我们感谢实验室的成员，为帮助这些细胞培养特征，蒂娜杰弗里Adijanto班松，李蓉，秦环，从小张，赵菁，康妮志，Awais齐亚，纳塔利娅Strunnikova。特别感谢赵菁，康妮志，他们在帮助维持细胞培养的大量库存和Tina班松。 这项工作是由美国国立卫生院内研究计划研究院的支持。

1。人类胎儿组织所有的人体组织的相关研究如下的"赫尔辛基宣言"和美国国立卫生研究院的机构审查委员会的信条。胎儿的眼睛是由一个独立的皮条客，先进的生物科技资源（ABR，阿拉米达，CA）获得的，随意放置在RPMI - 1640含管（ABR提供）的媒体，捐助者在16至22周妊娠，装在冰，交付隔夜优先递送服务。组织仍然可行眼球摘除后长达48小时。 2。细胞培养液 MEM -α修饰培养基（​​Sigma - Aldrich公司）作为基介质准备5％和15％的含血清培养的视网膜色素上皮细胞（RPE细胞培养基;下面的表1）媒体。 <table border="1"><tbody><tr><td>名称</td><td>西格玛</td><td> Gibco公司</td><td>金额</td><td>存储</td></tr><tr><td>纪念，阿尔法修改</td><td> M - 4526 </td><td></td><td> 500毫升</td><td> 4 ° C </td></tr><tr><td> N1的补充</td><td> N - 6530 </td><td></td><td> 5毫升</td><td> 4 ° C </td></tr><tr><td>青霉素链霉素</td><td></td><td> 15140-148 </td><td> 5毫升</td><td> -20℃ </td></tr><tr><td> GlutaMax - 我</td><td></td><td> 35050 </td><td> 5毫升</td><td> -20℃ </td></tr><tr><td>非必需氨基酸</td><td> M - 7145 </td><td></td><td> 5毫升</td><td> 4 ° C </td></tr><tr><td> THT * </td><td></td><td></td><td></td><td> -80℃ </td></tr><tr><td>牛磺酸</td><td> T - 0625 </td><td></td><td> 125毫克</td><td></td></tr><tr><td>氢化可的松</td><td> H - 0396 10 </td><td></td><td> 10微克</td><td></td></tr><tr><td>三碘thyronin </td><td> T - 5516 </td><td></td><td> 0.0065微克</td><td></td></tr><tr><td>小牛血清** </td><td></td><td></td><td> 5％或15％ </td><td> -80℃ </td></tr></tbody></table> 表1。人胚RPE中等组件500毫升培养基的制备 * THT是由溶解在1 1.5毫升PBS中期之前，牛磺酸氢化可的松三碘- thyronin。多个等份并保存于-80 ° C至简化培养基中培养的准备。 **胎牛血清未获得来自Sigma - Aldrich公司或Gibco公司。 胎牛血清培养基制备中的应用是来自亚特兰大的生物制品（诺克罗斯，GA）获得。每一瓶血清热灭活（56℃为1小时），使用前。为了确保细胞培养的一致性，从同一批次血清大量购买，并保存在-20℃，直到它们被用于媒体准备。 24 - 或12 - 孔板是生长在人烟稀少的从一个特定的批次hfRPE细胞种子细胞在含有20％胎牛血清的不同批次的媒体的几个星期。最经典的RPE细胞形态（鹅卵石），增长最快的melanated细胞，可用于细胞培养表明适当的血清很多。 培养液中还包含:N1补充（Sigma - Aldrich公司）1:100毫升/毫升，GlutaMax /青霉素，链霉素1:100毫升/毫升（GIBCO），和不必要的氨基酸液（Sigma - Aldrich公司）1:100毫升/毫升。此外，氢化可的松（20微克/升），牛磺酸（250 mg / L的）和三碘thyronin（0.013微克/升）（THT）提前准备这三个组成部分溶解在PBS到终浓度为1:500 （毫升/毫升）。 THT等分储存在80 °彗星，直到添加到视网膜色素上皮介质。 3。细胞培养在收到完整的地球抗生素antimycotic溶液冲洗（稀释到10倍;。猫没有15240-096; Invitrogen公司）加庆大霉素（1毫克/毫升）为3〜5分钟（图1）。 <img src="/files/ftp_upload/2032/2032fig1.jpg" alt="图1" /> 图1。