猪视网膜色素上皮细胞原代培养
1Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, School of Medicine,Xiamen University, 2Department of Ophthalmology, Xiang’an Hospital of Xiamen University, School of Medicine,Xiamen University, 3Xiamen University Affiliated Xiamen Eye Center, School of Medicine,Xiamen University
Summary
本文介绍了一种易于遵循的体 外 培养原代猪视网膜色素上皮细胞的方法。
Abstract
视网膜色素上皮（RPE）是单层极化色素上皮细胞，位于视网膜的脉络膜和神经视网膜之间。多种功能，包括吞噬作用，营养/代谢物运输，维生素A代谢等，由RPE每天进行。RPE细胞是终末分化的上皮细胞，具有很少或没有再生能力。RPE细胞的缺失会导致多种眼部疾病，导致视力障碍，例如年龄相关性黄斑变性。因此，建立原代RPE细胞的 体外 培养模型，其与 体内 RPE比细胞系更相似，对于RPE细胞的特征和机理研究至关重要。考虑到人类眼球的来源有限，我们创建了一个培养原代猪RPE细胞的方案。通过使用该方案，RPE细胞可以很容易地从成年猪眼球中解离。随后，这些解离的细胞附着在培养皿/插入物上，增殖形成汇合的单层，并在2周内快速重建 体内 上皮组织的关键特征。通过qRT-PCR，证明原代猪RPE细胞表达的多个特征基因与天然RPE组织相当，而这些基因中的大多数在人RPE样细胞ARPE-19中丢失/高度降低。此外，免疫荧光染色显示紧密连接、组织极性和细胞骨架蛋白的分布，以及培养的原代细胞中存在对维生素 A 代谢至关重要的异构酶 RPE65。总之，我们开发了一种易于遵循的方法来培养具有高纯度和天然RPE特征的原代猪RPE细胞，这可以作为了解RPE生理学，研究细胞毒性和促进药物筛选的良好模型。
Introduction
视网膜色素上皮 （RPE） 位于视网膜1 外层的感光器和脉络膜毛细血管之间，具有多种功能，包括形成血液-视网膜屏障、运输和交换营养物质和视网膜代谢物、回收维生素 A 以维持正常的视觉周期，以及吞噬和清除脱落的感光器外段 （POS）2，3.由于POS需要不断的自我更新才能产生视力，因此RPE细胞需要不断吞噬分离的POS以维持视网膜稳态4。因此，RPE功能障碍导致许多致盲性眼病，如年龄相关性黄斑变性（AMD）4，视网膜色素变性（RP）5，Leber先天性黑朦6，糖尿病视网膜病变7等。到目前为止，大多数这些疾病的确切发病机制仍然难以捉摸。因此，建立了RPE细胞培养来研究RPE细胞生物学，病理变化和潜在机制。
作为研究细胞生物学的最简单模型，RPE细胞的培养早在1920年代就开始了8。尽管ARPE-19被广泛用作RPE细胞，但色素沉着的丧失，鹅卵石形态，特别是该细胞系中的屏障功能引起了很多关注9。相比之下，原代人RPE细胞的培养为生理和病理研究提供了更现实的场景9。然而，相对有限的可用性限制了它们的使用，道德问题始终存在。此外，几个小组使用小鼠模型培养RPE细胞。但小鼠眼睛尺寸小，单一培养通常需要多只小鼠，不方便9.最近，科学家们开发了利用人类胚胎干细胞或诱导多能干细胞来衍生RPE细胞的新方法。尽管该技术在治疗遗传性RPE疾病方面具有特殊潜力，但它非常耗时，通常需要几个月才能产生成熟的RPE细胞10。为了克服这些问题，我们在这里介绍了一种易于遵循的方案，用于在实验室中常规分离和培养高纯度RPE细胞。在合适的培养条件下，这些细胞可以显示出典型的RPE功能并表现出典型的RPE形态。因此，该培养方法可为了解RPE生理学、研究细胞毒性、研究相关眼病的病理机制、进行药物筛选提供良好的模型。
Protocol
实验动物的使用符合视觉与眼科研究协会（ARVO）的规定，并得到厦门大学实验动物管理伦理委员会的批准。 1. 实验手术器械、组织消化酶、细胞培养缓冲液的制备 准备实验性手术装置，在眼球解剖前一天清洁并高压灭菌两对眼科手术剪刀和镊子，然后在65°C的通用方案烤箱中用手术器械干燥盒子过夜。 培养基制备。制备补充有 10% （v/v） 胎…
Representative Results
将原代猪RPE（pRPE）细胞在含有10S的DMEM/碱性培养基中培养，并在接种后2天（图2A）、6天（图2B）和10天（图2C）在光学显微镜下拍摄细胞形态。1周后，观察到具有鹅卵石形态的单层色素沉着pRPE细胞汇合。 为了更好地表征原代pRPE细胞，将传代3（P3）18和ARPE-19细胞的原代人RPE细胞（hRPE）在DME…
Discussion
在这里，描述了用于从猪眼球中分离，培养和表征RPE细胞的详细和优化方案，该方案为RPE细胞的体外表征和RPE相关疾病研究提供了良好的模型。从人，小鼠和大鼠眼睛中分离RPE的方法已在前面描述过23，24，25。然而，在一些实验室中很难获得人类的眼球，并且通常会引发伦理问题。小鼠和大鼠的RPE组织相对较小，细胞在…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者希望对所有在这项研究中贡献细胞的动物表示感谢和尊重。这项研究部分得到了中国国家重点研发计划（2019YFA0111200，Yi Liao & Yuan Gao和Grant Nos. 2018YFA0107301，Wei Li）的资助。作者感谢厦门大学医学院中心实验室的黄静茹和尤翔在共聚焦成像方面的技术支持。
Materials
| ARPE-19 cells | CCTCC | GDC0323 | |
| Bovine serum albumin | Yeasen | 36101ES60 | |
| Confocal microscopy | Zeiss | LSM 880 with Airyscan | |
| ChemiDoc Touch | Bio-Rad | 1708370 | |
| Cell scraper | Sangon | F619301 | |
| 10 cm culture dish | NEST | 121621EH01 | |
| 12-well culture plate | NEST | 29821075P | |
