Name: performing subretinal injections in rodents to deliver retinal pigment epithelium cells in suspension
Uploaded: 2015-01-23T13:00:00
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....

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.

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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

Süspansiyon Retina pigment epiteli Hücreleri sunun Kemirgenler Subretinal Enjeksiyonları Sahne

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 ...

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