Name: engineering transplantation-suitable retinal pigment epithelium tissue derived from human embryonic stem cells
Uploaded: 2018-09-06T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells at JoVE.com

作者要感谢 Jérôme Larghero 和 valériemarcel Vanneaux (Hôpital 圣路易斯, 巴黎, 法国) 在这里所描述的方法的建立过程中的投入。
这项工作得到了来自情报局的赠款的支持 [GPiPS: ANR-2010-RFCS005;SightREPAIR: ANR-16-CE17-008-02], 基金会倒 la 研究所 Médicale [生物工程项目 DBS20140930777] 和从 LABEX 复活 [ANR-10-LABX-73] 到奥利弗 Goureau 和 Christelle Monville。它得到了 NeurATRIS (Investissements d ' 艾文莉) 的支持, 为神经 [ANR-11-INBS-0011] 和 INGESTEM 的 biotherapies, 国家基础设施 (Investissements d ' 艾文莉) 工程为多潜能和分化的干细胞 [ANR-11-INBS-000] Christelle Monville。Barek 是由暗淡的 Stempole 和 LABEX 复兴 [ANR-10-LABX-73] 的研究金支持。Biotherapies 是法国反肌病 (AFM)-Téléthon 协会支持的罕见疾病研究所的一部分。

本议定书使用的所有人类材料都是按照欧洲联盟的规定使用的。本研究使用的人类 ES 细胞系来源于一个独特的胚胎。捐出胚胎的夫妇完全知情, 并同意匿名捐赠。一个临床级的人类 ES 细胞系来源于这个胚胎, 储存, 合格, 并正确记录的罗丝琳细胞 (英国)。在产妇剖宫产期间, 根据医院的指导原则 (APHP, Hôpital 圣路易斯) 签署了知情同意的胎盘捐献, 火腿是在无菌条件下采购的。
1、培养基和试剂的制备
<ol><li>
视网膜色素上皮细胞培养培养基的制备
	<ol>
<li>为了准备视网膜色素细胞培养培养基, 添加4% 血清替代品 (20 毫升的最终体积500毫升), 1,000x 稀释 2-巯基乙醇 (500 µL 为最终体积500毫升), 1% Eagle′s 最低基本培养基 (记忆体) 非必需氨基酸溶液 (5 毫升为最终体积500毫升) Dulbecco 的改良鹰的培养基 (DMEM) 高葡萄糖 (475 毫升的最终体积500毫升)。</li>
	</ol>
</li>
	<li>
运输/养护介质的制备
	<ol>
<li>要准备运输/保护培养基, 添加1% 青霉素-链霉素 (5 毫升的最终体积500毫升), 以 CO2独立的培养基 (495 毫升的最终体积500毫升), 并保持在4°c。</li>
	</ol>
</li>
	<li>
thermolysin 酶的泥沙
	<ol>
<li>thermolysin 库存液的制备<ol>
<li>要准备一个 thermolysin 的库存溶液, 解冻 thermolysin 粉, 在室温下储存在-20 摄氏度。</li>
			<li>由于酶活性可能是从批次到批次的变量, 计算此活动的基础上提供的分析证书与 thermolysin 粉的批次将使用。将 "活性中性蛋白酶计算 = ANPC" (在分析证明书中注明) 除以 181 (这是酪氨酸的分子量)。所得结果与所提供的 thermolysin 粉 (U/瓶) 的总酶活性有关。</li>
			<li>通过添加对应于总酶活性 (u/瓶) 的水量除以 200 (u/毫升), 在 200 U/毫升处准备一个库存溶液。</li>
			<li>用吹打的水来溶解酶。涡流解决方案三十年代, 并检查是否已完全暂停粉末。完全解散可能需要更多的吹打。</li>
			<li>整除库存解决方案, 并存储在-20 摄氏度。</li>
		</ol>
</li>
		<li>thermolysin 工作液的制备 注意: thermolysin 的解决方案必须在火腿治疗当天准备好。<ol>
<li>解冻的 thermolysin 的股票整除数在 200 U/毫升存储在-20 摄氏度。稀释在磷酸盐缓冲盐水 (PBS) 的库存溶液 200x, 以获得最终的酶活性的 1 U/毫升。准备40毫升治疗第1-4 火腿补丁。</li>
			<li>在使用前通过0.2 µm 过滤器筛选 thermolysin 解决方案。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
明胶泥沙
	<ol>
<li>20% 明胶溶液及块体的制备 注: 20% 明胶溶液及块可在使用前1周制备。<ol>
<li>温暖 CO2独立媒介到42°c 在水浴为30分钟 (至多 1 h)。在50毫升管, 加入10克明胶到40毫升温暖 CO2独立介质和漩涡的解决方案。</li>
			<li>将明胶 30–60 42 摄氏度溶解。涡旋每10分钟融汇溶液。</li>
			<li>一旦溶液是均匀的, 添加4毫升的20% 明胶溶液到四6厘米培养皿。避免气泡。添加一个塑料石蜡膜, 以保护菜肴, 并允许溶液凝固4摄氏度。</li>
		</ol>
</li>
		<li>8% 明胶溶液的制备 注: 8% 明胶溶液可在使用前一天制备, 贮存于4摄氏度。<ol>
<li>温暖 CO2独立媒介到42°c 为30分钟 (至多 1 h)。在50毫升管, 加入4克明胶到46毫升温暖 CO2独立介质和漩涡的解决方案。</li>
			<li>将明胶 30–60 42 摄氏度溶解。添加一个塑料石蜡膜, 以保护管, 并保持在水浴在37°c, 如果它将使用同一天, 或存储它在4°c, 如果它将使用后。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 人羊膜的 Thermolysin 治疗
