这项研究由 BrightFocus 基金会 (DS) 部分资助。

涉及动物问题的程序已获得匹兹堡大学机构动物保育和使用委员会 (IACUC) 的批准。
1. 准备解决方案
<ol><li>通过补充 Dulbecco 修饰的鹰培养基 (DMEM)、高葡萄糖、10% 胎牛血清 (血清)、1% 青霉素/链霉素、2.5 毫米 l-谷氨酰胺和1x 内存不必要的氨基酸, 制备生长培养基。预热的媒体在37摄氏度孵化器使用前。</li>
	<li>准备 2% (酶) II 工作解决方案:<ol>
<li>在300µL HEPES 缓冲盐水 (50 毫米 HEPES/KOH pH 7.4, 150 毫米氯化钠) 中溶解30毫克的酶 ii 酶, 制备100毫克/毫升的库存溶液。这个体积是 3-4 小鼠, 它可以放大或下降的基础上的小鼠数量的实验)。该股票可以储存至少1周在4摄氏度。</li>
		<li>2% 酶的工作解决方案, 加入300µL 100 毫克/毫升酶 II 库存到1.2 毫升的无菌 DMEM, 高葡萄糖和过滤溶液通过0.22 µm 过滤器。此步骤应在无菌条件下层流柜内进行。预热前准备酶 II 在37°c 孵化器使用前。</li>
	</ol>
</li>
</ol>2. 获取鼠标眼睛
<ol><li>使用任何已批准的方法弄死鼠标, 并将其放在吸水垫上。</li>
	<li>戴上手套, 把拇指和食指放在眼睛周围, 轻轻地推着皮肤 proptose 眼球。</li>
	<li>在皮肤和眼球之间插入尖角剪刀, 小心地剪掉眼睛。为了避免污染, 在70% 乙醇中简单地蘸上眼睛。</li>
	<li>立即把眼睛在磷酸盐缓冲盐水 (PBS) 的培养皿。</li>
	<li>重复步骤 2.2-2.4 以移除第二只眼睛。</li>
</ol>3. 眼部解剖
<ol><li>在解剖显微镜下小心地从眼睛中取出结缔组织。这可以做在 PBS 或干培养皿。<ol>
<li>使用缝合钳从眼球中提起结缔组织, 用 Vannas 剪刀将其切开。不要切开巩膜, 因为如果巩膜被切开, 几乎不可能获得完整的后眼罩。</li>
		<li>清洗结缔组织后, 将所产生的眼球放在 PBS 中进行下一个洗涤步骤。 注: 以下步骤应在无菌条件下层流柜内进行。</li>
	</ol>
</li>
	<li>两次洗眼睛前预热37°c DMEM (高葡萄糖)。用镊子小心地将眼球转到预热的37摄氏度 DMEM (高葡萄糖)。在新的盘子中吸入培养基并将眼球转移到新鲜的培养基上。</li>
	<li>吸 DMEM (高葡萄糖) 培养基和孵化的眼睛在预热 2% (重量/卷) 酶 II 工作解决方案45分钟在37°c 孵化器。 注意: 在以下所有步骤中, 在生长培养基中放置和操作眼睛或眼罩, 这将保护组织并为解剖提供良好的培养基。</li>
	<li>移除酶 II 溶液, 通过吸旧介质并加入新的预热37°c 生长介质 (图 1a), 在生长培养基中两次冲洗眼睛。 注: 酶后, 眼球变软。</li>
	<li>用钳夹住眼睛, 用 Vannas 剪刀在层流柜内的解剖显微镜下切开每只眼睛周围的青锯。<ol>
<li>保持眼球在溶液中, 小心地移除前角膜通过扩大切口周围的锯缘, 并拉前角膜离开后, 切口已经完成。</li>
		<li>用齿钳 (图 1b, 1c) 轻轻拉出眼罩, 取出晶状体囊和相关的虹膜色素上皮。</li>
	</ol>
</li>
	<li>孵化后眼罩在生长培养基中的20分钟, 在37摄氏度, 以促进分离的神经视网膜从视网膜色素。</li>
	<li>在层流柜内放置解剖显微镜下, 将后眼罩切成4瓣。切口应该足够长以使眼罩平坦, 但短到足以保持花瓣连接 (图 1d)。<ol>
<li>扁平化的眼罩和消除神经视网膜通过非常轻轻地拉它与钳, 而持有其余的 RPE-脉络膜-巩膜复合体与另一个钳 (图 1e)。从边缘而不是从中心开始拉。</li>
	</ol>
</li>
	<li>将所有的被扎营的组织转移到一个新的无菌培养皿中, 预热37摄氏度生长培养基。</li>
</ol>4. 原发性视网膜色素上皮细胞的分离
<ol><li>在解剖显微镜下, 用显微外科月牙刀在刷的基底膜 (膜) 上轻轻剥离出完整的视网膜色素片, 用超细钳夹住一瓣。放置在层流流柜中 (图 1f)。<ol>
<li>使用 P200 微将视网膜色素表转移到含有生长培养基的新型无菌培养皿中。当转移视网膜色素表时, 通过吹打多次湿润吸管提示和生长培养基, 以防止组织在尖端内黏附。视网膜色素从脉络膜可以明显区分: 视网膜色素看起来更褐色, 而脉络膜更暗, 看起来黏糊糊和蓬松。</li>
		<li>或者, 使用酶消化和温和离解没有钳, 以分离视网膜色素从脉络膜14。</li>
	</ol>
</li>
	<li>用预热的37°c 生长培养基在无菌培养皿中冲洗 2-3 倍的 RPE 片。每个洗涤步骤后, 仔细收集视网膜色素表, 同时避免其他组织, 如视网膜碎片或脉络膜。在最后洗涤的结尾收集视网膜色素表与 P200 微并且安置他们在一个新的1.5 毫升离心管。 注: 没有必要离心的视网膜色素表: 他们将在1分钟内的重力沉积。</li>
	<li>使用 P200 微移除额外的生长介质。并用重悬新鲜的预预热生长培养基中的细胞, 并用 P200 微轻轻 triturate 它们。避免气泡的形成。单细胞悬浮是理想的, 但也避免太多的研磨, 否则视网膜色素细胞无法生存。我们建议非常温和的研磨 40-50 倍。</li>
</ol>5. 培养视网膜色素上皮细胞
<ol><li>将细胞印在培养板上。<ol>
<li>如果视网膜色素上皮细胞的下游应用需要保留其极性, 则将2只小鼠的视网膜色素上皮细胞从12毫米聚酯膜插入预加载到12井培养板中。电镀细胞高密度是需要的, 因为在这种情况下, RPE 细胞能够保持原有的性质, 如六角形和色素沉着。</li>
	</ol>
</li>
	<li>不要移动板块或改变培养前72小时的生长培养基。3天后, 用新鲜的预热生长培养基取代旧培养基。以后那改变文化媒介每隔一天。一旦视网膜色素细胞得到融合, 将生长培养基中的血清减少到2%。 注: 细胞可以使用0.25% 胰蛋白酶分裂。然而, 分裂后的细胞可能会失去其六角形和色素沉着在随后的段落。我们不建议分裂细胞, 但如果它是需要的下游应用的文化, 请记住, 主要细胞不能无限期传代: 他们被称为消除区分后 5-7 通道。</li>
</ol>

