Name: development of a refined protocol for trans-scleral subretinal transplantation of human retinal pigment epithelial cells into rat eyes
Uploaded: 2017-08-12T13:00:00
Description: Watch this Scientific Journal Video about Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes at JoVE.com

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

All procedures involving animals have been approved by the Institutional Animal Care and Use Committee (IACUC) at the State University of New York at Albany.
1. Pre-injection Preparation
<ol>
	<li>Preparation of a hRPESC-RPE cell suspension 
	NOTE: All the following steps are performed in sterile tissue culture hood and familiarity with basic sterile technique is required.
	<ol>
		<li>Isolate primary hRPE cells from human donor eyes aged 50-90 years and culture cells in...

<ol>
	<li>De Jong, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration.">Age-related macular degeneration.</a> N Engl J Med. 355, 1474-1485 (2006).</li><li>Wong, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+prevalence+of+age-related+macular+degeneration+and+disease+burden+projection+for+2020+and+2040:+a+systematic+review+and+meta-analysis.">Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis.</a> Lancet Global Health. 2 (2), e106-e116 (2014).</li><li>Ambati, J., Fowler, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+agerelated+macular+degeneration.">Mechanisms of agerelated macular degeneration.</a> Neuron. 75, 26-39 (2012).</li><li>Abdelsalam, A., Del Priore, L. V., Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drusen+in+age-related+macular+degeneration:+Pathogenesis,+natural+course,+and+laser+photocoagulation-induced+regression.">Drusen in age-related macular degeneration: Pathogenesis, natural course, and laser photocoagulation-induced regression.</a> Surv Ophthalmol. 44 (1), 1-29 (1999).</li><li>Jager, R. D., Mieler, W. F., Miller, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration.">Age-related macular degeneration.</a> N Engl J Med. 358 (24), 2606-2617 (2008).</li><li>Lund, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+cells+rescue+visual+function+in+dystrophic+RCS+rats.">Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.</a> Cloning Stem Cells. 8 (3), 189-199 (2006).</li><li>Vugler, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+and+retinal+repair.">Embryonic stem cells and retinal repair.</a> Mech Dev. 124 (11-12), 807-829 (2007).</li><li>Schwartz, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+trials+for+macular+degeneration:+a+preliminary+report.">Embryonic stem cell trials for macular degeneration: a preliminary report.</a> Lancet. 379 (9817), 713-720 (2012).</li><li>Schwartz, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+retinal+pigment+epithelium+in+patients+with+age-related+macular+degeneration+and+Stargardt's+macular+dystrophy:+follow-up+of+two+open-label+phase+1/2+studies.">Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt's macular dystrophy: follow-up of two open-label phase 1/2 studies.</a> Lancet. 385 (9967), 509-516 (2015).</li><li>Stanzel, B. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+RPE+Stem+Cells+Grown+into+Polarized+RPE+Monolayers+on+a+Polyester+Matrix+Are+Maintained+after+Grafting+into+Rabbit+Subretinal+Space.">Human RPE Stem Cells Grown into Polarized RPE Monolayers on a Polyester Matrix Are Maintained after Grafting into Rabbit Subretinal Space.