小鼠原代视网膜色素上皮细胞的分离
Summary
本手稿描述了一种逐步从小鼠眼睛中分离视网膜色素上皮（RPE）细胞的简化方案。该方案包括小鼠眼睛的剜除和解剖，然后是RPE细胞的分离，接种和培养。
Abstract
视网膜色素上皮（RPE）层紧邻光感受器后面，并拥有一个复杂的代谢系统，在维持光感受器的功能中起着几个关键作用。因此，RPE结构和功能对于维持正常视力至关重要。该手稿介绍了原代小鼠RPE细胞分离的既定方案。RPE分离是研究不同眼部疾病小鼠模型中RPE病理学分子机制的好工具。此外，RPE分离可以帮助比较从野生型和转基因小鼠中分离的原代小鼠RPE细胞，以及测试可以加速视觉障碍治疗开发的药物。手稿提出了一个循序渐进的RPE隔离方案;整个过程，从眼球摘除到播种，大约需要4个小时。接种后5-7天内不应更换培养基，以使分离的细胞不受干扰地生长。该过程之后通过免疫荧光 表征 细胞中的形态、色素沉着和特异性标志物。细胞最多可以传代三到四次。
Introduction
视网膜色素上皮（RPE）细胞位于脉络膜和神经视网膜之间，形成位于感光器（PR）细胞后面的简单单层立方体细胞1。RPE在维持PR细胞的健康环境中起着关键作用，主要是通过减少活性氧（ROS）的过度积累和随之而来的氧化损伤1。RPE细胞监督许多功能，例如类视黄醇的转化和储存，散射光的吸收，液体和离子运输以及脱落的PR外段膜2，3的吞噬作用。RPE（形态/功能）的改变会损害其功能，导致视网膜病变，这是许多眼部疾病的共同特征4。许多眼部疾病与RPE细胞形态和功能的改变有关，包括一些遗传性疾病，如色素性视网膜炎、Leber先天性黑朦和白化病4，5，6，以及与年龄相关的眼部疾病，如糖尿病视网膜病变（DR）和年龄相关性黄斑变性（AMD）7，8.人类细胞是最理想的，因此研究原代人RPE细胞中的RPE疾病以形成RPE单层是理想的。然而，伦理问题和人类供体的可用性有限，因为大多数这些疾病导致发病率9，但不一定导致死亡率10，从而阻止了原代人RPE细胞的分离。这使得培养来自非人类动物供体的RPE细胞成为首选的替代方案。啮齿动物，特别是小鼠，被认为是研究不同眼部疾病的绝佳模型，因为转基因技术在这些物种中得到了更广泛的建立11。尽管使用培养的原代RPE细胞具有许多优点，但很难维持生长的细胞进行多次传代，或者储存和重复使用细胞。该协议的主要限制是小鼠的年龄;用于RPE分离的小鼠应该非常年轻（18-21天大是最佳的），因为很难从成年小鼠11，12，13培养RPE细胞。RPE细胞可以在任何年龄从小鼠眼睛中分离出来，但是多达四次细胞传代仅在年轻小鼠（18-21天大）中成功。使用C57BL6小鼠和在RPE细胞上缺失N-甲基-D-天冬氨酸受体（NMDARs）的转基因小鼠从小鼠视网膜中分离RPE以研究升高的氨基酸同型半胱氨酸对AMD14发育和进展的影响。此外，分离的原代RPE细胞通过抑制RPE细胞的NMDAR来帮助提出AMD的治疗靶点14。有一些NMDAR阻滞剂已获得美国食品和药物管理局（FDA）的批准，目前用于治疗与阿尔茨海默病（AD）相关的中度至重度混乱（痴呆），例如美金刚16，这可能是AMD14的潜在治疗靶点。此外，分离的原代小鼠RPE细胞用于检测炎症标志物，并使用转基因小鼠（CBS）诱导炎症作为同型半胱氨酸诱导的AMD和AD特征的潜在机制，其表现出高水平的同型半胱氨酸16，17。
该方案用于从我们实验室开发的野生型C57BL / 6小鼠和转基因小鼠中分离RPE细胞，作为其他已发布的分离方案13，18，19的简化改编，以达到易于应用和可靠的方案。该协议中没有性别偏好。虽然小鼠年龄对分离过程至关重要，但年轻，年龄的小鼠（18-21天大）和任何年龄（长达12个月）的老年小鼠用于RPE分离。然而，我们注意到从年轻小鼠中分离的RPE细胞寿命更长，最多可以进行四次传代。从老年小鼠中分离的RPE细胞可以传代一次或两次，然后它们将停止以正常速率生长并改变其形状以更细长（成纤维细胞样细胞）。还观察到色素沉着丧失和对组织培养板的粘附降低，随后出现脱落。
Protocol
Representative Results
Discussion
当前协议是从小鼠眼睛中分离RPE的报告，修改和简化的详细程序。该协议包括从小鼠眼睛分离的RPE细胞的剜除，解剖，收集，接种，培养和表征。
成功进行RPE分离必须满足一些限制和关键步骤，例如小鼠年龄，解剖的眼睛数量，组织培养板或培养皿的大小以及接种，储存和传代后的注意事项。为了能够传代分离的细胞多达三到四次，最好的小鼠年龄在18-21天之间。为了获得合…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家眼科研究所（NEI），国家眼科研究所（NEI）基金R01 EY029751-04的支持。
Materials
| Beaker : 100mL | KIMAX | 14000 | |