孵育后，抗生素两次中等的HBSS或PBS冲洗掉。一个在当时的眼球转移到10厘米的培养皿Sylgard - 184（WPI），涂层和27G针头担保。使用皆伐板载刀（爱尔康）切口通过巩膜睫状体（从眼球赤道前表面的距离的1 / 3）以下。这个切口是用来启动一个圆形切割拆除眼前部分。采用的是同一个刀片锯齿（FST）的一个碳化钨涂层弧形光圈剪刀剪开的。眼球前部的搬迁之前，降息是通过玻璃体，以避免分离的Retina从后极部视网膜色素上皮。眼的前部被删除后，后极是在5％dispase，我的解决方案（2 U /毫升，猫没有04942086001;罗氏诊断，印第安纳波利斯，在。）孵育40-60分钟的血清培养基在37 ° C - 5％的CO 2。 dispase治疗后，后极硅填充（Sylgard 184，道康宁，美联，心肌梗死）的培养皿转移到的HBSS到象限或大块足够的扁平化组织解剖。然后用镊子轻轻除去视网膜。单细胞的RPE层成片剥落，直接收集到冷的胰蛋白酶- EDTA（GIBCO，＃25200-056）解决方案。后的RPE收集，组织中的胰蛋白酶- EDTA管密封，转移到为10-15分钟的水洗澡，在37 ° C。孵育10分钟后，管大力动摇，分成小簇的RPE。如果分离不彻底，管放入水浴，再过5分钟。胰蛋白酶- EDTA的潜伏期后，联合国可能溶解混合细胞群管检查。任何观察到的集群都将被删除，用细尖玻璃巴斯德吸管。纺（1.4临床离心机转速为4分钟）后，hfRPE细胞重新悬浮在15％的视网膜色素上皮媒体，然后把Primaria瓶（例如:猫没有08-772-45;费舍尔科学，宾夕法尼亚州匹兹堡。 ）。这种介质更换后1天用5％的血清中含有的RPE中等，并随后进行了更改，每2至3天。 3〜4周后，细胞融合和统一色素。然后，他们在0.25％胰蛋白酶- EDTA为10至15分钟胰酶消化，重悬在15％血清中含有色素上皮细胞培养液中，每口井在150到200K细胞（Transwell小，康宁中光学到清晰的细胞培养插入种子，康宁，NY），使用12毫米直径的插入，0.4微米的毛孔，聚酯膜（例如:猫，Fisher Scientific则没有07-200-161）。播种前，与人类细胞外基质（在150μL的HBSS，每孔10微克，猫没有354237;。BD公司，新泽西州富兰克林湖，）涂井，并在2小时的引擎盖UV光固化。在某些情况下，胰蛋白酶消化过程重复第二次，收集细胞，没有分离后的第一个胰蛋白酶。相同的协议（不包括涂层与ECM）是用于培养细胞的烧瓶，生成细胞的P1人口。这些细胞进行实验，当他们≥200Ω•2厘米的一个总的组织性和均匀色素。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/2032/2032fig1full.jpg">请点击这里为一个较大的数字</a> 1 。 4。一步步过程<ol><li>准备了多种解决方案（每个4口井需要眼睛）12孔板: <ol class="lalpha"><li>添加10X抗生素antimycotic解决方案 - 1井/眼</li><li>添加冲洗PBS / HBSS解决方案 - 2井/眼</li><li>添加dispase解决方案 - 1井/眼</li></ol></li><li>解开5个固定针和填充的HBSS，准备解剖​​培养皿</li><li>拆开的眼睛和抗生素antimycotic的解决方案，为3-5分钟（在第1步准备） </li><li>在两口井（在第1步准备），用PBS / HBSS冲洗眼睛，然后将它们传输到解剖盘。 </li><li>修剪过多的眼部周围的肌肉和结缔组织</li><li>在解剖盘对准角膜面临的方式使用27G的针头安全眼（牵制硅基地）。 </li><li>使用板载刀下面的角膜切口，圆形切割将开始</li><li>使用虹膜剪刀，减少眼部周围，通过玻璃体切割和解除眼的前部，然后使用相同的剪刀。 注意:在步骤3至8，避免任何过大的机械压力眼球。 </li><li> dispase解决方案转移到开放眼睛（在第1步准备） </li><li>眼罩孵育40-60分钟，在37℃，5％的CO 2 </li><li>替换在解剖与新鲜菜之一的HBSS解决方案。 </li><li> dispase解决方案传输眼睛解剖盘。 </li><li>在培养皿中的位置眼（眼球杯朝上）和安全两个27G的针头。 </li><li>轻轻抬起部分分离视网膜和视网膜剪刀切断视神经视网膜。丢弃视网膜。 </li><li>使用虹膜剪一个切口从外围对视神经的眼。 </li><li>使用所有5个27G的针头很好地拉长眼RPE层扁平化。 </li><li>使用视网膜剪刀，使视神经周围的圆形切割分离挂职视神经RPE层。 注意:步骤13-17可以做低倍率或无立体显微镜</li><li>调整体视显微镜250倍的放大倍率或以上，并找到密切沿视神经，虹膜剪刀切割边缘的RPE表。 </li><li>利用两个钳单独的视网膜色素上皮布鲁赫的脉络膜组织层膜。它可能需要几个试图找到沿边缘区视网膜色素上皮及脉络膜是最弱的。 </li><li>进入寒冷的胰蛋白酶- EDTA溶液放置的RPE张在15毫升管</li><li>后的RPE收集到水浴组织中的胰蛋白酶- EDTA为10 - 15分钟，管帽和转让，在37 ° C。 </li><li>孵育10分钟后，大力摇晃15毫升管，分成小簇的RPE。如果分离是不完整的，放入水浴管另一个5分钟。 </li><li>联合国可能溶解混合细胞群的检查管。用细尖玻璃巴斯德吸管，应删除任何观察到的集群。 </li><li>离心（1.4临床离心机转速为4分钟）hfRPE细胞，去除上清液，重新悬浮细胞在15％的视网膜色素上皮媒体（9毫升总） </li><li> Primaria烧瓶中加入2 ml新鲜的15％的视网膜色素上皮媒体，放入孵化器的烧瓶中，直到第二天（37℃，5％CO 2）放入3 ml的细胞悬液</li></ol> 5。代表性的成果 hfRPE细胞培养的功能验证在以前的实验中，我们已经使用了这些原代培养，以确定质膜转运蛋白的表达，多极化和功能，并确定具体的信号转导通路，调节视网膜色素上皮功能1-11的这些特点，为他们积累提供属性，可应用于设置一个验证每个细胞的产生。总结如下实验确定的蛋白质，确定跨上皮流体输送和完整性的paracellular途径。这些在体外实验中已证实在动物模型的视网膜重新复位6。 <img src="/files/ftp_upload/2032/2032fig2.jpg" alt="图2" /> 图2。本地化hfRPE细胞CFTR的 左上面板:CFTR的检测中使用膜富集提取的hfRPE细胞。男，分子量标记; 1巷 ，主要成熟（C波段）和不成熟（带A和B）中心较低的面板:CFTR的免疫荧光定位（绿色标签）右上角的面板 :扩大交叉通过的 Z平面显示剖面视图。通过Z轴的最大强度投影。 ZO - 1（红色标记）作为一个紧密连接的标记划定的RPE心尖和基底双方的供应。 DAPI染色（蓝色）标签的原子核靠近基底膜。 ZO - 1的显示为黄色/橙色，因为它的红色标签重叠CFTR的绿色荧光。 <img src="/files/ftp_upload/2032/2032fig3.jpg" alt="图3" /> 图3。 γ干扰素的诱导生理变化，在 hfRPE答:IFNγ基础浴除了增加整个小学，培养hfRPE细胞单层的跨上皮流体输送（J V）J V是作为一个在顶部跟踪功能的时间和净液绘制。吸收（根尖基础浴）表示正面的价值观;跨上皮电位（TEP）和总的组织电阻（R T）绘制的时间在底部的痕迹功能。乙:基础浴抑制CFTRinh - 172（5微米）的γ干扰素刺激J v增加。 下面是一个动物模型实验，在体外研究结果证实hfRPE的例子。在这个实验中，人为地造成视网膜脱离除了IFNG外部眼球表面（图4，A，B）后显着降低。这种效应可部分阻止:（1）除营阻滞剂（图4D）;（2）完全的JAK +营阻滞剂后除了封锁。下面的图片是使用华侨城扫描仪获得。 <img src="/files/ftp_upload/2032/2032fig4.jpg" alt="图4" /> 图4。 体内实验的程序， 这些在体内视网膜脱离的实验 。 以前 6详细描述。在这些实验中，视网膜脱离0.5-3μL修改PBS液注射到视网膜下腔（SRS），单独或与相结合的JAK - STAT和PKA通路抑制剂在大鼠眼球。每次实验后，视网膜脱离创造一个初步控制在40-70分钟时间。在此期间，大疱体积的变化率测量，以确保气泡稳定。光学相干断层扫描成像（应用物理研究所，俄罗斯科学院，下诺夫哥罗德，俄罗斯）是用来衡量SRS体积变化的时间过程。 重新附件卷的时间当然UME变化是衡量光学相干断层扫描（OCT）。 OCT图像显示在支队尺寸变化（箭头在A，B和D）的IFNγ除了40-70分钟的前表面，从4个不同的实验（公元在左边的面板）。 A和B显示，IFNγ的增加其控制率的合资企业（≈2μL•厘米•HR -1）2至14和12μL•厘米2•HR - 1，分别为。除了​​JAK - STAT通路和PKA抑制剂，γ干扰素（C和D） -引起的吸收率显着降低为0.2和7.9μL•2•HR - 1厘米，分别。箭头指示的气泡的边界内虚线包围的区域，开始卷比较。图:右侧顶部面板摘要测量JV率从几个实验;中间面板电影（ <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/2032/Bleb%205.wmv">点击这里</a> ）;下部面板- 3D B.蓝色的伪彩色总结实验的部分表示在t = 0的空间支队程度和40分钟后INFγ此外。 所有的动物实验与研究，在视觉与眼科声明协会进行遵守。该协议是由美国国立卫生研究院的动物护理和使用委员会批准。

