原发性小鼠视网膜色素上皮细胞的有效解剖与培养
Summary
该协议最初由费尔南德斯-戈迪诺等人于2016年1月报告，描述了一种有效分离和培养小鼠RPE细胞的方法，该细胞在一周内在Transwell板上形成功能和极化的RPE单层。手术大约需要3个小时。
Abstract
眼疾影响全球数百万人，但人体组织的有限可用性阻碍了他们的研究。小鼠模型是了解眼科疾病病理生理学的有力工具，因为它们与人体解剖学和生理学相似。视网膜色素上皮（RPE）的改变，包括形态和功能的变化，是许多眼部疾病共有的共同特征。然而，成功隔离和培养原鼠RPE细胞是非常具有挑战性的。本文是费尔南德斯-戈迪诺等人之前在 2016 年发布的协议的更新视听版本，旨在有效隔离和培养主要鼠标 RPE 细胞。这种方法具有高度可重复性，并导致高度极化和色素化的 RPE 单层的坚固培养物，可在 Transwells 上保持数周。该模型为研究眼部疾病背后的分子和细胞机制开辟了新的途径。此外，它提供了一个平台，用于测试治疗方法，可用于治疗重要的眼部疾病与未满足的医疗需求，包括遗传性视网膜疾病和黄斑变性。
Introduction
该协议最初由费尔南德斯-戈迪诺等人于2016年1月报告，描述了一种有效隔离和培养小鼠视网膜色素上皮（RPE）细胞的方法，该细胞在一周内在Transwell板块上形成功能性和极性RPE单层。RPE 是位于神经视网膜和布鲁赫膜之间的眼睛中的单层。这一层由高度极化和色素化的上皮细胞组成，由紧密的结点连接，呈现出类似于蜂巢2的六边形形状。尽管这种明显的组织学简单，RPE执行各种功能的关键视网膜和正常的视觉周期2，3，4。RPE单层的主要功能包括光吸收、光感受器的滋养和更新、代谢端产品的去除、亚视网膜空间中的离子平衡控制以及血视网膜屏障2、3的维护。RPE在眼睛5、6、7、8、9、10、11的局部调节中也起着重要的作用。RPE的退化和/或功能障碍是许多眼部疾病的共同特征，如视网膜炎色素沉着症，勒伯先天性动脉瘤，白化病，糖尿病视网膜病变，和黄斑变性12，13，14，15。不幸的是，人体组织的可用性是有限的。鉴于小鼠与人类高度保存的遗传同源性，小鼠模型是研究眼部疾病16、17、18、19的合适和有用的工具。此外，使用培养的原发性RPE细胞提供的优势，如基因操纵和药物测试，可以加快开发新的疗法，这些危及视力的疾病9，11。
可用于鼠标 RPE 隔离和文化的现有方法缺乏可重复性，并且无法以足够的可靠性在体内重新概括 RPE 功能。细胞往往在几天内失去色素化，六角形和跨皮电阻（TER）在培养13，20。由于从小鼠身上建立这些初级RPE细胞培养是一个具有挑战性的过程，这个优化的协议已经创建基于其他协议，从大鼠和人眼分离RPE细胞21，22，23解剖小鼠的眼睛，收集RPE和培养小鼠RPE细胞在体外。
Protocol
遵循了《在眼科和视力研究中使用动物的ARVO声明》的准则。 注：这种方法已被证明对不同遗传背景的小鼠成功，包括C57BL/6J，B10。D2-Hco H2d H2-T18 c/oSnJ和白化病小鼠，在不同年龄。最好使用8到12周大的小鼠来获得RPE细胞。来自老老鼠的RPE细胞在培养中繁殖较少，而幼鼠的细胞越来越小，这就要求汇集不同动物的眼睛才能有可行的培养物。 <p class="jove_…
Representative Results
该协议已被用来隔离和培养RPE细胞从转基因小鼠1。没有观察到小鼠菌株或性别之间的差异。研究结果有助于了解眼部疾病机制的一些重要方面，如与年龄有关的黄斑变性，这是老年人视力丧失的最常见原因。在此协议后分离的 RPE 细胞在播种后 24 小时内完全连接到膜插入上，并在 72 h1后显示典型的 RPE 大小、形态和色素沉着。一周后，一个高?…
Discussion
虽然在1、13、20、22、26、27之前已经开发出几种小鼠RPE细胞分离和培养方法，但费尔南德斯-戈迪诺的方法首先使用膜插入，使RPE细胞在培养中的有效生长长达1周，即9周。他们的协议1，9…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了麻省眼耳眼科基因组研究所的支持。
Materials
| 10 ml BD Luer-Lok tip syringe, disposable | BD Biosciences | 309604 | |
| 15 ml centrifuge tube | VWR International | 21008-103 | |
| 50 ml centrifuge tube | VWR International | 21008-951 | |
| Alpha Minimum Essential Medium | Sigma-Aldrich | M4526-500ML | |
| Angled micro forceps | WPI | 501727 | |
| Bench-top centrifuge | any | ||
