isolating mouse lens epithelial cells to study lens-related disorders

水晶体上皮細胞の培養と特性評価

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.

著者スポットライト:白内障の秘密を解き明かす – 水晶体上皮細胞における酸化還元修復酵素の調査

マウス一次水晶体上皮細胞培養の最適化:トリプシン処理の包括的ガイド

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.

マウス水晶体上皮細胞の単離による水晶体関連疾患の研究

まず、安楽死させたマウスを手術台に置きます。手術用ハサミを使用して、まぶたを繊細に取り外します。次に、湾曲したピンセットの助けを借りて、眼窩の反対側に穏やかな圧力をかけ、目を外側に突き出させます。白内障ナイフを使用して角膜を慎重に切開し、湾曲したピンセットを使用して水晶体を慎重に引き抜きます。次に、先端が鈍い湾曲したピンセットを使用して、5ミリリットルの滅菌DPBSとゲンタマイシンが入った60ミリメートルのプラスチック培養皿にレンズを移します。ゲンタマイシンを含むDPBS溶液でレンズを静かにすすぎます。すすぎプロセスが完了したら、レンズをろ紙の上に置き、乾かします。レンズが十分に乾いたら、慎重にペトリ皿のカバーに移し、レンズカプセルを取り外す準備をします。次に、レンズを上向きに回転させ、前眼部が上を向いていることを確認します。前嚢をピンセットで保持しながら、利き手で被膜ヘキシス鉗子を使用して小さな裂け目を作成します。2つのツールを反対方向にそっと引いて、カプセルを取り外します。すべての水晶体の解剖が完了するまで、カプセルをDPBSに入れます。その後、レンズカプセルを6ウェルプレートに慎重に移します。1ミリリットルの0.05%トリプシン溶液を各ウェルに加え、酵素消化プロセスを開始します。トリプシン溶液を静かに攪拌して、均一な浸透を確保します。プレートを細胞培養インキュベーターに入れ、カプセルを摂氏37度で8〜10分間消化させます。次に、解剖ハサミを使用して、消化された水晶体カプセルを慎重に細かく刻み、細胞分離を促進します。10%FBSを含む培地0.5ミリリットルを加えて、トリプシンをクエンチします。組織サンプルを遠心分離チューブに移し、1000 Gで5分間遠心分離します。細胞ペレットを乱すことなく、上清を慎重に除去します。1ミリリットルの培地を使用して細胞を再懸濁し、細胞を24ウェルプレートに播種します。培養した水晶体上皮細胞の位相コントラスト画像は、3日目から5日目の間に細胞が増殖期を開始したことを示しました。急速な成長と対数増殖の段階は、7日目から10日目の間に観察されました。.

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, ...