Name: large-scale purification of porcine or bovine photoreceptor outer segments for phagocytosis assays on retinal pigment epithelial cells
Uploaded: 2014-12-12T13:00:00.000+00:00
Duration: 10 min 2 s
Description: Watch this Scientific Journal Video about Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells at JoVE.com

这项工作是由国立法新社德拉RECHERCHE（Chercheuses的Jeunes / Chercheurs的Jeunes到EFN），基金会VOIR等Entendre和贝当古基金会舒尔勒（青年研究补助金，以EFN），中心法国国家科学研究（CNRS，为EFN永久位置）支持和健康（R01-EY13295到SCF）全国学院的国立眼科研究所。此外，研究所德拉视觉是由国家研究所德拉桑特等德拉RECHERCHE MEDICALE，UNIVERSITE皮埃尔与玛丽居里 - 巴黎6，中心法国国家科学研究和百货巴黎资助。

这个POS隔离实验是耗时且可能需要长达12小时来完成，如果POS存储之前被标记。该协议是改编自1987年31 1997年10由RS Molday和同事发表和修改SC Finnemann和同事的论文。 动物根据研究协会视觉与眼科（ARVO）声明的眼科和视觉研究使用动物的处理。协议进行了审查并批准了查尔斯·达尔文的道德委员会从大学的皮埃尔和玛丽·居里 - 巴黎06和Fordham大学机构动物护理和使用委员会。 1.一般设置<ol><li>屠宰尽可能获得后新鲜一旦80鲜猪或牛的眼睛，让他们冷冻在黑暗中。提供指令给供应商，以便该公司进行的方式相同。该协议是优化80眼。到进行更多的目光并获得更大的股票（ 例如，从160眼），双液量并进行2轮超速离心的;游泳池样品后来在收集步骤（见步骤2.2.2）。 </li><li>设置在板凳上的红色安全指示灯，吸水垫，湿巾（ 材料表 ）。准备15厘米菜肴和生物危害袋垃圾收集（未用眼睛部位，玻璃体...）。穿紧身labcoat，手套，护袖和护目镜。 </li><li>注意事项: <ol><li>预冷所有的解决方案，然后在所有的步骤让他们感冒。保持眼睛和梯度冷冻冰尽可能。 </li><li> 保持在黑暗中尽可能直到超速离心后收集步骤，以避免光漂白视色素和感光色素的活性形式的橙粉色的丧失的眼睛和衍生组织 。 </li></ol></li></ol> 2.协议操作<ol><li> 准备解决方案和渐变: <ol><li>解冻牛磺酸股票完全采用37℃水浴。 </li><li>准备从原液所有解决方案（ 表1，储备溶液），并充分混合，用磁力搅拌器对每个溶液中的其它成分的蔗糖。 <ol><li>制备15ml的所述均质溶液至20％蔗糖的最终浓度，20毫摩尔Tris pH7.2的醋酸盐，2mM的MgCl 2的，10mM葡萄糖和5mM牛磺酸。 </li><li>制备25％的蔗糖溶液，以25％的蔗糖（使用70％的蔗糖的库存），20毫摩尔Tris乙酸盐pH为7.2，10mM葡萄糖和5mM牛磺酸的最终浓度。 </li><li>制备的60％的蔗糖溶液，以60％的蔗糖（使用蔗糖粉末）的终浓度，20毫摩尔Tris pH7.2的醋酸盐，10mM葡萄糖和5mM牛磺酸。 </li><li>制备洗涤溶液1至20毫摩尔Tris乙酸盐pH为7.2和5mM牛磺酸的最终浓度。 </li><li>准备洗2解决方案至10％的蔗糖，20毫摩尔Tris乙酸盐pH为7.2和5mM牛磺酸的最终浓度。 </li><li>制备洗3溶液至10％的蔗糖，20mM磷酸钠pH为7.2和5mM牛磺酸的最终浓度。 </li></ol></li><li>平稳梯度制造者磁力搅拌器上并插入在最靠近其中60％的溶液将被插入所述出口腔室中的搅拌棒。使用的截止P200吸管尖放置在管道的出口处，以达到适当的铸造速度。 </li><li>微妙的稀释和搅拌与一个25％使用12毫升每个解决方案（ 即共6管80眼）预冷超速离心管的60％的蔗糖溶液浇铸线性渐变。搅逐渐-稀释60％的蔗糖溶液好。平稳梯度流的速度，浇注溶液顺畅且不能太快。 注意:为了得到一个连续的，线性梯度，保持吸管尖开口就在整个梯度formatio溶液的表面ñ。 </li><li>让梯度坐在冰上约1小时的稳定。保持冷冻，直到加载的梯度。