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.

注：所有动物均按照由斯克里普斯研究所成立了道德准则处理。 1.准备材料，为注射（约20分钟） <ol style="margin-left: 40px;"><li>预暖细胞解离溶液（优选一个是通过稀释灭活，不与血清），无菌PBS，并培养介质（ 表1）。 </li><li>通过拆卸和它煮沸份在水中15分钟消毒用钝针头注射器。 </li></ol> 2.制备的视网膜色素上皮细胞的注射（〜30分钟至1小时） <ol style="margin-left: 40px;"><li>分离用5-8分钟预温的细胞解离溶液的视网膜色素上皮细胞在37℃。 </li><li>刮细胞轻轻地释放任何仍附加。 </li><li>稀释细胞具有大体积的培养基（补满15毫升管）以失活的离解溶液和计数。 </li><li>离心在800×g离心5分钟以沉淀细胞。 </li><li>重悬的细胞以200,000个细胞/微升（提供100,000个细胞在0.5微升体积）在无菌的预温热的PBS，并将它们转移到一个1.5ml微量管中。 </li><li>任选地，添加活细胞瞬态荧光标记物，并培育在37℃下30-45分钟。 </li><li>装入注射器的钝针用0.5微升细胞。尽快注入细胞。 </li></ol> 3.子视网膜注射（〜每次注射5分钟） 注意：如果可能的话，学习技术有成年白鼠自角膜缘船只更容易形象化。当学习更容易促进注射部位的可视化（试图注入细胞前）注入坚牢绿溶液。 <ol style="margin-left: 40px;"><li>麻醉的啮齿动物。使用100mg / ml的氯胺酮和10mg / ml的甲苯噻嗪（20微升/ 10克的机身瓦特腹膜内注射8）以上异氟烷吸入由于难以操纵的啮齿动物，并注入到眼睛中与在吸入器的炉鼻。 <ol><li>确保动物深受捏它的爪子1麻醉。如果退缩，等待几个分钟，开始视网膜下注射前再试一次。 </li></ol></li><li>啮齿动物的位置倒向一侧，这样，将被注入的眼睛对着天花板。 </li><li>在解剖显微镜下轻轻舒展皮肤，使眼部弹出小幅上涨了插座（临时突眼），并通过举办它的头用两个手指正上方的耳朵，它的下巴，轻轻地舒展平行于眼睑皮肤变得更容易让眼睛弹出小幅上涨从插槽中（参见图1C）。不掌握啮齿动物太近喉咙。 </li><li>用30升G一次性预消毒针头，立即使角膜缘下方的孔（如果船只被击中，流血会BË显著和它是以后难以找到该孔），并以一定的角度，以避免与所述针（ 图1D）接触透镜。避免接触与锐（或钝）针或立即白内障的形成将发生镜头。 注意：注射工作两个人好。这样一个人就可以通过注射器的针头钝他们所创建的第一个孔后，用锋利的一次性针保持专注于那里的孔进行注射的人。 </li><li>从眼睛缩回一次性尖锐的针，同时保持在头部的把手。记得确切位置的孔。 </li><li>无论是后安装的预装注射器上的显微钝针或者手握住它，将通过孔的钝针注射器的尖端，再小心不要接触到镜头，轻轻地通过眼睛推轻轻地，直到感觉阻力（ 图1D）。 </li><li> ķeeping所有运动到最低限度，精心注入RPE细胞慢慢进入视网膜下腔。 注：RPE /视网膜脱离会引起;这是不可避免的。然而，清洁器喷射最小的分离，极大地提高了再附着（ 图1E）的机会。任何夸张的动作可能将针回视网膜和侧身的动作可以损伤视网膜。使用注射泵是可选的，但允许非常精确的递送。 </li><li>收回注射器缓慢。敷眼滴保湿，保持眼部水分。 </li><li>继续监测动物，直到它重新获得胸骨斜卧。不要让动物无人值守或返回到与其他动物的警戒笼子里，直到它重新获得胸骨斜卧。 </li></ol>

None of the authors have any commercial disclosures to declare.

在这篇文章中，我们描述了在大鼠和小鼠进行RPE细胞的视网膜下注射悬浮一个比较简单的方法。该协议是简单易学和更多的经验与技术将在较少创伤平移（ 图3;这代表了更好注射1），特别是如果一个微操作时（ 图1A）。任何创伤可以在体内与CSLO和OCT系统（ 图2），如果可用来监测。如果更高的分辨率和更低噪声图像被需要，是本领域的成像平台的状态...

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

光转换成电脉冲发生在外层视网膜和在很大程度上是由杆和视锥感光细胞和视网膜色素上皮细胞（RPE）细胞中来实现的。 RPE提供感光细胞和死亡或视网膜色素上皮细胞的功能障碍是年龄相关性黄斑变性（AMD），永久性视力丧失的人55岁及以上的主要原因特性关键支持。而没有治疗AMD的已被确定的，健康的RPE在患病眼中植入可能被证明是一种有效的治疗，以及大量的RPE细胞可以从多能干细胞可以容易地产生。几个关于RPE细胞递送的安全性和有效性有趣的问题仍然可以检查在动物模型中，与用于注入的RPE广为接受的协议已被开发出来。此处所描述的技术中的各种研究已经使用由多个组，并且包括首先创建在眼的孔用锋利的针头。然后用注射器BLUnt的针装有细胞通过孔插入并通过玻璃体，直到它轻轻地接触到视网膜色素上皮。采用这种注射方法，该方法相对简单，并且需要最小的设备，我们实现干细胞衍生的视网膜色素上皮细胞的一致和有效的整合在宿主的RPE防止在动物模型显著量光感受器变性的之间。而实际的协议不一部分，我们还描述了如何确定诱发的注射，以及如何验证的细胞注射入使用体内成像方式视网膜下腔的创伤的程度。最后，使用该协议的应用不限于视网膜色素上皮细胞;它可以用于任何化合物或细胞进入视网膜下的空间注入。

我们可以提供RPE细胞的悬浮液进入啮齿动物的视网膜下腔迅速，稳定地使用本手稿中描述的技术。虽然不是必需的，创伤可以大大使用具有图1A和B中的显微所示的设置最小化。持啮齿类如图1C临时突眼。的步骤是相同的​​，如果与微操作或由手执行;这些描绘在图1D卡通。当从标记为视网膜色素上皮细胞中进行干净荧光可以利用CSLO和诱导视网膜脱离可使用OCT?...

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.

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

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

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JoVE Journal

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17.1K 次观看.  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.