</a> Stem Cell Reports. 2 (1), 64-77 (2014).</li><li>Blenkinsop, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adult+retinal+pigment+epithelial+stem+cell-derived+RPE+monolayers+exhibit+key+physiological+characteristics+of+native+tissue.">Human adult retinal pigment epithelial stem cell-derived RPE monolayers exhibit key physiological characteristics of native tissue.</a> Invest Ophthalmol Vis Sci. 56 (12), 7085-7099 (2015).</li><li>Salero, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+RPE+can+be+activated+into+a+multipotent+stem+cell+that+produces+mesenchymal+derivatives.">Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives.</a> Cell Stem Cell. 10 (1), 88-95 (2012).</li><li>Davis, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+RPE+Stem+Cell-Derived+RPE+Preserves+Photoreceptors+in+the+Royal+College+of+Surgeons+Rat:+Method+for+Quantifying+the+Area+of+Photoreceptor+Sparing.">Human RPE Stem Cell-Derived RPE Preserves Photoreceptors in the Royal College of Surgeons Rat: Method for Quantifying the Area of Photoreceptor Sparing.</a> Journal of Ocular Pharmacology and Therapeutics. 32 (5), 304-309 (2016).</li><li>Westenskow, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+Subretinal+Injections+in+Rodents+to+Deliver+Retinal+Pigment+Epithelium+Cells+in+Suspension.">Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension.</a> J Vis Exp. (95), e52247 (2015).</li><li>Lopez, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplanted+Retinal+Pigment+Epithelium+Modifies+the+Retinal+Degeneration+in+the+RCS+Rat.">Transplanted Retinal Pigment Epithelium Modifies the Retinal Degeneration in the RCS Rat.</a> Invest Ophthalmol Vis Sci. 30 (3), 586-588 (1989).</li><li>Eberle, D., Santos-Ferreira, T., Grahl, S., Ader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+Transplantation+of+MACS+Purified+Photoreceptor+Precursor+Cells+into+the+Adult+Mouse+Retina.">Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina.</a> J Vis Exp. (84), e50932 (2014).</li><li>Nair, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Common+Anesthetics+on+Eye+Movement+and+Electroretinogram.">Effects of Common Anesthetics on Eye Movement and Electroretinogram.</a> Doc Ophthalmol. 122 (3), 163-176 (2011).</li><li>McGill, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+central+nervous+system+stem+cells+-+neuroprotection+in+retinal+degeneration.">Transplantation of human central nervous system stem cells - neuroprotection in retinal degeneration.</a> Eur J Neurosci. 35, 468-477 (2012).</li><li>Al-Hussaini, H., Kam, J. H., Vugler, A., Semo, M., Jeffery, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mature+retinal+pigment+epithelium+cells+are+retained+in+the+cell+cycle+and+proliferate+in+vivo.">Mature retinal pigment epithelium cells are retained in the cell cycle and proliferate in vivo.</a> Mol Vis. 14, 1784-1791 (2008).</li><li>Wang, S., Lu, B., Wood, P., Lund, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafting+of+ARPE-19+and+Schwann+Cells+to+the+Subretinal+Space+in+RCS+Rats.">Grafting of ARPE-19 and Schwann Cells to the Subretinal Space in RCS Rats.</a> Invest Ophthalmol Vis Sci. 46 (7), 2552-2560 (2005).</li><li>Fabian, R. J., Bond, J. M., Drobeck, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+corneal+opacities+in+the+rat.">Induced corneal opacities in the rat.</a> Br J Ophthalmol. 51 (2), 124-129 (1967).</li></ol>

