我们希望感谢帕蒂吉莱德曼在手术和苏珊. 波顿为 RPE 细胞准备的帮助。我们也承认 NYSTEM C028504 为这个项目提供资金。贾斯汀· d ·米勒得到 NIH 授予 F32EY025931 的支持。

所有涉及动物的程序都得到了纽约州立大学奥尔巴尼分校动物保育和使用委员会 (IACUC) 的批准。
1. 预注射制剂
<ol><li>
hRPESC 视网膜色素上皮细胞悬浮液的制备 注意: 所有以下步骤都在无菌组织培养罩中执行, 并且需要熟悉基本的无菌技术。<ol>
<li>从50-90 岁的人眼中分离出主要的 hRPE 细胞, 并在24井板中培养细胞12。冷冻的细胞在通道 1, 解冻的需要和文化通道 2 (P2) 细胞4-5 周 (图 1A) 注射。</li>
		<li>移除培养基12 , 用500µL 5ml 1X Dulbecco 磷酸盐缓冲盐水, 不含钙 & 镁 (1x DPBS-CMF), 用 1000 DPBS 吸管将 1X CMF µL 加入井中, 然后用真空吸尘器将其取出。</li>
		<li>每个井加300µL 胰蛋白酶/dnasei。孵育 hRPESC-RPE 细胞在胰蛋白酶/dnasei (4 kU dnasei 每1毫升0.25% 胰蛋白酶-EDTA) 4 分钟, 在37° c 离解细胞。</li>
		<li>检查显微镜下的细胞, 看它们是否已经围捕。继续孵育的细胞在胰蛋白酶/dnasei 的额外2分钟, 如果他们还没有四舍五入。</li>
		<li>一旦细胞被围捕, 使用1000µL 吸管研制分离的细胞从井和转移的胰蛋白酶/dnasei 包含分离细胞成一个15毫升锥形管与同等体积的5ml 培养基, 使胰蛋白酶/dnasei 灭活。</li>
		<li>用 5ml 1X DPBS CMF 的冲洗井, 通过轻轻移上下, 尤其是在井边;然后将这些细胞添加到前锥管中。</li>
		<li>离心锥形管在 286 x g 为 5 min 在4° c, 颗粒细胞。</li>
		<li>用1毫升培养基去除上清和重细胞。</li>
		<li>使用例计数单元格。</li>
		<li>离心机在 286 x g 为5分钟在4° c 的颗粒细胞。</li>
		<li>在5万细胞/µL 的无菌平衡盐溶液 (BSS) 中去除上清和重细胞 (在视网膜注射过程中, 在1µL 体积内提供5万细胞)。</li>
		<li>将最终的细胞悬浮液 (CS) 转化为1.7 毫升的离心管, 并将其放入冰水混合物中, 直至注射使用。</li>
	</ol>
</li>
	<li>
细胞注射器的制备
	<ol>
<li>插入一个无菌的33口径斜角针注入注射器和螺丝紧紧地组装注射器。</li>
		<li>冲洗注射器与100% 乙醇5-6 次。</li>
		<li>冲洗注射器与70% 乙醇5-6 次。</li>
		<li>用 BSS 冲洗注射器5-6 次。</li>
		<li>在解剖显微镜 (图 1B) 下, 用不育的黑色记号笔在600µm 的位置上标记注射器针。</li>
		<li>将注射器放在微上进行注射。</li>
	</ol>
</li>
</ol>2. 视网膜注射液
<ol><li>
手术区和动物制剂
	<ol>
<li>称一个4-5 周大的 RCS 大鼠 (60-100 克), 并麻醉它使用异氟醚蒸汽输送系统。 注意:诱导麻醉使异氟醚的流动率保持在5%。通过按压爪子来确认麻醉深度, 然后在手术过程中将麻醉维持的流速降低到2-3%。</li>
		<li>在解剖显微镜的舞台上放置一个不育的外科悬垂, 上面有一个加热垫, 以建立一个不育的手术区。</li>
		<li>将大鼠转移到手术区, 将大鼠置于与异氟醚系统相连的鼻锥中, 以维持麻醉。</li>
		<li>用纱布盖住老鼠的身体。捏住老鼠的脚趾以确认全身麻醉。</li>
	</ol>
</li>
	<li>
显微镜下反式巩膜视网膜注射液
	<ol>
<li>在老鼠的 unoperated 眼上涂抹一滴眼润滑剂。</li>
		<li>将老鼠的左眼朝向右侧, 将它的头部朝向外科医生的右手, 将它的背部朝向外科医生。</li>
		<li>修剪任何用小剪刀遮住眼睛的胡须。</li>
		<li>从左眼的颞侧滴下少量的眼部洗净, 用棉签将多余的部分收集在鼻侧, 冲洗眼睛。</li>