<ol><li>
人羊膜洗膜
	<ol>
<li>有火腿供应作为小片断 (大约30毫米 x 30 毫米) 固定在尼龙脚手架, 要么已经在4°c 在 PBS 或冷冻。如果提供冷冻, 解冻的膜在37°c 在一个孵化器30分钟。</li>
		<li>洗膜:<ol>
<li>将第1-4 膜放置在含有80毫升 PBS 的250毫升瓶中。根据需要使用尽可能多的瓶子。</li>
			<li>以高速5分钟的速度在平板振动筛中摇动每瓶, 然后丢弃 PBS。添加80毫升 PBS 和重复。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Thermolysin 治疗
	<ol>
<li>添加40毫升的工作溶液的 thermolysin (1 U/毫升) 每瓶含膜 (第1-4 膜每瓶, 每次最多3瓶)。</li>
		<li>摇瓶5分钟, 在 450 rpm, 然后涡旋他们三十年代. 重复1x。</li>
		<li>丢弃 thermolysin 解决方案并添加80毫升的 PBS。</li>
		<li>在450转每分钟摇动瓶子5分钟。丢弃 pbs 并添加80毫升 pbs。重复3x。</li>
	</ol>
</li>
</ol>3. 人羊膜在培养物上的固定
<ol><li>使用长的无菌钳 (可以达到瓶子的底部) 一个一个地去除火腿, 并将每个火腿转移到含有10毫升 PBS 的10厘米培养皿中。</li>
	<li>在一个新的 10 cm 培养皿包含10毫升 PBS, 放置其中一个膜与尼龙朝下。将用于固定膜的四个剪辑中的两个分离到尼龙。</li>
	<li>在尼龙和膜之间插入文化插入的小环。</li>
	<li>确保膜完全覆盖小环。将区域性的第二部分插入到膜的顶部。火腿的基底膜正朝内的文化插入。分离最后两个剪辑。</li>
	<li>切, 用无菌剪刀, 多余的膜外的文化插入如有必要。使用镊子, 将固定在培养基中的膜转移到12井板上, 添加 PBS, 并将该板块存储在37摄氏度和 5% CO2的孵化器中。</li>
	<li>重复这些操作 (从步骤3.2 到 3.5) 与其他膜。</li>
</ol>4. 人羊膜中视网膜色素上皮细胞的解冻和播种
<ol><li>
视网膜色素上皮细胞的解冻
	<ol>
<li>视网膜色素上皮细胞库被冷冻在100万细胞每低温小瓶在液氮。根据需要解冻尽可能多的低温瓶 (每膜35万个细胞)。</li>
		<li>准备一个15毫升管包含3毫升的视网膜色素介质每低温瓶。一旦瓶子解冻, 把细胞转移到每管。对其他瓶子重复此操作。</li>
		<li>融汇 (吸管向上和向下几次并用重悬细胞在 RPE 培养基), 并采取10µL 的细胞溶液和转移到1.5 毫升管。加入10µL 的台盼蓝。将此解决方案的15µL 放在一个计数室中, 然后在显微镜下用4X 目标计算活细胞的数量 (不是蓝色)。对其他瓶子重复此操作。</li>
		<li>同时, 离心机的15毫升管在 110 x g为5分钟. 丢弃上清液并将细胞并用重悬70万细胞/毫升。</li>
	</ol>
</li>
	<li>
视网膜色素上皮细胞在人羊膜中的播种
	<ol>
<li>从孵化器中取出含有膜的12井板。</li>
		<li>吸入 PBS添加500µL 的细胞悬浮液中的步骤4.1.4 在文化插入中心。添加1毫升的视网膜色素培养基外的文化插入 (井内)。重复其他膜。</li>
		<li>将含有膜的12井板放在孵化器中。</li>
	</ol>
</li>
</ol>5. 人羊膜视网膜色素上皮细胞培养的维持
<ol><li>每星期更新中等 2x, 通常在星期一和星期五, 直到至少30天的文化。</li>
	<li>吸入培养基内外的文化插入为每个井和更新它与新鲜的培养基 (1 毫升外的文化插入和500µL 内)。将盘子放在孵化器的37°c 和 5% CO2。</li>
</ol>6. 视网膜色素上皮修补术的制备
注: 从培养的30天开始, 组织就可以移植了。
<ol><li>将8% 的明胶溶液在37摄氏度的水浴中加热30分钟. 用冷 CO2独立介质填充 vibratome 的内腔 (4 °c)。</li>
	<li>
将20% 明胶块放入 vibratome。
	<ol>
<li>从冰箱里拿出6厘米的培养皿, 里面有20% 块明胶。使用手术刀, 切出一个5厘米 x 5 厘米块。用氰胶水或其等效物将块的顶部粘贴到支架上。</li>
		<li>在 vibratome 上放一把新刀片。</li>
		<li>调整块的水平, 直到它在刀片的同一水平。</li>
		<li>用新鲜 CO2-独立媒介在4°c 填满浴在支持附近。使用中等速度切割 vibratome 块, 直到块均匀切割, 从而使表面光滑。设置位置0。</li>
		<li>在凝胶块周围吸出培养基, 直到完全干燥, 然后从孵化器的37摄氏度将含有组织的12井培养皿吸入。</li>
		<li>使用镊子, 小心打开在明胶块顶部的文化插入。用剪刀切开, 不含细胞的细胞膜的所有部分 (细胞未培养的环外)。吸入整个残余介质。</li>
		<li>添加1毫升的液体8% 明胶溶液在37摄氏度, 以覆盖膜。小心地去除多余的。等5–8分钟, 让明胶凝固。</li>
		<li>添加新鲜 CO2-独立介质 (在4°c) 到浴缸, 直到膜覆盖。</li>
		<li>切割, 与 vibratome 在-100 µm 的位置, 以中等速度, 仍然知道如何该区块的行为。在该节的末尾, 要注意保持组织的方向。</li>
		<li>用手术刀, 切一个角落, 以确定该部分的方向。</li>
		<li>用刮刀将所嵌入的明胶中的膜收集起来, 将其放置在6井板中, 填充在4摄氏度的保护培养基上, 直到将其嫁接到接受者眼中。</li>
		<li>在移植时, 在手术显微镜下调整植入物的大小以接受者眼的大小 (1–3为大鼠, 10–15毫米2为非人类灵长类)。</li>
	</ol>
</li>
</ol>

