Name: performing subretinal injections in rodents to deliver retinal pigment epithelium cells in suspension
Uploaded: 2015-01-23T13:00:00.000+00:00
Duration: 6 min 4 s
Description: Watch this Scientific Journal Video about Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension at JoVE.com

We wish to thank Alison Dorsey for helping to develop the subretinal injection technique. We also acknowledge the National Eye Institute (NEI grants EY11254 and EY021416), California Institute for Regenerative Medicine (CIRM grant TR1-01219), and the Lowy Medical Research Institute (LMRI) for very generous funding for this project.

NOT: Tüm hayvanlar Scripps Araştırma Enstitüsü tarafından kurulan etik kurallara uygun olarak tedavi edildi. Enjeksiyon için Materyallerin hazırlanması 1. (~ 20 dk) <ol style="margin-left: 40px;"><li> Ön sıcak hücre ayrışma çözeltisi (tercihen serumu ile seyreltme yoluyla durdurulmuş olduğu bir), steril PBS ve kültür ortamı (Tablo 1). </li><li> Demontaj ve 15 dakika için su içinde parça kaynatılmasıyla bir kör iğne ile şırınga sterilize edin. </li></ol> Enjeksiyon için RPE Hücreler 2. Hazırlık (30 dakika saat 1) <ol style="margin-left: 40px;"><li> 37 ° C&#39;de 5-8 dakika boyunca ön ısıtılmış hücre ayrışma çözeltisi kullanılarak RPE hücreleri ayırmak. </li><li> Hala bağlı olduğu herhangi bir serbest bırakmak için hafifçe kazıyın hücreleri. </li><li> Kültür ortamı büyük bir hacmi (15 ml tüp doldurmak) ayrışma çözümü inaktive ve onları saymak hücreleri seyreltin. </li><li> Pelet hücreleri 5 dakika boyunca 800 x g&#39;de santrifüjleyin. </li><li> Steril, önceden ısıtılmış PBS / ml 200,000 hücreleri (0.5 ul hacim içinde 100,000 hücre sağlamak için) hücreleri yeniden süspanse edin ve bir 1.5 ml mikrosantrifüj tüpü içine transfer edin. </li><li> İsteğe bağlı olarak, bir canlı hücre geçici floresan markörü ekleyebilir ve 30-45 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Hücrelerden 0.5 ul, kör bir iğne şırınga yerleştirin. Kısa sürede hücreleri enjekte edilir. </li></ol> 3. Alt-retinal Enjeksiyon (~ Enjeksiyon başına 5 dakika) NOT: Mümkünse Limbustan damarları görselleştirmek için çok daha kolay olduğundan, erişkin albino sıçanlar ile tekniğini öğrenmek. Daha kolay enjeksiyon yerinde görülmesini kolaylaştırmak için (hücreleri enjekte denemeden önce) öğrenme sırasında Hızlı Yeşil çözüm enjekte edilir. <ol style="margin-left: 40px;"><li> Kemirgen anestezisi. 100 mg / ml ketamin ve 10 mg / ml ksilazin (20 ul / 10 g vücut ağırlık intraperitoneal enjeksiyon kullanınizofluoran inhalasyon üzerinde sekiz) bu kemirgen manevra ve inhaler içinde burnu ile göz içine enjekte etmek zor olduğundan. <ol><li> Hayvan derinden pençeleri birini kısma anestezi emin olun. O geri çekildiğinde ise, birkaç dakika daha bekleyin ve subretinal enjeksiyon başlamadan önce tekrar deneyin. </li></ol></li><li> Enjekte edilecek göz tavan bakacak şekilde onun yüzüne kemirgen yerleştirin. </li><li> Göz soketi (geçici propitozisin) biraz dışarı açılır ve sadece kulak üstünde ve çene ile ve yavaşça göz kapaklarının deri paralel germek iki parmak ile başını tutarak daha erişilebilir hale böylece mikroskop altında cildi nazikçe germek çok göz hafifçe soketten açılır ki (Şekil 1C bakınız). Boğaz çok yakın kemirgen tutmayın. </li><li> 30 G keskin tek ön-sterilize iğne ile, gemiler isabet eğer, kanama olacak b (hemen limbusun altında bir delik yapmakönemli e ve daha sonra delik bulmak zor olabilir) ve bir açıyla iğne (Şekil 1D) ile lensi dokunmamak için. Meydana gelecek keskin (ya da künt) iğne veya acil katarakt oluşumu ile lensi dokunmaktan kaçının. NOT: Enjeksiyon iki kişi ile daha iyi çalışır. Bu şekilde bir kişi onlar delik olduğu odaklanmak korumak için keskin tek iğne ile ilk delik oluşturduktan sonra enjeksiyon yapan kişiye künt iğne ile şırınga iletebilirsiniz. </li><li> Kafasına kavrama korurken göz atılabilir keskin iğne geri çekin. Delik tam olarak nerede unutmayın. </li><li> Ya bir micromanipulator bir künt bir iğne ile ön-yüklü şırınga montaj veya elle tutarak, lensi dokunma ve hafifçe göz aracılığıyla itmek için değil yine dikkat çekici, delikten künt iğne ile şırınga ucu ekledikten sonra çok yavaşça duygu direnci (Şekil 1D) kadar. </li><li> Kminimumda tüm hareketleri eeping, dikkatle yavaş yavaş subretinal boşluğa RPE hücreleri enjekte. NOT: RPE / retinanın ayrılması bağlı olacak; Bu kaçınılmazdır. Bununla birlikte, daha temiz bir enjeksiyon dekolmanı en aza indirir ve büyük ölçüde yeniden takılması (Şekil 1E) şansını arttırır. Herhangi abartılı hareketler retina geri iğneyi hareket edebilir, ve yanlara hareketler retinaya zarar edebilirsiniz. bir enjeksiyon pompasının kullanılması isteğe bağlıdır fakat çok hassas bir teslimat sağlar. </li><li> Yavaş yavaş şırınga geri çekin. Göz nemlendirici uygulayın hidrate göz tutmak düşer. </li><li> Sternal yatma kavuşur kadar hayvan izlemeye devam edin. Gözetimsiz bırakmayın hayvan veya sternum yatma kavuşur kadar diğer uyarı hayvanlarla bir kafes iade etmeyin. </li></ol>

