我们感谢劳拉克拉克，她的宝贵援助。这项工作是由罗富国:干细胞网络，CIHR和美国国立卫生研究院支持。

1。准备解剖的解决方案和酶解决方案<ol><li>设为人工脑脊髓液（学联）和无血清培养基提前和冷藏。 </li><li>称量Kynurenic酸（0.2毫克/毫升），溶解在10毫升的希洛学联在37℃水浴时间提前，因为它不容​​易溶解。 </li><li>称出的胰蛋白酶（1.33毫克/毫升）和透明质酸（0.67毫克/毫升）和15毫升管的地方，并保持在-20 ° C，直到需要。添加的Kynurenic柠檬酸溶液管过滤器使用22微米的注射器过滤器在使用前。 </li><li>称取胰蛋白酶抑制剂（Ovamucoid 1毫克/毫升），溶解在温暖的无血清培养基和使用22μm注射器过滤器的过滤。 </li><li>使电镀媒体:无血清培养基含有FGF2组（10毫微克/毫升）和肝素（2微克/毫升）。 </li></ol> 2。隔离从鼠眼视网膜干细胞<ol><li>视网膜干细胞的分离是在一个特定的无菌室专用原代培养实验。开始此过程之前，成立剖析镜和冷光源，然后铺陈无菌剖析仪器。每个罩有一个热珠灭菌消毒步骤之间的文书。 </li><li>我们会从眼睛的视网膜干细胞已立即放置在无菌的Petri菜后，他们根据协议批准的动物伦理牺牲的小鼠去除含有人工脑脊髓液（学联）隔离。 </li><li>解剖显微镜下，干净的镊子的眼睛:摆脱头发和结缔组织附着在角膜/巩膜边界。然后，将眼睛与学联的新菜。 </li><li>同时轻轻按住眼与锯齿钳固定，使用角度微解剖剪刀去除眼部肌肉。取出视神经以及如果它仍是贴在眼睛上。在此过程中尽量不要压扁眼。将眼睛与学联的新菜。 </li><li>接下来，使用弧形微解剖剪刀将削减一半的眼睛:开始在视神经及角膜中间穿过，会议开始削减在视神经。它是理想的眼两片的大小大致相等，因为这将使下一步更容易。 </li><li>除了控股的眼角膜用两个镊子，轻轻剥离两眼半。删除和丢弃的镜头，内脏，神经视网膜从眼壳。将炮弹与学联的新菜。 </li><li>现在，我们已经准备好隔离纤毛上皮。首先，东方的眼睛外壳，使您的权利，角膜和视网膜色素上皮（RPE）是在左边。轻轻针的眼壳下的RPE方直钳。 </li><li>接下来，使用手术刀削减，从眼睛的睫状肌上皮角膜和虹膜，轻轻推了手术刀，而不是用锯议案。 </li><li>之后，切离的视网膜色素上皮的纤毛上皮。使用无锯齿钳纤毛上皮细胞的地带转移到一个新的35毫米的菜含有学联。 </li><li>以同样的方式，从另一只眼睛壳隔离的纤毛上皮细胞。 </li><li>一旦双方的纤毛上皮条已收集，转移到一个35毫米，含2毫升的Dispase盘条。放置10分钟，在37℃培养箱条菜。 </li><li> 10分钟后，从Dispase转移到含有2毫升过滤胰蛋白酶，透明质酸酶和Kynurenic酸性，菜条。菜放置在37 ° C为10分钟。 </li><li>现在，返回到解剖显微镜。虽然直钳巩膜，使用底部的弧形无锯齿钳轻轻刮去远离巩膜睫状体上皮。 </li><li>从盘中取出巩膜。应该在现在剩下的菜的兴趣和酶液的细胞。 </li><li>使用一个火抛光棉插吸管，这个解决方案转移到14毫升的管。磨碎这个解决方案的30倍，掰开的解决方案和移液器轻轻迫使上皮细胞。 </li><li> 5分钟1500转离心管。当离心完成后，取出离心管小心，因为细胞很容易在这个阶段的试管底部。 </li><li>轻轻吸去大部分上清，使用火抛光的吸管，然后在无血清培养基中添加1毫升的胰蛋白酶抑制剂的细胞。使用一个小的钻孔棉花插吸管磨碎的样品约50倍，直到它是一种单细胞悬液。 </li><li>再次为5分钟1500转离心管。 </li><li>去除上清，用1毫升电镀中型替换。轻轻磨碎使用火抛光的玻璃吸管悬浮CELLS。 </li><li>计数后所需的密度在24孔板中的细胞，板。我们通常盘10个细胞/微升灌装与介质，然后每孔加入细胞悬液500μL媒体得到的最终成交量。 </li><li>它不会移动，直到你算7天之后出现的领域，将在37℃CO 2培养箱板。 </li></ol> 3。视网膜干细胞的分离结果<ol><li>当此过程是成功执行，解剖纤毛上皮细胞看起来应该像这样，在低密度（图1）的分离和镀后。 </li><li>经过7天中文化，出现视网膜干细胞领域也计算在内。一个球体，应超过75微米，直径作为干细胞来源的领域内自由浮动。 （图2）。然而，某些细胞将有限的扩散，并形成球体不符合大小的标准，不会干细胞衍生的领域内;，这样一个球体的示例如下所示（图2）。 </li></ol> 4。代表性的成果<img src="/files/ftp_upload/2209/2209fig1.jpg" alt="图1" / > 图1。 <img src="/files/ftp_upload/2209/2209fig2.jpg" alt="图2" / > 图2。