不要摇晃试管，防止梯度的破坏。 </li></ol></li><li> 组织收集: <ol><li>收集在昏暗的红灯组织。取的眼睛成一方面与戳前端与刀片边缘，同时保持眼距（以避免飞溅; 图1）。使用剃刀刀片切割的前眼球成两半（角膜加上至少5mm到巩膜）。取下透镜和里面翻转眼罩出来，使得它可以保持在一个手指从而暴露在视网膜的小费。 </li><li>使用剃刀刀片以一个角度，轻轻刮去视网膜，拆卸容易，并显示为粉红色层，脱绒毡层表面和切断视神经头（ 图1）。收集所有的视网膜中2×50毫升各含15ml中在冰上均化溶液管中。 </li><li> 视网膜同源genization: <ol><li>为了扰乱不同的细胞层，折断的POS落公关细胞，片段的POS的其余部分，均质视网膜悬浮摇动该悬浮液剧烈用手2分钟。 </li><li>滤波器3倍通过双层纱布清洁，以除去大的组织片段，并收集流过清洁50ml试管（ 图1）。视网膜悬挂是很厚，最大化收益，轻轻按下每个纱布过滤后释放剩余的零散组织。 </li></ol></li></ol></li><li> 光感受器外段片段（POS）隔离: <ol><li>轻轻地将粗视网膜准备，约6 - 7毫升每管中，在6×30毫升超速离心管各包含24个毫升新鲜冷冻连续25 - 60％的蔗糖梯度（ 图1）。平衡相对管以适合所使用的转子。超速，在106000 XG为50分钟，在4℃。 </li&gt; <li>吸（ 图2A）去除大部分上面的橘粉色的带坡度的上三分之一的解决方案。用一个截止P1000尖到烧杯中收集橙粉色带。使用漂白剂（生物垃圾）中和后，放弃休息。 </li><li>稀释〜4 - 5卷冰冷的洗涤1解决方案。分成许多30毫升管需要。离心机在3000×g离心在4℃下10分钟。小心弃去上清液。 </li><li>在10毫升重悬粒料洗净2溶液并旋在3000×g离心10分钟，具体如上所述。在15毫升重悬粒料洗净3，并再次旋转以3,000×g离心10分钟。组合来自几个粒料悬浮，以减少在洗涤2离心前需要管的数目和清洗3的解决方案。 </li></ol></li><li> POS等分无标签: <ol><li>悬浮POS 10 - 20毫升DMEM。预稀释10微升在490微升DMEM（1:50）和计数细长以及在一个旋转着的POS细胞计数室（ 图2B）。计算产量和浓度在每毫升的POS颗粒。 注:产量变化，取决于主要的年龄和应变的猪/奶牛和眼睛的大小， 例如，良好的制剂的范围内，从5 - 8×10 9的POS 80的猪眼。 </li><li>决定最终的DMEM体积上（一般为10 - 20ml）中，并添加蔗糖以产生2.5％的蔗糖的最终浓度。 </li><li>制备所需大小的等分试样， 例如，每管5×10 7的POS为2ml的悬浮体积为96孔试验，在40微升/孔。自旋1等分试样5分钟，在2300×g离心在RT和评价粒料尺寸。准备各种尺寸的试样为不同的应用量为POS不应被冻结，解冻两次。 </li><li>冻结POS等分在-80°C，直到进一步使用。 注:等份稳定许多个月。在我们手中，未标记的或标记的POS可以存储至少一年。 </li></ol></li><li> POS标记和分装 : 注意:我们通常使用的FITC标记的POS为吞噬作用测定法的定量，但其它的染料也可以使用，以及根据需要。 <ol><li>重悬1 10毫克的FITC小瓶在2.5毫克/毫升的浓度在0.1M碳酸钠缓冲液pH 9.5通过混合2.6毫升的0.1M的NaHCO 3 pH8.4的与1.4毫升的0.1M的Na 2 CO 3的pH 11.5（ 表2，储备溶液）。旋转1小时在室温下，同时从光及自旋保护5分钟，在最大速度下，以沉淀的非重悬于FITC的固体。 </li><li>重悬在POS沉淀在10ml洗涤3溶液（或DMEM）中，并加入2毫升FITC溶液80的眼睛。存储剩余FITC冷冻在-80℃。旋转为至少1.5小时，在室温。用铝箔避光。 </li><li>直到几乎没有游离的FITC被认为是在上清液F之类在第2.3.4节中所述洗净用洗净3溶液两次标记的POS，然后一次或两次的DMEMraction。悬浮在POS DMEM并继续为未标记的POS计数和分装（ 第2.4节 ）。 </li></ol></li><li> 在实验中使用POS: <ol><li>解冻的POS在RT，并保持在黑暗中尽可能如果它们是荧光标记的。旋转5分钟，在2300 XG在室温。吸出上清液。 </li><li>立即悬浮在测定溶液适当体积决定了实验。的情况下的POS挑战不同的时间，存储在时间点之间的暗的POS悬浮于室温进行数小时。 </li></ol></li></ol>

