用于 体外 建模的原代猪视网膜色素上皮细胞的分离
Summary
该方案概述了从当地来源的猪眼中获取和培养原代视网膜色素上皮（RPE）细胞的程序。这些细胞可作为干细胞的高质量替代品，适用于 体外 视网膜研究。
Abstract
视网膜色素上皮 （RPE） 是视网膜外层负责支持光感受器的关键单层。RPE 变性通常发生在以进行性视力丧失为特征的疾病中，例如年龄相关性黄斑变性 （AMD）。AMD的研究通常依赖于人类供体的眼睛或诱导多能干细胞（iPSC）来代表RPE。然而，这些RPE来源需要更长的分化期和大量的培养专业知识。此外，一些研究机构，特别是农村地区的研究机构，很难获得捐赠者的眼睛。虽然存在市售的永生化 RPE 细胞系 （ARPE-19），但它缺乏基本的 体内 RPE 特征，并且在许多眼科研究出版物中没有被广泛接受。迫切需要从更容易获得和更具成本效益的来源获得具有代表性的原代 RPE 细胞。该方案阐明了从猪眼睛死后获得的原代RPE细胞的分离和传代培养，这些细胞可以从商业或学术供应商处本地采购。该协议需要组织培养实验室中常见的常见材料。其结果是 iPSC、人类供体眼睛和 ARPE-19 细胞的主要、差异化且具有成本效益的替代品。
Introduction
视网膜色素上皮 （RPE） 是位于 Bruch 膜和光感受器之间的视网膜外层的单层1。RPE 细胞与闭塞带-1 （ZO-1） 等蛋白质形成紧密连接，并具有以色素沉着和六边形形态为特征的独特表型 2,3。这些细胞有助于血液-视网膜屏障，从而支持光感受器健康并维持视网膜稳态 4,5。此外，RPE 细胞通过吸收光和回收光感受器的基本成分在视觉中发挥关键作用6.例如，RPE65 是一种在 RPE 细胞中高度表达的蛋白质，可将全反式视黄酯转化为 11-顺式视黄醇 7,8。鉴于 RPE 细胞执行的多种功能，它们的功能障碍与各种疾病有关，包括年龄相关性黄斑变性和糖尿病视网膜病变 9,10。为了加强对视网膜病变的理解并开发新的治疗方法，经常采用视网膜的体外模型。
为了生成健康或患病视网膜的代表性模型，必须使用模拟 RPE 细胞类型。市售的 ARPE-19 细胞系缺乏天然表型，例如色素沉着，而 iPSC 可能需要数月才能分化 11,12,13。尽管人类供体的眼睛可能是理想的，但许多研究实验室通常不容易获得它们。
在这里，我们设计了一种利用猪眼的方法，猪眼与人眼有许多相似之处14，以获得原代RPE细胞。这些原代猪 RPE 细胞已用于多种视网膜模型15,16。这些细胞不仅具有成本效益，而且与iPSC或供体眼睛相比，它们需要更少的时间来获取。此外，它们还表现出天然特征，例如色素沉着和微绒毛。虽然存在类似的猪RPE提取方案17,18,19，但这种简单而详细的技术进一步验证了酶解离，并采用了大多数细胞培养实验室中常见的材料。
Protocol
该程序中使用的眼睛是从当地经美国农业部检查的肉店死后获得的，并且不使用活体动物进行任何工作。在动物被处死后，大约2小时后眼睛被摘除。由于组织腐烂可能开始发生，因此在运输过程中保持眼睛凉爽以防止进一步腐烂非常重要。在此过程中，眼睛在摘除后立即放入冰箱中。随后，将眼袋放置在 1000 mL 聚丙烯烧杯内，并在 8 L 冷却器内被冰包围。重要的是不要将眼睛直接放在冰上。一旦?…
Representative Results
使用该程序，从猪眼中成功分离出原代RPE细胞。 图 1A 显示了分离后 3 天具有特征性色素沉着的 RPE 细胞。生长1周后，细胞完全融合并形成健康的单层（图1B）。然后将细胞转移到细胞培养插入物中，在那里它们保持其色素沉着和形态（图1C），进一步支持分离程序的功效。没有表现出这些特征，而是显示出变异形态和过度色素沉?…
Discussion
该协议描述了如何从猪眼中分离RPE细胞。在分离后 7 天内可以看到色素沉着和鹅卵石形态（图 1B）。此外，TEER 数据表明紧密结形成22 和健康的单层（图 5）。这些结果表明，用这种方法分离的RPE细胞与人RPE相似，在视网膜细胞培养模型中是有益的。
这份手稿中使用的眼睛是死后从当地一家肉店获得的，但也可以?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者要感谢 Farhad Farjood 在猪 RPE 细胞培养和分离方面的帮助，以及 Thomas Harris 在 SEM 方面的帮助。作者感谢犹他州立大学显微镜核心设施对SEM分析的支持。该设施维护着通过美国国家科学基金会主要研究仪器资助（CMMI-1337932）获得的扫描电子显微镜。这项研究的资金由美国国立卫生研究院通过拨款 1R15EY028732 （Vargis） 和 BrightFocus 基金会拨款M2019109 （Vargis） 提供。犹他州立大学研究办公室的本科生研究和创意机会补助金（Weatherston）和犹他州立大学阿尔茨海默病和痴呆症研究中心的种子补助金（Vargis）提供了额外的资金。
Materials
| 6 Micro-well glass bottom plate with 14 mm micro-well #1 cover glass | Cellvis | P06-14-1-N | |
| Antibiotic-Antimycotic (100x) | Gibco | 15240062 | |
| Biosafety Cabinet | |||
| Calcium Chloride, Dried, Powder, 97% | Alfa Aesar | L13191.30 | |
| Cell Strainer | Fisher Scientific | 22-363-548 | one per eye |
| Centrifuge | |||
| Centrifuge Tubes, 15 mL | Fisher Scientific | 05-539-12 | |
| Cooler, 8 L | Igloo | 32529 | |
| Corning Transwell Multiple Well Plate with Permeable Polyester Membrane Inserts | Fisher Scientific | 07-200-154 | Culture inserts |
| Cut Resistant Glove | Dowellife | 712971375857 | |