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DS 已获得拜耳医疗保健, 德国和 f. 霍夫曼-罗氏, 瑞士的研究资金。其余的作者声明没有竞争的财政利益。这项工作是支持的研究, 以防止失明 (不受限制的赠款威尔默眼科研究所和匹兹堡大学)。 这项工作还由匹兹堡大学眼科的初创基金支持。

所提出的详细协议能够可靠地建立在1周后到达融合的小鼠原发性视网膜色素培养物, 并在2周后呈现主要的视网膜色素特征, 如六角形和色素沉着。所获得的 RPE 细胞可用于一些下游应用, 如维生素 a 代谢9, 感光外段的吞噬功能10和离子传输11, 上皮细胞极性2, 溶酶体稳态和自噬12,13。?...

视网膜色素上皮 (RPE) 是一个单一层的极化上皮细胞位于神经视网膜和脉络膜之间的眼睛。视网膜色素上皮细胞的功能和 rpe 单层的完整性对视力是至关重要的, 因为视网膜色素上皮在维持外血-视网膜屏障、水和离子在视网膜之间的传输等多个过程中起着重要作用。脉络膜, 光吸收, 保护免受氧化应激, 控制甲酸代谢, 和吞噬的外部部分的感光细胞1,2。视网膜色素在眼睛后的位置, 以及它的屏障功能, 防止药物管理系统地从血液传递到玻璃体的幽默, 使人们很难研究视网膜色素功能在体内的复杂性。因此, 很有必要建立视网膜色素细胞培养, 以研究在一个灵活的, 受控环境中的 rpe 细胞3,4。
有许多已建立的 RPE 细胞线存在, 提供了一种方便和方便的方式获取和储存的细胞;但是, 与主单元格2、3、4相比, 传代单元格有一些缺点。首先, 它们通常以细胞形态学的变化为特征。例如, 由于细胞极性表型的丧失和紧连接的部分消失4, 现有的细胞系都没有发现适合于对 RPE 屏障特性进行可靠的研究。除了极性的丧失和细胞对细胞的正常连接外, 视网膜色素上皮细胞系由于在成人视网膜色素沉着5中没有关键的黑酶而迅速失去色素。色素沉着可以恢复, 但对重新着色机制的综合分析, 将包括透射电镜, 基因表达分析和化学化验的结合, 以确认黑色素的存在从来没有已执行6。还有一个限制是视网膜色素上皮细胞系延长了细胞的寿命 (有时是不朽的), 在某些条件下, 可以转化为自更新的多能干细胞, 分离出基质, 形成漂浮的菌落7,8. 这一限制使得无法使用细胞系进行移植实验3。
考虑到已建立的视网膜色素细胞系的缺点, 从新鲜组织获得的原发性视网膜色素上皮细胞培养可能成为研究视网膜色素的一种更具生物学意义的模型。主要的视网膜色素细胞不仅用于研究视网膜色素特异功能, 如维生素 A 代谢9, 感光外段的吞噬作用10和离子传输11, 但也研究基本细胞生物学, 如上皮细胞极性2 , 溶酶体稳态和自噬12,13。
在过去的几年中, 有许多关于建立主要视网膜色素培养的出版物, 表明对这一研究领域的兴趣越来越浓厚3,14,15。许多人类视网膜色素细胞和非人类视网膜色素上皮细胞的协议, 如牛和猪视网膜色素上皮细胞被发布了16,17,18,19。然而, 由于它们的体积小得多, 所以处理小鼠视网膜色素细胞更加困难。尽管相当多的出版物已经描述了将 rpe 细胞从鼠标14、20、21中隔离出来的协议, 但仍有许多研究人员在努力隔离 rpe 细胞而不神经视网膜碎片对脉络膜细胞或细胞的污染。在这里, 我们提出了建立主要的小鼠 RPE 细胞培养的协议, 包括从老鼠的眼睛, 眼睛的解剖和视网膜色素表的分离, 以获得细胞培养。该视频协议将特别有用的研究员谁开始工作的小鼠主要视网膜色素培养和需要指导的解剖技术。

<table><tbody><tr><td>&lt;strong&gt;animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2~3-week old wildtype mice</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM), high glucose</td><td>GIBCO</td><td>11965092</td><td></td></tr><tr><td>Dispase II</td><td>Sigma</td><td>D4693</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F4135</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>GIBCO</td><td>15140122</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100X)</td><td>GIBCO</td><td>11140050</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS), 1X, pH 7.4</td><td>GIBCO</td><td>10010023</td><td></td></tr><tr><td>HEPES</td><td>Cellgro</td><td>61-034</td><td></td></tr><tr><td>KOH</td><td>Sigma-Aldrich</td><td>1310-58-3</td><td></td></tr><tr><td>NaCl</td><td>Ambion</td><td>AM9759</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>GIBCO</td><td>25030-081</td><td></td></tr><tr><td>Ethyl Alcohol</td><td>Fisher