视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

Summary

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.

Abstract

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。

Introduction

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 elderly^1,2.

While no cure has been discovered for AMD, several clinical studies have shown that RPE cell replacement may be a promising therapeutic option^3-13. 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 anatomically^14-26. 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 degeneration^{16,18,21,22,25,28,29}. 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 ongoing^30,31.

RPE can be readily derived from hES and/or hiPS and implanted in the subretinal space of rodents using various derivation and injection techniques^32,33. (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 skill^{16,18,21,29,36,37}. 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.

Protocol

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） 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）。 通过拆卸和它煮沸份在水中15分钟消毒用钝针头注射器。 2.制备的视网膜色素上皮细胞的注射（〜30分钟至1小时）…

Representative Results

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?…

Discussion

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…

Materials

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