作者要感谢 Farhad Farjood 在猪 RPE 细胞培养和分离方面的帮助，以及 Thomas Harris 在 SEM 方面的帮助。作者感谢犹他州立大学显微镜核心设施对SEM分析的支持。该设施维护着通过美国国家科学基金会主要研究仪器资助（CMMI-1337932）获得的扫描电子显微镜。这项研究的资金由美国国立卫生研究院通过拨款 1R15EY028732 （Vargis） 和 BrightFocus 基金会拨款M2019109 （Vargis） 提供。犹他州立大学研究办公室的本科生研究和创意机会补助金（Weatherston）和犹他州立大学阿尔茨海默病和痴呆症研究中心的种子补助金（Vargis）提供了额外的资金。

该程序中使用的眼睛是从当地经美国农业部检查的肉店死后获得的，并且不使用活体动物进行任何工作。在动物被处死后，大约2小时后眼睛被摘除。由于组织腐烂可能开始发生，因此在运输过程中保持眼睛凉爽以防止进一步腐烂非常重要。在此过程中，眼睛在摘除后立即放入冰箱中。随后，将眼袋放置在 1000 mL 聚丙烯烧杯内，并在 8 L 冷却器内被冰包围。重要的是不要将眼睛直接放在冰上。一旦眼睛到达实验室，RPE细胞的分离就会在生物安全柜内进行。在 3-5 小时内完成此过程至关重要。该协议已针对处理 10 只眼睛进行了优化。
1. 制备 DNase 原液等分试样
注意:建议在隔离之前执行此步骤。
<ol><li>用无菌去离子水制备5mM氯化钙溶液。 注意: 氯化钙粉末会引起严重的眼睛刺激或损伤。穿戴适当的个人防护装备。</li>
	<li>向 DNase 粉末中加入 5 mM 氯化钙溶液（参见 材料表），得到最终 DNase 浓度为 3 mg/mL。确保溶液充分混合。 注意:DNase 是一种呼吸道致敏危害。不要吸入粉末/物质。</li>
	<li>将小体积（例如 1 mL）等分装到微量离心管中。</li>
	<li>将等分试样冷冻在-20°C直至使用。</li>
</ol>2. 设置解剖区域
<ol><li>将无菌解剖仪器和细胞培养设备转移到生物安全柜中:手术窗帘、培养皿、细胞过滤器、孔板、纱布、镊子、手术刀柄、手术刀刀片和虹膜剪刀（见 材料表）。</li>
	<li>使用镊子，铺开手术布。将一块 6 孔板放在手术悬垂物上，然后取下盖子。</li>
	<li>将等分试样的无菌杜尔贝科磷酸盐缓冲盐水 （DPBS） 放在冰上并放入生物安全柜中。</li>
	<li>将 6 孔板的一个孔填充一半（约 6 mL）的 10% 聚维酮碘溶液。</li>
	<li>用冷却的DPBS填充剩余的孔。</li>
	<li>从其中一个培养皿上取下盖子，将其倒置在手术窗帘上。将培养皿底座作为废物容器放在一边。</li>
	<li>将等分试样的细胞培养基（Dulbecco改良的Eagle培养基（DMEM），10%胎牛血清（FBS），1%抗生素/抗真菌剂）和0.25%胰蛋白酶/ EDTA的等分试样放入37°C水浴中。</li>
	<li>从冰箱中取出DNase储备溶液（有关详细信息，请参阅方案的其余部分）。</li>
</ol>3. 去除外部组织
注意:外部组织去除可以在生物安全柜外进行。切割前检查眼睛，确保巩膜没有被刺穿，瞳孔没有雾化，并且存在视神经。丢弃不符合这些标准的眼睛。
<ol><li>用一只手握住附着在眼睛上的肌肉，用另一只手用手术刀轻轻切掉巩膜周围的组织。小心不要压伤眼睛或意外切开巩膜。如果巩膜被切开并露出黑色内部，请丢弃眼睛。 注意:强烈建议握着肌肉的手戴上防割手套，以防止意外割伤。</li>
	<li>用弯曲的虹膜剪刀修剪视神经，长度不超过 3 毫米。如果视神经太长，眼罩以后将无法正确放置在细胞过滤器中。</li>
	<li>将眼睛放在冰上，角膜朝下。</li>
	<li>重复步骤 3.1-3.3，直到从每只眼睛上取出外部组织。</li>