| Collagenase from Clostridium histolyticum | Sigma-Aldrich | C7657-25MG | For working enzyme, A |
| Disposable Graduated Transfer Pipettes :3.2mL Sterile | 13-711-20 | ||
| DMEM/F12 | gibco | 11330 | Media to grow RPE cells |
| Fetal Bovine Serum (FBS) | gibco | 26140079 | For complete RPE cell culture media |
| Gentamicin Reagent Solution | gibco | 15750-060 | For complete RPE cell culture media |
| Hanks' Balanced Salt Solution (HBSS) | Thermo Scientific | 88284 | For working enzymes (A&B) |
| Heracell VISO 160i CO2 Incubator | Thermo Scientific | 50144906 | |
| Hyaluronidase from bovine testes | Sigma-Aldrich | H3506-500MG | For working enzyme A |
| Kimwipes | Kimberly-Clark | 34155 | |
| Luer-Lok Syringe with attached needle 21 G x 1 1/2 in., sterile, single use, 3 mL | B-D | 309577 | |
| Micro Centrifuge Tube: 2 mL | Grainger | 11L819 | |
| Mouse monoclonal anti-RPE65 antibody | Abcam, Cambridge, MA, USA | ab78036 | For IF staining |
| Pen Strep | gibco | 15140-122 | For complete RPE cell culture media |
| Positive Action Tweezers, Style 5/45 | Dumont | 72703-DZ | |
| Scissors Iris Standard Straight 11.5cm | GARANA INDUSTRIES | 2595 | |
| Sorvall St8 Centrifuge | ThermoScientific | 75007200 | |
| Stemi 305 Microscope | Zeiss | n/a | |
| Surgical Blade, #11, Stainless Steel | Bard-Parker | 371211 | |
| Suspension Culture Dish 60mm x 15mm Style | Corning | 430589 | |
| Tissue Culture Dish : 100x20mm style | Corning | 353003 | |
| Tornado Tubes: 15mL | Midsci | C15B | |
| Tornado Tubes: 50mL | Midsci | C50R | |
| Trypsin EDTA (1x) 0.25% | gibco | 2186962 | For working enzyme B |
| Tweezers 5MS, 8.2cm, Straight, 0.09×0.05mm Tips | Dumont | 501764 | |
| Tweezers Positive Action Style 5, Biological, Dumostar, Polished Finish, 110 mm OAL | Electron Microscopy Sciences Dumont | 50-241-57 | |
| Underpads, Moderate : 23" X 36" | McKesson | 4033 | |
| Vannas Spring Scissors – 2.5mm Cutting Edge | FST | 15000-08 | |
| Zeiss AxioImager Z2 | Zeiss | n/a | |
| Zeiss Zen Blue 2.6 | Zeiss | n/a |
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