这项研究支持的研究, 以防止失明, 纽约, www.rpbusa.org, 和更大的纽约视网膜退行性疾病基金会的战斗失明, www.blindness.org 中心。作者要感谢 Luanna 博士对这篇手稿的评论。

以下议定书是遵守《赫尔辛基宣言》和《耶鲁大学人类研究道德委员会准则》的原则执行的。
1. 采购人类捐献者的眼睛
<ol><li>从国家疾病研究交流 (NDRI, 费城, PA) 获得人类捐赠地球。根据项目, 为每个实验组准备不少于三对捐献者的眼睛。</li>
	<li>Enucleate 捐赠者的眼睛在 10 h 之内和船在 48 h以后验尸。然后将眼睛传送到实验室, 在无菌 Dulbecco 的改良鹰培养基 (DMEM) 的冰上培养基。先前报告的研究表明, 以这种方式收获和运输的捐赠者眼睛保存刷的膜的生物特征和活动25,26,29。</li>
</ol>2. 人类刷膜外植体的收获
<ol><li>将每只眼睛放在一个 1.5 x 1.5 英寸的纱布浸泡与二氧化碳独立的 DMEM 媒体在一个100毫米培养皿, 并使用细刀片剪刀使切口通过巩膜3毫米后的角膜缘和圆周延伸在任一方向。</li>
	<li>做一个全厚的周向切口 (穿透到玻璃体) 1 毫米后, 青缘青蟹。取出并丢弃前段 (角膜、晶状体和虹膜)、玻璃体和视网膜。</li>
	<li>使用精细的外科剪刀在巩膜和脉络膜之间做四个径向切口, 然后将巩膜从外围剥离到视神经, 同时注意避免撕裂脉络膜/刷的细胞膜复合物。</li>
	<li>其次, 通过沿脉络空间进行圆周切口, 并从巩膜上剥离视网膜色素刷的细胞膜脉络膜复合向后. </li>
	<li>在室温下, 用0.02 米的氢氧化铵在 Dulbecco 磷酸盐缓冲盐水 (DPBS) 中的 60 x 15 毫米聚苯乙烯培养皿中, 去除本机 RPE 细胞, 然后在 DPBS 中清洗三次 (图 1)。</li>
	<li>将刷在无二氧化碳介质中的膜复合外植体漂浮在 unlaminated、疏水性65µm 厚的聚四氟乙烯膜上, 具有0.2 µm 毛孔, 与所面对膜的每个外植体的基底叶片层。</li>
	<li>在玻璃微分析真空过滤器系统中, 将聚四氟乙烯膜与刷的膜外植体放在烧结玻璃支架上。</li>
	<li>扁平的卷曲的边缘, 从脉络膜一侧的细钳涂上聚四氟乙烯, 注意不要接触刷的膜基层粘连蛋白侧。</li>
	<li>加热15毫升琼脂糖 (4% DPBS), 然后冷却到37摄氏度。然后将15毫升的液化凝胶从脉络膜一侧倒入刷的膜脉络复合物中, 同时应用温和吸力保证外植体保持平坦。保持组织在4°c 2-3 分钟, 以巩固琼脂糖, 然后剥去聚四氟乙烯膜。</li>
	<li>将刷的膜外植体 (直径1.5 英寸, 在固化琼脂糖凝胶内) 放在 60 x 15 毫米聚苯乙烯细胞培养皿中 (刷的膜朝上), 在盘子底部放上温热的液体琼脂糖, 固定刷的膜在井底外植体与植块基底层粘连蛋白的面朝上。</li>
	<li>琼脂糖将在室温 2-3 分钟内凝固, 以稳定刷在培养皿中的膜外植体。用 DPBS 将刷的膜外植体覆盖在培养皿中, 并将菜盘保存在4摄氏度, 供将来使用。图 2A演示了在 60 x 15 mm 培养皿中隔离的刷的膜外植体。</li>
	<li>如果所需的养殖系统位于96井板中, 则将刷的膜外植体 (直径1.5 英寸, 用固化琼脂糖凝胶) 放在与层粘连蛋白侧的扁平聚四氟乙烯板上。</li>
	<li>使用骨髓, 切割五或六, 6 毫米-圆形按钮从刷的膜复合体外植块和放置在4% 琼脂糖在37°c, 在一个未处理的聚苯乙烯井的96井板。琼脂糖将在室温2-3 分钟内凝固, 以固定刷的膜外植体 (纽扣) 在每个井的底部。 注意:图 2B演示了一个96井板中 trephined 刷的膜按钮。通常, 9-11 按钮或外植体可以从每只眼睛中收获。</li>
</ol>3. 视网膜色素上皮 (RPE) 细胞在人刷膜外植体中的培养
<ol><li>收到捐献者的眼睛后, 用 DPBS 清洁眼外组织。在虹膜-巩膜接触点 (切口) 下方5毫米的圆周切口进入视网膜下腔.放弃前段, 玻璃体幽默和视网膜。</li>
	<li>洗眼罩, 其中含有刷的膜和视网膜色素表, 与冷 DPBS。分离视网膜色素上皮细胞与5毫升0.25% 胰蛋白酶不超过10分钟的每一个眼睛样本。用25毫升的 DMEM 完全培养基与15% 胎牛血清 (血清) 在50毫升管中加热37摄氏度时, 对胰蛋白酶反应进行猝灭。</li>
	<li>离心细胞在 200 x g 10 分钟在24°c, 并并用重悬颗粒在5毫升的 DMEM 完全培养基 (15% 血清)。</li>
	<li>将细胞和种子1.5万可存活的 RPE 细胞计算在96井板中每个不同年龄的刷膜外植体 (纽扣) 的基底叶片上), 其中200µL 无血清最低基本培养基 (记忆体), 含抗生素 (100 毫克/毫升青霉素 G, 100 mg/链霉素, 5 毫克/毫升庆大霉素, 2.5 毫克/毫升两性霉素 B)。假设细胞直径为20µm, 镀层在这个密度将允许 RPE 细胞覆盖约15% 的电镀面积。</li>
	<li>将单元格板到按钮上, 据此可以增加细胞密度, 从而逐步提高到100% 汇流, 这取决于每个参数对细胞行为的影响。</li>
	<li>在 95% air/5%co2的湿润气氛中, 允许细胞附着在24小时的外植体上, 37 摄氏度。用温暖完整的 DMEM 轻轻地改变培养基。</li>
</ol>

