Name: culturing of retinal pigment epithelial cells on an ex vivo model of aged human bruch's membrane
Uploaded: 2018-04-12T14:00:00
Duration: 8 min 32 s
Description: 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

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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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

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