flow. GFs can arrive from the choroid to the retinal cells, interact with their membrane receptors, and finally activate an intracellular pathway. The presented data suggest that LRRT can increase choroidal perfusion and photoreceptor trophism not only through bFGF-receptor interactions, but also through stimulation by M&#252;ller cells, RPEs, and retinal photoreceptors. As a result, gene expression changes and the final antiapoptotic effect could explain the neuroenhancement. This cellular mechanism underlies the ability to increase visual performance, as highlighted in the clinical findings in the grafted group. In summary, LRRT could be useful to preserve the visual function of dry AMD-affected patients in the long term.
However, as we have demonstrated in previous studies, cone-rod ERG and rod ERG show a highly significant correlation with RTA, while this is not the case of cone ERG. This can be explained by the fact that fovea function appears to be compromised, although the macular volumes in dry AMD continues to be regular, at least in the initial stages26. In this pathology, residual retinal trophism measured by RTA can be a prognostic criterion for LRRT treatment, since better outcomes are more frequent in patients with RTA equal to or greater than 250 &#181;m26. The available GF set could result in neuroenhancement, the extent of which is proportional to the presence of areas with greater cellularity, as recorded by electrical activity26. At a later stage, poor tissue cellularity would not give the therapeutic effect that is sought after with the procedure, due to the scarce GF-membrane receptor interactions.
The next steps of this research will require the recruitment of a larger number of subjects with greater VA and central fixation by statistically assessing all the indispensable tests needed to confirm that the technique is valid and to study the biochemical effects. It can be argued that the increase in cell trophism is reflected in the cell visual activity, measured objectively by ERG, BCVA, and MY11. GF-based therapy could provide an up-to-date, selective, safe, and reasonable treatment in ophthalmologic diseases.

Cell therapy, consisting of the systemic or local injection of stem/progenitor cells in the injured area to treat multiple chronic disorders, has drawn close attention in the last decade1. Since the 1990s, growth factors (GFs) have been studied for their potentially therapeutic role in retinal atrophy2. In fact, many human cells can produce GFs, which are specific proteins that are able to block or slow down apoptosis, i.e., the programmed death of cells3.
It is known that dry age-related macular degeneration (AMD) is an atrophic retinal disease where gradual and irreversible cell death involves injury to the photoreceptor layer and, consequently, the loss of central visual function4. AMD is the leading cause of blindness in people over 55 years of age in developed countries and accounts for 80% of all macular degenerations, which lack an effective treatment to date.
Several studies have shown that there are various sources from which autologous GFs can be obtained. These comprise different cell types, including adipose stromal cells derived from orbital fat, platelets derived from platelet-rich plasma (PRP), and adipose-derived stem cells (ADSCs) included in the stromal vascular fraction (SVF) of adipose tissue5,6,7. The current GF set ensures retinal neuroenhancement, and research conducted by Filatov, Meduri, Pelaez, and Limoli has demonstrated that autologous fat transplantation (AFT) is effective8,9,10.
Moreover, a prior study showed significant improvements in electroretinogram (ERG) data, recorded post suprachoroidal autologous graft, in dry AMD-affected eyes11. The surgically grafted tissue in the suprachoroidal space modulated the paracrine secretion of retinal cells, delaying their apoptosis6,7,12. Considering outer nuclear layer thickness, the histological examination of the retina of guinea pigs has shown that GFs could have a trophic effect on the retina. Therefore, the direct or indirect use of GFs can potentially bring therapeutic benefits through a balanced relation between molecular inducers and inhibitors6,7,12.
The purpose of this method is to assess whether the suprachoroidal graft of adipocytes, ADSCs in SVF and PRP can improve best corrected visual acuity (BCVA) and microperimetry (MY) responses in dry AMD-affected eyes. This study aims to demonstrate the therapeutic effect of autograft on the basis of its GF production, according to the cited literature6,7,12,13.

<table><tbody><tr><td>Blunt cannula, 3 mm.&amp;nbsp;</td><td>Mentor, Santa Barbara, CA.</td><td></td><td></td></tr><tr><td>Luer-LokTM syringe.&amp;nbsp;</td><td>BD Biosciences, Franklin Lakes, NJ.</td><td></td><td></td></tr><tr><td>Regen-BCT tube.&amp;nbsp;</td><td>RegenKit; RegenLab, Le Mont-sur-Lausanne, CH.</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>&amp;nbsp;RegenPRP Centri. RegenLab, Le Mont-sur-Lausanne, CH.</td><td></td><td></td></tr><tr><td>BD Venflon Pro Safety 22G x 1.00 inch (0.9 mm x 25 mm).&amp;nbsp;</td><td>BD Biosciences, Franklin Lakes, NJ.</td><td></td><td></td></tr><tr><td>SPSS Statistics Version 19.0</td><td>IBM Corp., Armonk, NY, USA.</td><td></td><td></td></tr><tr><td>Confocal scanning laser ophthalmoscope&amp;nbsp;</td><td>Nidek Inc, Fremont, CA</td><td>Nidek F10&amp;nbsp;</td><td></td></tr><tr><td>Cirrus 5000 Spectral Domain-Optical Coherence Tomography</td><td>Carl Zeiss Meditec AG, Jena, Germany</td><td>&amp;nbsp;SD-OCT&amp;nbsp;</td><td></td></tr><tr><td>Maia 100809 Microperimetry&amp;nbsp;</td><td>CenterVue S.p.A., Padua, Italy</td><td></td><td></td></tr><tr><td>Ocular electrophysiology electromedical system,</td><td>C.S.O., S.r.l., Scandicci, Italy</td><td></td><td>&amp;nbsp;Retimax for ERG&amp;nbsp;</td></tr></tbody></table>