None of the authors have any commercial disclosures to declare.

Bu yazıda sıçanlarda ve farelerde süspansiyon RPE hücrelerinin subretinal enjeksiyonları gerçekleştirmek için nispeten basit bir yöntem tarif. protokol az travmalarda tercüme edecek tekniği ile öğrenmesi kolay ve daha deneyim (Şekil 3, bu iyi enjeksiyon birini temsil) bir mikromanipülatör (Şekil 1A) kullanıldığında, özellikle. Herhangi bir travma, bir cSLO ve Ekim sistemi (Şekil 2) varsa in vivo izlenebilir. Yüksek çözünürlük ve da...

The sensory retina is organized in functional tiers of neurons, glia, and endothelial cells. Photoreceptors at the back of the retina are activated by light; through phototransduction they convert photons into electrical signals that are refined by interneurons and transmitted to the visual cortex in the brain. Phototransduction cannot occur without the coordinated efforts of Mueller glia and retinal pigment epithelium (RPE) cells. RPE are organized in a monolayer directly behind the photoreceptors and perform multiple and diverse functions integral to photoreceptor function and homeostasis. In fact, RPE and photoreceptors are so co-dependent that they are considered to be one functional unit. Death or dysfunction of RPE results in devastating secondary effects on photoreceptors and is associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly1,2.
While no cure has been discovered for AMD, several clinical studies have shown that RPE cell replacement may be a promising therapeutic option3-13. With the advent of stem cell technology, it is now possible to generate large numbers of RPE cells in vitro from embryonic and induced pluripotent stem cells (hES and hiPS) that strongly resemble their somatic counterparts functionally and anatomically14-26. Stem cell-derived RPE have also been shown to function in vivo by multiple independent groups, including our own, to significantly slow retinal degeneration in rat and mouse lines with spontaneous retinal degeneration16,18,21,22,25,28,29. This combination of clinical and preclinical supporting evidence is so compelling that several clinical trials to prevent retinal degeneration using stem cell-derived RPE cells are now ongoing30,31. 
RPE can be readily derived from hES and/or hiPS and implanted in the subretinal space of rodents using various derivation and injection techniques32,33. (See Westenskow et al. for a methods paper in multimedia format demonstrating the directed differentiation protocol we employ)34. There are critical remaining questions regarding the safety, survival, and functional capacity of exogenously delivered RPE cells upon implantation, therefore the ability to perform subretinal injections in rodents is a critical skill16,18,21,29,36,37. The delivery of RPE is not trivial, and the field is divided on the most effective injection technique. The protocol we describe here is a simple and effective way to deliver of bolus of RPE cells subretinally, and was used in the first clinical trial for stem cell-derived RPE transplantation31. (The reader may also refer to another JoVE article by Eberle et al. for an alternative depiction of subretinal injections in rodents.38) 
The technique outlined in this manuscript cannot be visualized and trauma is unavoidable (as with any subretinal injection technique). It is performed by making a hole just under the limbus vessels and inserting a blunt needle along a transscleral route to inject a bolus of cells under the diametrically opposed retina. The person doing the injection will feel resistance as the blunt needle touches the retina. The cells may be directly visualized after the injection, however, and the degree of the induced retinal detachment can be determined by labeling the RPE cells with a transient fluorescent marker and detecting them with a confocal scanning ophthalmoscope (cSLO). An optical coherence tomography (OCT) system can also be used to monitor the trauma and easily identify the injection site.