<ol>
	<li>Bharti, K., Miller, S. S., Arnheiter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+paradigm:+Retinal+pigment+epithelium+cells+generated+from+embryonic+stem+cells+or+induced+pluripotent+cells.">The new paradigm: Retinal pigment epithelium cells generated from embryonic stem cells or induced pluripotent cells.</a> Pigment Cell &#38; Melanoma Research. , .</li><li>Strunnikova, N. V., Maminishkis, A., Barb, J. J., Wang, F., Zhi, C., Sergeev, Y., Chen, W., Edwards, A. O., Stambolian, D., Abecasis, G., Swaroop, A., Munson, P. J., Miller, S. S., S, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+and+molecular+signature+of+human+retinal+pigment+epithelium.">Transcriptome analysis and molecular signature of human retinal pigment epithelium.</a> Hum Mol Genet. 19 (12), 2468-2486 (2010).</li><li>Miller, S. S., Maminishkis, A., Li, R., Adijanto, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+184:+Phototransduction:+RPE+transport+Retina+Phototransduction:+RPE+transport.">Chapter 184: Phototransduction: RPE transport Retina Phototransduction: RPE transport.</a> Encyclopedia of the Eye. , 2540-2540 (2010).</li><li>Bryant, D. M., Mostov, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cells+to+organs:+Building+polarized+tissue.">From cells to organs: Building polarized tissue.</a> Nature Reviews. Molecular Cell Biology. 9 (11), 887-901 (2008).</li><li>Maminishkis, A., Chen, S., Jalickee, S., Banzon, T., Shi, G., Wang, F. E., Ehalt, T., Hammer, J. A., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confluent+monolayers+of+cultured+human+fetal+retinal+pigment+epithelium+exhibit+morphology+and+physiology+of+native+tissue.">Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.</a> Invest Ophthalmol Vis Sci. 47, 3612-3624 (2006).</li><li>Shi, G., Maminishkis, A., Banzon, T., Jalickee, S., Li, R., Hammer, J., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+chemokine+gradients+by+the+retinal+pigment+epithelium.">Control of chemokine gradients by the retinal pigment epithelium.</a> Invest Ophthalmol Vis Sci. 49, 4620-4630 (2008).</li><li>Economopoulou, M., Hammer, J., Wang, F. E., Fariss, R., Maminishkis, A., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+localization,+and+function+of+junctional+adhesion+molecule-C+(JAM-C)+in+human+retinal+pigment+epithelium.">Expression, localization, and function of junctional adhesion molecule-C (JAM-C) in human retinal pigment epithelium.</a> Invest Ophthalmol Vis Sci. 50 (3), 1454-1463 (2008).</li><li>Li, R., Maminishkis, A., Banzon, T., Wan, Q., Jalickee, S., Chen, S., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN{gamma}+Regulates+Retinal+Pigment+Epithelial+Fluid+Transport.">IFN{gamma} Regulates Retinal Pigment Epithelial Fluid Transport.</a> Am J Physiol Cell Physiol. 297, C1452-C1465 (2009).</li><li>Li, R., Maminishkis, A., Wang, F. E., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDGF-C+and+-D+induced+proliferation/migration+of+human+RPE+is+abolished+by+inflammatory+cytokines.">PDGF-C and -D induced proliferation/migration of human RPE is abolished by inflammatory cytokines.</a> Invest Ophthalmol Vis Sci. 48, 5722-5732 (2007).</li><li>Maminishkis, A., Jalickee, S., Blaug, S. A., Rymer, J., Yerxa, B. R., Peterson, W. M., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P2Y(2)+receptor+agonist+INS37217+stimulates+RPE+fluid+transport+in+vitro+and+retinal+reattachment+in+rat.">The P2Y(2) receptor agonist INS37217 stimulates RPE fluid transport in vitro and retinal reattachment in rat.</a> Invest Ophthalmol Vis Sci. 43, 3555-3566 (2002).</li><li>Wang, F. E., Zhang, C., Maminishkis, A., Dong, L., Zhi, C., Li, R., Zhao, J., Majerciak, V., Gaur, A. B., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-204/211+alters+epithelial+physiology.">MicroRNA-204/211 alters epithelial physiology.</a> FASEB J. 24, (2010).</li><li>Adijanto, J., Banzon, T., Jalickee, S., Wang, N. S., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CO2-induced+ion+and+fluid+transport+in+human+retinal+pigment+epithelium.">CO2-induced ion and fluid transport in human retinal pigment epithelium.</a> J Gen Physiol. 133, 603-622 (2009).</li><li>Li, R., Maminishkis, A., Zahn, G., Vossmeyer, D., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+alpha5beta1+mediates+attachment,+migration,+and+proliferation+in+human+retinal+pigment+epithelium:+relevance+for+proliferative+retinal+disease.">Integrin alpha5beta1 mediates attachment, migration, and proliferation in human retinal pigment epithelium: relevance for proliferative retinal disease.</a> Invest Ophthalmol Vis Sci. 50, 5988-5996 (2009).</li><li>Peterson, W. M., Meggyesy, C., Yu, K., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+activates+calcium+signaling,+ion,+and+fluid+transport+in+retinal+pigment+epithelium.">Extracellular ATP activates calcium signaling, ion, and fluid transport in retinal pigment epithelium.</a> J Neurosci. 17, 2324-2337 (1997).</li><li>Voloboueva, L. A., Liu, J., Suh, J. H., Ames, B. N., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R)-alpha-lipoic+acid+protects+retinal+pigment+epithelial+cells+from+oxidative+damage.">R)-alpha-lipoic acid protects retinal pigment epithelial cells from oxidative damage.</a> Invest Ophthalmol Vis Sci. 46, 4302-4310 (2005).</li></ol>

