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 border="0" cellpadding="0" cellspacing="0" width="1219">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:599px;">Human donor globes</td>
			<td style="width:305px;">National Disease Research Interchange, NDRI</td>
			<td style="width:148px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carbon-dioxide independent media</td>
			<td>Thermo Fisher Scientific</td>
			<td>18045-088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60-mm polystyrene petri dish</td>
			<td>Corning Inc.</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s phosphate buffered saline, PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrophobic 65 &#181;m-thick polytetrafluoroethylene membrane with 0.2 &#181;m pores</td>
			<td>Merck Millipore</td>
			<td>JGWP04700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand glass microanalysis vacuum filter holder system&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>09-753-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A2576-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 &#215; 10mm culture dish</td>
			<td>VWR International</td>
			<td>25373-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trephine</td>
			<td>Accutome</td>
			<td>AM0570 60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well plate</td>
			<td>Corning Inc.</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum essential media (MEM)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11095-080&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin G</td>
			<td>Sigma-Aldrich</td>
			<td>P3032-1MU</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>S6501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G1914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>1397-89-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium hydroxide solution</td>
			<td>Sigma-Aldrich</td>
			<td>1336-21-6 
			&#160;</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/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/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/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

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

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

优化<em&gt;前OVO</em&gt;鸡胚培养，以发展的高级阶段

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

科研

发育生物学

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 ...

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

The objective of this report is to describe the protocols to derive the retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells using different sizes of embryoid bodies.

从诱导多能干得出视网膜色素上皮（RPE）（IPS），由胚体大小不同的细胞

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

建立临床相关<em&gt;离体</em&gt;调查上皮创伤修复的微环境本地人模拟白内障手术模型

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

一个<em&gt;离体</em&gt;方法培养大鼠肠系膜微血管网络的时间推移成像

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

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 ...

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

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

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

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

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...