<table><tbody><tr><td>2-Mercaptoethanol (55 mM)</td><td>Gibco&amp;nbsp;</td><td>21985-023</td><td>50 ml x 1&amp;nbsp;</td></tr><tr><td>Cell Scapers</td><td>VWR</td><td>89260-222</td><td>Case x 1</td></tr><tr><td>CellTracker Green CMFDA</td><td>Molecular Probes</td><td>C34552</td><td>50 &amp;micro;g x 20</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190-144</td><td>500 ml x 1&amp;nbsp;</td></tr><tr><td>Fast Green</td><td>Sigma-Aldrich</td><td>F7258</td><td>25 g x 1&amp;nbsp;</td></tr><tr><td>Genteal Geldrops Moderate to Severe Lubricant Eye Drops&amp;nbsp;</td><td>Walmart</td><td>4060941</td><td>25 ml x 1</td></tr><tr><td>Hamilton Model 62 RN SYR</td><td>Hamilton</td><td>87942</td><td>Syringe x 1&amp;nbsp;</td></tr><tr><td>Hamilton Needle 33 G, 0.5&quot;, point 3 (304 stainless steel)</td><td>Hamilton</td><td>7803-05</td><td>Needles x 6</td></tr><tr><td>Knockout DMEM</td><td>Gibco</td><td>10829-018</td><td>500 ml x 1&amp;nbsp;</td></tr><tr><td>KnockOut Serum Replacement</td><td>Gibco</td><td>10828-028</td><td>500 ml x 1&amp;nbsp;</td></tr><tr><td>L-Glutamine 200 mM</td><td>Gibco</td><td>25030-081</td><td>100 ml x 1</td></tr><tr><td>Magnetic Stand</td><td>Leica Biosystems</td><td>39430216</td><td>Stand x 1</td></tr><tr><td>MEM Non-Essential Amino Acids Solution 100X&amp;nbsp;</td><td>Gibco</td><td>11140-050</td><td>100 ml x 1</td></tr><tr><td>Micromanipulator</td><td>Leica Biosystems</td><td>3943001</td><td>Manipulator x 1</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Gibco</td><td>15140-122</td><td>100 ml x 1</td></tr><tr><td>Slip Tip Syringes without Needles BD&amp;nbsp; (3 ml)&amp;nbsp;&amp;nbsp;</td><td>VWR</td><td>BD309656</td><td>Pack x 1</td></tr><tr><td>Specialty-Use Needles BD&amp;nbsp; (30 G, 1&quot;)</td><td>VWR</td><td>BD305128</td><td>Box x 1</td></tr><tr><td>TrypLE Express Enzyme (1X), no phenol red</td><td>Gibco</td><td>12604013</td><td>100 ml x 1</td></tr></tbody></table>

performing subretinal injections in rodents to deliver retinal pigment epithelium cells in suspension