</ol>4.眼球解剖
<ol><li>用镊子将眼睛转移到生物安全柜和含有10%聚维酮碘溶液的孔中，并旋转眼睛以确保其完全涂覆在碘溶液中。为了尽可能保持工具无菌，这些镊子只能用于触摸眼睛的外部。</li>
	<li>当眼睛坐在碘溶液中时，将无菌纱布放在先前准备好的培养皿的浅倒盖上。在单独的培养皿中，放置细胞过滤器。仅使用细胞过滤器来支撑眼罩，而不是用于细胞分离。</li>
	<li>让眼睛在碘溶液中静置约 30 秒后，将眼睛浸入含有 DPBS 的孔中约 5 秒，从一个孔移动到另一个孔以逐渐稀释碘溶液，然后将眼睛转移到无菌纱布上。</li>
	<li>用手术刀在锯齿肌下方做一个小切口，或从虹膜边缘向下大约1/3处做一个切口。</li>
	<li>切开后，用虹膜剪刀沿眼球圆周均匀切割，平行于角膜。操作眼睛时，只能使用镊子触摸眼睛外侧或纱布以保持无菌。</li>
	<li>用镊子抓住视神经，轻轻地将球体从纱布上抬起。玻璃体应该从地球上掉下来。如果需要，轻轻摇晃眼睛以帮助去除玻璃体。 注意: 如果玻璃体很难去除，请尝试在球体上切得更低。</li>
	<li>轻轻地将眼罩放入准备好的细胞过滤器中，眼罩内部朝上。如果不均匀，请使用镊子调整眼罩，使切口尽可能水平。</li>
	<li>将冷却的 DPBS 轻轻添加到眼罩中，使液位刚好低于切口的最低点。该 DPBS 将在神经视网膜切除后移除。 注意: 添加的 DPBS 体积会因眼睛大小和切口位置而异。</li>
	<li>重复步骤4.1-4.8，直到所有眼睛都被解剖。</li>
</ol>5. 神经视网膜切除和RPE解离
注意:以下部分更容易分阶段执行。在这种设置中，此过程一次在一批（通常为 3 个眼罩）上执行。对于步骤 5.1-5.7，在进入下一步之前，应对整个批次执行每个步骤。
<ol><li>在手电筒下，找到神经视网膜（白色/粉红色）开始与 RPE（黑色）分离的任何区域。然后，用钝镊子轻轻抓住抬起的神经视网膜并将其剥离。 注意: 如果没有神经视网膜分离的区域，请用镊子轻轻抓住它靠近眼罩顶部。使用这种方法，如果对 RPE/脉络膜造成损害，这些细胞在 RPE 收集过程中不会解离。</li>
	<li>轻轻收集视盘附近的神经视网膜。</li>
	<li>使用连接到真空抽吸系统的巴斯德移液管吸出神经视网膜。建议羽化吸气以帮助去除过程。 注意:移液器吸头可以放在巴斯德移液器的末端以减少吸力。</li>
	<li>小心地添加额外的冷却 DPBS 以替换吸气的 DPBS。</li>
	<li>通过再次吸气去除剩余的神经视网膜。这一次，在不干扰暴露的 RPE 的情况下尽可能多地删除 DPBS。</li>
	<li>将胰蛋白酶轻轻加入眼罩中。将体积水平保持在切口线和受损 RPE 的任何部分以下，以减少污染。 注意:胰蛋白酶是一种酶，接触时会引起皮肤和眼睛刺激。穿戴适当的个人防护装备。</li>
	<li>将胰蛋白酶添加到每只眼睛后，确保更换培养皿盖。接下来，将培养皿转移到37°C和5%CO2 的加湿培养箱中30分钟。</li>
	<li>对每批重复步骤5.1-5.7，直到处理完所有眼罩。</li>
</ol>6. RPE收集
<ol><li>从每批眼睛（2-3 只眼睛）中收集 RPE 细胞，并放入单个 15 mL 离心管中，使接种过程简单明了。<ol><li>在手电筒下，使用 1000 μL 移液管轻轻研磨以去除 RPE 细胞。如果RPE细胞未分离，则加入新鲜胰蛋白酶并再孵育10-15分钟。小心不要刮擦视网膜。</li>
		<li>将 RPE 细胞收集在 15 mL 离心管中。</li>
		<li>用细胞培养基清洗眼罩以收集任何剩余细胞并中和胰蛋白酶。确保 15 mL 离心管中培养基的体积至少为胰蛋白酶的两倍。</li>
	</ol></li>
	<li>重复步骤6.1，直到从所有眼罩中收集RPE细胞。</li>
