Name: primary culture of porcine retinal pigment epithelial cells
Uploaded: 2022-09-23T14:00:00
Description: Watch this Scientific Journal Video about Primary Culture of Porcine Retinal Pigment Epithelial Cells at JoVE.com

作者希望对所有在这项研究中贡献细胞的动物表示感谢和尊重。这项研究部分得到了中国国家重点研发计划（2019YFA0111200，Yi Liao & Yuan Gao和Grant Nos. 2018YFA0107301，Wei Li）的资助。作者感谢厦门大学医学院中心实验室的黄静茹和尤翔在共聚焦成像方面的技术支持。

实验动物的使用符合视觉与眼科研究协会（ARVO）的规定，并得到厦门大学实验动物管理伦理委员会的批准。
1. 实验手术器械、组织消化酶、细胞培养缓冲液的制备
<ol><li>准备实验性手术装置，在眼球解剖前一天清洁并高压灭菌两对眼科手术剪刀和镊子，然后在65°C的通用方案烤箱中用手术器械干燥盒子过夜。</li>
	<li>培养基制备。<ol><li>制备补充有 10% （v/v） 胎...

<ol>
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在这里，描述了用于从猪眼球中分离，培养和表征RPE细胞的详细和优化方案，该方案为RPE细胞的体外表征和RPE相关疾病研究提供了良好的模型。从人，小鼠和大鼠眼睛中分离RPE的方法已在前面描述过23，24，25。然而，在一些实验室中很难获得人类的眼球，并且通常会引发伦理问题。小鼠和大鼠的RPE组织相对较小，细胞在...

