优化小鼠原晶状体上皮细胞培养：胰蛋白酶消化综合指南
Summary
本手稿概述了用于培养原晶状体上皮细胞 （LEC） 的详细视频方案，旨在提高可重复性并帮助白内障和后囊混浊 （PCO） 的研究。它提供了有关晶状体解剖、LEC 分离和验证的分步说明，可作为有价值的指南，尤其是对于该领域的新手而言。
Abstract
晶状体上皮细胞（LECs）在维持晶状体的稳态和正常功能方面发挥着多种重要作用。LEC 决定了晶状体的生长、发育、尺寸和透明度。相反，功能失调的 LEC 可导致白内障形成和后囊混浊 （PCO）。因此，建立强大的原代LEC培养系统对于从事晶状体开发、生物化学、白内障治疗和PCO预防的研究人员非常重要。然而，由于原代LECs的可用性有限、增殖速度慢且性质脆弱，其培育长期以来一直面临挑战。
本研究通过提出原代 LEC 培养的综合方案来解决这些障碍。该方案包含基本步骤，例如配制优化的培养基、晶状体胶囊的精确分离、胰蛋白酶消化技术、传代培养程序、收获方案以及储存和运输指南。在整个培养过程中，使用相差显微镜监测细胞形态。
为了确认培养的LEC的真实性，进行了免疫荧光测定以检测关键晶状体蛋白（即αA和γ晶状体蛋白）的存在和亚细胞分布。这一详细的方案为研究人员提供了培养和表征原发性 LEC 的宝贵资源，从而促进了我们对晶状体生物学的理解和晶状体相关疾病治疗策略的开发。
Introduction
眼睛的晶状体通过将入射光聚焦到视网膜上，在视觉中起着至关重要的作用。它由透明的无血管结构组成，由特化细胞组成，其中晶状体上皮细胞 （LEC） 是关键参与者。LEC 位于晶状体的前表面，负责保持其透明度、调节水平衡并参与晶状体生长和发育 1,2。LEC 是位于晶状体前部的一种独特类型的细胞，通过在整个生命周期中持续产生晶状体纤维，在维持晶状体清晰度和功能方面发挥着关键作用。
白内障的特征是晶状体逐渐混浊，导致光线失真和散射，导致视力受损 3,4。白内障形成的确切机制是复杂和多因素的，涉及各种细胞和分子过程，如紫外线辐射、氧化损伤和糖基化 5,6。已发现 LEC 对白内障的发展有显着贡献，使其成为研究的重要焦点 1,2,7,8,9。
此外，当今眼科最紧迫的问题之一是后囊混浊 （PCO） 的发生率相对较高，也称为继发性白内障。PCO 仍然是白内障手术后最常见的并发症，在手术后 5 年内影响多达 20-40% 的成人患者和 100% 的儿童10.PCO主要是由白内障摘除术后残留在囊袋中的LEC引起的。这些细胞经历多方面的病理生理学转化，不仅涉及上皮-间充质转化 （EMT），还涉及 LEC 向晶状体纤维的分化，从而形成 LEC、纤维和肌成纤维细胞的混合物细胞群 11,12,13。转化的细胞增殖并迁移到晶状体后囊，导致视力障碍。了解培养模型中 LEC 的行为和控制机制可以为 PCO 的预防和管理提供有价值的见解。因此，这种培养 LEC 的方案为眼科研究人员提供了一个重要的工具，旨在研究、理解并最终对抗这种普遍的术后并发症。
为了揭示LEC生物学的复杂性及其在白内障形成和PCO中的作用，必须建立强大且可重复的 体外 原代细胞培养系统。原代 LEC 培养为研究人员提供了一个受控环境来研究 LEC 的功能、信号传导和分子特征。此外，它还允许研究细胞过程和不同实验条件的影响，为晶状体生理学和病理学提供有价值的见解。
先前的研究丰富了我们对LEC培养技术的理解14,15,16,17,18,19,20。尽管这些研究采用了各种方法，并在LEC行为和特征方面取得了重要发现，但目前的文献中缺乏用于培养LEC的全面且可访问的视频记录方案。这种局限性会阻碍新手研究人员准确重现技术的能力，并可能导致实验结果的不一致和变化。通过提供视频录制协议，本文旨在弥合这一差距，并提供一种标准化资源，以提高LEC文化领域的可重复性并促进知识转移。
Protocol
Representative Results
Discussion
本文介绍的方案为成功分离、培养和传代 LEC 提供了全面的分步指南，并附有视频文档。详细的视觉指南和书面说明增强了协议的清晰度和可访问性，促进了该领域研究人员的使用和可重复性。最终目的是促进围绕 LEC 在白内障形成和 PCO（白内障手术后普遍存在的并发症）中的作用的知识体系的扩展。
当将原代 LEC 与晶状体上皮细胞系（如 HLE-B3 和 SRA01/04）进行比较时，每种?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NEI R21EY033941（对吴洪利）的支持;国防部W81XWH2010896（致吴洪利）;R15GM123463-02 （致Kayla Green和Hongli Wu）
Materials
| 0.05% Trypsin-EDTA | Thermo Fisher | #25300054 | For LECs dissociation |
| Alexa Fluor 488 Secondary Antibody | Jackson ImmunoResearch | #715-545-150 | For cell validation |
| Alexa Fluor 647 AffiniPure Goat Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 111-605-003 | For cell validation |
| Antibody dilution buffer | Licor | #927-60001 | For cell validation |
| Beaver safety knife | Beaver-Visitec International | #3782235 | For lens dissection |
| Blocking buffer | Licor | #927-60001 | For cell validation |
| Capsulorhexis forceps | Titan Medical Instruments | TMF-124 | For lens capsule isolation |
| DMEM | Sigma Aldrich | D6429 | For LECs culture medium |
| DMSO | Sigma Aldrich | #D2650 | For making freezing medium |
| Dulbecco's Phosphate Buffered Saline | Thermo Fisher | #J67802 | For lens dissection |
| Dumont tweezers | Roboz Surgical Instrument | RS-4976 | For lens capsule isolation |
| EpiCGS-a (optional) | ScienCell | 4182 | For LECs culture medium |
| FBS | Sigma Aldrich | F2442 | For LECs culture medium |
| Gentamicin (50 mg/mL) | Sigma-Aldrich | G1397 | For LECs culture medium |
| Hoechst 33342 solution | Thermo Fisher | #62249 | For cell validation |
| Micro-dissecting scissors | Roboz Surgical Instrument | RS-5983 | For lens dissection |
| Micro-dissecting tweezers | Roboz Surgical Instrument | RS5137 | For lens dissection |
| PROX1 antibody | Thermo Fisher | 11067-2-AP | For cell validation |
| Vannas micro-dissecting spring scissors | Roboz Surgical Instrument | RS-5608 | For lens capsule isolation |
| αA-crystallin antibody | Santa Cruz | sc-28306 | For cell validation |
| γ-crystallin antibody | Santa Cruz | sc-365256 | For cell validation |
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