		<li>扩大瞳孔与1% 卡和2.5% 肾上腺素 (新鲜的10% 肾上腺素, 稀释它在不育0.9% 生理盐水的手术当天) 为一个后 OCT 考试通过应用一滴每一个。</li>
		<li>轻轻地将眼部周围的皮肤拉4-6 次以打开眼睑, 使眼睛稍微 proptosed, 以便更方便地进入角膜缘后部。</li>
		<li>应用一滴眼洗, 保持眼睛湿润。</li>
		<li>轻轻研制 cs (准备在步骤 1.1.12) 和负载的注射器与1.2 µL cs。额外的0.2 µL 用于补偿注入回流。 注意:根据我们的测量, 大约 5,000-8 000 细胞在回流中丢失, 注射了5万细胞/µL, 相当于大约10-16% 的细胞丢失, 另外还注射了0.2 µL CS 以补偿这种细胞损失。</li>
		<li>放置在一个微 (或有一个助理持有它) 垂直的注射器填充, 因为 RPE 细胞往往容易下沉悬浮。</li>
		<li>应用一滴0.5% 丙 (局部外用麻醉剂) 的眼睛和删除过剩的棉花涂抹剂。 注意:此步骤应抑制角膜反射, 防止眼睛在后续步骤中闪烁。</li>
		<li>用镊子夹住角膜缘后部的结膜, 旋转眼睛鼻, 抬起结膜作 "帐篷"。</li>
		<li>用剪刀剪下顶部的 "帐篷", 使一个小开口在结膜和暴露的基础巩膜。</li>
		<li>使用镊子握住角膜缘旁边剩余的结膜边缘的边缘, 并旋转眼睛鼻, 使瞳孔轴的角度相对于桌面 (图 1C) 大约30度。继续抓取的结膜边缘是需要提供一个反在针插入和保持眼睛的最佳角度。</li>
		<li>要做一个试验孔为细胞注射, 位置末端一个无菌的斜面31测量胰岛素针在 1,200-1, 500 µm 后到角膜缘与开头的尖朝向。</li>
		<li>调整胰岛素针的角度, 使它是10-15 度以上的巩膜 (切线到一个假想的平面在预期的注射地点)。慢慢穿透巩膜-脉络膜复合物到针深度约500µm。在色素大鼠, 针端将 ' 消失 ' 下色素脉络膜。在这里使用的胰岛素针的牌子, 从针尖到斜面的距离是500µm。</li>
		<li>小心地取出胰岛素针 (可以看到一个非常小的血液渗出)。</li>
		<li>如果注意到过多的出血, 可以使用眼枪清除孔, 如果需要。在矛应用后持续出血表明一艘船可能已经损坏。</li>
		<li>引导视网膜色素上皮细胞加载注射器针进入先导孔, 与开口朝下, 并在大约10-15 度的角度相对于巩膜的局部表面。</li>
		<li>轻轻地将注射器针插入先导孔, 深度约500µm, 进入视网膜空间。应该有大约100µm 的边缘之间的黑色钢笔标记和点的针是覆盖的有色脉络膜 (图 1C和1D)。</li>
		<li>请助手轻轻压低注射器的柱塞, 注入适当的细胞容积 (约1.2 µL)。准备提供一些反作为助手按下柱塞。 注意:事先与助手的模拟练习可以为外科医生和助手提供必要的经验。</li>
		<li>当视觉上专注于钢笔标记边缘, 按住注射器到位25-30 秒, 然后慢慢收回注射器。通常观察到少量的回流。 注意:如果没有观察到回流, 可能有一个玻璃注射。如果你看到通过密封或在巩膜下填充细胞的回流, 注射太浅。</li>
		<li>冲洗注射部位的细胞外排, 用无菌眼洗3次, 用棉签收集多余的。</li>
		<li>在操作眼睛上涂抹一滴眼部润滑剂, 将大鼠转移到 OCT 站, 检查移植细胞的位置和视网膜疱疹的大小。</li>
	</ol>
</li>
</ol>3. 注射后处理
<ol><li>
抗炎镇痛药治疗
	<ol>
<li>在生理盐水皮下注射0.1 毫克/千克体重的丁丙诺啡以减少疼痛。</li>
		<li>通过腹腔注射 (ip) 在生理盐水中注射地塞米松1.6 毫克/千克的体重以控制炎症。</li>
	</ol>
</li>
	<li>
动物康复
	<ol>
<li>将大鼠返回到热灯下的回收笼中, 以保持体温。</li>
		<li>观察手术眼出血的迹象。</li>
		<li>观察鼠, 确保它从麻醉中出来。</li>
		<li>将被恢复的动物带回一个新的笼子, 用手术卡将笼子标记起来, 每天监测任何遇险、眼部出血或角膜混浊的迹象。如果有问题, 立即通知兽医。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