在目前的实验中，我们描述了我们先前公布的技术，旨在简化产生一致的hfRPE一个较高的数字每眼细胞的原代培养所需的多个步骤3的其他修改。在多种生理和分子生物学实验中的每个改变原来的程序，经过严格的测试，以确保变更没有引入文物，并不断被许多其他实验室用这些细胞进行测试。最后，进行了实验动物模型，在体外实验结果比较类似的扰动。

仪器需要清扫协议<ol><li>立体显微镜 - 任何需要放大解剖X250 </li><li>解剖菜 - 如金布尔100 x 20毫米＃23062（每清扫一）任何来源</li><li> Sylgard 184 - WPI的猫。 ＃SYLG184 * *为了准备解剖盘后续Sylgard 184包括混合指示，倒入培养皿混合液体弹性体，形成5 - 8MM层。允许弹性治愈至少24小时。菜固化的弹性体，可以使用70％乙醇和重复使用多次（100倍）的消毒</li><li> 27 G针头从屋宇署PrecissionGlide 1 ¼长度（每清扫5）任何来源</li><li> 1X的HBSS溶液中含有钙，镁等盐类 - GIBCO猫＃14025500毫升（好多个解剖）。 </li><li>虹膜剪刀硬质合金锯齿刀片，弧形 - 福斯特猫＃14559-11（一）。 </li><li>视网膜剪刀 - Katena猫＃K4 - 5300（一）。 </li><li>钳 - 如杜蒙镊子与WPI的猫打磨提示＃5＃500085（2件）。 </li><li>板载刀 - 爱尔康，皆伐1毫米，双斜面，角度猫＃8065921540。 </li><li>离心管15毫升 - （每眼）任何来源</li><li>巴斯德玻璃吸管 - （每清扫一）任何来源</li><li>图版12（每夹层）任何来源</li><li> Primaria25厘米2烧瓶- （每眼2-3）任何来源</li></ol>

