Name: efficient dissection and culture of primary mouse retinal pigment epithelial cells
Uploaded: 2021-02-10T14:30:00
Description: Watch this Scientific Journal Video about Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells at JoVE.com

这项工作得到了麻省眼耳眼科基因组研究所的支持。

遵循了《在眼科和视力研究中使用动物的ARVO声明》的准则。
注：这种方法已被证明对不同遗传背景的小鼠成功，包括C57BL/6J，B10。D2-Hco H2d H2-T18 c/oSnJ和白化病小鼠，在不同年龄。最好使用8到12周大的小鼠来获得RPE细胞。来自老老鼠的RPE细胞在培养中繁殖较少，而幼鼠的细胞越来越小，这就要求汇集不同动物的眼睛才能有可行的培养物。
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A., Pereira, A., Saxon, J., Peachey, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+retinal+pigment+epithelium+dysfunction+is+concomitant+with+hyperglycemia+in+mouse+models+of+type+1+and+type+2+diabetes.">Early retinal pigment epithelium dysfunction is concomitant with hyperglycemia in mouse models of type 1 and type 2 diabetes.</a> Journal of Neurophysiology. 113 (4), 1085-1099 (2015).</li><li>Garland, D. 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=Mouse+genetics+and+proteomic+analyses+demonstrate+a+critical+role+for+complement+in+a+model+of+DHRD/ML,+an+inherited+macular+degeneration.">Mouse genetics and proteomic analyses demonstrate a critical role for complement in a model of DHRD/ML, an inherited macular degeneration.</a> Human Molecular Genetics. 23 (1), 52-68 (2014).</li><li>Fu, 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=The+R345W+mutation+in+EFEMP1+is+pathogenic+and+causes+AMD-like+deposits+in+mice.">The R345W mutation in EFEMP1 is pathogenic and causes AMD-like deposits in mice.</a> Human Molecular Genetics. 16 (20), 2411-2422 (2007).</li><li>Greenwald, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Models+of+NMNAT1-Leber+Congenital+Amaurosis+(LCA9)+Recapitulate+Key+Features+of+the+Human+Disease.">Mouse Models of NMNAT1-Leber Congenital Amaurosis (LCA9) Recapitulate Key Features of the Human Disease.</a> American Journal of Pathology. 186 (7), 1925-1938 (2016).</li><li>Gupta, P. R., 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=Ift172+conditional+knock-out+mice+exhibit+rapid+retinal+degeneration+and+protein+trafficking+defects.">Ift172 conditional knock-out mice exhibit rapid retinal degeneration and protein trafficking defects.</a> Human Molecular Genetics. 27 (11), 2012-2024 (2018).</li><li>Gibbs, D., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+primary+mouse+retinal+pigmented+epithelial+cells.">Isolation and culture of primary mouse retinal pigmented epithelial cells.</a> Advances in Experimental Medicine and Biology. 533, 347-352 (2003).</li><li>Bonilha, V. L., Finnemann, S. C., Rodriguez-Boulan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ezrin+promotes+morphogenesis+of+apical+microvilli+and+basal+infoldings+in+retinal+pigment+epithelium.">Ezrin promotes morphogenesis of apical microvilli and basal infoldings in retinal pigment epithelium.</a> Journal of Cell Biology. 147 (7), 1533-1547 (1999).</li><li>Nandrot, E. F., 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=Loss+of+synchronized+retinal+phagocytosis+and+age-related+blindness+in+mice+lacking+&#945;v&#946;5+integrin.">Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking &#945;v&#946;5 integrin.</a> Journal of Experimental Medicine. 200 (12), 1539-1545 (2004).</li><li>Maminishkis, A., 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=Confluent+monolayers+of+cultured+human+fetal+retinal+pigment+epithelium+exhibit+morphology+and+physiology+of+native+tissue.">Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.</a> Investigative Ophthalmology and Visual Science. 47 (8), 3612-3624 (2006).</li><li>Maminishkis, A., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+for+study+of+retinal+pigment+epithelial+physiology+and+pathophysiology.">Experimental models for study of retinal pigment epithelial physiology and pathophysiology.</a> Journal of Visualized Experiments. (45), (2010).</li><li>Brydon, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV-Mediated+Gene+Augmentation+Therapy+Restores+Critical+Functions+in+Mutant+PRPF31+/&#8722;+iPSC-Derived+RPE+Cells.">AAV-Mediated Gene Augmentation Therapy Restores Critical Functions in Mutant PRPF31+/&#8722; iPSC-Derived RPE Cells.</a> Molecular Therapy - Methods and Clinical Development. 15, 392-402 (2019).</li><li>Shang, P., Stepicheva, N. A., Hose, S., Zigler, J. 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虽然在1、13、20、22、26、27之前已经开发出几种小鼠RPE细胞分离和培养方法，但费尔南德斯-戈迪诺的方法首先使用膜插入，使RPE细胞在培养中的有效生长长达1周，即9周。他们的协议1，9...

