Name: a simplified method for isolation and culture of retinal pigment epithelial cells from adult mice
Uploaded: 2024-05-24T14:50:00.000+00:00
Duration: 5 min 4 s
Description: Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

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. 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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><tbody><tr><td>0.009 RD Single-Edge Blades</td><td>Personna</td><td>941202</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;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&#039;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。

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?...

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

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。

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 ...

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Research

JoVE Journal

Neuroscience

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

972 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

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

评估和量化年龄相关性黄斑变性小鼠模型中视网膜色素上皮病理的协议

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

科研

遗传学

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

生物工程

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

小鼠视网膜内皮细胞分离用于二代测序

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

发育生物学

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 ...

神经科学

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

两种剥离方法，用于分离小鼠视网膜中的光感受器细胞区室以进行蛋白质分析

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

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 ...

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 ...

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.