isolating mouse lens epithelial cells to study lens-related disorders

Coltivazione e caratterizzazione delle cellule epiteliali del cristallino

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.

Autore in primo piano: Svelare i segreti della cataratta - Indagine sugli enzimi di riparazione redox nelle cellule epiteliali del cristallino

Ottimizzazione della coltura di cellule epiteliali primarie del cristallino di topo: una guida completa alla tripsinizzazione

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.

Isolare le cellule epiteliali del cristallino di topo per studiare i disturbi correlati al cristallino

Per iniziare, posiziona il topo sottoposto a eutanasia sulla piattaforma chirurgica. Usa le forbici chirurgiche per rimuovere delicatamente le palpebre. Quindi, con l'aiuto di una pinzetta curva, applica una leggera pressione sui lati opposti dell'orbita, facendo sporgere l'occhio verso l'esterno.Usa il coltello per la cataratta per praticare un'accurata incisione sulla cornea e utilizza una pinzetta curva per estrarre con cura il cristallino. Successivamente, utilizzando una pinzetta curva con punte smussate, trasferire le lenti in un piatto di coltura di plastica da 60 millimetri contenente cinque millilitri di DPBS sterile e gentamicina. Risciacquare delicatamente le lenti con una soluzione DPBS contenente gentamicina.Dopo aver completato il processo di risciacquo, posizionare l'obiettivo su un pezzo di carta da filtro e lasciarlo asciugare. Una volta che la lente è adeguatamente asciutta, trasferirla con cura sul coperchio di una capsula di Petri per preparare la rimozione della capsula della lente. Quindi ruotare la lente verso l'alto, assicurandosi che il segmento anteriore sia rivolto verso l'alto.Mentre tieni la capsula anteriore con una pinzetta, usa una pinza per capsuloressi nella mano dominante per creare una piccola lacrima. Tirare delicatamente i due strumenti in direzioni opposte per rimuovere la capsula. Posizionare la capsula in DPBS fino al completamento di tutte le dissezioni della lente.Successivamente, trasferire con cautela la capsula dell'obiettivo su una piastra a sei pozzetti. Aggiungere un millilitro di soluzione di tripsina allo 0,05% in ciascun pozzetto per avviare il processo di digestione enzimatica. Agitare delicatamente la soluzione di tripsina per garantire una permeazione uniforme.Mettere la piastra in un incubatore per colture cellulari e lasciare digerire la capsula per 8-10 minuti a 37 gradi Celsius. Quindi utilizzare le forbici da dissezione per tritare con cura la capsula del cristallino digerita per promuovere la separazione cellulare. Aggiungere 0,5 millilitri del terreno di coltura contenente il 10% di FBS per estinguere la tripsina.Trasferire i campioni di tessuto in una provetta da centrifugazione e centrifugare a 1000 G per cinque minuti. Rimuovere con cautela il surnatante senza disturbare il pellet cellulare. Utilizzare un millilitro di terreno di coltura per risospendere le cellule e seminare le cellule in una piastra a 24 pozzetti.L'immagine di contrasto in fase delle cellule epiteliali del cristallino in coltura ha mostrato che tra il terzo e il quinto giorno, le cellule hanno iniziato la loro fase di proliferazione. Tra il settimo e il decimo giorno si sono potute osservare fasi di crescita rapida e proliferazione logaritmica.

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, ...