Scientific</td><td>111000200</td><td></td></tr><tr><td>RPE65 antibody</td><td>A gift from Dr. Michael Redmond</td><td></td><td></td></tr><tr><td>Fluorescein Phalloidin&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Invitrogen</td><td>F432</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L), Alexa Flour 568&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Invitrogen</td><td>A11011&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Goat serum</td><td>Sigma-Aldrich</td><td>G9023</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Polysciences, Inc</td><td>00380</td><td></td></tr><tr><td>ZO-1 Polyclonal Antibody</td><td>ThermoFisher Scientific</td><td>40-2200</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;instruments and equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Laminar flow cabinet</td><td>Baker</td><td>SterilGARD SG403A</td><td></td></tr><tr><td>Dissecting microscope (Zoom stereomicroscope)</td><td>Nikon</td><td>SMZ1500</td><td></td></tr><tr><td>CO2 incubator with hot air sterilization</td><td>Binder</td><td>C150</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher Scientific</td><td>0875712</td><td></td></tr><tr><td>12 mm Polyester Membrane Inserts Pre-Loaded in 12-Well Culture Plates, Pore Size: 0.4 &amp;micro;m, Sterile</td><td>Corning Incorporated</td><td>COR-3460</td><td></td></tr><tr><td>Westcott tenotomy scissors, std blades, sharp</td><td>STEPHENS instruments</td><td>S7-1320</td><td></td></tr><tr><td>Castroviejo suturing forceps 0.12mm</td><td>Stroz Ophthalmic Instruments</td><td>E1796</td><td></td></tr><tr><td>Crescent straight knife</td><td>Beaver-Visitec International</td><td>373808</td><td></td></tr><tr><td>Dumont Tweezers #5, 11 cm, Straight, 0.1x0.06 mm Tips, Dumostar</td><td>World Precision Instruments</td><td>500233</td><td></td></tr><tr><td>Vannas Scissors, 8 cm, 45&amp;deg; Angle, Standard</td><td>World Precision Instruments</td><td>500260</td><td></td></tr><tr><td>Millex-GS Syringe Filter Unit, 0.22 &amp;micro;m</td><td>Millipore</td><td>SLGL0250S</td><td></td></tr><tr><td>Syringe, 5 mL</td><td>BD</td><td>309632</td><td></td></tr><tr><td>Inverted Laboratory Microscope&amp;nbsp;Leica DM IL LED</td><td>&amp;nbsp;Leica&amp;nbsp;</td><td></td><td></td></tr><tr><td>Pipette</td><td>Gilson</td><td></td><td></td></tr><tr><td>Barrier and non-filtered pipette tips</td><td>Thermo Scientific</td><td></td><td></td></tr></tbody></table>