regenerative therapy by suprachoroidal cell autograft in dry age-related macular degeneration: preliminary in vivo report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Using the procedure presented here, two groups of dry AMD-affected patients, with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR), were enrolled in the study. Group A, including 11 eyes of 11 patients, received suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT), whereas group B, including 14 eyes of 14 patients, was used as a control group.
Student&#39;s t-test and the chi square test were used to compare, respectively, mean age and gender distribution between the two study groups (Table 2). Statistical analyses and data visualization were performed before and after LRRT surgery.
A Wilcoxon-Mann-Whitney signed rank test was carried out to determine whether the pre- and post-treatment differences were significant. This non-parametric statistical hypothesis test was applied to compare two dependent samples when the population cannot be assumed to be normally distributed, as in this case. VA values were measured at each step of the analysis. Statistical significance was set at a p value &#60;0.05.
Eleven eyes (6 right and 5 left eyes) of 11 patients (7 male and 4 female) with the clinical diagnosis of dry AMD were examined in this study. Patient age ranged from 62 to 84 years, with a mean age of 71.5 years (&#177; 3.8 SD).
Table 2 provides an overview of the clinical profiles of the patients treated with LRRT, and the average values recorded at 0 (T0), 90 (T90), and 180 (T180) days after surgery. Adverse effects were always reported to guarantee maximum safety. Mean values of intraocular pressure recorded at 0 (T0), 90 (T90), and 180 (T180) days after surgery did not record any significant change.
Results after LRRT surgery were as follows: 
In group A, BCVA changed from 0.581 (T0) preoperatively to 0.504 at 90 days (T90), and to 0.376 logMAR at 180 days (T180) with a significant increase of 35.20% (p&#160;&#60;0.01). In control group B, including 14 eyes of 14 patients, 7 males and 7 females, with a mean age of 80.4 years, SD &#177; 2.3, BCVA changed from 0.573 (T0) to 0.587 (T90), and to 0.601 logMAR (T180) with a non-significant mean decrease of 4.72% (Table 2) (Figure 3). In group A, MY test increased significantly from 11.44 dB (T0) to 12.59 dB (T180) (+9.58%) (Figure 4), whereas there was no significant improvement in postoperative values in group B.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56469/56469fig1.jpg" /> 
Figure 1: Representation of suprachoroidal autologous graft. Growth factors (GFs) produced by adipose cells, platelet-rich plasma (PRP), and adipose-derived stem cells (ADSCs) reach the choroidal and retinal tissues through the retinal pigment epithelium (RPE). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56469/56469fig1large.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/56469/56469fig2.jpg" /> 
Figure 2: Technical Procedure. Cannula and subsequent withdrawal of adipose tissue from the abdominal area (Panel&#160;A). The cannula moves subcutaneous fat with mild aspiration by aspirating fat cells into its own lumen (Panel B). After centrifugation, there are three-layers of adipose tissue: oil (high layer), homogeneous fat (intermediate layer), and blood fluid (lower layer) (Panel C). Observe the tube with blood immediately after centrifugation. There are three layers: platelets poor plasma (PPP), platelets rich plasma (PRP), and erythrocytes (Panel D). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56469/56469fig2large.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/56469/56469fig3.jpg" /> 
Figure 3: Best corrected visual acuity (BCVA) in dry age-related macular degeneration (AMD). Change in group A after suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT), and in the control group (Check), measured at time 0 (T0), 90 (T90), and 180 (T180) days. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56469/56469fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56469/56469fig4.jpg" /> 
Figure 4: Microperimetry (MY) in a patient of group A. Dry age-related macular degeneration (AMD) six months after Limoli Retinal Restoration Technique (LRRT). MY increased from 11.44 dB (T0) to 12.59 dB (T180) (+9.58%). Color scale from 0 to 36 in dB. Fixation stability: stable, relatively unstable, unstable. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56469/56469fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Inclusion Criteria</td>
			<td>Exclusion Criteria</td>
		</tr>
		<tr>
			<td>Caucasian subjects</td>
			<td>Refractive errors with spherical equivalent &#62;6 D</td>
		</tr>
		<tr>
			<td>Well-nourished participants</td>
