本出版物中报告的研究得到了 NIH Grants R01EY018341 and R01EY019250 （CSS）、NIH Grant F31EY035156 （AH） 和 P30 EY011373 的支持。资助组织在这项研究的设计或实施中没有任何作用。

本研究中动物受试者的使用得到了凯斯西储大学机构动物护理和使用委员会 （IACUC） 的批准。
1. 试剂制备
<ol><li>通过补充 Hank 平衡盐溶液 （HBSS）、无钙、无镁、无酚红和 10 mM HEPES 缓冲溶液来制备洗涤缓冲液。将溶液保持在 4 °C 直至使用。</li>
	<li>通过在 Dulbecco 改良 Eagle 培养基中补充 4.5 g/L 葡萄糖、1.25x GlutaMAX 补充剂（储备液浓度 100 倍或 200 mM;终浓度为 2.5 mM L-谷氨酰胺）、10% 胎牛血清、1% 青霉素/链霉素和 1x MEM 非必需氨基酸溶液 （100x） 来制备 RPE 培养基。使用前，在水浴中将培养基预热至 37 °C。</li>
</ol>2. 小鼠眼睛摘除
<ol><li>使用 IACUC 批准的安乐死方法对小鼠实施安乐死，最好在 3 至 14 周龄之间（在麻醉下使用氯胺酮/甲苯噻嗪混合物以每 20 克小鼠 0.1 mL [87.5 mg/kg 氯胺酮和 12.5 mg/kg 甲苯噻嗪] 是本研究中使用的方法）。 注意：随着时间的推移，从年轻小鼠收集的眼睛将有助于增加视网膜色素上皮细胞的产量，并延长连续传代的能力。</li>
	<li>将鼠标平放在一侧，眼睛朝上。</li>
	<li>将食指和拇指分别放在眼睛的上方和下方。轻轻按压眼睛周围的骨骼结构，以诱导眼球突出。</li>
	<li>将略微张开的剪刀的尖端插入眼睛下方，轻轻地将手腕从眼睛向外旋转 90°，直到眼睛从眼窝上脱落。 注意： 不要用剪刀剪伤眼睛或视神经，因为这会使眼罩的解剖更加困难。</li>
	<li>立即将眼睛放入 70% 乙醇中并转动不超过 5 秒，然后将其转移到保存在冰上的洗涤缓冲液培养基中。</li>
	<li>在解剖显微镜下，一次将一只眼睛转移到装有洗涤缓冲液和浸泡纱布条的培养皿中。</li>
	<li>用镊子握住视神经来稳定眼睛。使用 3.00 毫米 45° 手术刀或 0.009 英寸剃须刀片在锯齿孔水平轻轻切开。</li>
	<li>使用 Vannas 剪刀，轻轻地将剪刀插入切口，并在锯齿孔的圆周周围剪断，直到可以去除和丢弃前段和玻璃体。</li>
	<li>使用系留镊子轻轻地从眼罩上剥离视网膜，小心不要干扰 RPE 层。 注意：为了使视网膜更容易去除，请在移液管中加入洗涤缓冲液，然后小心地将其涂抹在眼罩的边缘，以轻轻提起视网膜。</li>
	<li>最后，用剪刀从眼罩上去除视神经和多余的结缔组织。小心不要刺穿眼罩。</li>
</ol>3. 原发性 RPE 的分离
<ol><li>将眼罩转移到含有 1 mL 0.25% 胰蛋白酶 + 0.02% EDTA 的 1.5 mL 微量离心管中。 注意：可以将两个眼罩添加到一份胰蛋白酶-EDTA 中，以提高可培养 RPE 的产量。</li>
	<li>将微量离心管转移到设定为 37 °C 的水浴中并孵育 10 分钟。每 2 分钟，从水浴中取出试管，然后将试管底部用力敲击台面 40 次。</li>
	<li>10 分钟后，使用 P1000 或 2 mL 血清移液管上下移液不超过 3 次，轻轻地机械地破坏眼罩。 注意：RPE 片材的碎裂是必不可少的，因为大片材无法正确粘附。</li>
	<li>通过将分离的 RPE 片而不是眼罩分层到 15 mL 锥形管中的 0.5 mL 胎牛血清 （FBS） 上，立即中和胰蛋白酶。</li>
	<li>此外，通过将 3 mL RPE 培养基滴加到 FBS 和 RPE 片层上来稀释胰蛋白酶。</li>
	<li>将 RPE 以 340 x g 离心 3 分钟。</li>
	<li>弃去上清液，将细胞重悬于适量的 RPE 培养基中，用于 24 孔板 （1.9 cm2/孔） 或 48 孔板 （0.75 cm2/孔）。 注：RPE 可以接种到涂有细胞外基质蛋白（特别是层粘连蛋白、IV 型胶原或纤连蛋白）的孔板上，以提高粘附性11。RPE 也可以重悬于 1 mL RPE 培养基中，并分层到 40% 密度分离梯度上，以进一步分离纯 RPE 细胞。此外，以 340 x g 旋转板 3-5 分钟可能会增加细胞粘附13。</li>
	<li>在 37 °C 下，5% CO2 孵育。</li>
</ol>4. 培养 RPE
<ol><li>至少 3 天内不要破坏分离的 RPE。72 小时后，轻轻去除旧培养基，并用新鲜的预热培养基替换。</li>
	<li>前 3 天后每 48 小时更换一次培养基。</li>
	<li>一旦细胞达到汇合，使用 0.25% 胰蛋白酶 + 0.02% EDTA 传代细胞，并将 RPE 培养基中的 FBS 降低至 2%。 注意：先前报道原发性 RPE 在 5-7 代后开始去分化，并将开始失去其六边形和色素沉着14。</li>
</ol>