视网膜色素上皮 （RPE） 位于视网膜1 外层的感光器和脉络膜毛细血管之间，具有多种功能，包括形成血液-视网膜屏障、运输和交换营养物质和视网膜代谢物、回收维生素 A 以维持正常的视觉周期，以及吞噬和清除脱落的感光器外段 （POS）2，3.由于POS需要不断的自我更新才能产生视力，因此RPE细胞需要不断吞噬分离的POS以维持视网膜稳态4。因此，RPE功能障碍导致许多致盲性眼病，如年龄相关性黄斑变性（AMD）4，视网膜色素变性（RP）5，Leber先天性黑朦6，糖尿病视网膜病变7等。到目前为止，大多数这些疾病的确切发病机制仍然难以捉摸。因此，建立了RPE细胞培养来研究RPE细胞生物学，病理变化和潜在机制。
作为研究细胞生物学的最简单模型，RPE细胞的培养早在1920年代就开始了8。尽管ARPE-19被广泛用作RPE细胞，但色素沉着的丧失，鹅卵石形态，特别是该细胞系中的屏障功能引起了很多关注9。相比之下，原代人RPE细胞的培养为生理和病理研究提供了更现实的场景9。然而，相对有限的可用性限制了它们的使用，道德问题始终存在。此外，几个小组使用小鼠模型培养RPE细胞。但小鼠眼睛尺寸小，单一培养通常需要多只小鼠，不方便9.最近，科学家们开发了利用人类胚胎干细胞或诱导多能干细胞来衍生RPE细胞的新方法。尽管该技术在治疗遗传性RPE疾病方面具有特殊潜力，但它非常耗时，通常需要几个月才能产生成熟的RPE细胞10。为了克服这些问题，我们在这里介绍了一种易于遵循的方案，用于在实验室中常规分离和培养高纯度RPE细胞。在合适的培养条件下，这些细胞可以显示出典型的RPE功能并表现出典型的RPE形态。因此，该培养方法可为了解RPE生理学、研究细胞毒性、研究相关眼病的病理机制、进行药物筛选提供良好的模型。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ARPE-19 cells</td>
			<td>CCTCC</td>
			<td>GDC0323</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Yeasen</td>
			<td>36101ES60</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td>Zeiss</td>
			<td>LSM 880 with Airyscan</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Touch</td>
			<td>Bio-Rad</td>
			<td>1708370</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Sangon</td>
			<td>F619301</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish</td>
			<td>NEST</td>
			<td>121621EH01</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well culture plate</td>
			<td>NEST</td>
			<td>29821075P</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F12 Medium</td>
			<td>Gibco</td>
			<td>C11330500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM basic Medium</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM2</td>
			<td>World Precision Instruments</td>
			<td>EVOM2</td>
			<td>For TER measurement</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ExCell Bio</td>
			<td>FSP500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Mouse IgG (H/L):HRP</td>
			<td>Bio-Rad</td>
			<td>0300-0108P</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Rabbit IgG (H/L):HRP</td>
			<td>Bio-Rad</td>
			<td>5196-2504</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrocortisone</td>
			<td>MCE</td>
			<td>HY-N0583/CS-2226</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342 solution (20 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 96 Instrument</td>
			<td>Roche</td>
			<td>5815916001</td>
			<td></td>
		</tr>
		<tr>
			<td>Liothyronine</td>
			<td>MCE</td>
			<td>HY-A0070A/CS-4141</td>
			<td></td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM(1X)+GlutaMAX Medium</td>
			<td>Gibco</td>
			<td>10566-016</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA(100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP syringe filter unit</td>
			<td>Millipore</td>
			<td>SLGPR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>N1</td>
			<td>Sigma-Aldrich</td>
			<td>SLCF4683</td>
			<td></td>
		</tr>
		<tr>
			<td>NcmECL Ultra</td>
			<td>New Cell&#38;Molecular Biotech</td>
			<td>P10300</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat Powdered Milk</td>
			<td>Solarbio</td>
			<td>D8340</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>SparkJade</td>
			<td>SJ-MV0061</td>
			<td></td>
		</tr>
		<tr>
			<td>Na+-K+ ATPase antibody</td>
			<td>Abcam</td>
			<td>ab76020</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>PAGE Gel Fast Preparation Kit(10%)</td>
			<td>Epizyme</td>
			<td>PG112</td>
			<td></td>
		</tr>
		<tr>
			<td>primary Human RPE cells&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>Generous gift from Shoubi Wang lab&#160;</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad by Dotmatics</td>
			<td>version 8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktails</td>
			<td>APExBIO</td>
			<td>K1024</td>
			<td></td>
		</tr>
		<tr>
			<td>PRE65 antibody</td>
			<td>Proteintech</td>
			<td>17939-1-AP</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>PEDF antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-390172</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>100 x penicillin/streptomycin&#160;</td>
			<td>Biological Industries</td>
			<td>03-031-1BCS</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>RARBIO</td>
			<td>RA-9005</td>
			<td></td>
		</tr>
		<tr>
			<td>ReverTra Ace qPCR RT Master Mix</td>
			<td>Toyobo</td>
			<td>FSQ-201</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile centrifuge tubes</td>
			<td>NEST</td>
			<td>601052</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tubes</td>
			<td>NEST</td>
			<td>602052</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Damas-beta</td>
			<td>107-35-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo-Fisher&#160;</td>
			<td>15596026</td>
			<td>RNA extraction solution</td>
		</tr>
		<tr>
			<td>TB Green Fast qPCR Mix</td>
			<td>Takara</td>
			<td>RR430A</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well transwell inserts</td>
			<td>Labselect</td>
			<td>14212</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF antibody</td>
			<td>Proteintech</td>
			<td>19003-1-AP</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>VEGF ELISA kit</td>
			<td>Novusbio</td>
			<td>VAL106</td>
			<td></td>
		</tr>
		<tr>
			<td>ZO-1 antibody</td>
			<td>ABclonal</td>
			<td>A0659</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
	</tbody>
</table>