本文所描述的视网膜注射技术是通过跨巩膜通路, 注射器针穿透眼壁的外层 (巩膜-脉络膜-RPE 复合体) 而不损害神经视网膜或扰乱玻璃腔。一个替代的反式 vitreal 方法有潜在的风险, 导致白内障的晶状体损伤, 因为啮齿目动物的镜头占据了大多数的玻璃腔。与这种方法相比, 我们的技术是低风险和造成最小的创伤, 因为注射器针不需要穿过整个玻璃腔, 以达到视网膜空间。事实上, 在我们的研究中, OCT 检查显示非常罕见的视网膜穿透事件, 并在动物的后续考试中, 没有持续的视网膜脱离。此外, 注射部位是非常小 (< 200 µm 直径) 时使用33口径注射器针, 因此结构干扰的巩膜-脉络膜-RPE 复合体是非常有限的。针回缩后, 注射孔自动 self-seals, 所以不需要缝合或组织胶。
手术并发症包括在注射区周围或在试验孔内过度出血。当使用钳子来抓住角膜缘的结膜缘时, 只有轻柔的力量才能避免捏血管和减少可能的出血。在创建先导孔之前, 检查预期的注射部位, 以避免表面血管的穿透。通过使用矛, 先导孔可以清除血液前插入的细胞注射针。如果在矛的几个应用之后, 从先导孔中流血, 可能会被破坏。我们观察到的另一项并发症是小比例的 RCS 大鼠发展角膜混浊术。在某些情况下, 混浊是慢性的, 其他是短暂的。有持久混浊的动物被从研究组中移除。角膜混浊可能开发由于眼睛干燥, 物理损伤, 炎症, 药物或化学刺激21。为了减少他们的形成, 眼睛应该保持湿润, 保持充足的眼睛润滑, 并避免接触的角膜与棉花涂抹器或其他工具在操作过程中。
这里概述的注射协议使用定义的方法角度和针深度相对于眼部标志, 和眼睛角度相对于手术表在我们的设置。利用后 OCT 扫描对改善这些注射参数, 以提供高准确度的大鼠视网膜空间的视网膜色素上皮细胞移植的重现性控制是很重要的。一旦被反复练习掌握, 该方法就很容易完成。特别是在训练中, 建议进行后的眼科检查以确定结果。注射针的角度和深度可能需要根据手术的设置, 动物的年龄, 和/或如果使用其他动物种类 (例如, 鼠标) 的反馈相应调整。
为了评估注射质量和确定纳入或排除的研究, 视网膜的存在, 从 OCT 观察出血是至关重要的。在没有 OCT 的实验室中, 以下所述的标准可用于外科医生快速筛查, 排除疑似注射失败: (1) 在注射前: (a) 先导孔太深 (即, 严重深度 > 500 µm;品牌的胰岛素针在这里使用, 从针尖到中间的斜角的距离是500µm)。(b) 先导孔过度出血 (即, 在用眼矛对孔施加外力时不能阻止出血)。(c) 注射针太深 (即, 明显深度 > 500 µm 或注射针上的标记进入巩膜/脉络膜/RPE 复合组织)。(2) 注射过程中: (a) 在注射过程中不能保持注射针在先导孔内。(b) 注射针注射时周围立即发生渗漏。(c) 注射针太深 (即, 注射针上的标记进入巩膜/脉络膜/RPE 复合组织), 这是外科医生在注射过程中注意到的。(d) 在注射后5秒内无法维持注射针的位置。(e) 在缩回注射针时过多的血液。(f) 与步骤2结合后, 在注射针缩回后不出现回流/流出。
同时, 通过对针的位置、角度和深度的细致管理, 视网膜注射技术具有高度的可靠性、准确性, 并能进行最小的手术创伤。有了这些好处, 这项技术不仅可以用于视网膜色素上皮细胞的传递, 也可用于其他细胞类型、化合物或基因治疗。