培养的原代鼠 RPE 的表征和形态动力学

<ol>
	<li>Strauss, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+pigment+epithelium+in+visual+function.">The retinal pigment epithelium in visual function.</a> Physiol Rev. 85 (3), 845-881 (2005).</li><li>Lakkaraju, 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=The+cell+biology+of+the+retinal+pigment+epithelium.">The cell biology of the retinal pigment epithelium.</a> Prog Retin Eye Res. 100846, (2020).</li><li>Lambros, M. L., Plafker, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+the+Nrf2+anti-oxidant+transcription+factor+in+age-related+macular+degeneration.">Oxidative stress and the Nrf2 anti-oxidant transcription factor in age-related macular degeneration.</a> Adv Exp Med Biol. 854, 67-72 (2016).</li><li>Ferrari, S., 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=Retinitis+pigmentosa:+genes+and+disease+mechanisms.">Retinitis pigmentosa: genes and disease mechanisms.</a> Curr Genomics. 12 (4), 238-249 (2011).</li><li>Xia, T., Rizzolo, L. 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O., Clark, V., Abrams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+isolation+and+culture+of+rat+retinal+pigment+epithelial+cells.">An improved method for isolation and culture of rat retinal pigment epithelial cells.</a> Invest Ophthalmol Vis Sci. 26 (11), 1599-1609 (1985).</li><li>Chang, C. W., Roque, R. S., Defoe, D. M., Caldwell, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+isolation+and+culture+of+pigment+epithelial+cells+from+rat+retina.">An improved method for isolation and culture of pigment epithelial cells from rat retina.</a> Curr Eye Res. 10 (11), 1081-1086 (1991).</li><li>Wang, N., Koutz, C. A., Anderson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+of+retinal+pigment+epithelial+cells+from+adult+rats.">A method for the isolation of retinal pigment epithelial cells from adult rats.</a> Invest Ophthalmol Vis Sci. 34 (1), 101-107 (1993).</li><li>Sakagami, K., 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=A+rapid+method+for+isolation+of+retinal+pigment+epithelial+cells+from+rat+eyeballs.">A rapid method for isolation of retinal pigment epithelial cells from rat eyeballs.</a> Ophthalmic Res. 27 (5), 262-267 (1995).</li><li>Heller, J. P., Kwok, J. C., Vecino, E., Martin, K. R., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+and+culture+of+adult+rat+retinal+pigment+epithelial+(RPE)+cells+to+study+retinal+diseases.">A method for the isolation and culture of adult rat retinal pigment epithelial (RPE) cells to study retinal diseases.</a> Front Cell Neurosci. 9, 449 (2015).</li><li>Shen, J., He, J., Wang, F. <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+pigment+epithelial+(RPE)+cells+with+Rho-Kinase+and+TGFbetaR-1/ALK5+inhibitor.">Isolation and culture of primary mouse retinal pigment epithelial (RPE) cells with Rho-Kinase and TGFbetaR-1/ALK5 inhibitor.</a> Med Sci Monit. 23, 6132-6136 (2017).</li><li>Hood, E. M. S., Curcio, C., 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 Protoc. 3 (4), 101758 (2022).</li><li>Naylor, A., Hopkins, A., Hudson, N., Campbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+Junctions+of+the+outer+blood+retina+barrier.">Tight Junctions of the outer blood retina barrier.</a> Int J Mol Sci. 21 (1), 211 (2019).</li><li>Ban, B., Rizzolo, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+culture+model+of+development+reveals+multiple+properties+of+RPE+tight+junctions.">A culture model of development reveals multiple properties of RPE tight junctions.</a> Mol Vis. 3, 18 (1997).</li><li>Fernandez-Godino, R., Garland, D. L., Pierce, E. 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在本文中，我们概述了一种分离和培养小鼠视网膜色素上皮的简化方案。从成年小鼠的眼睛中分离的 RPE 细胞表达 RPE 特异性标志物 RPE65 和细胞间连接标志物 ZO-1。此外，培养的细胞在培养物中发育成有色的六边形片材。
之前已经发表了几种在啮齿动物中分离 RPE 的方法6、7、8、9<...