该协议最初由费尔南德斯-戈迪诺等人于2016年1月报告，描述了一种有效隔离和培养小鼠视网膜色素上皮（RPE）细胞的方法，该细胞在一周内在Transwell板块上形成功能性和极性RPE单层。RPE 是位于神经视网膜和布鲁赫膜之间的眼睛中的单层。这一层由高度极化和色素化的上皮细胞组成，由紧密的结点连接，呈现出类似于蜂巢2的六边形形状。尽管这种明显的组织学简单，RPE执行各种功能的关键视网膜和正常的视觉周期2，3，4。RPE单层的主要功能包括光吸收、光感受器的滋养和更新、代谢端产品的去除、亚视网膜空间中的离子平衡控制以及血视网膜屏障2、3的维护。RPE在眼睛5、6、7、8、9、10、11的局部调节中也起着重要的作用。RPE的退化和/或功能障碍是许多眼部疾病的共同特征，如视网膜炎色素沉着症，勒伯先天性动脉瘤，白化病，糖尿病视网膜病变，和黄斑变性12，13，14，15。不幸的是，人体组织的可用性是有限的。鉴于小鼠与人类高度保存的遗传同源性，小鼠模型是研究眼部疾病16、17、18、19的合适和有用的工具。此外，使用培养的原发性RPE细胞提供的优势，如基因操纵和药物测试，可以加快开发新的疗法，这些危及视力的疾病9，11。
可用于鼠标 RPE 隔离和文化的现有方法缺乏可重复性，并且无法以足够的可靠性在体内重新概括 RPE 功能。细胞往往在几天内失去色素化，六角形和跨皮电阻（TER）在培养13，20。由于从小鼠身上建立这些初级RPE细胞培养是一个具有挑战性的过程，这个优化的协议已经创建基于其他协议，从大鼠和人眼分离RPE细胞21，22，23解剖小鼠的眼睛，收集RPE和培养小鼠RPE细胞在体外。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 ml BD Luer-Lok tip syringe, disposable</td>
			<td>BD Biosciences</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>VWR International</td>
			<td>21008-103</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube</td>
			<td>VWR International</td>
			<td>21008-951</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium</td>
			<td>Sigma-Aldrich</td>
			<td>M4526-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled micro forceps</td>
			<td>WPI</td>
			<td>501727</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench-top centrifuge</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo</td>
			<td>HERA VIOS 160I CO2 SST TC 120V</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phospate Buffered Saline no Calcium, no Magnesium</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 45&#176; Medical Biology tweezers, 0.05 x 0.01 mm tip, 11 cm length</td>
			<td>WPI</td>
			<td>14101</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Easy-Grip Clear Polystyrene Cell Culture Dish, 35mm</td>
			<td>BD Biosciences</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td>Heat inactivated.</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution plus Calcium and Magnesium, no Phenol Red</td>
			<td>Life Technologies</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution plus Calcium and Magnesium, no Phenol Red B6</td>
			<td>Life Technologies</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1M</td>
			<td>Gibco</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma-Aldrich</td>
			<td>H-3506 1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H-0396</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Thermo</td>
			<td>CLASS II A2 4 115V PACKAGECLA</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin 1mg/ml</td>
			<td>Sigma-Aldrich</td>
			<td>L2020-1 MG</td>
			<td>Dilute in PBS at 37C to 1mg/ml</td>
		</tr>
		<tr>
			<td>McPherson-Vannas Micro Scissors 8 cm long</td>
			<td>WPI</td>
			<td>503216</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids 100X</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>N1 Supplement 100X</td>
			<td>Sigma-Aldrich</td>
			<td>N6530-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-148</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Bard-Parker Carbon steel surgical blade size 11</td>
			<td>Fisher-Scientific</td>
			<td>08-914B</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T-0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture treated 12-well plates</td>
			<td>Fisher-Scientific</td>
			<td>08-772-29</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture treated 6-well plates</td>
			<td>Fisher-Scientific</td>
			<td>14-832-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell supports 6.5 mm</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3470-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodo-thyronin</td>
			<td>Sigma-Aldrich</td>
			<td>T-5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer, Dumont #5 Medical Biology 11 cm, curved, stainless steel 0.02 x 0.06 mm Mod tips</td>
			<td>WPI</td>
			<td>500232</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors 8cm long, stainless steel</td>
			<td>WPI</td>
			<td>501790</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Puradisc 25mm Syringe Filters 0.45&#956;m pore size</td>
			<td>Fisher-Scientific</td>
			<td>6780-2504</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient dissection and culture of primary mouse retinal pigment epithelial cells