| DMEM F12 Medium | Gibco | C11330500BT | |
| DMEM basic Medium | Gibco | C11995500BT | |
| EVOM2 | World Precision Instruments | EVOM2 | For TER measurement |
| Fetal bovine serum | ExCell Bio | FSP500 | |
| Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | ThermoFisher Scientific | A-11034 | |
| Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 | ThermoFisher Scientific | A-11012 | |
| Goat anti Mouse IgG (H/L):HRP | Bio-Rad | 0300-0108P | |
| Goat anti Rabbit IgG (H/L):HRP | Bio-Rad | 5196-2504 | |
| hydrocortisone | MCE | HY-N0583/CS-2226 | |
| Hoechst 33342 solution (20 mM) | ThermoFisher Scientific | 62249 | |
| LightCycler 96 Instrument | Roche | 5815916001 | |
| Liothyronine | MCE | HY-A0070A/CS-4141 | |
| laminin | Sigma-Aldrich | L2020-1MG | |
| MEM(1X)+GlutaMAX Medium | Gibco | 10566-016 | |
| MEM NEAA(100X) | Gibco | 11140-050 | |
| Millex-GP syringe filter unit | Millipore | SLGPR33RB | |
| N1 | Sigma-Aldrich | SLCF4683 | |
| NcmECL Ultra | New Cell&Molecular Biotech | P10300 | |
| Non-fat Powdered Milk | Solarbio | D8340 | |
| Nicotinamide | SparkJade | SJ-MV0061 | |
| Na+-K+ ATPase antibody | Abcam | ab76020 | Recognize both human and porcine proteins |
| PAGE Gel Fast Preparation Kit(10%) | Epizyme | PG112 | |
| primary Human RPE cells | – | – | Generous gift from Shoubi Wang lab |
| Pierce BCA Protein Assay Kit | ThermoFisher Scientific | 23225 | |
| Prism | GraphPad by Dotmatics | version 8.0 | |
| Protease Inhibitor Cocktails | APExBIO | K1024 | |
| PRE65 antibody | Proteintech | 17939-1-AP | Recognize both human and porcine proteins |
| PEDF antibody | Santa Cruz Biotechnology | sc-390172 | Recognize both human and porcine proteins |
| 100 x penicillin/streptomycin | Biological Industries | 03-031-1BCS | |
| Phosphate buffered saline (PBS) | RARBIO | RA-9005 | |
| ReverTra Ace qPCR RT Master Mix | Toyobo | FSQ-201 | |
| RIPA buffer | ThermoFisher Scientific | 89900 | |
| 15 mL sterile centrifuge tubes | NEST | 601052 | |
| 50 mL sterile centrifuge tubes | NEST | 602052 | |
| 0.25% Trypsin-EDTA | Gibco | 25200-056 | |
| Taurine | Damas-beta | 107-35-7 | |
| Trizol | Thermo-Fisher | 15596026 | RNA extraction solution |
| TB Green Fast qPCR Mix | Takara | RR430A | |
| 12-well transwell inserts | Labselect | 14212 | |
| VEGF antibody | Proteintech | 19003-1-AP | Recognize both human and porcine proteins |
| VEGF ELISA kit | Novusbio | VAL106 | |
| ZO-1 antibody | ABclonal | A0659 | Recognize both human and porcine proteins |
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Citer Cet Article
Wen, F., Wang, Y., He, D., Liao, C., Ouyang, W., Liu, Z., Li, W., Liao, Y. Primary Culture of Porcine Retinal Pigment Epithelial Cells. J. Vis. Exp. (187), e64244, doi:10.3791/64244 (2022).