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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+RPE+stem+cells+grown+into+polarized+RPE+monolayers+on+a+polyester+matrix+are+maintained+after+grafting+into+rabbit+subretinal+space.">Human RPE stem cells grown into polarized RPE monolayers on a polyester matrix are maintained after grafting into rabbit subretinal space.</a> Stem Cell Reports. 2 (1), 64-77 (2014).</li><li>Ilmarinen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin+polyimide+membrane+as+cell+carrier+for+subretinal+transplantation+of+human+embryonic+stem+cell+derived+retinal+pigment+epithelium.">Ultrathin polyimide membrane as cell carrier for subretinal transplantation of human embryonic stem cell derived retinal pigment epithelium.</a> PloS One. 10 (11), e0143669 (2015).</li><li>Thumann, G., Schraermeyer, U., Bartz-Schmidt, K. 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J., Bharti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+into+the+future:+Using+induced+pluripotent+stem+cells+to+build+two+and+three+dimensional+ocular+tissue+for+cell+therapy+and+disease+modeling.">Looking into the future: Using induced pluripotent stem cells to build two and three dimensional ocular tissue for cell therapy and disease modeling.</a> Brain Research. 1638 (Pt A), 2-14 (2016).</li><li>Ramsden, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+retinal+regeneration:+Past,+present+and+future).">Stem cells in retinal regeneration: Past, present and future).</a> Development. 140 (12), 2576-2585 (2013).</li><li>da Cruz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+1+clinical+study+of+an+embryonic+stem+cell-derived+retinal+pigment+epithelium+patch+in+age-related+macular+degeneration.">Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration.</a> Nature Biotechnology. 36 (4), 328-337 (2018).</li><li>Kashani, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioengineered+retinal+pigment+epithelial+monolayer+for+advanced,+dry+age-related+macular+degeneration.">A bioengineered retinal pigment epithelial monolayer for advanced, dry age-related macular degeneration.</a> Science Translational Medicine. 10 (435), (2018).</li><li>Binder, S., Stanzel, B. V., Krebs, I., Glittenberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+the+RPE+in+AMD.">Transplantation of the RPE in AMD.</a> Progress in Retinal and Eye Research. 26 (5), 516-554 (2007).</li><li>Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., Hjelmeland, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARPE-19,+a+human+retinal+pigment+epithelial+cell+line+with+differentiated+properties.">ARPE-19, a human retinal pigment epithelial cell line with differentiated properties.</a> Experimental Eye Research. 62 (2), 155-169 (1996).</li><li>Salero, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+RPE+can+be+activated+into+a+multipotent+stem+cell+that+produces+mesenchymal+derivatives.">Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives.</a> Cell Stem Cell. 10 (1), 88-95 (2012).</li></ol>