	<li>在室温下以250× g 离心细胞悬浮液5分钟。</li>
</ol>7. 制备DNase工作溶液
<ol><li>计算添加到细胞培养基中所需的 3 mg/mL DNase 储备溶液（在步骤 1 中制备）的体积，最终浓度为 100 μg/mL，每管细胞的最终体积为 3 mL（即，对于三个离心管的细胞，需要 9 mL 的 100 μg/mL DNase 溶液）。</li>
	<li>混合适当体积的 DNase 储备溶液和细胞培养基。在工作溶液中使用 DNase 有助于防止细胞结块，并允许细胞均匀接种。</li>
</ol>8. RPE细胞接种
<ol><li>在步骤6.3中离心后，从每个15mL离心管中取出上清液，注意不要破坏细胞沉淀。</li>
	<li>将每个细胞沉淀重悬于 3 mL DNase 工作溶液中。</li>
	<li>将含有重悬细胞的15mL离心管放入37°C和5%CO2 的湿润培养箱中15分钟。</li>
	<li>孵育后，在室温下以150× g 离心细胞悬浮液5分钟。</li>
	<li>取出上清液，注意不要破坏细胞沉淀。</li>
	<li>将每个细胞沉淀重悬于 3 mL 新鲜细胞培养基中。</li>
	<li>对于每个含有 2-3 个眼睛的 RPE 细胞的离心管，接种 6 孔板的一个孔（传代 0）。</li>
	<li>小心地将接种的6孔板转移到37°C和5%CO2的加湿培养箱中。</li>
</ol>9. 原代猪RPE细胞培养
<ol><li>在移动平板之前，让新接种的RPE细胞附着48-72小时。在第一次更换培养基期间，可以用新鲜的细胞培养基进行洗涤，以帮助去除多余的细胞碎片。</li>
	<li>每48小时更换一次细胞培养基。</li>
	<li>当细胞达到汇合时，过渡到FBS浓度为2%的细胞培养基中，并保持直至实验。</li>
</ol>10. RPE单元验证
<ol><li>免疫细胞化学（ICC）染色<ol><li>在室温下将细胞固定在DPBS中的4%甲醛中10分钟。</li>
		<li>用DPBS洗涤两次。</li>
		<li>在室温下使用0.2%Triton-X 100在DPBS中透化细胞（如果需要）10分钟。</li>
		<li>用DPBS洗涤两次。</li>
		<li>在室温下在DPBS中施用3%（w / v）牛血清白蛋白（BSA）封闭溶液1小时。</li>
		<li>加入1:100稀释度（或推荐的制造商浓度）的一抗（参见 材料表），并在室温下静置1小时或在4°C下静置过夜。</li>
		<li>用DPBS洗涤三次，每次5分钟。</li>
		<li>加入 2 μg/mL（或推荐的制造商浓度）的二抗（如果需要）（参见 材料表），并在室温下静置 1 小时。</li>
		<li>如果应用二抗，则用 DPBS 洗涤 3 次，每次 5 分钟。</li>
		<li>加入制造商浓度的核染色探针（参见 材料表），并在室温下静置25分钟。</li>
		<li>用PBS洗涤三次，每次5分钟。</li>
		<li>如果在细胞培养插入物上，则使用Rickabaugh和Weatherston等人20概述的封固剂，用盖玻片安装到显微镜载玻片上。否则，请在 DPBS 中对单元格进行映像。</li>
	</ol></li>
	<li>扫描电子显微镜（SEM）<ol><li>遵循 Harris 等人 21 概述的程序。</li>
	</ol></li>
	<li>反式上皮电阻 （TEER）<ol><li>按照制造商的TEER测量方案（参见 材料表），在基底室15中加入1 mL培养基。</li>
	</ol></li>
	<li>酶联免疫吸附试验（ELISA）<ol><li>按照制造商的方案，使用多重人酶联免疫吸附测定试剂盒进行ELISA15 （参见 材料表）。</li>
	</ol></li>
</ol>