<ol>
	<li>Tropepe, V., Coles, B. L., Chiasson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells+in+the+adult+mammalian+eye.">Retinal stem cells in the adult mammalian eye.</a> Science. 287, 2032-2036 (2000).</li><li>Ahmad, I., Tang, L., Pham, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+progenitors+in+the+adult+mammalian+eye.">Identification of neural progenitors in the adult mammalian eye.</a> Biochem Biophys Res Commun. 270, 517-521 (2000).</li><li>Coles, B. L., Angenieux, B., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+isolation+and+the+characterization+of+human+retinal+stem+cells.">Facile isolation and the characterization of human retinal stem cells.</a> Proc Natl Acad Sci U S A. 101, 15772-15777 (2004).</li><li>Cicero, S. A., Johnson, D., Reyntjens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+previously+identified+as+retinal+stem+cells+are+pigmented+ciliary+epithelial+cells.">Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells.</a> Proc Natl Acad Sci U S A. 106, 6685-6690 (2009).</li><li>Lamba, D., Karl, M., Reh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+regeneration+and+cell+replacement:+a+view+from+the+eye.">Neural regeneration and cell replacement: a view from the eye.</a> Cell Stem Cell. 2 (6), 538-549 (2008).</li><li>Coles, B. L., Horsford, D. J., McInnes, R. R., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+retinal+progenitor+cells+leads+to+an+increase+in+the+retinal+stem+cell+population+in+vivo.">Loss of retinal progenitor cells leads to an increase in the retinal stem cell population in vivo.</a> Eur J Neurosci. 23, 75-82 (2006).</li><li>Ang&#233;nieux, B., Schorderet, F. D., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+is+a+neuronal+differentiation+factor+for+retinal+stem+cells+in+vitro.">Epidermal growth factor is a neuronal differentiation factor for retinal stem cells in vitro.</a> Stem Cells. 24 (3), 696-706 (2006).</li><li>Xu, S., Sunderland, M. E., Coles, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proliferation+and+expansion+of+retinal+stem+cells+require+functional+Pax6.">The proliferation and expansion of retinal stem cells require functional Pax6.</a> Dev Biol. 304, 713-721 (2007).</li><li>Canola, K., Ang&#233;nieux, B., Tekaya, M., Quiambao, A., Naash, M. I., Munier, F. L., Schorderet, D. F., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells+transplanted+into+models+of+late+stages+of+retinitis+pigmentosa+preferentially+adopt+a+glial+or+a+retinal+ganglion+cell+fate.">Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate.</a> Invest Ophthalmol Vis Sci. 48 (1), 446-4454 (2007).</li><li>Canola, K., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cells+committed+towards+the+photoreceptor+fate+for+retinal+transplantation.">Generation of cells committed towards the photoreceptor fate for retinal transplantation.</a> Neuroreport. 18 (9), 851-855 (2007).</li><li>Djojosubroto, M. W., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells:+promising+candidates+for+retina+transplantation.">Retinal stem cells: promising candidates for retina transplantation.</a> Cell Tissue Res. 331 (1), 347-357 (2008).</li><li>Inoue, T., Coles, B. L., Dorval, K., Bremner, R., Bessho, Y., Kageyama, R., Hino, S., Matsuoka, M., Craft, C. M., McInnes, R. R., Tremblay, F., Prusky, G. T., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+functional+photoreceptor+differentiation+from+adult+human+retinal+stem+cells.">Maximizing functional photoreceptor differentiation from adult human retinal stem cells.</a> Stem Cells. 28 (3), 489-500 (2010).</li><li>Tropepe, V., Sibilia, M., Ciruna, B. G., Rossant, J., Wagner, E. F., Kooy, D. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+neural+stem+cells+proliferate+in+response+to+EGF+and+FGF+in+the+developing+mouse+telencephalon.+Dev+Biol.">Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol.</a> , 208-201 (1999).</li><li>Coles-Takabe, B. L., Brain, I., Purpura, K. A., Karpowicz, P., Zandstra, P. W., Morshead, C. M., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Don't+look:+growing+clonal+versus+nonclonal+neural+stem+cell+colonies.">Don't look: growing clonal versus nonclonal neural stem cell colonies.</a> Stem Cells. 26 (11), 2938-2944 (2008).</li></ol>