primary cell cultures from the mouse retinal pigment epithelium

视网膜色素上皮 (RPE) 是一种高度极化的多功能上皮, 位于神经视网膜和眼脉络之间。这是一个单一的色素细胞, 是六边形包装和连接紧密交界处。视网膜色素的主要功能包括光的吸收、棚感光外段的吞噬、离子的空间缓冲、养分的输送、离子和水以及在视觉循环中的积极参与。对视网膜色素上皮细胞的生物学研究具有重要而多样的作用。已经建立了一些视网膜色素上皮细胞系;然而, 传代和永生化细胞很快就失去了自然视网膜色素细胞的一些形态学和生理特性。因此, 主细胞更适合研究视网膜色素上皮细胞生物学和功能的不同方面。小鼠原发性视网膜色素上皮细胞培养对研究人员非常有用, 因为小鼠模型在生物研究中被广泛应用, 然而从老鼠那里采集视网膜色素细胞也很有挑战性, 因为它们的体积小。在这里, 我们提出了建立主要的小鼠 RPE 细胞培养的协议, 包括眼睛的摘除和解剖和视网膜色素表的分离, 以产生细胞培养。此方法可实现有效的单元恢复。从两个小鼠获得的 RPE 细胞可以达到融合在一个12毫米聚酯膜插入预加载的文化板块后, 一个星期的文化, 并显示一些原始的性质, 如六角形和色素沉着在两个周的文化。

2只小鼠在12毫米聚酯膜插入物中建立的小鼠原发性视网膜色素培养在1周的培养后达到 90-95% 融合。经过2周的培养, 细胞达到100% 融合, 并开始形成一个镶嵌的六角, 有色和双核细胞。3周的文化, 细胞继续形成的形状和色素沉着, 然而, 4 周后, 部分细胞得到了 hyperpigmented (图 2)。
主要视网膜色素细胞培养的纯?...

Watch this Scientific Journal Video about Primary Cell Cultures from the Mouse Retinal Pigment Epithelium at JoVE.com

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

视网膜色素上皮 (RPE) 是一种多功能的眼睛上皮。在这里, 我们提出了建立从小鼠视网膜色素形成的主要细胞培养的协议。

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 ...

primary-cell-cultures-from-the-mouse-retinal-pigment-epithelium

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

21.0K 次观看.  University of Pittsburgh School of Medicine. 视网膜色素上皮 (RPE) 是一种多功能的眼睛上皮。在这里, 我们提出了建立从小鼠视网膜色素形成的主要细胞培养的协议。

视频: Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

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

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

生物学

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

长期，无血清培养有机小鼠视网膜外植与完整的视网膜色素上皮

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

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 ...

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 ...

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.

小鼠视网膜色素上皮细胞的原代培养

摘要

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视网膜色素上皮的生理与病理生理学研究的实验模型

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长期，无血清培养有机小鼠视网膜外植与完整的视网膜色素上皮

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

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

猪视网膜色素上皮细胞原代培养

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