奥利弗. Goureau 是与人类多潜能干细胞产生视网膜细胞有关的未决专利的发明者。其他作者没有什么可透露的。

我们描述了一种生物支架上的 RPE 细胞培养方法及其在动物模型中的植入准备。该协议的关键步骤之一是保持火腿的方向一直沿着过程, 直到它纳入到明胶。事实上, 膜的本机上皮被去除, 其基底膜暴露9。视网膜色素上皮细胞必须在基底膜的顶端播种。在制备明胶时, 必须在规定的温度下与所有产品配合使用。事实上, 明胶的性质是刚性在4°c 和液体在体温 (37 °c)9。如果温度不受尊重, 明胶可以在不需要这种效果的步骤中凝固或液化。
一些生物支架已经提出, 像后弹力层的膜25或火腿26。特别是, 从剖宫产27的火腿, 被证明是良好的耐受性在视网膜下空间, 造成有限的炎症和减少脉络膜新生血管26。该膜成功地支持人类视网膜色素上皮细胞的培养9,28。此外, 这些膜也有悠久的历史, 在29诊所, 使他们的良好候选支架的视网膜色素细胞治疗。其他生物支持可以很容易地实现与本议定书。基于聚合物的合成脚手架已经是刚性的, 在植入13、30、31、32、33之前可能不需要凝胶嵌入。其他植入的系统最近已经开发, 以视网膜下分娩的人眼中的人类胚胎干细胞衍生的 RPE 单层在刚性聚酯脚手架34或在合成派瑞林基板设计模仿刷的膜35.尽管这些策略是有希望的, 但我们相信生物支架可以为视网膜色素组织工程提供最佳的平台。
本协议采用自发分化的方法, 从人类 ES 细胞中提取出种子视网膜色素上皮细胞。然而, 不同类型的 RPE 细胞可以使用, 无论是区别于人类诱导的多潜能干细胞或从从尸体获得的主要视网膜色素细胞, 甚至从视网膜色素细胞19,36,37, 38。此外, 如果使用多能干细胞, 在过去几年中开发了一些协议, 以获得基于自发分化的 RPE 细胞或使用小分子引导分化18,22。从这些不同的分化协议获得的 RPE 细胞也可以用这种方法进行组织工程。
这种方法的一个限制是在4°c 的嵌入组织的稳定性。由于它被包括在明胶刚刚装运到手术现场, 它必须保持在这个温度, 直到植入。在这方面, 手术应在48小时内进行。
这种方法可以很容易地转移到诊所。在2独立培养基中, 嵌入在明胶中的 RPE 组织保存在4摄氏度。如果需要, 此种媒介可以被美国食品和药物管理局 (FDA) 批准, 用于保护经验尸后获得的角膜, 并用于移植到人类体内。我们成功地证明了这一策略的有效性, 移植在一个概念验证的研究到啮齿类动物模型9, 我们目前正在验证的手术方法在非人类灵长类, 然后实施它的临床试验治疗特定形式的 RP 影响视网膜色素的患者。