眼疾影响全球数百万人，但人体组织的有限可用性阻碍了他们的研究。小鼠模型是了解眼科疾病病理生理学的有力工具，因为它们与人体解剖学和生理学相似。视网膜色素上皮（RPE）的改变，包括形态和功能的变化，是许多眼部疾病共有的共同特征。然而，成功隔离和培养原鼠RPE细胞是非常具有挑战性的。本文是费尔南德斯-戈迪诺等人之前在 2016 年发布的协议的更新视听版本，旨在有效隔离和培养主要鼠标 RPE 细胞。这种方法具有高度可重复性，并导致高度极化和色素化的 RPE 单层的坚固培养物，可在 Transwells 上保持数周。该模型为研究眼部疾病背后的分子和细胞机制开辟了新的途径。此外，它提供了一个平台，用于测试治疗方法，可用于治疗重要的眼部疾病与未满足的医疗需求，包括遗传性视网膜疾病和黄斑变性。

该协议已被用来隔离和培养RPE细胞从转基因小鼠1。没有观察到小鼠菌株或性别之间的差异。研究结果有助于了解眼部疾病机制的一些重要方面，如与年龄有关的黄斑变性，这是老年人视力丧失的最常见原因。在此协议后分离的 RPE 细胞在播种后 24 小时内完全连接到膜插入上，并在 72 h1后显示典型的 RPE 大小、形态和色素沉着。一周后，一个高?...

Watch this Scientific Journal Video about Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells at JoVE.com

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

该协议最初由费尔南德斯-戈迪诺等人于2016年1月报告，描述了一种有效分离和培养小鼠RPE细胞的方法，该细胞在一周内在Transwell板上形成功能和极化的RPE单层。手术大约需要3个小时。