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没有一个作者有任何商业披露申报。

老年人类刷的细胞膜 (细胞外基质) 有助于 AMD 的疾病进展, 因此有必要了解这种改变基质是如何导致视网膜色素上皮细胞功能障碍的。由于缺乏足够的动物模型来研究 AMD 的年龄相关变化, 因此, 模拟疾病影响的体外模型系统可以作为了解病理生理学的宝贵工具. 本手稿中描述的方法可用于一致隔离人刷的膜外植体和培养 rpe 细胞, 包括主要的, 永生的, 或干细胞衍生的视网膜色素上皮细胞系。
如前所述, 人类刷的膜外植体来源于 NDRI。与 NDRI 建立了一个协议, 它指定了为捐赠者的眼睛所设定的条件是可以接受的。例如, 捐赠者必须没有已知的视网膜眼病, 眼睛/地球在10小时之内被检索在死亡和地球在48小时之内到达实验室在死亡以后 (通过隔夜包裹交付在冰)。指定适当的年龄范围和对研究的统计分析所需的眼睛数, e. g, 五对地球从捐赠者在 20-49 年和五个地球对之间从捐赠者在50年的年龄之间。
眼睛地球仪的可用性各不相同, 平均每个月可提供十对球体。眼睛包裹到达与基本, 被取消辨认的捐赠者信息: 年龄、种族、死亡时间、死因和简要地过去医学历史 (疾病名单或并发症)。
最重要的是, 收割人类刷的膜外植体需要去除眼睛和玻璃体的前段, 允许隔离视网膜色素刷的膜脉络复合物。一旦刷的膜被隔离, 视网膜色素细胞就可以在不同浓度的脱细胞外植体上播种, 并培养用于下游实验。本文所述的制备和程序处理是至关重要的, 注意隔离刷膜的方向, 确保层流表面在不接触表面的同时, 避免破坏细胞外基质表面结构。
还应指出的是, 在使用刷的膜外植体系统时, 有一些变量可能会影响下游实验的结果, 如单个捐献者基因组背景、刷膜的年龄或刷的死后时间。膜, 以及刷膜的位置, i. e, 刷膜外植体的中央或外围部分。与任何其他人体组织研究一样, 你必须招募合适的捐献者眼样本数, 以获得必要的统计能力, 并在适当的时候匹配捐赠者的眼睛年龄。建立严格的眼睛接收标准是一个关键的参数。只接受无核少于10小时的眼睛, 刷的膜制剂可以在死后 24-48 小时内收获。视神经部位可作为定位标记, 并用于实验和对照研究组的相同位置。通过采取这些措施, 你可以减少实验性的变异性。
总而言之, 对视网膜色素刷的膜脉络综合体的理解以及它是如何受到年龄和疾病的影响, 对于了解其对 AMD 病理生理学的贡献至关重要。使用 "体外" 技术的模型系统是利用人体组织来调查这些影响的宝贵工具. 在这里, 我们描述了一种方法, 利用人类捐献者外植体作为相关的前体内模型的老年刷的膜。这项技术允许使用老化和/或患病的人类刷的膜作为研究工具, 以调查如何结构变化的矩阵可能影响覆盖 RPE 细胞行为, 包括依恋, 增殖和基因表达25,27. 类似的前体模型系统的成功开发将进一步了解 AMD 并促进新的治疗方案的发展。