primary culture of porcine retinal pigment epithelial cells

视网膜色素上皮（RPE）是单层极化色素上皮细胞，位于视网膜的脉络膜和神经视网膜之间。多种功能，包括吞噬作用，营养/代谢物运输，维生素A代谢等，由RPE每天进行。RPE细胞是终末分化的上皮细胞，具有很少或没有再生能力。RPE细胞的缺失会导致多种眼部疾病，导致视力障碍，例如年龄相关性黄斑变性。因此，建立原代RPE细胞的 体外 培养模型，其与 体内 RPE比细胞系更相似，对于RPE细胞的特征和机理研究至关重要。考虑到人类眼球的来源有限，我们创建了一个培养原代猪RPE细胞的方案。通过使用该方案，RPE细胞可以很容易地从成年猪眼球中解离。随后，这些解离的细胞附着在培养皿/插入物上，增殖形成汇合的单层，并在2周内快速重建 体内 上皮组织的关键特征。通过qRT-PCR，证明原代猪RPE细胞表达的多个特征基因与天然RPE组织相当，而这些基因中的大多数在人RPE样细胞ARPE-19中丢失/高度降低。此外，免疫荧光染色显示紧密连接、组织极性和细胞骨架蛋白的分布，以及培养的原代细胞中存在对维生素 A 代谢至关重要的异构酶 RPE65。总之，我们开发了一种易于遵循的方法来培养具有高纯度和天然RPE特征的原代猪RPE细胞，这可以作为了解RPE生理学，研究细胞毒性和促进药物筛选的良好模型。

将原代猪RPE（pRPE）细胞在含有10%FBS的DMEM/碱性培养基中培养，并在接种后2天（图2A）、6天（图2B）和10天（图2C）在光学显微镜下拍摄细胞形态。1周后，观察到具有鹅卵石形态的单层色素沉着pRPE细胞汇合。
为了更好地表征原代pRPE细胞，将传代3（P3）18和ARPE-19细胞的原代人RPE细胞（hRPE）在DME...

Watch this Scientific Journal Video about Primary Culture of Porcine Retinal Pigment Epithelial Cells at JoVE.com

Primary Culture of Porcine Retinal Pigment Epithelial Cells

本文介绍了一种易于遵循的体 外 培养原代猪视网膜色素上皮细胞的方法。

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. ...