从猪眼中分离原代 RPE 细胞的经济高效方法

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C., Schnichels, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+primary+porcine+retinal+pigment+epithelium+monolayer+to+complement+retinal+ex+vivo+cultures.">Establishment of a primary porcine retinal pigment epithelium monolayer to complement retinal ex vivo cultures.</a> STAR Protocols. 4 (3), 102443 (2023).</li><li>Hood, E. M. S., Curcio, C. A., Lipinski, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+cryosectioning+of+primary+porcine+retinal+pigment+epithelium+on+transwell+cell+culture+inserts.">Isolation, culture, and cryosectioning of primary porcine retinal pigment epithelium on transwell cell culture inserts.</a> STAR Protocols. 3 (4), 101758 (2022).</li><li>Toops, K. A., Tan, L. X., Lakkaraju, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+three-step+protocol+for+live+imaging+of+intracellular+traffic+in+polarized+primary+porcine+RPE+monolayers.">A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers.</a> Experimental Eye Research. 124, 74-85 (2014).</li><li>Rickabaugh, E., Weatherston, D., Harris, T. I., Jones, J. A., Vargis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+biomimetic+in+vitro+model+of+bruch's+membrane+using+hagfish+slime+intermediate+filament+proteins.">Engineering a biomimetic in vitro model of bruch's membrane using hagfish slime intermediate filament proteins.</a> ACS Biomaterials Science &amp; Engineering. 9 (8), 5051-5061 (2023).</li><li>Harris, T. I., 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=Utilizing+recombinant+spider+silk+proteins+to+develop+a+synthetic+bruch's+membrane+for+modeling+the+retinal+pigment+epithelium.">Utilizing recombinant spider silk proteins to develop a synthetic bruch's membrane for modeling the retinal pigment epithelium.</a> ACS Biomaterials Science &amp; Engineering. 5 (8), 4023-4036 (2019).</li><li>Zou, X. 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=Protection+of+tight+junction+between+RPE+cells+with+tissue+factor+targeting+peptide.">Protection of tight junction between RPE cells with tissue factor targeting peptide.</a> International Journal of Ophthalmology. 11 (10), 1594-1599 (2018).</li></ol>

该协议描述了如何从猪眼中分离RPE细胞。在分离后 7 天内可以看到色素沉着和鹅卵石形态（图 1B）。此外，TEER 数据表明紧密结形成22 和健康的单层（图 5）。这些结果表明，用这种方法分离的RPE细胞与人RPE相似，在视网膜细胞培养模型中是有益的。
这份手稿中使用的眼睛是死后从当地一家肉店获得的，但也可以从其他来源获得，例如与使用猪产品的合作者的学术环境。重要的是在动物死亡后尽快开始手术，并在手术前保持眼睛凉爽。在分离过程中，在胰蛋白酶消化后研磨之前，必须不要破坏 RPE。在剥离神经视网膜时，这种护理最为关键，因为缺乏谨慎可能会导致用镊子抓住布鲁赫膜1 并将其他细胞类型引入溶液中。
虽然此程序已针对我们的条件进行了优化，但可以对方法进行一些更改。例如，如果需要特定的接种密度，则可以在 DNase 孵育后对细胞进行计数。此外，如果真空抽吸不起作用，可以通过用弯曲的小剪刀仔细修剪来去除神经视网膜。在为实验接种时，可以使用高接种密度（例如 2.5 x 105 个细胞/cm2 ）来防止过度的细胞分裂。此外，不建议重复传代，因为细胞可能开始失去其天然表型。虽然这项研究使用了传代 1 细胞进行实验，但如果 RPE 表型和特征得以维持，则可能会使用额外的传代。最后，如果需要，可以使用浓度为1%的FBS代替2%进行传代培养或实验。
这种方法有一些缺点。首先，难以复制或获得动物的年龄、性别、品种和死亡时间，这些都是原代细胞分离中常见的问题。其次，过度胰蛋白酶消化会迅速导致细胞异常，Fietz 等人17 也注意到了这一点。然而，最终，这种方法提供了一种廉价且有效的方法来获得原代 RPE 细胞。这些细胞不像 iPSC 那样需要较长的分化时间。这些猪 RPE 天生也具有关键结构，如微绒毛和色素沉着15,16，这与未分化的对应物不同。因此，这些细胞为视网膜研究提供了更具代表性的细胞类型。最后，这种方法可能非常有益于更多无法获得人类供体眼睛的农村地区或机构，以产生类似于人类模型的视网膜模型，以了解视力和疾病。