elektriksel uyarıların içine ışık dönüşümü dış retina oluşur ve çubuk ve koni fotoreseptör ve retina pigment epiteli (RPE) hücreleri tarafından büyük ölçüde gerçekleştirilir. RPE fotoreseptör ve ölüm ya da RPE hücrelerinin disfonksiyonu yaşa bağlı makula dejenerasyonu (YBMD), insanlar yaş 55 ve üzeri kalıcı görme kaybının önde gelen nedeni karakteristik için kritik destek sağlamaktadır. AMD için bir tedavi tespit edilmiş olmakla birlikte, hasta göze sağlıklı RPE implantasyonu etkili bir tedavi olduğu ortaya çıkabilir ve RPE hücreleri, çok sayıda hali hazırda, pluripotent kök hücreleri elde edilebilir. RPE hücre teslim ve güvenliğine ilişkin birçok ilginç sorular hala hayvan modellerinde incelenebilir ve RPE enjekte için kullanılan kabul görmüş protokoller geliştirilmiştir. Burada açıklanan teknik çeşitli çalışmalarda çoklu gruplar tarafından kullanılan ve ilk keskin iğne ile göz delik oluşturarak içerir. Bir blu ile daha sonra bir şırıngaHücre yüklü nt iğne deliğinden sokulur ve hafifçe RPE temas edene kadar vitröz geçirilir. Nispeten basittir ve çok az ekipman gerektirmektedir, bu enjeksiyon yöntemi kullanarak, hayvan modellerinde fotoreseptör dejenerasyonu önemli miktarda önleyen ana RPE arasında kök hücre türetilmiş RPE hücreleri, tutarlı ve etkili bir entegrasyon sağlamak. Gerçek protokolünün bir parçası değildir, biz de enjeksiyon, ve nasıl hücreler in vivo görüntüleme yöntemleri kullanarak subretinal boşluğa enjekte edildi doğrulamak için neden olduğu travmanın boyutlarını belirlemek için nasıl açıklar. Son olarak, bu protokolün kullanılması RPE hücreleri ile sınırlı değildir; Bu retina altına doğru herhangi bir bileşik ya da bir hücre enjekte etmek için kullanılabilmektedir.

Biz bu yazıda anlatılan tekniği kullanarak hızlı ve tutarlı kemirgenler subretinal boşluğa RPE hücrelerinin bir süspansiyon sunabilirsiniz. Gerekli olmamakla beraber, travmalar büyük ölçüde Şekil 1A ve B, bir mikromanipülatör ile gösterilen düzeneği kullanılarak en aza indirilebilir. Geçici propitozlar Şekil 1C&#39;de gösterildiği gibi, kemirgen tutun. mikromanipülatör veya elle yapıldığı takdirde adımlar aynıdır; Bu Şekil 1D karikat?...

Watch this Scientific Journal Video about Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension at JoVE.com

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

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.

Süspansiyon Retina pigment epiteli Hücreleri sunun Kemirgenler Subretinal Enjeksiyonları Sahne

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

performing-subretinal-injections-rodents-to-deliver-retinal-pigment

Research

JoVE Journal

Medicine

17.3K Görüntüleme.  The Scripps Research Institute. 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.

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

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.

Fare Göz içine Gen Terapisi Vektörler ve Kök Hücre Subretinal Enjeksiyon

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 ...

Tıp

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.

Fareler Retina Pigment Epitel Gen Tedavisi için Viral Vektörler Limbal Yaklaşım-Subretinal Enjeksiyon

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 ...

Nörobilim

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Subretinal Space erişim için bir alternatif ve Onaylı Enjeksiyon Yöntemi<i&gt; yoluyla</i&gt; Bir transkleral Posterior Yaklaşım

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

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.

Sıçan gözlerinin içine insan retina Pigment epitel hücrelerinin Trans-scleral Subretinal nakli için rafine bir protokol geliştirilmesi

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

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

Ultrahigh çözünürlük fare optik koherens tomografi göz içi enjeksiyon Retina gen terapisi araştırma yardımcı olmak için

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

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

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

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

Minipiglerde RPE'nin taşıyıcı üzerine subretinal implantasyonu: Preoperatif preparatlar, cerrahi teknikler ve postoperatif bakım için kılavuzlar

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

Biyomühendislik

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.

Retinal Dejenerasyon Fare Modelinde Subretinal Greftlerin Transpupiller Kılavuzluğunda Transskleral Transplantasyonu

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

Development of a Surgical Technique for Subretinal Implants in Rats

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

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

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

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

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

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

Büyük gözlü manken coğrafi Atrofi, içine insan embriyonik kök hücre elde edilen retina Pigment epitel hücrelerinin subretinal nakli

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

Biyoloji

Canlandırıcı Görme: Retina Dejenerasyonu için Kök Hücre Tedavisi

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.

Yazar Spotlight: Görme Restorasyonunu İlerletmek - Retina Hastalıkları için Kök Hücre Bazlı Tedavi 

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.

Rd10 Farelerde İnsan Embriyonik Kök Hücre Kaynaklı Fotoreseptör Progenitörlerinin Sub-Retinal Verilmesi

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