人视网膜位于眼睛的后面功能作为一个轻的感觉组织并且在视觉知觉扮演一个关键的角色。因此视网膜细胞功能障碍或细胞死亡导致视力问题或永久性失明。在视网膜不同层次的细胞变性或功能紊乱的疾病被称为退行性视网膜疾病, 其中 AMD 是最常见的类型, 也是发达国家老年人不可逆转性失明的主要原因之一1,2。AMD 的病理过程与视网膜色素上皮层与底层赫膜之间的 "疣" 堆积有关, 从而损害了视网膜色素上皮支持的感光生理, 导致了神经系统的萎缩和视力丧失3, 4,5。到目前为止, 没有治疗高级干 (non-neovascular) AMD。干细胞治疗作为再生医学的一个新范式的出现, 带来了用干细胞衍生的健康细胞取代功能失调或死亡的 RPE 细胞的希望。事实上, 广泛的临床前研究的移植干细胞 (如, 人类胚胎干细胞) 衍生 rpe 细胞到 rpe-变性动物模型已经执行6,7, 其中一些已取得进展临床试验8,9 (NCT01344993, ClinicalTrials.gov)。最近, 我们的实验室发现了人类视网膜色素上皮细胞 (hRPESCs) 中的一种替代的干细胞来源, 目前正被用于 hRPESC 衍生-rpe 细胞 (hRPESC-rpe) 移植治疗 AMD 的前临床研究。10,11,12,13。
视网膜注射技术应用于上述多个群体, 包括本组的前临床研究。动物视网膜注射有两种一般的方法: 反 vitreal 和反式巩膜。vitreal 方法的优势是, 外科医生能够直接观察针端, 因为它穿透了前眼, 越过整个 vitreal 腔毗邻的镜头, 并穿透视网膜在后面的眼睛, 以达到视网膜空间14,15,16。然而, 它需要扰乱视网膜在两个位置 (前部和后部), 承担损害晶状体的风险, 并可能导致细胞回流到玻璃体时, 针被收回。相反, 跨巩膜的方法, 原则上, 避免介入视网膜和玻璃体, 和回流退出眼睛。在有色啮齿目动物中, 外科医生最初可以观察到巩膜的穿透, 但在进入色素脉络后, 针端不再可见。没有直接观察, 破坏视网膜是常见的, 并可能导致视网膜解剖和交付细胞和/或血液进入玻璃体。此外, 由于眼睛表面是弯曲的, 很难知道哪种针的角度和深度是最有效的反式巩膜注射。
在这一可视化的文章中, 我们介绍了一个跨巩膜视网膜注射方法, 通过使用后评价与光学相干断层扫描 (OCT), 这使得对注射场的详细检查。我们的反式巩膜注射技术利用定义的位置, 角度和深度注射针, 以产生非常低的外科创伤和高可靠性。在这里, 我们特别演示了 hRPESC-RPE 细胞注入视网膜空间的 RCS 大鼠, 一个临床前模型的人 AMD。采用这种注射方法, 成功、一致地将 hRPESC 视网膜色素上皮细胞导入到 RCS 大鼠眼的视网膜空间中, 成功率很高。注入的细胞以前发现, 导致保存 RCS 光至少2月后, 注入13。本程序在解剖显微镜下进行, 易于学习。它需要两个人 (一个外科医生和一个助理) 来执行注射和平均注射时间为每种动物少于5分钟。定义的角度和深度为注射针, 使之成为可能的实验室, 在那里 OCT 是无法获得成功的视网膜注射液。它允许高度可再生的视网膜通路, 不仅可以用于细胞移植, 也可用于药物传递和基因治疗。