视网膜色素上皮 （RPE） 是位于 Bruch 膜和光感受器之间的视网膜外层的单层1。RPE 细胞与闭塞带-1 （ZO-1） 等蛋白质形成紧密连接，并具有以色素沉着和六边形形态为特征的独特表型 2,3。这些细胞有助于血液-视网膜屏障，从而支持光感受器健康并维持视网膜稳态 4,5。此外，RPE 细胞通过吸收光和回收光感受器的基本成分在视觉中发挥关键作用6.例如，RPE65 是一种在 RPE 细胞中高度表达的蛋白质，可将全反式视黄酯转化为 11-顺式视黄醇 7,8。鉴于 RPE 细胞执行的多种功能，它们的功能障碍与各种疾病有关，包括年龄相关性黄斑变性和糖尿病视网膜病变 9,10。为了加强对视网膜病变的理解并开发新的治疗方法，经常采用视网膜的体外模型。
为了生成健康或患病视网膜的代表性模型，必须使用模拟 RPE 细胞类型。市售的 ARPE-19 细胞系缺乏天然表型，例如色素沉着，而 iPSC 可能需要数月才能分化 11,12,13。尽管人类供体的眼睛可能是理想的，但许多研究实验室通常不容易获得它们。
在这里，我们设计了一种利用猪眼的方法，猪眼与人眼有许多相似之处14，以获得原代RPE细胞。这些原代猪 RPE 细胞已用于多种视网膜模型15,16。这些细胞不仅具有成本效益，而且与iPSC或供体眼睛相比，它们需要更少的时间来获取。此外，它们还表现出天然特征，例如色素沉着和微绒毛。虽然存在类似的猪RPE提取方案17,18,19，但这种简单而详细的技术进一步验证了酶解离，并采用了大多数细胞培养实验室中常见的材料。