experimental models for study of retinal pigment epithelial physiology and pathophysiology

我们已经开发出可以产生大量的初级人类胚胎视网膜色素上皮（hfRPE），母语的人RPE的特点的形态，生理和遗传，文化融合的单层细胞培养过程。这些hfRPE细胞培养表现出沉重的色素沉着，电子显微镜显示广泛的顶膜微绒毛。交界各种紧密连接蛋白的免疫荧光标记复合物。这些容易复制的原代培养的上皮极性和功能酷似以前学过本土的RPE细胞，包括人类的哺乳动物模型。这些结果是延长治疗干预在几个人的眼睛疾病的动物模型的发展。我们把重点放在消除异常液体积聚在视网膜或视网膜下腔的战略。视网膜细胞外空间分隔的感光细胞外段的视网膜色素上皮的顶膜和视网膜附件和维护整个主机的RPE /视网膜之间的相互作用的关键。

Watch this Scientific Journal Video about Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology at JoVE.com

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

我们提供了一个汇合单层培养的人胚胎视网膜色素上皮细胞（hfRPE）细胞重现的方法，表现出形态，生理，极性，和成人自然组织的蛋白质和基因表达模式。这项工作已经延伸到一些眼部疾病的动物模型。

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 ...

experimental-models-for-study-retinal-pigment-epithelial-physiology

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

19.2K 次观看.  National Institutes of Health. 我们提供了一个汇合单层培养的人胚胎视网膜色素上皮细胞（hfRPE）细胞重现的方法，表现出形态，生理，极性，和成人自然组织的蛋白质和基因表达模式。这项工作已经延伸到一些眼部疾病的动物模型。

视频: Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms remain elusive mainly due to the lack of a good in vivo model for studying BEST1 and its mutations under physiological conditions. BEST1 encodes an ion channel, namely BESTROPHIN1 (BEST1), which functions in retinal pigment epithelium (RPE); however, the extremely limited accessibility to native human RPE cells represents a major challenge for scientific research. This protocol describes how to generate human RPEs bearing BEST1 disease-causing mutations by induced differentiation from human pluripotent stem cells (hPSCs). As hPSCs are self-renewable, this approach allows researchers to have a steady source of hPSC-RPEs for various experimental analyses, such as immunoblotting, immunofluorescence, and patch clamp, and thus provides a very powerful disease-in-a-dish model for BEST1-associated retinal conditions. Notably, this strategy can be applied to study RPE (patho)physiology and other genes of interest natively expressed in RPE.