视网膜色素上皮 （RPE） 是位于 Bruch 膜上的一种特殊细胞单层，位于光感受器和脉络膜1 之间。RPE 细胞在视网膜的正常功能中起着关键作用。RPE 细胞将葡萄糖和维生素 A 运输到光感受器，通过将全反式视网膜再异构化为 11-顺式视网膜来促进视力，并通过吞噬脱落的外段来维持光感受器的外段，从视网膜下空间去除水分，通过紧密连接的存在形成外血视网膜屏障并分泌嗜神经性生长因子（如色素上皮衍生因子、 和碱性成纤维细胞生长因子）支持光感受器2。RPE 细胞功能障碍参与各种视网膜病变的发病机制，包括年龄相关性黄斑变性、色素性视网膜炎和糖尿病性视网膜病变 3,4,5。使用 RPE 细胞的体外研究对于提高我们对这些疾病发病机制的理解至关重要。原代 RPE 细胞是这些研究的首选，因为 RPE 细胞系虽然很容易获得，但缺乏原代 RPE 细胞的关键特征。
虽然各种物种已被用作原代 RPE 细胞的来源，但小鼠具有使用基因修饰来帮助了解视网膜病变发病机制的优势。先前描述的从啮齿动物中分离 RPE 细胞的方案要么需要使用新生动物，要么冗长，需要技术技能，要么不适合培养 6,7,8,9,10,11,12。我们描述了一种从成年小鼠中分离 RPE 细胞的简单快速方法，可产生这些细胞的高纯度培养物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.009 RD Single-Edge Blades</td>
			<td>Personna</td>
			<td>941202</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td>with 4.5 g/L glucose, L-glutamine, sodium pyruvate</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Corning</td>
			<td>35010CV</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX, 100x</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td>no Calcium, no magnesium, no phenol red</td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution (1M)</td>
			<td>Gibco</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids, 100x</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Unitome Knife</td>
			<td>BVI Beaver</td>
			<td>377546</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100x</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene Microplates</td>
			<td>Falcon</td>
			<td>08-772-1</td>
			<td>24-well or 48-well</td>
		</tr>
		<tr>
			<td>Regular Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>with phenol red</td>
		</tr>
		<tr>
			<td>Vannas scissors</td>
			<td>Fine Science Tools</td>
			<td>10091-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simplified method for isolation and culture of retinal pigment epithelial cells from adult mice

视网膜色素上皮细胞 （RPE） 对于视网膜的正常功能至关重要。RPE 功能障碍参与重要视网膜疾病的发病机制，例如年龄相关性黄斑变性、色素性视网膜炎和糖尿病性视网膜病变。我们提出了一种从小鼠成人眼中分离 RPE 的简化方法。与以前报道的方法相比，这种方法能够从成年小鼠中分离和培养高纯度 RPE。这种简单快速的方法不需要广泛的技术技能，并且可以通过基本的科学工具和试剂来实现。通过摘除眼睛，然后去除眼前节，从 3 至 14 周龄的 C57BL/6 背景小鼠中分离出原发性 RPE。酶促胰蛋白酶消化和离心用于从眼罩中解离和分离 RPE。总之，这种方法为 RPE 在视网膜功能和疾病研究中的使用提供了一种快速有效的方案。

The retinal pigment epithelium (RPE) is a specialized cell monolayer lining the Bruch&#39;s membrane located between photoreceptors and the choroid1. RPE cells play a critical role in the proper function of the retina. RPE cells transport glucose and vitamin A to photoreceptors, promote vision by re-isomerization of all-trans retinal into 11-cis retinal and maintain outer segments of photoreceptor through phagocytosis of shed outer segments, remove water from the subretinal space, form the outer blood-retinal barrier through the presence of tight junctions and secrete neurotropic growth factors (such as Pigment Epithelium Derived Factor, and Basic Fibroblast Growth Factor) that support photoreceptors2. Dysfunction of RPE cells is involved in the pathogenesis of various retinopathies, including age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy3,4,5. In vitro studies using RPE cells are critical to improving our understanding of the pathogenesis of these diseases. Primary RPE cells are much preferred for these studies since RPE cell lines, while readily available, lack key characteristics of primary RPE cells.
Whereas various species have been utilized as sources of primary RPE cells, mice have the advantage of using genetic modifications to help understand the pathogenesis of retinopathies. Previously described protocols to isolate RPE cells from rodents either require the use of neonatal animals, are lengthy, require technical skill, or are not suitable for culture6,7,8,9,10,11,12. We describe a simple and fast method to isolate RPE cells from adult mice that yield highly pure cultures of these cells.