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

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

退行性视网膜疾病, 如年龄相关的黄斑变性 (AMD) 是导致不可逆转的视力丧失全球。AMD 的特点是视网膜色素上皮 (RPE) 细胞的退化, 这是一个单层的细胞功能支持和解剖包裹周围的神经视网膜。目前的药理治疗 non-neovascular amd (干 amd) 只减缓疾病进展, 但不能恢复视力, 必须研究, 以确定新的治疗策略。用健康细胞取代退行性视网膜色素上皮细胞有望在未来治疗干性 AMD。广泛的临床前研究的干细胞替代疗法的 AMD 涉及移植干细胞衍生 RPE 细胞进入视网膜空间的动物模型, 其中视网膜注射技术的应用。在这些临床前动物研究中最常用的方法是通过经巩膜路线, 这是由于缺乏直接可视化的针端, 往往会导致视网膜损伤而变得困难。通过玻璃体的另一种方法可以直接观察针端位置 , 但是由于更多的眼部组织扰 , 它有很高的手术创伤风险。我们已经开发了一个低风险和可重复性的改良反式巩膜注射法, 使用定义的针角和深度, 成功地和一贯地提供 RPE 细胞进入大鼠视网膜空间, 并避免过多的视网膜损伤。以这种方式传送的细胞在皇家外科医生 (RCS) 大鼠中已经被证明是有效的, 至少2月。该技术不仅可用于细胞移植, 也可用于小分子或基因治疗的传递。

利用本文所描述的技术, 我们通过精确地控制插入到组织中的注射器针的位置、角度和深度, 将 hRPESC 视网膜色素上皮细胞送到 RCS 大鼠的视网膜空间中 (图 1B-D).在移植后立即进行 OCT 检查, 观察注射部位和移植细胞产生的视网膜疱疹。术后 OCT 评估是评估注射质量和视网膜损伤或出血监测的检查工具。在 OCT 扫描下, 可以清楚地看到视网膜疱疹 (图 2A、 C和D) 和注入站点 (图 2A和B)。视网膜疱疹通常在注射后24小时内解决。虽然泡的大小测量是困难的使用 OCT, 我们可以估计的疱疹面积假设它等于感光保存区的细胞移植。我们以前证明, 1 µL 注射5万细胞可能导致节省约6-7% 区域的 RCS 视网膜周围的注射站点13。如图 2A、 C和D所示, 在注射部位的视网膜层是完好的, 在疱疹中没有发现血, 在玻璃体中没有观察到细胞, 表明注射造成的创伤极小。此外, 有代表性的 OCT 图像的失败注射也包括在参考 (图 2E和F) 中。
利用 OCT 作为反馈工具, 优化了注射针插入组织的角度和深度。一旦优化, 这种方法使我们能够达到90.8% 视网膜访问的成功率, 只有5.7% 手术失败, 根据在我们其他研究13 (表 1) 中执行的超过300以前的视网膜注射的结果。在剩下的 3.5%, oct 没有执行由于几个原因, 包括眼睛没有在适当的位置由于异氟醚麻醉相关的眼睛滚动17。
移植后7天, 手术后的大鼠眼核, 固定和切片进行免疫分析。人体细胞核标记 (Hunu)18和 RPE 细胞标记 (OTX2)19用于检测移植细胞。图 3显示了在视网膜空间中移植的视网膜色素上皮细胞的厚层, 在注射后一周, 被两个标记物所染色, 证实了移植的身份和成功的分娩。在注射后一个星期内, 大量的细胞, 如图 3C所示, 由于宿主免疫应答, 甚至在免疫 RCS 大鼠20中, 可能会迅速下降到一个小数目。尽管如此, 如前所述, RCS 大鼠眼的退化感光层可以被发现在移植后至少2月内获救, hRPESC-RPE13。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55220/55220fig1.jpg"> 图 1:4 周前 P2 hRPESC 视网膜色素上皮细胞的图像, 并演示注射器针头在注射过程中使用的角度和深度.(a) 用于注射的4周 P2 hRPESC-RPE 细胞的相衬图像。刻度栏 = 100 µm. (B) 示意图显示了标记边缘与注射器针尖尖端之间的600µm 距离。最小的微尺度的毕业是100µm. (C) 一卡通显示大鼠眼的解剖结构的横断面, 以及注射器针插入眼球壁的角度和深度的侧面。鼠眼的瞳孔轴是30度相对于表顶, 注射器针是15度相对于眼球的局部表面。(D) 一卡通显示注射器针上标记的起始点和注射部位的顶部视图, 其中500µm 注射器针插入组织, 在射入孔的开口和市场边缘之间留有100µm 空间r. 洞的位置是 1,200-1, 500 µm 后的角膜缘。针尖是显示在其一侧, 但应朝在注射。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55220/55220fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55220/55220fig2.jpg"> 图 2:在注射后立即操作大鼠眼球的 OCT 图像.(a) OCT B 扫描图像, 显示视网膜疱疹和注射部位, 无玻璃出血。(b) b 扫描系列的 OCT 体积强度投影 (VIP) 图像, 表示注入区域的 enface 眼底图像。小注射地点是可见的 VIP 图像显示最小的创伤。(C) 扩大 OCT 图像 (A) 显示视网膜空间中的移植细胞, 所有视网膜层均有标记。这一图像表明, 移植细胞位于视网膜空间。(D) OCT B 扫描图像, 显示平均大小视网膜疱疹。(E) OCT B 扫描图像, 显示在玻璃空间中的 CS 视网膜注入失败。(F) OCT B 扫描图像, 显示一个失败的视网膜注射液与整个视网膜戳通过在注射现场。缩放栏 = 100 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55220/55220fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55220/55220fig3.jpg"> 图 3: hRPESC-RPE 细胞移植后视网膜冰冻切片的免疫染色.(A) 人体细胞核标记 (Hunu) 染色, 表明对移植的人视网膜色素上皮细胞的检测。(B、E)细胞核计数器染色 (4, 6-diamidino-2-吲;DAPI) 显示视网膜层, 移植, 和 RPE 层。(C) Hunu 和 DAPI 的合并图像, 表明移植的 hRPE 细胞位于视网膜空间。移植与视网膜色素上皮层之间的分离是一种与冰冻保护的 RCS 眼有关的加工工件。(D) rpe 细胞标记物 (OTX2) 染色的人视网膜色素上皮细胞移植。(F) OTX2 和 DAPI 的合并图像。缩放栏 = 20µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55220/55220fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>全注射 RCS 大鼠眼</td>
			<td>OCT 下的好视网膜泡</td>
			<td>OCT 下的小视网膜泡</td>
			<td>OCT 下的总并发症视网膜泡</td>
			<td>Complicatd 视网膜泡在 OCT (即气泡或视网膜出血) 下</td>
			<td>没有执行 OCT</td>
			<td>手术失败 (OCT 下无视网膜疱疹)</td>
		</tr>
<tr>
<td>数</td>
			<td>314</td>
			<td>260</td>
			<td>25</td>
			<td>285</td>
			<td>5</td>
			<td>6</td>
			<td>18</td>
		</tr>
<tr>
<td>总射入的眼睛的%</td>
			<td>------</td>
			<td>82.80%</td>
			<td>7.96%</td>
			<td>90.76%</td>
			<td>1.59%</td>
			<td>1.91%</td>
			<td>5.73%</td>
		</tr>
<tr>
<td colspan="5">(这些数据从十组的视网膜注射的 RCS 大鼠总结)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>表 1: RCS 大鼠十实验组视网膜注射液的总结。

