isolating mouse lens epithelial cells to study lens-related disorders

Odling och karakterisering av linsepitelceller

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.

Författare Spotlight: Låsa upp hemligheterna med grå starr - Undersöka redoxreparationsenzymer i linsepitelceller

Optimering av mus primära linsepitelcellkultur: En omfattande guide till trypsinisering

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.

Isolering av epitelceller från muslinser för att studera linsrelaterade sjukdomar

Börja med att placera den avlivade musen på den kirurgiska plattformen. Använd en kirurgisk sax för att försiktigt ta bort ögonlocken. Använd sedan en böjd pincett för att trycka försiktigt på motsatta sidor av ögonhålan, vilket gör att ögat sticker ut utåt.Använd gråstarrkniven för att göra ett försiktigt snitt på hornhinnan och använd en böjd pincett för att försiktigt dra ut linsen. Använd sedan en böjd pincett med trubbiga spetsar och överför linserna till en 60 millimeter plastodlingsskål som innehåller fem milliliter steril DPBS och Gentamicin. Skölj försiktigt linserna med DPBS-lösning som innehåller gentamicin.När du är klar med sköljningen, placera linsen på en bit filterpapper och låt den torka. När linsen är tillräckligt torr, överför den försiktigt till locket på en petriskål för att förbereda borttagningen av linskapseln. Vrid sedan linsen uppåt och se till att det främre segmentet är vänt uppåt.Medan du håller den främre kapseln med pincett, använd kapsulorhexis pincett i den dominanta handen för att skapa en liten tår. Dra försiktigt de två verktygen i motsatta riktningar för att ta bort kapseln. Placera kapseln i DPBS tills alla linsdissektioner är klara.Överför sedan försiktigt linskapseln till en platta med sex brunnar. Tillsätt en milliliter 0,05 % trypsinlösning till varje brunn för att initiera den enzymatiska matsmältningsprocessen. Rör försiktigt om trypsinlösningen för att säkerställa jämn genomträngning.Placera plattan i en cellodlingsinkubator och låt kapseln smälta i åtta till 10 minuter vid 37 grader Celsius. Använd sedan dissekeringssax för att försiktigt hacka den smälta linskapseln för att främja cellseparation. Tillsätt 0,5 ml av odlingsmediet som innehåller 10%FBS för att släcka trypsinet.Överför vävnadsproverna till ett centrifugeringsrör och centrifugera vid 1000 G i fem minuter. Ta försiktigt bort supernatanten utan att störa cellpelleten. Använd en milliliter odlingsmedium för att återsuspendera cellerna och så cellerna i en 24-hålsplatta.Den fasade kontrastbilden av de odlade linsepitelcellerna visade att cellerna mellan den tredje och femte dagen initierade sin proliferationsfas. Snabba tillväxt- och logaritmiska spridningsfaser kunde observeras mellan den sjunde och tionde dagen.

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, ...