| Cytiva HyClone Dulbecco's Phosphate Buffered Saline, Solution | Fisher Scientific | SH3026401 | for ICC dilutions only |
| Deionized Water | |||
| DMEM, 1x with 4.5 g/L glucose, L-glutamine & sodium pyruvate | Corning | 10-013-CV | |
| DNase I from Bovine Pancreas | Sigma Aldrich | DN25 | |
| DPBS/Modified – calcium – magnesium | Cytiva | SH3002B.02 | stored at 4 °C |
| ELISA kit, Q-Plex Human Angiogenesis (9-Plex) | Quansys Biosciences, Logan, UT | ||
| ENDOHM 6 TEER device | World Precision Instruments | ||
| Fetal Bovine Serum (FBS) | Avantor | 232B20 | |
| Fisher BioReagents Bovine Serum Albumin (BSA) DNase- and Protease-free Powder | Fisher Scientific | BP9706100 | |
| Flashlight | |||
| Formaldehyde, ACS Grade, 36.5% (w/w) to 38.0% (w/w), LabChem | Fisher Scientific | LC146501 | |
| Gauze Sponges | Fisher Scientific | 22-415-504 | One per eye |
| Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647, Invitrogen | Thermo Scientific | A32728 | RPE65 secondary antibody |
| Ice | Crushed prefered | ||
| Inverted Phase Contrast Microscope | |||
| Invitrogen NucBlue Live ReadyProbes Reagent (Hoechst 33342) | Fisher Scientific | R37605 | |
| Iris Fine Tip Scissors, Standard Grade, Curved, 4.5" | Cole-Palmer | EW-10818-05 | |
| Iris Scissors, 11 cm, Straight, Tungsten Carbide | Fisher Scientific | 50-822-379 | |
| LSM-710 Confocal Microscope | Zeiss | ||
| Petri Dish, 100 mm x 20 mm | Corning | 430167 | one per 2-3 eyes and one for dissection surface/waste |
| Povidone-Iondine Solution, 10% | Equate | 49035-050-34 | |
| RPE65 Monoclonal Antibody (401.8B11.3D9), Invitrogen | Thermo Scientific | MA116578 | RPE65 primary antibody |
| Scalpel Blades Size 10 | Fisher Scientific | 22-079-683 | |
| Scalpel Handles Style 3 | Fisher Scientific | 50-118-4164 | |
| Surgical Drape, 18 x 26" | Fisher Scientific | 50-209-1792 | |
| Tissue Culture Incubator | 37 °C, 5% CO2, 95% Humidity | ||
| Tissue Culture Plates, 6 Wells | VWR | 10062-892 | One for eye wash and one for seeding |
| Tri-Cornered Polypropylene Beaker, 1000 mL | Fisher Scientific | 14-955-111F | |
| Triton X-100 | Sigma Aldrich | T8787 | |
| Trypsin 0.25%, 2.21 mM EDTA in HBSS; w/o Ca, Mg, Sodium Bicarbonate | Corning | 25053Cl | |
| Tweezers Style 20A | Fisher Scientific | 17-467-231 | |
| Tweezers Style 2A | Fisher Scientific | 50-238-47 | for removing neural retina |
| Tweezers Style 5-SA-PI | Fisher Scientific | 17-467-168 | |
| Vacuum Aspiration System | |||
| Water Bath, 37 °C | |||
| ZO-1 Monoclonal Antibody (ZO1-1A12), FITC, Invitrogen | Fisher Scientific | 33-911-1 | ZO-1 conjugated primary antibody |
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Citer Cet Article
Paterson, C. A., Weatherston, D., Teeples, T., Vargis, E. Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling. J. Vis. Exp. (207), e66079, doi:10.3791/66079 (2024).