isolating mouse lens epithelial cells to study lens-related disorders

Kultivierung und Charakterisierung von Linsenepithelzellen

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.

Autor im Rampenlicht: Den Geheimnissen des Grauen Stars auf die Spur kommen – Untersuchung von Redox-Reparaturenzymen in Linsenepithelzellen

Optimierung der Epithelzellkultur der Maus-Primärlinse: Ein umfassender Leitfaden zur Trypsinisierung

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.

Isolierung von Epithelzellen von Mauslinsen zur Untersuchung linsenbedingter Erkrankungen

Positionieren Sie zunächst die euthanasierte Maus auf der Operationsplattform. Verwenden Sie eine chirurgische Schere, um die Augenlider vorsichtig zu entfernen. Üben Sie dann mit Hilfe einer gebogenen Pinzette sanften Druck auf gegenüberliegende Seiten der Augenhöhle aus, wodurch das Auge nach außen ragt.Verwenden Sie das Kataraktmesser, um einen vorsichtigen Schnitt in die Hornhaut zu machen, und verwenden Sie eine gebogene Pinzette, um die Linse vorsichtig herauszuziehen. Als nächstes geben Sie die Linsen mit einer gebogenen Pinzette mit stumpfen Spitzen in eine 60-Millimeter-Kunststoffkulturschale mit fünf Millilitern sterilem DPBS und Gentamicin. Spülen Sie die Linsen vorsichtig mit einer DPBS-Lösung ab, die Gentamicin enthält.Legen Sie die Linse nach Abschluss des Spülvorgangs auf ein Stück Filterpapier und lassen Sie sie trocknen. Sobald die Linse ausreichend trocken ist, legen Sie sie vorsichtig auf die Abdeckung einer Petrischale, um das Entfernen der Linsenkapsel vorzubereiten. Drehen Sie dann die Linse nach oben und achten Sie darauf, dass der vordere Augenabschnitt nach oben zeigt.Während Sie die vordere Kapsel mit einer Pinzette halten, verwenden Sie eine Kapselzange in der dominanten Hand, um einen kleinen Riss zu erzeugen. Ziehen Sie die beiden Werkzeuge vorsichtig in entgegengesetzte Richtungen, um die Kapsel zu entfernen. Legen Sie die Kapsel in DPBS, bis alle Linsendissektionen abgeschlossen sind.Übertragen Sie anschließend die Linsenkapsel vorsichtig auf eine Sechs-Well-Platte. Geben Sie einen Milliliter 0,05%ige Trypsinlösung in jede Vertiefung, um den enzymatischen Verdauungsprozess einzuleiten. Rühren Sie die Trypsin-Lösung vorsichtig um, um eine gleichmäßige Permeation zu gewährleisten.Legen Sie die Platte in einen Zellkultur-Inkubator und lassen Sie die Kapsel acht bis 10 Minuten bei 37 Grad Celsius verdauen. Verwenden Sie dann eine Präparierschere, um die verdaute Linsenkapsel vorsichtig zu zerkleinern, um die Zelltrennung zu fördern. Fügen Sie 0,5 Milliliter des Kulturmediums hinzu, das 10 % FBS enthält, um das Trypsin zu löschen.Übertragen Sie die Gewebeproben in ein Zentrifugationsröhrchen und zentrifugieren Sie sie fünf Minuten lang bei 1000 g. Entfernen Sie vorsichtig den Überstand, ohne das Zellpellet zu stören. Verwenden Sie einen Milliliter Kulturmedium, um die Zellen zu resuspendieren und die Zellen in einer 24-Well-Platte zu säen.Das Phasenkontrastbild der kultivierten Linsenepithelzellen zeigte, dass die Zellen zwischen dem dritten und fünften Tag ihre Proliferationsphase einleiteten. Schnelles Wachstum und logarithmische Proliferationsphasen konnten zwischen dem siebten und 10. Tag beobachtet werden.

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, ...