RPE 是至关重要的生存和稳态的感光细胞, 它是紧密关联1。一些病理状况改变其功能和/或生存, 包括 RP 和 AMD。
RP 是一组遗传性的基因突变, 影响感光细胞或视网膜色素的功能, 或2,3。据估计, 特别影响视网膜色素上皮细胞的突变占5% 的 RP2。AMD 是另一种情况下, 视网膜色素层被改变, 最终导致中心视力损失。AMD 是由遗传和环境因素复杂的相互作用引起的, 影响老年人4,5,6。根据预测, AMD 将是全球1亿9600万名患者的关注, 到 2020年7。对于这些疾病, 目前还没有有效的治疗方法, 建议的策略之一是移植新的视网膜色素细胞, 以弥补死/非功能性视网膜色素上皮细胞8。
最终产品的配方, 是必不可少的, 以确保最佳的功能效果。RPE 细胞作为细胞悬浮液注入, 尽管是一种简单而直接的分娩方法, 但对其生存、整合和功能9、10、11、12等问题提出了关注。,13. 科学家现在正在开发更复杂的配方, 以提供工程化的视网膜组织9、13、14、15、16。在这种情况下, 我们开发了一种原始的方法来生成体外视网膜色素组织, 可用于移植9。
从人类胚胎干细胞中提取的 RPE 细胞库用于本协议。然而, 不同细胞源 (人诱导的多潜能干细胞、原发性视网膜色素上皮细胞等) 的替代视网膜色素细胞库也适用于该协议。它包括定向分化协议使用细胞因子和/或小分子17,18,19,20,21,22。
要移植, 工程组织应在脚手架上准备。在过去的几年中, 不同的脚手架是基于聚合物或生物起源13,23,24的矩阵开发的。在这里, 使用的生物基质是火腿, 但其他基质, 如裸露的刷膜, 可以实施。此处描述的方法具有使用与 RPE 本地环境更相关的生物支架的优点。
人类 ES 细胞源性视网膜色素上皮细胞培养至少4周, 以充分组织为鹅卵石单层。在该阶段, 获得的上皮细胞功能和极化9。最后, 由于这种组织容易皱纹, 它嵌入在薄层的水凝胶载体, 使其更刚性和弹性, 并在注射过程中保护它。该产品然后存储在4°c, 直到嫁接。