<table><tbody><tr><td>6 Micro-well glass bottom plate with 14 mm micro-well #1 cover glass</td><td>Cellvis</td><td>P06-14-1-N</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>Biosafety Cabinet</td><td></td><td></td><td></td></tr><tr><td>Calcium Chloride, Dried, Powder, 97%</td><td>Alfa Aesar</td><td>L13191.30</td><td></td></tr><tr><td>Cell Strainer</td><td>Fisher Scientific&amp;nbsp;</td><td>22-363-548</td><td>one per eye</td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Centrifuge Tubes, 15 mL</td><td>Fisher Scientific&amp;nbsp;</td><td>05-539-12</td><td></td></tr><tr><td>Cooler, 8 L</td><td>Igloo</td><td>32529</td><td></td></tr><tr><td>Corning&amp;nbsp;Transwell Multiple Well Plate with Permeable Polyester Membrane Inserts</td><td>Fisher Scientific&amp;nbsp;</td><td>07-200-154</td><td>Culture inserts</td></tr><tr><td>Cut Resistant Glove</td><td>Dowellife</td><td>712971375857</td><td></td></tr><tr><td>Cytiva&amp;nbsp;HyClone Dulbecco&#039;s Phosphate Buffered Saline, Solution</td><td>Fisher Scientific&amp;nbsp;</td><td>SH3026401</td><td>for ICC dilutions only&amp;nbsp;</td></tr><tr><td>Deionized Water</td><td></td><td></td><td></td></tr><tr><td>DMEM, 1x with 4.5 g/L glucose, L-glutamine &amp;amp; sodium pyruvate</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>DNase I from Bovine Pancreas</td><td>Sigma Aldrich</td><td>DN25</td><td></td></tr><tr><td>DPBS/Modified - calcium - magnesium</td><td>Cytiva</td><td>SH3002B.02</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>ELISA kit, Q-Plex Human Angiogenesis (9-Plex)&amp;nbsp;</td><td>Quansys Biosciences, Logan, UT</td><td></td><td></td></tr><tr><td>ENDOHM 6 TEER device</td><td>World Precision Instruments</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Avantor</td><td>232B20</td><td></td></tr><tr><td>Fisher BioReagents&amp;nbsp;Bovine Serum Albumin (BSA) DNase- and Protease-free Powder</td><td>Fisher Scientific&amp;nbsp;</td><td>BP9706100</td><td></td></tr><tr><td>Flashlight</td><td></td><td></td><td></td></tr><tr><td>Formaldehyde, ACS Grade, 36.5% (w/w) to 38.0% (w/w), LabChem</td><td>Fisher Scientific&amp;nbsp;</td><td>LC146501</td><td></td></tr><tr><td>Gauze Sponges</td><td>Fisher Scientific&amp;nbsp;</td><td>22-415-504</td><td>One per eye</td></tr><tr><td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647, Invitrogen</td><td>Thermo Scientific</td><td>A32728</td><td>RPE65 secondary antibody</td></tr><tr><td>Ice&amp;nbsp;</td><td></td><td></td><td>Crushed prefered</td></tr><tr><td>Inverted Phase Contrast Microscope</td><td></td><td></td><td></td></tr><tr><td>Invitrogen&amp;nbsp;NucBlue Live ReadyProbes Reagent (Hoechst 33342)</td><td>Fisher Scientific&amp;nbsp;</td><td>R37605</td><td></td></tr><tr><td>Iris Fine Tip Scissors, Standard Grade, Curved, 4.5&quot;</td><td>Cole-Palmer</td><td>EW-10818-05</td><td></td></tr><tr><td>Iris Scissors, 11 cm, Straight, Tungsten Carbide</td><td>Fisher Scientific&amp;nbsp;</td><td>50-822-379</td><td></td></tr><tr><td>LSM-710 Confocal Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Petri Dish, 100 mm x 20 mm&amp;nbsp;</td><td>Corning</td><td>430167</td><td>one per 2-3 eyes and one for dissection surface/waste&amp;nbsp;</td></tr><tr><td>Povidone-Iondine Solution, 10%</td><td>Equate</td><td>49035-050-34</td><td></td></tr><tr><td>RPE65 Monoclonal Antibody (401.8B11.3D9), Invitrogen</td><td>Thermo Scientific</td><td>MA116578</td><td>RPE65 primary antibody</td></tr><tr><td>Scalpel Blades Size 10</td><td>Fisher Scientific&amp;nbsp;</td><td>22-079-683</td><td></td></tr><tr><td>Scalpel Handles Style 3</td><td>Fisher Scientific&amp;nbsp;</td><td>50-118-4164</td><td></td></tr><tr><td>Surgical Drape, 18 x 26&quot;</td><td>Fisher Scientific&amp;nbsp;</td><td>50-209-1792</td><td></td></tr><tr><td>Tissue Culture Incubator</td><td></td><td></td><td>37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;, 95% Humidity</td></tr><tr><td>Tissue Culture Plates, 6 Wells</td><td>VWR</td><td>10062-892</td><td>One for eye wash and one for seeding&amp;nbsp;</td></tr><tr><td>Tri-Cornered Polypropylene Beaker, 1000 mL</td><td>Fisher Scientific&amp;nbsp;</td><td>14-955-111F</td><td></td></tr><tr><td>Triton&amp;nbsp;X-100</td><td>Sigma Aldrich</td><td>T8787</td><td></td></tr><tr><td>Trypsin 0.25%, 2.21 mM EDTA in HBSS; w/o Ca, Mg, Sodium Bicarbonate</td><td>Corning</td><td>25053Cl</td><td></td></tr><tr><td>Tweezers Style 20A</td><td>Fisher Scientific&amp;nbsp;</td><td>17-467-231</td><td></td></tr><tr><td>Tweezers Style 2A</td><td>Fisher Scientific&amp;nbsp;</td><td>50-238-47</td><td>for removing neural retina</td></tr><tr><td>Tweezers Style 5-SA-PI</td><td>Fisher Scientific&amp;nbsp;</td><td>17-467-168</td><td></td></tr><tr><td>Vacuum Aspiration System</td><td></td><td></td><td></td></tr><tr><td>Water Bath, 37 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>ZO-1 Monoclonal Antibody (ZO1-1A12), FITC, Invitrogen</td><td>Fisher Scientific&amp;nbsp;</td><td>33-911-1</td><td>ZO-1 conjugated primary antibody</td></tr></tbody></table>

isolation of primary porcine retinal pigment epithelial cells for in vitro modeling