The authors declare that they have no competing financial interest.

三个步骤或条件是一个优化的纯化至关重要:梯度铸造细腻梯度管操纵质量，保持组织冷冻并在黑暗中直到收集步骤，抖视网膜匀浆的强度，从PR的其余部分获得适当的POS隔离细胞。如果有些问题出现在正确看到橙色的乐队，他们是由于三个原因大于1（另见结果部分的第二段），最有可能的。某些条件做一些修改是可能的，就像使用27 - 50％的蔗糖浓度梯度32或Optiprep步梯度3...

在视网膜，视力是由光敏分子，称为视蛋白，被转化成可在神经元之间向上传送到视觉区域在大脑中的信号之前的异构触发。这些分子被嵌入在叠层膜的磁盘类似，构成感光细胞外节的部分（PRS）的煎饼。受到恒定曝光，因此，相当大的水平的氧化应激，收受不断更新它们的外节段，以限制潜在的氧化损伤。光感受器外节是与相邻的视网膜色素上皮（RPE）细胞的顶端微绒毛紧密接触。视网膜色素上皮细胞构成的血-视网膜屏障的外部部分，并确保许多任务，这对于光感受器的健康和功能1是至关重要的，例如经由黑色素颜料，再异构化光反应视蛋白成分的视网膜，提供营养物和克淬火光线rowth因素，参与PR​​代谢产物处置。 此外，RPE细胞消除花费POS和回收它们的组件，一个由昼夜节律在哺乳动物视网膜2,3调节每日占领。棚的POS的间隙为PR生存绝对必要的。当它被完全废除，POS碎片累积和永久退化造成快速视力下降4,5。如果有节奏的配置文件丢失，通过不断的活动取代，PR和RPE缺陷积累与6岁。因此，这是非常重要的，以便了解表型与它的功能障碍以表征RPE吞噬作用的分子调控体外 。有趣的是，在视网膜色素上皮细胞的分子机制非常类似于使用由巨噬细胞清除凋亡细胞的1和两者都依赖于识别暴露磷脂酰丝氨酸上吞噬碎片7-9。仍然，RPE细胞和巨噬细胞调节pH值agocytosis不同，如巨噬细胞相遇时选择立即消除凋亡细胞的同时RPE细胞有节奏，每天吞噬POS只有一次，尽管他们与外节永久接触。这表明，还没有完全理解特定调节机制。 许多牵连的视网膜色素上皮细胞吞噬机械的分子已被确定或确认由于使用分离的POS和细胞培养物的吞噬作用测定法。与其配体MFG-E8位于RPE顶端细胞表面上的alphavbeta5整联蛋白受体，在协调，特异性结合的POS 10-12，其然后经由MERTK酪氨酸激酶受体13-15内在化。的CD36的清道夫受体已经显示参与POS摄取并影响其速度16,17，和可作为氧化磷脂在POS表面18的传感器。内在需要F-肌动蛋白细胞骨架屁股招聘ociated蛋白例如膜联 ​​蛋白2 19，肌球蛋白II 20和肌球蛋白VIIA 21,22。 体外天然或氧化的POS也利用来理解RPE细胞的体内连接于消化的不良氧化的POS 23-28中累积的衰老表现型。从干细胞的RPE细胞的产生已经开始对被用来证明细胞的功能它们被移植到动物或患者29,30,27之前孤立的POS一个新的应用程序。 首先由Molday和他的同事在1987年31所述，协议牛POS隔离结合连续蔗糖梯度与观察漂白视网膜感光色素的特点橙色外观的视网膜匀浆的超速离心步骤（携带11- 顺式视黄醛）。在过去的10年里，由于为了减少疯牛病的风险采取的预防措施，利用猪的眼睛已成为increasingly突出。这里所描述的协议说明如何获得从猪或牛的眼睛可以被等分并储存延长的时间段的大量的POS的。这样就无需从啮齿动物的眼睛，这需要使用大量POS每个准备和检测32,33,21,22动物准备POS。另外，使用荧光分子贮存前约POS标注细节给出，量化和可视化的POS中用于相对于吞噬测定32,10后的POS标签一些应用的简化和可比的方式。因此，这些大股允许再现性和易用性在许多不同类型的实验。