人类刷膜的年龄相关结构变化是由多种因素引起的, 包括 nitrosative 和氧化应激, 对视网膜色素上皮 (RPE) 细胞的功能产生多种有害影响, 有助于与年龄相关的黄斑变性 (AMD)的病理学1,2,3,4, 5,6,7,8,9.在考虑细胞置换治疗晚期萎缩性 AMD 或地理萎缩时, 治疗可能需要将细胞移植到视网膜色素上皮细胞萎缩的床上。由于细胞外基质的损伤6,9,10,11 ,人类刷膜内与年龄相关的变化会对移植的 RPE 细胞移植的成功产生不利影响。,12,13,14,15,16,17,18,19,20,21,22. 研究人体刷的膜生物学以及基质内部的结构变化如何促进 AMD 的发展, 对了解疾病病理学至关重要。因此, 在与年龄相关的眼部领域的调查人员迫切需要制定一些协议来描述老年人和/或患病的人类刷膜的前体收获。
历史上, 很难建立与年龄相关的疾病的模型, 如地理萎缩和人类刷在动物体内的细胞膜23。这一困难产生于多种因素, 包括人类的长寿与啮齿类动物和其他经常用于疾病建模的物种相比, 以及大多数脊椎动物中缺乏黄斑24。本文所描述的方法的优点是, 细胞可以直接在刷的膜上进行检测, 从老年和/或患病的人的死后眼中提取。本文的总目标是为老年和/或患病的人刷膜的体外模型提供一个详细的方法, 包括从人类捐献者中分离人刷的膜外植体和视网膜色素细胞的播种下游实验。该模型可以作为一个相关的模型来研究细胞外基质损伤对视网膜色素上皮细胞功能和病理的贡献20,25,26,27,28。

<table><tbody><tr><td>Human donor globes</td><td>National Disease Research Interchange, NDRI</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle medium (DMEM)</td><td>Thermo Fisher Scientific</td><td>11995-065</td><td></td></tr><tr><td>Carbon-dioxide independent media</td><td>Thermo Fisher Scientific</td><td>18045-088</td><td></td></tr><tr><td>60-mm polystyrene petri dish</td><td>Corning Inc.</td><td>351007</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline, PBS</td><td>Thermo Fisher Scientific</td><td>14190144</td><td></td></tr><tr><td>Hydrophobic 65 &amp;micro;m-thick polytetrafluoroethylene membrane with 0.2 &amp;micro;m pores</td><td>Merck Millipore</td><td>JGWP04700</td><td></td></tr><tr><td>Fisherbrand glass microanalysis vacuum filter holder system&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>09-753-102</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A2576-5G</td><td></td></tr><tr><td>35 &amp;times; 10mm culture dish</td><td>VWR International</td><td>25373-041</td><td></td></tr><tr><td>Trephine</td><td>Accutome</td><td>AM0570 60</td><td></td></tr><tr><td>96 well plate</td><td>Corning Inc.</td><td>3595</td><td></td></tr><tr><td>Minimum essential media (MEM)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11095-080&amp;nbsp;</td><td></td></tr><tr><td>Penicillin G</td><td>Sigma-Aldrich</td><td>P3032-1MU</td><td></td></tr><tr><td>Streptomycin</td><td>Sigma-Aldrich</td><td>S6501</td><td></td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1914</td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>1397-89-3</td><td></td></tr><tr><td>Ammonium hydroxide solution</td><td>Sigma-Aldrich</td><td>1336-21-6&lt;br/&gt; &amp;nbsp;</td><td></td></tr></tbody></table>