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

视网膜注射液已广泛应用于干细胞置换治疗老年性黄斑变性的临床前研究。在这一可视化的文章中, 我们描述了一个低风险, 可重复和精确修改的视网膜注射技术, 通过巩膜的方法传递细胞到大鼠的眼睛。

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

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

development-refined-protocol-for-trans-scleral-subretinal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.5K 次观看.  Neural Stem Cell Institute. 视网膜注射液已广泛应用于干细胞置换治疗老年性黄斑变性的临床前研究。在这一可视化的文章中, 我们描述了一个低风险, 可重复和精确修改的视网膜注射技术, 通过巩膜的方法传递细胞到大鼠的眼睛。

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

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

MACS纯化光感受器前体细胞的亚视移植到成年小鼠视网膜中

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

科研

医学

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

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

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 ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

一种用于兔视网膜下手术分步协议

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

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.

小型猪载体上 RPE 的视网膜下植入:术前准备、手术技术和术后护理指南

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

生物工程

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

猫大型动物模型中人类胚胎干细胞来源的视网膜组织的视网膜下移植

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

神经科学

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

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.

经瞳孔引导下视网膜下移植物的视网膜下移植物经巩膜移植视网膜变性小鼠模型

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.

<meta charset="utf8"></head><body>大鼠视网膜下植入物手术技术的开发

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.

大鼠视网膜下植入物手术技术的开发

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.

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

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

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

摘要

探索更多视频

MACS纯化光感受器前体细胞的亚视移植到成年小鼠视网膜中

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

一种用于兔视网膜下手术分步协议

小型猪载体上 RPE 的视网膜下植入:术前准备、手术技术和术后护理指南

猫大型动物模型中人类胚胎干细胞来源的视网膜组织的视网膜下移植

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

经瞳孔引导下视网膜下移植物的视网膜下移植物经巩膜移植视网膜变性小鼠模型

大鼠视网膜下植入物手术技术的开发

大鼠视网膜下植入物手术技术的开发

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