The subretinal injection technique depicted in this article is via the trans-scleral pathway, where the injector needle penetrates the outer layers (sclera-choroid-RPE complex) of the eye wall without harming the neural retina or disturbing the vitreous cavity. An alternative trans-vitreal approach has a potential risk of lens damage leading to cataract, since rodents&#39; lens occupies the majority of the vitreous cavity. Compared to this method, our technique is less risky and causes minimal trauma as the injector need...

The human retina located at the back of the eye functions as a light sensory tissue and plays a critical role in vision perception. Retinal cell dysfunction or cell death therefore causes vision problems or permanent blindness. Disorders involving degeneration or dysfunction of cells in different layers of the retina are known as degenerative retinal diseases, among which AMD is the most common type and the leading cause of irreversible blindness in the elderly in developed countries1,2. The pathological process of AMD is associated with &#34;drusen&#34; accumulation between the RPE layer and the underlying Bruch&#39;s membrane, which in turn impairs RPE support of photoreceptor physiology, leading to neural retinal atrophy and vision loss3,4,5. Thus far, there is no cure for advanced dry (non-neovascular) AMD. The emergence of stem cell therapy as a new paradigm in regenerative medicine brings the hope of replacing the dysfunctional or dead RPE cells with stem cell-derived healthy cells. Indeed, extensive preclinical studies of transplanting stem cells (e.g., human embryonic stem cell)-derived RPE cells into RPE-degenerative animal models have been performed6,7, some of which have progressed to clinical trials8,9 (NCT01344993, ClinicalTrials.gov). Recently, an alternative source of stem cells resident in the human RPE layer, the human RPE stem cells (hRPESCs), was identified by our lab and is currently being used in preclinical studies of hRPESC derived-RPE cell (hRPESC-RPE) transplantation therapy for AMD10,11,12,13.
The subretinal injection technique is applied in the preclinical studies mentioned above by multiple groups, including our group. There are two general approaches for subretinal injection in animals: trans-vitreal and trans-scleral. The trans-vitreal approach has the advantage of the surgeon being able to directly observe the needle end as it penetrates the anterior eye, crosses the entire vitreal cavity adjacent to the lens, and penetrates the retina at the back to the eye to reach the subretinal space14,15,16. However, it requires disrupting the retina in two locations (anterior and posterior), carries the risk of damaging the lens, and can result in backflow of cells into the vitreous when the needle is retracted. In contrast, the trans-sclera approach, in principle, avoids involvement of the retina and vitreous, and backflow exits the eye. In pigmented rodents, the surgeon can initially observe penetration of the sclera, but after passage into the pigmented choroid, the needle end is no longer visible. Without direct observation, breaching the retina is common and can result in retinal dissection and delivery of cells and/or blood into the vitreous. Moreover, because the eye surface is curved, it is very difficult to know which needle angles and depths are most effective for trans-scleral injections.
In this visualized article, we introduce a trans-scleral subretinal injection method informed by the use of post-surgical evaluations with Optical Coherence Tomography (OCT), which allows a detailed examination of the injection site. Our trans-scleral injection technique utilizes defined locations, angles, and depths for injection needles to produce very low surgical trauma and high reliability. Here, we specifically demonstrate the injection of hRPESC-RPE cells into the subretinal space of the RCS rat, a pre-clinical model of human AMD. With this injection method, we successfully and consistently delivered hRPESC-RPE cells into the subretinal space of RCS rat eyes with a very high success rate. Injection of cells was previously found to result in preservation of RCS photoreceptors at least 2 months after injection13. This procedure is performed under the dissecting microscope and is easy to learn. It requires two people (a surgeon and an assistant) to perform the injection and the average time of injection for each animal is less than 5 minutes. The defined angles and depths for injection needles make it possible for laboratories, where OCT is unavailable, to achieve successful subretinal injection. It allows for highly reproducible subretinal access and can be used not only for cell transplantation, but also for drug delivery and gene therapies.