本协议描述了如何从鼠标的眼睛1-3纤毛上皮细胞的视网膜干细胞的分离。隔离纤毛上皮细胞的条的方法可以有所不同，但是实现一个单细胞悬液，酶和方法已在本议定书的优化。同样重要的是纤毛上皮已被隔离带不包含大量的视网膜色素上皮或角膜，因为这些似乎有一个可从每只眼睛孤立的领域总数的负面影响。此外，我们脑部神经球13的最初制定的，在无血清培养基是日益增长的视网膜干细胞领域的理想选择。人们还应该确保这些细胞不受到干扰后，他们已镀上和放置在孵化器，使不断增长的领域不聚合在一起14。 一旦视网膜干细胞形成克隆领域，他们可以算作得到每眼干细胞的一个准数。球体，然后就可以被分离为单个细胞，并代确定的自我更新能力，或到不同的细胞类型的神经视网膜和视网膜色素上皮生长因子的不同组合和/或蛋白质有区别的。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stemi 2000 Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cold Light Source</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>KL1500 </td>
<td>dual arm optic light</td>
</tr>
<tr>
<td>Curved Mini Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15000-10</td>
<td>&nbsp;</td>
<tr>
<td>Angled Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15005-08</td>
<td>&nbsp;</td>
<tr>
<td>#7 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11272-30</td>
<td>non-serrated</td>
</tr>
<tr>
<td>#5 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11251-10</td>
<td>straight</td>
</tr>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11051-10</td>
<td>serrated curved</td>
</tr>
<tr>
<td>#3 Scalpel handle</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2586-M36-10</td>
<td>&nbsp;</td>
<tr>
<td>#10 Scalpel blades</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2580-M90-10</td>
<td>&nbsp;</td>
<tr>
<td>Hot Bead Sterilizer</td>
<td>&nbsp;</td>
<td>FST</td>
<td>18000-45</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>VWR/BD </td>
<td>CACB354235</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1005</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H6254</td>
<td>&nbsp;</td>
<tr>
<td>Kynurenic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>K3375</td>
<td>1000 IU</td>
</tr>
<tr>
<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10109878001</td>
<td>&nbsp;</td>
<tr>
<td>35 mm dishes</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA354235</td>
<td>&nbsp;</td>
<tr>
<td>24-well plate</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA73521-004</td>
<td>&nbsp;</td>
<tr>
<td>Cotton-plugged pipettes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14672-400</td>
<td>fire-polish</td>
</tr>
<tr>
<td>14 mL Tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352057</td>
<td>Polystyrene</td>
</tr>
<tr>
<td>Serum-free Media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ref# 13 for formulation</td>
</tr>
<tr>
<td>FGF2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0291</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3149</td>
<td>100 IU</td>
</tr>		</tbody>
		</table>
		</div>

isolation of retinal stem cells from the mouse eye

成年鼠的视网膜干细胞（RSC）是一种罕见的的静态细胞内的纤毛上皮细胞的哺乳动物的眼睛 1,2,3（CE） 。行政长官是由无色素的内层和色素外层细胞层，并从单一的行政长官的色素细胞克隆产生的RSC殖民地可分化的细胞色素和非色素形成的所有神经视网膜和色素上皮细胞类型。有一些争议，关于是否所有的领域内的细胞中都含有至少有一些色素4;然而，细胞仍然能够形成不同的细胞类型的神经视网膜1-3内发现。在一些物种，如两栖类和鱼类，他们的眼睛能在伤后5再生的，但是，哺乳动物的眼睛显示没有这样的再生性能。我们设法确定在体内的干细胞和理解的机制，使哺乳动物的视网膜干细胞的静态6-8，甚至伤后，以及使用它们作为一个潜在的细胞来源，以帮助修复眼外伤的物 ​​理或遗传模型通过移植9-12。在这里，我们介绍了如何以隔离从小鼠眼纤毛上皮细胞，并在文化中成长，才能形成克隆视网膜干细胞领域。既然有没有已知的标记的干细胞在体内 ，这些领域是唯一已知的方法来前瞻性地识别内眼睫状体上皮干细胞群。

Watch this Scientific Journal Video about Isolation of Retinal Stem Cells from the Mouse Eye at JoVE.com

Isolation of Retinal Stem Cells from the Mouse Eye

在这段视频中，我们将演示如何隔离鼠标眼睫状体上皮视网膜干细胞，并在文化中成长，他们形成克隆视网膜领域。被隔离的领域拥有的干细胞的大是大非特性:自我更新和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 ...

isolation-of-retinal-stem-cells-from-the-mouse-eye

Research

JoVE Journal

Biology

24.7K 次观看.  University of Toronto. 在这段视频中，我们将演示如何隔离鼠标眼睫状体上皮视网膜干细胞，并在文化中成长，他们形成克隆视网膜领域。被隔离的领域拥有的干细胞的大是大非特性:自我更新和multipotentiality。

视频: Isolation of Retinal Stem Cells from the Mouse Eye

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, ...

科研

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

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 ...

神经科学

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 ...

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 ...

生物学

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

体外细胞生理学研究中视网膜微动脉的分离

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

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.