culturing of retinal pigment epithelial cells on an ex vivo model of aged human bruch's membrane

除了与年龄相关的眼部疾病研究推荐的维生素和抗氧化剂外, 没有有效的治疗 "干" 或萎缩年龄相关的黄斑变性 (AMD), 代表90% 的病例。治疗需要减缓或延缓地理萎缩 (GA) 的发展, 了解刷的膜病理学是这一过程的一部分。人类刷膜的改变在 AMD 的进展之前, 导致视网膜色素上皮 (RPE) 细胞的损伤。由于缺乏足够的动物模型来研究 AMD, 老年人类刷膜的体外模型是研究永生化和原代细胞系视网膜色素上皮细胞行为和诱导多能干细胞产生的 rpe 线的有用工具. 细胞 (iPSCs)。在这里, 我们提出了一个详细的方法, 允许一个确定的视网膜色素上皮细胞行为的影响, 从人类的捐献者刷的膜外植体, 包括附着, 凋亡和增殖, 吞噬的能力片段, 极性的建立和基因表达。本试验提供了一种在老化/受损细胞外基质上对视网膜色素上皮细胞的功能特性进行评估的刷膜的前体模型。

当该协议正确执行时, 可以通过建立极性和外血视网膜屏障, 生长因子和细胞因子的极化分泌, 来确定衰老和/或患病的人刷膜对视网膜色素上皮细胞功能的影响,感光棒外段的吞噬功能。
我们已经生成了一些数据, 显示了老年人类刷膜上的一种疾病表型。例如, 在老年人类刷的膜外植体上培养 rpe 细胞会降低视网膜色素上皮细胞的再附着 (图 3)。在老化的人刷膜上培养的 RPE 细胞降低了这些细胞对吞噬棒外段 (ROS) 的能力, 这是一个重要的 rpe 功能 (图 4)。此外, 在这些按钮外植体上, 可以用终端 deoxynucleotidyl 转移酶 dUTP 尼克末端标记 (隧洞) 染色来识别和比较凋亡细胞, 如前一工作26所述。使用微阵列技术可以确定老年和/或患病的人刷膜对视网膜色素上皮细胞基因表达谱的影响。我们已经表明, 视网膜色素上皮细胞基因表达的变化时, 人类刷的膜, 从较年轻的个人外植体 (图 5)27。在统计分析中, 每组至少使用 3-5 人捐献者的外植体。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57084/57084fig1v2.jpg"> 图1。从供体眼中分离人刷膜的示意图.人类刷的细胞膜是通过去除巩膜, 前段, 视网膜, 玻璃体, 和本机视网膜色素上皮 (RPE) 细胞来收获的。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57084/57084fig1v2large.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/57084/57084fig2.jpg"> 图2。在培养皿中隔离的人类刷膜 (BM).(A)人体刷膜外植体的示意图 (直径每1.5 英寸, 凝固琼脂糖凝胶) 放置在 60 x 15 毫米聚苯乙烯细胞培养皿 (BM 面上), 在盘子底部装上温暖的液体琼脂糖。(B)一个人刷的膜外植体, 已被 trephined 成6毫米圆形按钮 (植体), 每一个, 然后放在4% 琼脂糖在37°c 在一个未处理的聚苯乙烯井的96井板。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57084/57084fig2large.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/57084/57084fig3.jpg"> 图3。青年和老年人刷膜 (BM) 视网膜色素上皮 (RPE) 细胞再附着率.最初的视网膜色素细胞被播种到人刷的膜外植体从年轻 (< 50 岁, n = 3) 或更老 (> 70 岁, n = 5) 捐献者眼睛三周。MTT 细胞活性测定法测定细胞附着率。老年人类刷的细胞膜降低了视网膜色素上皮细胞附着率 24% (年轻的 bm 100 +/-8.54 东南亚 vs 老 bm 76.65 +/-1.44 东南亚)。**p < 0.05, 东南亚 = 标准错误。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57084/57084fig4.jpg"> 图4。视网膜色素上皮 (RPE) 细胞吞噬功能受人类刷膜的年龄影响.在老年人刷的膜外植体上培养的原发性视网膜色素上皮细胞有降低吞噬棒外部段 (ROS) 的能力 (年轻的 bm 873 +/-9.3 东南亚, n = 5 vs 老 bm 608 +/-25.4 东南亚, n = 5)。p < 0.01, 东南亚 = 标准错误。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57084/57084fig5.jpg"> 图5。在年轻或年长的人类刷膜外植体上培养的每个样品中所表达的视网膜色素上皮 (RPE) 细胞基因的数量.有更多的散布在最初的视网膜色素细胞中的基因, 种子在老年与年轻的人类刷的膜 (6201 388 vs 6439 @ 80, 分别);对每个年龄组的五个外植体进行了测试。在所有 Cai 的作者的许可下提交, H et al。27.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57084/57084fig5large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>