<table><tbody><tr><td>Sterile biosafety cabinet</td><td>TechGen International</td><td>Not applicable</td><td></td></tr><tr><td>Liquid waste disposal system for aspiration</td><td>Vacuubrand</td><td>BVC 21</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-controlled +37&amp;nbsp;&amp;deg;C cell incubator</td><td>Thermo Electron Corporation</td><td>BVC 21 NT</td><td></td></tr><tr><td>200&amp;nbsp;&amp;micro;L pipette: P200</td><td>Gilson</td><td>F144565</td><td></td></tr><tr><td>1&amp;nbsp;mL pipette: P1000</td><td>Gilson</td><td>F144566</td><td></td></tr><tr><td>Pipet aid</td><td>Drummond</td><td>75001</td><td></td></tr><tr><td>+4&amp;nbsp;&amp;deg;C refrigerator</td><td>Liebherr</td><td>Not applicable</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000S</td><td></td></tr><tr><td>Fine scissors</td><td>WPI</td><td>501758</td><td></td></tr><tr><td>Forceps (x2)</td><td>WPI</td><td>555227F</td><td></td></tr><tr><td>Water bath</td><td>Grant subaqua pro</td><td>SUB6</td><td></td></tr><tr><td>Precision balance</td><td>Sartorius</td><td>CP225D</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorff</td><td>5804</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>SC30</td><td></td></tr><tr><td>Horizontal Rocking Shaker</td><td>IKA-WERKE</td><td>IKA MTS 214D</td><td></td></tr><tr><td>Vortex</td><td>VWR</td><td>LAB DANCER S40</td><td></td></tr><tr><td>Disposable Scalpel</td><td>WPI</td><td>500351</td><td></td></tr><tr><td>plastic paraffin film</td><td>VWR</td><td>PM992</td><td></td></tr><tr><td>0.200 &amp;micro;m single use syringe filter</td><td>SARTORIUS</td><td>16532</td><td></td></tr><tr><td>Syringe without needle 50 mL</td><td>Dutscher</td><td>50012</td><td></td></tr><tr><td>Bottles 250 mL</td><td>Dutscher</td><td>28024</td><td></td></tr><tr><td>15 mL sterile Falcon tubes</td><td>Dutscher</td><td>352097</td><td></td></tr><tr><td>50 mL sterile Falcon tubes</td><td>Dutscher</td><td>352098</td><td></td></tr><tr><td>culture insert</td><td>Scaffdex</td><td>C00001N</td><td></td></tr><tr><td>60&amp;nbsp;mm cell culture disches: B6</td><td>Dutscher</td><td>353004</td><td></td></tr><tr><td>12 well cell culture plate</td><td>Corning</td><td>3512</td><td></td></tr><tr><td>6-well culture plates</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Razor blades</td><td>Ted Pella, Inc</td><td>121-9</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Castorama</td><td>3178040670105</td><td></td></tr><tr><td>PBS 1x (500&amp;nbsp;mL)</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Thermolysine</td><td>Roche</td><td>5339880001</td><td></td></tr><tr><td>DMEM, high glucose, GlutaMAX</td><td>Invitrogen</td><td>61965-026</td><td></td></tr><tr><td>KSR CTS (KnockOut SR XenoFree CTS)</td><td>Invitrogen</td><td>12618-013</td><td></td></tr><tr><td>MEM-NEAA (100x)</td><td>Invitrogen</td><td>11140-035</td><td></td></tr><tr><td>b-mercaptoethanol (50&amp;nbsp;mM)</td><td>Invitrogen</td><td>31350-010</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140122</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-independent medium</td><td>GIBCO</td><td>18045-054</td><td></td></tr><tr><td>Gelatin</td><td>MERCK</td><td>104078</td><td></td></tr><tr><td>human amniotic membrane</td><td>Tissue bank St Louis hospital (Paris, France)</td><td>Not applicable</td><td></td></tr></tbody></table>

engineering transplantation-suitable retinal pigment epithelium tissue derived from human embryonic stem cells

眼睛的几个病理条件影响视网膜色素上皮 (RPE) 的功能和/或生存。这包括一些形式的视网膜色素变性 (RP) 和年龄相关的黄斑变性 (AMD)。细胞治疗是建议治疗这些疾病的最有希望的治疗策略之一, 在人类身上已经有了令人鼓舞的初步结果。然而, 移植物的制备方法对其在体内的功能结果有显著的影响。事实上, 作为细胞悬架移植的视网膜色素上皮细胞的功能比作为视网膜组织移植的细胞要少。在此, 我们描述了一个简单的和可重复的方法来设计视网膜色素组织和它的准备在体内植入。从人类多潜能干细胞中提取的视网膜色素细胞是在生物支持下播种的, 即人羊膜 (火腿)。与人工支架相比, 这种支持有一个基底膜接近刷的膜, 其中内源性视网膜色素细胞附加的优势。然而, 它的操作不容易, 我们制定了一些策略, 以适当的培养和准备移植在体内。