<table><tbody><tr><td colspan="4">Specific Material/Equipment</td></tr><tr><td>2 Chamber gradient maker</td><td></td><td></td><td>Gradient maker with 30 ml chambers</td></tr><tr><td>3 mm diameter silicone tubing</td><td></td><td></td><td>tubing for gradient casting</td></tr><tr><td>Small size magnetic stir bar</td><td></td><td></td><td>Stir bar fitting the gradient maker chamber</td></tr><tr><td>Red safelight lamp</td><td></td><td></td><td>Inactinic lamp for dissection in the dark</td></tr><tr><td>Ultra Clear 25 x 89 cm tubes</td><td>Beckman</td><td>344058</td><td>Ultracentrifugation tubes</td></tr><tr><td>PP Oak Ridge tubes</td><td>Nalgene</td><td>3119-0050</td><td>30 ml centrifugation tubes</td></tr><tr><td>Optima LE-80K</td><td>Beckman Coulter</td><td>365668</td><td>Ultracentrifuge</td></tr><tr><td>SW 32Ti swing rotor</td><td>Beckman Coulter</td><td>369694</td><td>Swing rotor for ultracentrifuge</td></tr><tr><td>Avanti J-26 XP</td><td>Beckman Coulter</td><td>393124</td><td>Centrifuge</td></tr><tr><td>JA-25.50 rotor</td><td>Beckman Coulter</td><td>363058</td><td>Rotor for Avanti J-26 XP centrifuge</td></tr><tr><td>FITC Isomer I</td><td>Life Technologies</td><td>F-1906</td><td>Fluorescent dye</td></tr><tr><td colspan="4">Other Material/Equipment</td></tr><tr><td>Counting chamber (such as Neubauer or Malassez)</td><td></td><td></td><td></td></tr><tr><td>Dark ice buckets with lids</td><td></td><td></td><td></td></tr><tr><td>Scales</td><td></td><td></td><td></td></tr><tr><td>Magnetic stirrer and upholding pole</td><td></td><td></td><td></td></tr><tr><td>Refrigated microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C water bath</td><td></td><td></td><td></td></tr><tr><td>-80 &amp;deg;C freezer</td><td></td><td></td><td></td></tr><tr><td colspan="4">Consumables</td></tr><tr><td>Labcoat</td><td></td><td></td><td>Health and safety</td></tr><tr><td>Gloves</td><td></td><td></td><td></td></tr><tr><td>Sleeve protectors</td><td></td><td></td><td></td></tr><tr><td>Goggles</td><td></td><td></td><td></td></tr><tr><td>Absorbent pads</td><td></td><td></td><td></td></tr><tr><td>Biohazard trash bags and bins</td><td></td><td></td><td></td></tr><tr><td>Weck-Prep blades</td><td></td><td>Dissection</td><td>60 mm/2.25 inch wide razor blades</td></tr><tr><td>15 cm plastic dish</td><td></td><td></td><td></td></tr><tr><td>Sterile gauze sheets</td><td></td><td></td><td></td></tr><tr><td>15 and 50 ml tubes</td><td></td><td></td><td>Common consumables</td></tr><tr><td>Microtubes</td><td></td><td></td><td></td></tr><tr><td>Aluminum foil</td><td></td><td></td><td></td></tr></tbody></table>