RPE相关疾病仍然是混合疾病的主要部分，但在原代RPE细胞的虚拟培养中没有有效的残留物，无法作为RPE相关疾病的生理和病理学研究的良好模型。在该协议中，我们提供了一种易于遵循的实验方案来培养初级IPC，可以快速恢复和维持以后的RP特征，我们相信通过使用这种方法，人们可以从无痕状态研究和保护性药物筛选中快速产生RP细胞，这可以促进RPE疾病的新治疗方法的开发。分步价值演示将使该方法更容易在想要研究RP细胞的不同实验室中复制。开始解冻组织消化酶马拉夸德并灭菌1X PBS。通过0.22微米注射器过滤装置过滤溶液，补充2%青霉素和2%链霉素。用1X PBS清洗培养板和跨孔插入物的孔。用移液管从孔和转孔中取出PBS，并向下室中加入一毫升新鲜的1X PBS，向上室中加入600微升每毫升10微克的层粘连蛋白溶液。将板和Transwell插入物在该螺旋培养箱中在37摄氏度和5%二氧化碳下孵育过夜。之后，从上室中取出层压板溶液，并在就位之前用一毫升冷的 1X PBS 洗涤两次，用 75% 乙醇和紫外线清洁层压板流罩。准备三个含有约25毫升75%乙醇的50毫升无菌离心管，三个含有约25毫升1X PBS的50毫升无菌离心管，以及三个含有约15毫升1X PBS的10厘米无菌细胞培养皿。将四个猪眼球浸泡在10厘米培养皿中约15毫升75%乙醇中，用一把剪刀和镊子切断所有残留的结缔组织和肌肉。要净化眼球，请依次浸泡并清洗三个装有75%乙醇的50毫升无菌离心管中的四个眼球。然后在三个装有1X PBS的50毫升无菌离心管中依次洗涤眼球。每分钟倒置一次管子以彻底清洗眼球。将四个眼球移动到含有1X PBS的10厘米无菌细胞培养皿中。再次修剪每个眼球的外表面，以去除视神经和小碎屑。用剪刀在角膜缘和巩膜的交叉处做一个小切口。然后去除角膜、虹膜、晶状体、玻璃体和神经视网膜。将RPE脉络膜巩膜复合物转移到新的10厘米细胞培养皿中，并进行四次切割，将眼杯平整成四叶草的形状。通过将四个 RPE 脉络膜巩膜复合物放入新的 10 厘米培养皿中并倒入 20 毫升新鲜胰蛋白酶 EDTA 溶液以合并 RPE 脉络膜巩膜复合物，在 EDTA 溶液消化中进行旅行。将培养皿放入37摄氏度的细胞培养箱中近30分钟。30分钟后，将培养皿从培养箱中取出，向培养皿中加入20毫升含有10%FBS的预热细胞培养基。为了中和EDTA溶液中的行程，请使用5毫升移液器通过轻轻移液数次来解离RPE细胞。将细胞悬浮液收集到15毫升离心管中，然后用FBS轻轻移液另外10毫升新鲜培养基，以洗涤培养皿上的RPE脉络膜巩膜复合物。通过在室温下以300G离心管5分钟来收集RPE细胞。使用移液管吸出超级剂，并用FBS再加入5毫升培养基以重新悬浮细胞。以 300 G 离心再离心五分钟。倒出超级试剂并用12毫升培养基重新悬浮细胞。接下来每孔接种100， 000至200， 000个细胞到12孔培养板或跨孔插入物中。每两天更换一次培养基，当细胞完全覆盖每个插入物的孔表面时，将血清浓度降低至1%。每两天更换一次培养基，直到收获细胞。此处显示了原代猪RPE细胞在第二天，第六天和第10天的代表性图像。通过该图形图像检测原代猪RPE细胞，原代人RPE细胞，ARPE 19细胞和猪RPE脉络膜组织中特征基因的mRNA水平的QRTPCR分析。在这里，GAP DH被用作RTPCR的管家基因。原代猪RPE细胞使用4个生物学重复，RPE 19细胞使用3个生物学重复，其中3个生物重复用于原代人RPE细胞和猪RPE脉络膜组织。将ARPE 19细胞中的基因表达水平设置为对照。ARPE 19和猪RPE细胞以及原代人RPE细胞和猪RPE脉络膜组织中RPE 65蛋白的西方血液分析如图所示。长春斑素在这里用作加载控制。该图显示了在含有1%FBS的DMEM基本培养基中猪RPE细胞的两周培养。用或不含层粘连蛋白培养的细胞用RPE 65，钠钾APTA，Z01和Hex染色。图中描述了用或不带层粘连蛋白培养的细胞片中的TR测量值。该图显示了原代猪RPE和ARPE 19细胞中血管内皮生长因子或VEGF和色素上皮衍生因子或PEDF的西方血液分析。这些图像显示了培养的原代猪RPE和ARPE 19细胞的部门功能。该协议的重要部分是从RPE神经胶质复合物中溶解RP细胞。我们建议每次使用新鲜行程和溶液，并适当控制消化时间，以溶解八个RP细胞。

primary-culture-of-porcine-retinal-pigment-epithelial-cells

Research

JoVE Journal

Biology

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

科研

生物学

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

人类iPS细胞的微小RNA表达谱，视网膜色素上皮衍生自IPS和胎儿视网膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

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 ...

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 ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

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 ...

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 ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

使用高分辨率呼吸测定法实时分析原代人视网膜色素上皮细胞中的生物能量学

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

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 ...

从人体组织中分离乳腺上皮细胞的方案

Isolation and Culture of Primary Human Mammary Epithelial Cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

作者聚焦：东西方医学结合治疗肉芽肿性乳腺炎

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

原代人乳腺上皮细胞的分离和培养

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...