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

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 or RPE are located between photoreceptors and the choroid. Dysfunction of RPE is important in the pathogenesis of diseases, such as age-related macular degeneration, diabetic retinopathy, and retinitis pigmentosa. The use of primary RPE is key to studies to improve the understanding of how disease is developed.While various species have been used as a source of primary RPE, mice have the advantage of allowing to use genetic manipulation to help understand how retinopathies develop. Prior methods of isolation of primary RPE from rodents require either the use of neonatal animals, are lengthy, require technical expertise, or are not suitable for cell culture. We describe a simple and fast method to isolate primary RPE from adult mice that yields highly pure cultures of the cells.

所述方案已用于 C57BL/6 背景小鼠。性别似乎并没有改变培养 RPE 的能力。与老年小鼠相比，6 周以下的小鼠产生的 RPE 片有限，可能需要更多的眼睛才能达到最佳汇合。分离后，RPE 细胞大约需要 3 天才能稳定并附着在细胞培养板上。分离后约 24 小时，看起来无核的圆形色素沉着细胞已经开始沉降，但尚未完全粘附在板上（图 1A）。在接下来的 48-72 小时内，细胞开始附着和扩?...

视网膜色素上皮 （RPE） 是脉络膜和视网膜之间的重要屏障，促进视网膜细胞类型（如光感受器）的健康和功能。在此，我们描述了一种简单有效的分离和培养成年小鼠 RPE 的方案。

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

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JoVE Journal

Neuroscience

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

642 次观看.  Case Western Reserve University. 视网膜色素上皮细胞或 RPE 位于光感受器和脉络膜之间。RPE 功能障碍在疾病的发病机制中很重要，例如年龄相关性黄斑变性、糖尿病性视网膜病变和色素性视网膜炎。原发性 RPE 的使用是提高对疾病发展方式的理解的研究的关键。虽然各种物种已被用作原发性 RPE 的来源，但小鼠的优势在于允许使用基因操作来帮助了解视网膜病变是如何发展的?...

视频: 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 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.

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.

科研

神经科学

Extraction and Cultivation of Retinal Pigment Epithelial Cells from Adult Mice Eyes

To begin, prepare the wash buffer by supplementing Hank's Balanced Salt Solution with 10 millimolar heaps buffer. Store the buffer at four degrees Celsius until use. Prepare retinal pigment epithelium or RPE medium in DMEM and prewarm to 37 degrees Celsius in a water bath.Lay the euthanized mouse flat on its side with the eye facing upwards. Place the index finger above the eye and the thumb below it. Apply gentle pressure on the bone structure around the eye to cause the eyeball to protrude.Insert the tip of slightly open scissors under the eye and gently rotate the wrist away from the eye 90 degrees until the eye detaches from the socket. Immediately place and roll the eye in 70%ethanol for five seconds. Then transfer the eye to a wash buffer kept on ice.Under a dissecting microscope, place the eye in a Petri dish filled with wash buffer and strips of soaked gauze. Stabilize the eye by holding the optic nerve with tweezers and using a surgical knife gently make an incision at the level of the Ora Serrata. Insert Vannas Scissors into the incision and cut around the circumference of the Ora Serrata until the anterior segment and vitreous are removed.Gently peel away the retina from the eye cup using tethered forceps without disturbing the RPE layer. With the help of scissors, remove the optic nerve and excess connective tissue from the eye cup without puncturing the eye cup. Transfer the eye cup to a 1.5 milliliter microcentrifuge tube containing one milliliter of 0.25%trypsin and 0.02%EDTA.incubate the eye cup in a 37 degrees Celsius water bath for 10 minutes. Every two minutes, remove the tube and firmly tap the bottom 40 times onto the countertop. After incubation, using AP-1000 pipette gently mix up and down three times to disrupt the eye cup.To neutralize the trypsin, immediately layer the detached RPE sheets, excluding the eye cup onto 0.5 milliliters of FBS in a 15 milliliter conical tube. Then dropwise, add RPE media onto the layers of FBS and RPE sheets to dilute the trypsin. Centrifuge the RPE at 340G for three minutes.Discard the supernatant and resuspend the cells in a suitable amount of RPE media for a 24 well plate. Incubate the RPE at 37 degrees Celsius with 5%carbon dioxide for three days. 24 hours after RPE isolation, a nucleate pigmented cells begin to settle without full adherence.Over the 48 to 72 hours, these cells attach and spread, retaining dark pigmentation in a transparent nucleus. After five days, RPE cells show increased co fluency in start forming cell to cell contacts shaping into a polarized monolayer with hexagonal structures. Isolated RPE showed purity as evidenced by the RPE specific marker and tight junction integrity after six days in culture.