large-scale purification of porcine or bovine photoreceptor outer segments for phagocytosis assays on retinal pigment epithelial cells

可以在体外进行的视网膜色素上皮（RPE）细胞，光感受器外节（POS）的花费老化远端片段的吞噬的重要功能之一的分析。光感受器外节与栈含有光转机械膜质盘被连续更新的在视网膜上。花了POS是由视网膜色素上皮细胞每天淘汰。啮齿动物，猪/牛和人RPE细胞识别以类似的方式，从不同物种的POS。为了便于进行大系列很少变性的实验中，一个大的股票的POS可以从猪眼来分离和冷冻储存在等分试样。该协议需要时蒙在鼓里中显示橙色的感光色素的特性优势。在昏暗的红灯，视网膜被收集在从切成两半打开眼罩的缓冲区。视网膜细胞悬浮液均化，过滤，并上样到一个连续的蔗糖梯度。岑后trifugation，POS位于一个离散的频带中具有特性橙色渐变的上部。 POS然后收集，纺丝，在洗涤缓冲液再悬浮依次计数并等分。可用于吞噬作用测定法和蛋白激活，定位或相互作用的POS激发后的不同时间分析得到这样的POS。此外，POS可以标记荧光， 例如 ，FITC，等分的POS随后的荧光定量结合或吞噬之前。其他可能的应用包括使用改性的POS或POS挑战结合胁迫条件，研究氧化应激或RPE细胞老化的作用。

线性蔗糖梯度和超速离心的组合允许视网膜悬浮液密度的不同组分的分离。重较大的视网膜碎片和RPE细胞沉到或接近渐变（ 图2A）的底部。来自视网膜打火机POS和较轻的单个细胞或细胞碎片迁移作为单独的频带通过离心期间结束到达梯度的上半部分。通过保持眼睛和样品在黑暗中直到离心步骤后，含有的POS频带可立即通过其明亮的橙色颜色识别。该频带的宽度和强度取决于梯度质量?...

Watch this Scientific Journal Video about Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells at JoVE.com

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

猪或牛光感受器外段的细胞吞噬分析对视网膜色素上皮细胞大规模纯化

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

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Research

JoVE Journal

Immunology and Infection

12.0K 次观看.  INSERM, U968. This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 °C.

视频: Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

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 ...

科研

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, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

哺乳动物原色素上皮细胞的分离、培养和基因工程用于非病毒基因治疗

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

猪视网膜色素上皮细胞原代培养

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

量化视网膜色素上皮吞噬作用和脂褐素积累

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

作者聚焦：改进的脂褐素模型和高度极化人类视网膜色素上皮培养物中外段吞噬作用能力的定量  

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

改进脂褐素模型和高极化人视网膜色素上皮培养物中外段吞噬能力的定量

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging ...

猪或牛光感受器外段的细胞吞噬分析对视网膜色素上皮细胞大规模纯化

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此视频中的章节

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

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

小鼠视网膜色素上皮细胞的原代培养

哺乳动物原色素上皮细胞的分离、培养和基因工程用于非病毒基因治疗

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

改进脂褐素模型和高极化人视网膜色素上皮培养物中外段吞噬能力的定量

改进脂褐素模型和高极化人视网膜色素上皮培养物中外段吞噬能力的定量