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">0.25% Trypsin-EDTA (1x)</td>
			<td style="width:256px;">Life Technologies</td>
			<td style="width:119px;">25200-072</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">DNAse I</td>
			<td style="width:256px;">Sigma</td>
			<td style="width:119px;">DN-25</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">1xDulbecco&#8217;s Phosphate Buffered Saline without Calcium &#38; Magnesium (1xDPBS-CMF)</td>
			<td style="width:256px;">Corning Cellgro</td>
			<td style="width:119px;">431219</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile Balanced Salt Solution (BSS)</td>
			<td style="width:256px;">Alcon</td>
			<td style="width:119px;">00065079550</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile eye wash</td>
			<td style="width:256px;">Moore Medical</td>
			<td style="width:119px;">75519</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile 0.9% saline</td>
			<td style="width:256px;">Hospira</td>
			<td style="width:119px;">488810</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Proparacaine Hydrochloride Ophthalmic Solution (0.5%)</td>
			<td style="width:256px;">Akorn</td>
			<td style="width:119px;">17478026312</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Tropicamide Ophthalmic Solution, USP (1%)</td>
			<td style="width:256px;">Bausch &#38; Lomb</td>
			<td style="width:119px;">24208058559</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Phenylepherine Ophtalmic Solution, USP (10%) stock</td>
			<td style="width:256px;">Bausch &#38; Lomb</td>
			<td style="width:119px;">42702010305</td>
			<td style="width:231px;">This is used to make 2.5% Phenylepherine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Buprenex</td>
			<td style="width:256px;">Patterson</td>
			<td style="width:119px;">433502</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Dexamethasone</td>
			<td style="width:256px;">APP Pharmaceuticals</td>
			<td style="width:119px;">63323051610</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">100% Ethanol</td>
			<td style="width:256px;">Thermo Scientific</td>
			<td style="width:119px;">615090040</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">70% Ethanol</td>
			<td style="width:256px;">Ricca Chemical Company</td>
			<td style="width:119px;">2546.70-5</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:259px;">Sterile GenTeal Lubricant Eye Gel</td>
			<td style="width:256px;">Novartis</td>
			<td style="width:119px;">78042947</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Sterile Systane Ultra Lubricant Eye Drops</td>
			<td style="width:256px;">Alcon</td>
			<td style="width:119px;">00065143105</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:259px;">hRPESC-RPE cells</td>
			<td style="width:256px;">Not available commercially</td>
			<td style="width:119px;"></td>
			<td style="width:231px;">Please refer to &#34;Reference #12&#34; for cell isolation and mainteinance.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">24-well plates</td>
			<td style="width:256px;">Corning</td>
			<td style="width:119px;">3526</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Conical tubes (15 ml)</td>
			<td style="width:256px;">Sarstedt</td>
			<td style="width:119px;">62554002</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Microcentrifuge cap with o-ring</td>
			<td style="width:256px;">LPS inc</td>
			<td style="width:119px;">L233126</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Capless Microcentrifuge tubes (1.7 ml)</td>
			<td style="width:256px;">LPS inc</td>
			<td style="width:119px;">L233041</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Centrifuge</td>
			<td style="width:256px;">Eppendorf</td>
			<td style="width:119px;">5804R</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile alcohol wipe</td>
			<td style="width:256px;">McKesson</td>
			<td style="width:119px;">58-204</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile cotton tip applicators</td>
			<td style="width:256px;">McKesson</td>
			<td style="width:119px;">24-106-2S</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile Weck-Cel spears</td>
			<td style="width:256px;">Beaver-Visitec International&#160;</td>
			<td style="width:119px;">0008680</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile surgical drapes&#160;</td>
			<td style="width:256px;">McKesson</td>
			<td style="width:119px;">25-515</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Gauze</td>
			<td style="width:256px;">McKesson</td>
			<td style="width:119px;">16-4242</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Nanofil syringe (10 ul)</td>
			<td style="width:256px;">World Precision Instruments</td>
			<td style="width:119px;">Nanofil</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Nanofil beveled 33-gauge needle</td>
			<td style="width:256px;">World Precision Instruments</td>
			<td style="width:119px;">NF33BV-2</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Insulin syringe needles 31-gauge</td>
			<td style="width:256px;">Becton Dickinson</td>
			<td style="width:119px;">328418</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Rat toothed forceps</td>
			<td style="width:256px;">World Precision Instruments</td>
			<td style="width:119px;">555041FT</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:259px;">Vannas Micro Dissecting Spring Scissors</td>
			<td style="width:256px;">Roboz</td>
			<td style="width:119px;">RS-5602</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Circulating water T pump&#160;</td>
			<td style="width:256px;">Stryker</td>
			<td style="width:119px;">TP700</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Heating pad</td>
			<td style="width:256px;">Kent Scientific</td>
			<td style="width:119px;">TPZ-814</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Animal anesthesia system</td>
			<td style="width:256px;">World Precision Instruments</td>
			<td style="width:119px;">EZ-7000</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Balance</td>
			<td style="width:256px;">Ohaus</td>
			<td style="width:119px;">PA1502</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Stereo microscope</td>
			<td style="width:256px;">Zeiss</td>
			<td style="width:119px;">Stemi 2000</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Microscope light source</td>
			<td style="width:256px;">Schott</td>
			<td style="width:119px;">ACE series</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:259px;">Bioptigen Envisu Spectral Domain Ophthalmic Imaging System</td>
			<td style="width:256px;">Bioptigen</td>
			<td style="width:119px;">R2210</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sterile black marker pen</td>
			<td style="width:256px;">Viscot Industries</td>
			<td style="width:119px;">1416S-100</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Miniature measuring scale</td>
			<td style="width:256px;">Ted Pella Inc</td>
			<td style="width:119px;">13623</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:259px;">Infrared Basking Spot Lamp&#160;</td>
			<td style="width:256px;">EXO-TERRA</td>
			<td style="width:119px;">PT2144</td>
			<td style="width:231px;">This is used as a heating lamp for animals during the post-surgical recovery&#160; phase</td>
		</tr>
	</tbody>
</table>

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

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

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

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

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

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

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

Research

JoVE Journal

Neuroscience

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

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

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

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

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

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

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 ...

Enrichment of Bruch's Membrane from Human Donor Eyes

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

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

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

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

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

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

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

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

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