| CO2 incubator | Thermo | HERA VIOS 160I CO2 SST TC 120V | |
| Dissecting microscope | Any | ||
| Dulbecco’s Phospate Buffered Saline no Calcium, no Magnesium | Gibco | 14190144 | |
| Dumont #5 45° Medical Biology tweezers, 0.05 x 0.01 mm tip, 11 cm length | WPI | 14101 | |
| Ethanol | Sigma-Aldrich | E7023-500ML | |
| Falcon Easy-Grip Clear Polystyrene Cell Culture Dish, 35mm | BD Biosciences | 353001 | |
| Fetal Bovine Serum | Hyclone | SH30071.03 | Heat inactivated. |
| Hank’s Balanced Salt Solution plus Calcium and Magnesium, no Phenol Red | Life Technologies | 14175095 | |
| Hank’s Balanced Salt Solution plus Calcium and Magnesium, no Phenol Red B6 | Life Technologies | 14025092 | |
| HEPES 1M | Gibco | 15630106 | |
| Hyaluronidase | Sigma-Aldrich | H-3506 1G | |
| Hydrocortisone | Sigma-Aldrich | H-0396 | |
| Laminar flow hood | Thermo | CLASS II A2 4 115V PACKAGECLA | |
| Laminin 1mg/ml | Sigma-Aldrich | L2020-1 MG | Dilute in PBS at 37C to 1mg/ml |
| McPherson-Vannas Micro Scissors 8 cm long | WPI | 503216 | |
| Non-essential amino acids 100X | Gibco | 11140050 | |
| N1 Supplement 100X | Sigma-Aldrich | N6530-5ML | |
| Penicillin-Streptomycin | Gibco | 15140-148 | |
| Sterile Bard-Parker Carbon steel surgical blade size 11 | Fisher-Scientific | 08-914B | |
| Taurine | Sigma-Aldrich | T-0625 | |
| Tissue culture treated 12-well plates | Fisher-Scientific | 08-772-29 | |
| Tissue culture treated 6-well plates | Fisher-Scientific | 14-832-11 | |
| Transwell supports 6.5 mm | Sigma-Aldrich | CLS3470-48EA | |
| Triiodo-thyronin | Sigma-Aldrich | T-5516 | |
| Trypsin-EDTA (0.25%), phenol red | Gibco | 25200056 | |
| Tweezer, Dumont #5 Medical Biology 11 cm, curved, stainless steel 0.02 x 0.06 mm Mod tips | WPI | 500232 | |
| Vannas Scissors 8cm long, stainless steel | WPI | 501790 | |
| Whatman Puradisc 25mm Syringe Filters 0.45μm pore size | Fisher-Scientific | 6780-2504 |
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Cite This Article
Chinchilla, B., Getachew, H., Fernandez-Godino, R. Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells. J. Vis. Exp. (168), e62228, doi:10.3791/62228 (2021).