Watch this Scientific Journal Video about Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane at JoVE.com

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

本协议的目的是展示视网膜色素上皮 (RPE) 细胞在老年和/或患病的人刷膜的培养。该方法适用于细胞外基质上皮细胞行为的研究。

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

culturing-retinal-pigment-epithelial-cells-on-an-ex-vivo-model-aged

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

10.1K 次观看.  Yale School of Medicine. 本协议的目的是展示视网膜色素上皮 (RPE) 细胞在老年和/或患病的人刷膜的培养。该方法适用于细胞外基质上皮细胞行为的研究。

视频: Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

视网膜色素上皮的生理与病理生理学研究的实验模型

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

科研

神经科学

Enrichment of Bruch's Membrane from Human Donor Eyes

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of the center&#160;of the retina, called the macula, resulting in the loss of central vision. Early AMD is characterised by the presence of small, yellowish lesions called soft drusen that can progress onto late AMD such as geographic atrophy (dry AMD) or neovascularisation (wet AMD). Although the clinical changes are well described, and the understanding of genetic influences on conferring AMD risk are getting ever more detailed, one area lacking major progress is an understanding of the biochemical consequences of genetic risk. This is partly due to difficulties in understanding the biochemistry of Bruch&#8217;s membrane, a very thin extracellular matrix that acts as a biological filter of material from the blood supply and a scaffold on which the retinal pigment epithelial (RPE) cell monolayer resides. Drusen form within Bruch&#8217;s membrane and their presence disrupts nutrient flow to the RPE cells. Only by investigating the protein composition of Bruch&#8217;s membrane, and indeed how other proteins interact with it, can researchers hope to unravel the biochemical mechanisms underpinning drusen formation, development of AMD and subsequent vision loss. This paper details methodologies for enriching either whole Bruch&#8217;s membrane, or just from the macula region, so that it can be used for downstream biochemical analysis, and provide examples of how this is already changing the understanding of Bruch&#8217;s membrane biochemistry.

Identifying proteins specifically associated with Bruch&#8217;s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

从人类供体眼富集布鲁赫的膜

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

长期，无血清培养有机小鼠视网膜外植与完整的视网膜色素上皮

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

新型人类组织特征视网膜培养技术的特征

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

医学

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 ...

培养的原代鼠 RPE 的表征和形态动力学

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

作者聚焦：从成年小鼠中有效分离原代视网膜色素上皮细胞用于视网膜病变研究

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

小鼠视网膜色素上皮细胞的原代培养

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

生物学

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

哺乳动物原色素上皮细胞的分离、培养和基因工程用于非病毒基因治疗

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

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此视频中的章节

视网膜色素上皮的生理与病理生理学研究的实验模型

从人类供体眼富集布鲁赫的膜

长期，无血清培养有机小鼠视网膜外植与完整的视网膜色素上皮

新型人类组织特征视网膜培养技术的特征

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

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

小鼠视网膜色素上皮细胞的原代培养

哺乳动物原色素上皮细胞的分离、培养和基因工程用于非病毒基因治疗