火腿含有上皮层, 应在视网膜色素细胞播种前除去。用 thermolysin 在震动下对膜进行酶处理。为了不失去膜的极性 (上皮是在一侧), 它是固定在一个支持, 组成可能会有所不同取决于提供者 (图 1A)。在本步骤中检查膜的附着力, 并在必要时添加剪辑。在固定的文化插入时, 要小心地避免在膜上制造孔, 并保持其极性与基底膜朝上 (图 1B)。当细胞被播种在细胞膜上时, 它们可以从这些孔中逸出, 最终细胞浓度会减少。孔的存在可以可视化显微镜下或添加 pbs 内的文化插入和检查 pbs 是否泄漏。任何有孔的膜都应丢弃。
在对培养基进行固定后, 对相衬显微镜中残留细胞的存在进行评估 (图 1C和1D)。经典的, 一些死细胞可能留在膜表面, 但这些细胞将被淘汰时, 培养基改变 (图 1C)。如果没有细胞残留, 基底膜的纤维可以在更高的放大倍数 (图 1D) 中看到。如果情况并非如此, 则可以调整与 thermolysin 的孵化时间。
在视网膜色素细胞播种后的日子里, 检查细胞是否坚持。根据所使用的显微镜, 在最初的几周内很难清楚地看到细胞, 但是如果细胞不粘, 细胞就会在细胞培养基中漂浮。一旦上皮形成并开始成熟, 它就会变得更容易在显微镜下看到它 (图 2A、 2B和2C)。在3周, 细胞形成一个完整的单层上皮典型的视网膜色素 (鹅卵石组织)。4周后, 上皮细胞足够成熟, 为其植入准备 (图 2D)。然而, 由于后勤方面的原因, 它可以在文化中保留更多的星期。
图 3描述了移植物的制备方法。在膜同位语20% 明胶块后, 吸入所有多余细胞培养基。这一步是重要的, 因为任何剩余的培养基可以阻止8% 明胶与视网膜色素和膜的黏附。事实上, 培养基会在明胶层之间形成液体膜。
用于埋植植入物的明胶可以有不同的强度, 这取决于其开花指数 (即由供应商进行的质量控制测试, 在标准温度下评估凝胶的强度和浓度)。如果所用的明胶在嫁接过程中没有足够的刚性和松散, 则应将所用的明胶参照改成另一种强度较高的。在实验的基础上, 也可以改变明胶的浓度, 以调整其强度和弹性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58216/58216fig1.jpg"> 图 1: 在 thermolysin 治疗和固定后的火腿的代表性图像在一个文化插入.(a) 组织银行提供的膜的代表性图像。(B) 固定在文化插入物上的膜的代表性图像。(C) thermolysin 用低放大倍数处理的膜的代表性图像。(D) thermolysin 用高放大倍数处理的膜的代表性图像。<a href="https://www.jove.com/files/ftp_upload/58216/58216fig1large.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/58216/58216fig2.jpg"> 图 2: 在给视网膜色素细胞播种时火腿的代表性图像.(A) 播种前膜的代表性图像。(B和C) 在播种后3周的细胞膜上视网膜色素上皮细胞的代表性图像。膜可能不是完全平面的, 如面板C所示。缩放条 = 100 µm (C), 20 µm 放大倍数。(D) 在播种后30天的膜上视网膜色素上皮细胞的代表性图像, 对应于将其嵌入明胶的时间。<a href="https://www.jove.com/files/ftp_upload/58216/58216fig2large.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/58216/58216fig3.jpg"> 图 3: 描述了在明胶膜内的火腿内含有视网膜色素上皮细胞层的工程化视网膜组织的顺序步骤的方案.<a href="https://www.jove.com/files/ftp_upload/58216/58216fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells at JoVE.com

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

从人胚胎干细胞中提取合适的视网膜色素上皮组织的工程移植

我们描述了一种由人类羊膜顶部培养的人多能干细胞的视网膜色素上皮细胞组成的视网膜组织的方法及其在动物模型中的移植准备。

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

engineering-transplantation-suitable-retinal-pigment-epithelium

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.6K 次观看.  Institut National de la Santé et de la Recherche Médicale (INSERM). 我们描述了一种由人类羊膜顶部培养的人多能干细胞的视网膜色素上皮细胞组成的视网膜组织的方法及其在动物模型中的移植准备。

视频: Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

视网膜色素上皮细胞的干细胞高效的推导

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

科研

发育生物学

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

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 ...

医学

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.

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 ...

神经科学

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 ...

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. ...

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, ...

生物学

焕活视力:干细胞疗法治疗视网膜变性

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.

作者聚焦:推进视力恢复 - 基于干细胞的视网膜疾病疗法 

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小鼠中人胚胎干细胞衍生的光感受器祖细胞的视网膜下递送

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