视网膜色素上皮 （RPE） 是视网膜外层负责支持光感受器的关键单层。RPE 变性通常发生在以进行性视力丧失为特征的疾病中，例如年龄相关性黄斑变性 （AMD）。AMD的研究通常依赖于人类供体的眼睛或诱导多能干细胞（iPSC）来代表RPE。然而，这些RPE来源需要更长的分化期和大量的培养专业知识。此外，一些研究机构，特别是农村地区的研究机构，很难获得捐赠者的眼睛。虽然存在市售的永生化 RPE 细胞系 （ARPE-19），但它缺乏基本的 体内 RPE 特征，并且在许多眼科研究出版物中没有被广泛接受。迫切需要从更容易获得和更具成本效益的来源获得具有代表性的原代 RPE 细胞。该方案阐明了从猪眼睛死后获得的原代RPE细胞的分离和传代培养，这些细胞可以从商业或学术供应商处本地采购。该协议需要组织培养实验室中常见的常见材料。其结果是 iPSC、人类供体眼睛和 ARPE-19 细胞的主要、差异化且具有成本效益的替代品。

使用该程序，从猪眼中成功分离出原代RPE细胞。 图 1A 显示了分离后 3 天具有特征性色素沉着的 RPE 细胞。生长1周后，细胞完全融合并形成健康的单层（图1B）。然后将细胞转移到细胞培养插入物中，在那里它们保持其色素沉着和形态（图1C），进一步支持分离程序的功效。没有表现出这些特征，而是显示出变异形态和过度色素沉着的培养物（图1D）被怀疑是细胞污染，并且没有用于实验。在本手稿中分离的 RPE 细胞表达 RPE65，这是一种在 RPE 细胞中高度表达的蛋白质（图 2）。RPE65表达较低的区域可能是由于色素沉着阻断了荧光，因为这些区域对应于色素沉着更严重的细胞（图2A）。分离的RPE细胞表达的另一个特征蛋白是VEGF。10 天后，VEGF 在 RPE 单层基底侧的表达量更高，浓度为 2612.5 ± 28.0 pg/mL，而顶端侧的浓度为 2141.2 ± 53.5 pg/mL（图 3）。原代猪RPE细胞还具有微绒毛和鹅卵石形态（图4）。此外，当在细胞培养插入物上生长时，TEER 读数从 7 天时的 166.1 ± 75.9 Ω∙cm2 增加到 14 天后的 363.4 ± 9.0 Ω∙cm 2 （图 5），表明随着时间的推移形成一致、健康的紧密连接22。为了进一步研究连接的形成，分析了 RPE 细胞的 ZO-1 表达，该表达定位于细胞边界，这意味着健康的连接形成和单层完整性（图 6）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66079/66079fig01.jpg"> 图 1:将初级 RPE 接种到孔板上并传代培养后。分离后 3 天 （A） 的健康 RPE 细胞，分离后 7 天完全融合的单层细胞 （B），RPE 成功传代到培养插入物 （C） 上，以及 RPE 培养物上的潜在细胞污染区域 （箭头） （D）。比例尺 = 100 μm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66079/66079fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66079/66079fig02.jpg"> 图 2:显示猪 RPE 细胞中 RPE65 表达的免疫细胞化学图像。 接种后 15 天细胞的明场 （A）、RPE65 （B）、细胞核 （C） 和合并 （D） 图像。请注意，明场图像中的颗粒状背景是多孔培养插入膜。比例尺 = 50 μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66079/66079fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66079/66079fig03v2.jpg"> 图 3:RPE 单层顶端和基底侧的 VEGF 表达。VEGF浓度通过在培养插入物上细胞生长10天后进行的ELISA测定。误差线表示 n = 2 的标准差。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66079/66079fig03largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66079/66079fig04.jpg"> 图 4:原代猪 RPE 细胞的扫描电子显微镜 （SEM） 图像。 细胞在成像前生长 30 天。比例尺 = 5 μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66079/66079fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66079/66079fig05v2.jpg"> 图 5:对在细胞培养插入物上培养的 RPE 细胞进行的 TEER 读数。 生长 7、10 和 14 天后的阻力值。误差线表示 n = 3 的标准差。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66079/66079fig05largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66079/66079fig06.jpg"> 图 6:显示猪 RPE 细胞中 ZO-1 表达的免疫细胞化学图像。 在玻璃底孔板上接种 6 天后细胞的明场 （A） ZO-1 （B）、细胞核 （C） 和合并 （D） 图像。比例尺 = 50 μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66079/66079fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Author Spotlight: Modeling Retinal Pathologies with Enhanced RPE Cell-Based Disease Models at JoVE.com