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

人视网膜色素上皮细胞的分化、维持和分析: 一种BEST1突变的碟形模型

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

科研

遗传学

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 ...

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain the overall health and structural integrity of the photoreceptors. The RPE is polarized, exhibiting apically and basally located receptors or channels, and performs vectoral transport of water, ions, metabolites, and secretes several cytokines.
In vivo noninvasive measurements of RPE function can be made using direct-coupled ERGs (DC-ERGs). The methodology behind the DC-ERG was pioneered by Marmorstein, Peachey, and colleagues using a custom-built stimulation recording system and later demonstrated using a commercially available system. The DC-ERG technique uses glass capillaries filled with Hank&#8217;s buffered salt solution (HBSS) to measure the slower electrical responses of the RPE elicited from light-evoked concentration changes in the subretinal space due to photoreceptor activity. The prolonged light stimulus and length of the DC-ERG recording make it vulnerable to drift and noise resulting in a low yield of useable recordings. Here, we present a fast, reliable method for improving the stability of the recordings while reducing noise by using vacuum pressure to reduce/eliminate bubbles that result from outgassing of the HBSS and electrode holder. Additionally, power line artifacts are attenuated using a voltage regulator/power conditioner. We include the necessary light stimulation protocols for a commercially available ERG system as well as scripts for analysis of the DC-ERG components: c-wave, fast oscillation, light peak, and off response. Due to the improved ease of recordings and rapid analysis workflow, this simplified protocol is particularly useful in measuring age-related changes in RPE function, disease progression, and in the assessment of pharmacological intervention.

Direct-Coupled Electroretinogram DC-ERG for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

直接耦合电雷电图（DC-ERG），用于记录小鼠视网膜颜料皮皮的光唤起电响应

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain ...

化学

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

医学

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离 体 模型

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

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 ...

生物学

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. ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

从人眼高效、一致地生成视网膜色素上皮/脉络膜平面安装，用于组织学分析

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

从豚鼠眼睛中分离视网膜色素上皮细胞

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

建模和分析视网膜变性

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients&#39; fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

作者聚焦:揭开年龄相关性黄斑变性的发病机制并发现潜在疗法 

Degenerative eye diseases that affect the retinal pigment epithelium layer of the eye have monogenic and polygenic origins. Several disease models and a software application, LipidUNet, have been developed to study mechanisms of disease, as well as potential therapeutic interventions.

视网膜色素上皮的生理与病理生理学研究的实验模型

摘要

探索更多视频

此视频中的章节

人视网膜色素上皮细胞的分化、维持和分析: 一种BEST1突变的碟形模型

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

直接耦合电雷电图（DC-ERG），用于记录小鼠视网膜颜料皮皮的光唤起电响应

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离体模型

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

从人眼高效、一致地生成视网膜色素上皮/脉络膜平面安装，用于组织学分析

从豚鼠眼睛中分离视网膜色素上皮细胞

基于脂质UNet-机器学习的脂质沉积物使用iPSC衍生的视网膜色素上皮表征和定量脂质沉积物的方法

视网膜色素上皮的生理与病理生理学研究的实验模型

摘要

探索更多视频

此视频中的章节

人视网膜色素上皮细胞的分化、维持和分析: 一种BEST1突变的碟形模型

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

直接耦合电雷电图（DC-ERG），用于记录小鼠视网膜颜料皮皮的光唤起电响应

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离 体 模型

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

从人眼高效、一致地生成视网膜色素上皮/脉络膜平面安装，用于组织学分析

从豚鼠眼睛中分离视网膜色素上皮细胞

基于脂质UNet-机器学习的脂质沉积物使用iPSC衍生的视网膜色素上皮表征和定量脂质沉积物的方法

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离体模型