isolating mouse lens epithelial cells to study lens-related disorders

晶状体上皮细胞的培养和表征

The lens of the eye plays a crucial role in vision by focusing incoming light onto the retina. It consists of a transparent, avascular structure composed of specialized cells, among which lens epithelial cells (LECs) are key players. LECs are located at the anterior surface of the lens and are responsible for maintaining its transparency, regulating water balance, and participating in lens growth and development1,2. LECs are a unique type of cells located at the anterior part of the lens, playing a critical role in maintaining lens clarity and function by continuously producing lens fibers throughout life.
Cataracts are characterized by the progressive clouding of the lens, resulting in the distortion and scattering of light, leading to compromised vision3,4. The precise mechanisms underlying cataract formation are complex and multifactorial, involving various cellular and molecular processes such as UV radiation, oxidative damage, and glycation5,6. LECs have been found to contribute significantly to the development of cataracts, making them a vital focus of research1,2,7,8,9.
Furthermore, one of the most pressing issues in ophthalmology today is the relatively high incidence of posterior capsule opacification (PCO), also known as secondary cataract. PCO remains the most common complication after cataract surgery, affecting up to 20-40% of adult patients and 100% of children within 5 years post surgery10. PCO is primarily caused by the residual LECs that remain in the capsular bag following cataract extraction. These cells undergo a multifaceted pathophysiological transformation involving not only epithelial-to-mesenchymal transition (EMT) but also the differentiation of LECs to lens fibers, resulting in a cell population that is a mixture of LECs, fibers, and myofibroblasts11,12,13. The transformed cells proliferate and migrate across the posterior lens capsule, leading to visual impairment. Understanding the behavior and control mechanisms of LECs in culture models can provide valuable insights into the prevention and management of PCO. Therefore, this protocol of culturing LECs presents a vital tool for ophthalmic researchers aiming to study, understand, and ultimately combat this prevalent postoperative complication.
To unravel the intricacies of LEC biology and its role in cataract formation and PCO, it is essential to establish robust and reproducible in vitro primary cell culture systems. Primary LEC culture provides researchers with a controlled environment to study the functions, signaling, and molecular characteristics of LECs. Furthermore, it allows for the investigation of cellular processes and the effects of different experimental conditions, providing valuable insights into lens physiology and pathology.
Prior research has enriched our understanding of LEC culture techniques14,15,16,17,18,19,20. Although these studies have employed various methodologies and yielded significant findings on LEC behavior and characteristics, a comprehensive and accessible video recording protocol for culturing LECs is absent in the current literature. This limitation can hinder novice researchers&#39; ability to accurately reproduce the techniques and can lead to inconsistencies and variations in experimental results. By providing a video recording protocol, this research paper aims to bridge this gap and provide a standardized resource that can enhance reproducibility and facilitate knowledge transfer in the field of LEC culture.

作者聚焦：揭开白内障的秘密 – 研究晶状体上皮细胞中的氧化还原修复酶

优化小鼠原晶状体上皮细胞培养：胰蛋白酶消化综合指南

Our research objective is to explore the underlying causes of cataracts with a particular emphasis on the function of redox repair enzymes. Our aim is to identify the critical role this enzyme play in lens protection, and to establish their potential as drug targets for cataract prevention and treatment. Among many exciting technologies, our research uses lens epithelial cells as a foundational platform.This approach is critical for unraveling the mechanism behind cataract formation, posterior capsule opacification, and the discovery of new anti-cataract drugs. Moving forward, we will focus on understanding the impact of antioxidant enzymes and aging on lens functionality. Our goal is to discover innovative antioxidant treatments that can delay lens aging, prevent cataracts, and provide effective intervention for PCO.

分离小鼠晶状体上皮细胞以研究晶状体相关疾病

首先，将安乐死的小鼠放在手术平台上。用手术剪刀轻轻取下眼睑。然后在弯曲的镊子的帮助下，轻轻按压眼窝的两侧，使眼睛向外突出。用白内障刀在角膜上小心切开，用弯曲的镊子小心地取出晶状体。接下来，使用带有钝头的弯曲镊子，将镜片转移到含有 5 毫升无菌 DPBS 和庆大霉素的 60 毫米塑料培养皿中。用含有庆大霉素的DPBS溶液轻轻冲洗镜片。完成漂洗过程后，将镜头放在一张滤纸上并使其干燥。镜片充分干燥后，小心地将其转移到培养皿的盖子上，以准备取出镜片胶囊。然后向上旋转晶状体，确保前节朝上。用镊子握住前囊的同时，用惯用手的囊镊子产生小撕裂。向相反方向轻轻拉动两个工具以取出胶囊。将胶囊放入DPBS中，直到所有晶状体解剖完成。之后，小心地将镜头囊转移到六孔板上。向每个孔中加入一毫升0.05%胰蛋白酶溶液以启动酶解过程。轻轻搅拌胰蛋白酶溶液以确保均匀渗透。将板放入细胞培养箱中，让胶囊在 37 摄氏度下消化 8 至 10 分钟。然后用解剖剪刀小心地将消化后的晶状体胶囊切碎，以促进细胞分离。加入0.5毫升含有10%FBS的培养基以淬灭胰蛋白酶。将组织样品转移到离心管中，并以1000G离心五分钟。小心地除去上清液，不要干扰细胞沉淀。使用一毫升培养基重悬细胞并将细胞接种在 24 孔板中。培养的晶状体上皮细胞的相位对比图像显示，在第三天和第五天之间，细胞开始增殖阶段。在第 7 天和第 10 天之间可以观察到快速生长和对数增殖阶段。

isolating-mouse-lens-epithelial-cells-to-study-lens-related-disorders

Research

JoVE Journal

Biology

Isolating Mouse Lens Epithelial Cells to Study Lens-Related Disorders

Optimizing Mouse Primary Lens Epithelial Cell Culture: A Comprehensive Guide to Trypsinization

Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, development, size, and transparency. Conversely, dysfunctional LECs can lead to cataract formation and posterior capsule opacification (PCO). Consequently, establishing a robust primary LEC culture system is important to researchers engaged in lens development, biochemistry, cataract therapeutics, and PCO prevention. However, cultivating primary LECs has long presented challenges due to their limited availability, slow proliferation rate, and delicate nature. 
This study addresses these hurdles by presenting a comprehensive protocol for primary LEC culture. The protocol encompasses essential steps such as the formulation of an optimized culture medium, precise isolation of lens capsules, trypsinization techniques, subculture procedures, harvest protocols, and guidelines for storage and shipment. Throughout the culture process, cell morphology was monitored using phase-contrast microscopy. 
To confirm the authenticity of the cultured LECs, immunofluorescence assays were conducted to detect the presence and subcellular distribution of critical lens proteins, namely &#945;A- and &#947;-crystallins. This detailed protocol equips researchers with a valuable resource for cultivating and characterizing primary LECs, enabling advancements in our comprehension of lens biology and the development of therapeutic strategies for lens-related disorders.

This manuscript outlines a detailed video protocol for culturing primary lens epithelial cells (LECs), aiming to improve reproducibility and aid research in cataracts and posterior capsule opacification (PCO). It offers step-by-step instructions on lens dissection, LECs isolation, and validation, serving as a valuable guide, especially for newcomers in the field.

Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, ...