作者聚焦:使用增强的 RPE 基于细胞的疾病模型对视网膜病理进行建模

该方案概述了从当地来源的猪眼中获取和培养原代视网膜色素上皮（RPE）细胞的程序。这些细胞可作为干细胞的高质量替代品，适用于 体外 视网膜研究。

用于 体外 建模的原代猪视网膜色素上皮细胞的分离

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly ...

我们设计疾病模型来模拟视网膜病变的各个方面。通过这些模型，我们可以根据RPE细胞对物理变化的反应来更好地了解RPE细胞在疾病中的作用，例如当单层上发生应变或Brex膜随着年龄的增长而增厚时。最大的挑战之一是拥有具有代表性的细胞类型。与常用的永生化细胞系相比，用这种技术分离的原代细胞使我们的疾病模型更真实地反映了我们眼中发生的事情。该技术为我们提供了一种无需大量分化时间即可获得高质量细胞的方法。此外，我们使用研究实验室通常可用的资源，这使得它更具成本效益。展望未来，我们想看看在衰老过程中眼睛发生的自然变化如何导致AMD的早期阶段。我们希望更好地了解这些变化，以便确定具体的治疗目标。

isolation-primary-porcine-retinal-pigment-epithelial-cells-for-vitro

Universidad San Sebastian

Research

JoVE Journal

Biology

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling

892 次观看.  Utah State University. 我们设计疾病模型来模拟视网膜病变的各个方面。通过这些模型，我们可以根据RPE细胞对物理变化的反应来更好地了解RPE细胞在疾病中的作用，例如当单层上发生应变或Brex膜随着年龄的增长而增厚时。最大的挑战之一是拥有具有代表性的细胞类型。与常用的永生化细胞系相比，用这种技术分离的原代细胞使我们的疾病模型更真实地反映了我们眼中发生的事情。该技术为...

视频: 作者聚焦:使用增强的 RPE 基于细胞的疾病模型对视网膜病理进行建模

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

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 ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

猪视网膜色素上皮细胞原代培养

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

小鼠原代视网膜色素上皮细胞的分离

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

从豚鼠眼睛中分离视网膜色素上皮细胞

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

Isolating RPE and IPE Cells from Bovine Eye: A Procedure to  Harvest and Culture Bovine Primary Pigment Epithelial Cells

Source: Bascuas, T. et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/62145">Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy.</a> J. Vis. Exp. (2021).
This video demonstrates the procedure to isolate the retinal pigment epithelial, or RPE, cells and iris pigment epithelial, or IPE, cells from harvested bovine eyes. The isolated cells can be cultured to study their role in the treatment of ocular diseases.

从牛眼中分离 RPE 和 IPE 细胞:收获和培养牛原代色素上皮细胞的程序

Source: Bascuas, T. et al., Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy. J. Vis. ...

用于体外建模的原代猪视网膜色素上皮细胞的分离

副本

摘要

探索更多视频

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

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

猪视网膜色素上皮细胞原代培养

小鼠原代视网膜色素上皮细胞的分离

从豚鼠眼睛中分离视网膜色素上皮细胞

从牛眼中分离 RPE 和 IPE 细胞:收获和培养牛原代色素上皮细胞的程序

用于 体外 建模的原代猪视网膜色素上皮细胞的分离

副本

摘要

探索更多视频

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

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

猪视网膜色素上皮细胞原代培养

小鼠原代视网膜色素上皮细胞的分离

从豚鼠眼睛中分离视网膜色素上皮细胞

从牛眼中分离 RPE 和 IPE 细胞:收获和培养牛原代色素上皮细胞的程序

用于体外建模的原代猪视网膜色素上皮细胞的分离