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

此协议允许对鼠标 RPE 细胞进行有效的隔离和培养。健康的原鼠RPE培养是了解眼部疾病的机理的宝贵模型。融合和可行的 RPE 文化在三天内获得，可以种植数周。该议定书已成功地用于了解与年龄早期有关的黄斑变性背后的机制，这是发达国家老年人失明的主要原因。可视化解剖步骤非常重要。细节对于协议的成功至关重要，例如，如何处理眼球，使硬皮球不被打，究竟应该在哪里进行切口，以及如何将眼杯嵌入到trypsin中，以便它们保持开放。首先为协议准备所有必要的试剂。然后，开始清洁眼球，小心地去除所有的结缔组织，血液和肌肉，使用杜蒙五号钳子和斜剪刀，而不会在硬皮上做任何切口。定期将眼球转移到含有三毫升无 HEPES HBSS 缓冲器的新鲜菜肴中，以保持眼睛新鲜和清洁，并避免任何污染。使用视神经作为手柄，以保持眼球，并在角膜中心用锋利的碳钢11号刀片打一个洞。用杜蒙五号剪刀在角膜上切开三个切口，确保有足够的空间去除镜片。握住视神经，用斜剪刀的底座对奥拉塞拉塔施加轻微压力，直到镜头完全出来。保持虹膜上皮完好无损，以防止潜伏期间神经网膜和 RPE 的分离。将眼睛放在无高音速的 HBSS 缓冲区中。在37摄氏度的透明质溶液中孵化眼睛，在5%的二氧化碳加气孵化器中45分钟，将神经视网膜从RPE分离出来。将每只眼睛放在一口新井中，每口井有1.5毫升的冷HBSS HEPES缓冲器，并在冰上孵育30分钟。洗涤后，将每只眼睛放入一个35毫米的文化菜肴与新鲜的HBSS HEPES缓冲器。用八厘米的Vannas剪刀切开角膜，直到到达奥拉塞拉塔，然后切到奥拉塞拉塔下面，去除虹膜上皮和角膜。用弯曲的钳子握住奥拉塞拉塔，使用倾斜的微钳子，拉开神经网膜，确保 RPE 层不被切割。然后，切断视神经的内部附件。切开视神经，将每个眼杯转移到不同的12个油井板中，每口井含有1.5毫升的新鲜trypsin-EDTA。确保眼杯保持打开，并完全淹没在试剂中。在37摄氏度下将眼杯孵化45分钟，在5%的二氧化碳孵化器中。将每个眼杯与在尝试蛋白孵化过程中分离的任何 RPE 片一起收集，并将其转移到包含每口油井 1.5 毫升 FBS 溶液的 12 个井板中。如果 RPE 表仍保留在试穿素溶液中，请使用微管将它们转移到带 FBS 解决方案的井中。将每个眼杯由视神经按住，朝下摇动到 12 个井板中，其中含有 1.5 毫升 20% FBS 的 HBS HEPES 缓冲器，直到 RPE 板完全分离。用微管收集任何 RPE 片和 RPE 聚类，避免任何可能污染培养物的白色鳞片或胆汁，并将它们放置在 15 毫升管中。把两只眼睛从同一只老鼠身上拉到一根管子里。然后，在室温下将 RPE 混合物在 340 x g 下离心两分钟，然后丢弃超母体。轻轻将 RPE 颗粒以 0.25% 的 trypsin-EDTA 混合物中重新注入，并在 37 摄氏度的水浴中孵育一分钟，将 RPE 片分解成单个细胞。然后轻轻地吹笛器上下10次，避免气泡形成。加入九毫升新制备的 RPE 介质，稀释并灭活 trypsin，并在室温下以 340 x g 将混合物离心两分钟。吸气超自然，并小心地用 5% FBS 将细胞颗粒重新喷入 150 微升 RPE 介质中。接下来，从先前准备的层膜插入的底腔中取出 PBS，并添加 700 微升 RPE 介质。从膜插入的上腔取出层蛋白，并顺便将 RPE 细胞悬架滴入并均匀地分配到腔室的中心。将膜在37摄氏度的5%二氧化碳孵化器中不受干扰地插入，至少24小时。然后检查显微镜下的膜插入，以确保大多数 RPE 细胞连接到插入物上，汇合率至少为 50% 在头 72 小时内不要更改介质。使用此协议分离的 RPE 细胞显示了典型的 RPE 大小、形态和色素化。一周后观察到一个高度极化的RPE单层，有两个核，由紧密的交汇点连接，以及存在杏微维利和基底折叠。RPE 单层的极化得到了 TER 值的确认，平均平方度高于 200 欧米厘米，随着时间的推移保持稳定。在 FITC 标记牛 POS 上执行的噬菌体检测中，观察到 POS 的吞没和消化，表明 RPE 细胞的功能汇合单层形成。可行的文化需要很短的解剖时间，这是通过经验实现的。一些 RPE 细胞可能保持附着在神经网状网膜上，但如果保留几个 RPE 表，协议将不会成功。必须每次使用新鲜的透明质酶来防止此问题。这些功能性原生小鼠RPE细胞培养是研究许多眼疾中RPE病理生物学的宝贵工具。例如，此协议使得在小鼠黄斑变性模型中研究 RPE 的病理生物学成为可能。

Research

JoVE Journal

Biology

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前初级人类输卵管上皮细胞的体外培养

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科研

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隔离和鼠标主动脉内皮细胞原代培养

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Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

小鼠视网膜色素上皮细胞的原代培养

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猪视网膜色素上皮细胞原代培养

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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 ...

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从人眼高效、一致地生成视网膜色素上皮/脉络膜平面安装，用于组织学分析

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...