			<td>Signs of exudative AMD by SD-OCT and FA</td>
		</tr>
		<tr>
			<td>Diagnosis by SD-OCT, AFI, and FA</td>
			<td>Ocular disorders: CT, GL, ON, MP, VM, CRD, etc.</td>
		</tr>
		<tr>
			<td>Measurable VA</td>
			<td>Ocular trauma</td>
		</tr>
		<tr>
			<td>BCVA &#8805;1 logMAR</td>
			<td>Systemic diseases: MS; PD; DM; RD; HD; vasculitis</td>
		</tr>
		<tr>
			<td>Normal IOP</td>
			<td>Hypertension, cancer, and other systemic diseases</td>
		</tr>
		<tr>
			<td>Good storage extrafoveal areas</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">LogMAR: logarithm of the minimum angle of resolution; SD-OCT: spectral domain-optical coherence tomography; AFI: autofluorescence imaging; FA: fluorescein angiography; VA: visual acuity; IOP: intraocular pressure; D: diopters; CT: cataract; GL: glaucoma; ON: optic neuritis; MP: macular pucker; VM: neovascular membranes; CRD: chorioretinal disease; MS: multiple sclerosis; PD: Parkinson disease; DM: diabetes mellitus; RD: renal diseases; HD: hepatic diseases.</td>
		</tr>
	</tbody>
</table>
Table 1: Inclusion and exclusion criteria for dry age-related macular degeneration (AMD) patients.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>PARAMETERS</td>
			<td>LRRT (n = 11)</td>
			<td>Control (n = 14)</td>
		</tr>
		<tr>
			<td>Age (average)</td>
			<td>71.5 &#177;3.8SD</td>
			<td>80.4 &#177;2.3SD</td>
		</tr>
		<tr>
			<td>Age (range)</td>
			<td>62 - 84</td>
			<td>73 - 79&#160;</td>
		</tr>
		<tr>
			<td>Sex</td>
			<td>F:4 M:7</td>
			<td>F:7 M:7</td>
		</tr>
		<tr>
			<td>BCVA T0</td>
			<td>0.581 logMAR</td>
			<td>0.573 logMAR</td>
		</tr>
		<tr>
			<td>(average)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BCVA T0 (range)</td>
			<td>0.301 - 1.0</td>
			<td>0.0 - 1.0</td>
		</tr>
		<tr>
			<td>BCVA T90 (average)</td>
			<td>0.504 logMAR</td>
			<td>0.587 logMAR</td>
		</tr>
		<tr>
			<td>BCVA T90 (range)</td>
			<td>0.222 - 1</td>
			<td>0.0 - 1</td>
		</tr>
		<tr>
			<td>BCVA T180 (average)</td>
			<td>0.376 logMAR</td>
			<td>0.601 logMAR</td>
		</tr>
		<tr>
			<td>BCVA T180 (range)</td>
			<td>0.046 - 0.699</td>
			<td>0.0 - 1.0</td>
		</tr>
		<tr>
			<td>Percentage</td>
			<td>35.19</td>
			<td>4.72</td>
		</tr>
		<tr>
			<td>change</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p value</td>
			<td>&#60;0.01</td>
			<td>&#62;0.5</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Age in years; n=patients and controls; SD=standard deviation; F=female; M=male; BCVA= best corrected visual acuity in logMAR; T0=baseline before surgery autograft; T90=90 days post surgical autograft; T180=180 days post&#160; surgical autograft.</td>
		</tr>
	</tbody>
</table>
Table 2: Clinical profiles of patients examined in the study. Average values recorded before (T0), 90 (T90), and 180 (T180) days after cell autograft by Limoli Retinal Restoration Technique (LRRT) in all patients.

Watch this Scientific Journal Video about Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report at JoVE.com

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

regenerative-therapy-suprachoroidal-cell-autograft-dry-age-related

Nanyang Technological University

Research

JoVE Journal

Medicine

10.1K Views.  Low Vision Research Centre of Milan. Het doel van deze studie is om te beoordelen of de suprachoroidal prothese van adipeus afkomstige stamcellen opgenomen in de stromale vasculaire Fractie en bloedplaatjes platelet-rich plasma door de Limoli techniek voor de restauratie van de retinale afgeleid gezichtsscherpte verbeteren kan en netvlies gevoeligheid reacties in ogen beïnvloed door droge leeftijdsgebonden Macula degeneratie.

Video: Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

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 ...

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 ...

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

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

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

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

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 ...

Neuroscience

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 ...

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

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

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

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

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

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

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

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

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 ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

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