isolating mouse lens epithelial cells to study lens-related disorders

Cultivo e caracterização de células epiteliais do cristalino

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 em destaque: Desvendando os segredos da catarata - investigando enzimas de reparo redox em células epiteliais do cristalino

Otimizando a cultura de células epiteliais primárias de lente de camundongo: um guia abrangente para tripsinização

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.

Isolando células epiteliais do cristalino de camundongo para estudar distúrbios relacionados ao cristalino

Para começar, posicione o camundongo eutanasiado na plataforma cirúrgica. Use tesoura cirúrgica para remover as pálpebras delicadamente. Em seguida, com a ajuda de pinças curvas, aplique uma pressão suave em lados opostos da cavidade ocular, fazendo com que o olho se projete para fora.Use a faca de catarata para fazer uma incisão cuidadosa na córnea e use pinças curvas para extrair cuidadosamente o cristalino. Em seguida, utilizando pinças curvas com pontas rombas, transfira as lentes para uma placa de cultura plástica de 60 milímetros contendo cinco mililitros de DPBS estéril e gentamicina. Enxaguar suavemente as lentes com solução de DPBS contendo gentamicina.Depois de concluir o processo de enxágue, coloque a lente em um pedaço de papel de filtro e deixe secar. Quando a lente estiver adequadamente seca, transfira-a cuidadosamente para a tampa de uma placa de Petri para se preparar para remover a cápsula da lente. Em seguida, gire a lente para cima, garantindo que o segmento anterior esteja voltado para cima.Ao segurar a cápsula anterior com uma pinça, use pinça capsulorrexis na mão dominante para criar uma pequena ruptura. Puxe suavemente as duas ferramentas em direções opostas para remover a cápsula. Coloque a cápsula em DPBS até que todas as dissecções da lente estejam completas.Depois, transfira cuidadosamente a cápsula da lente para uma placa de seis poços. Adicione um mililitro de solução de tripsina a 0,05% a cada poço para iniciar o processo de digestão enzimática. Agite suavemente a solução de tripsina para garantir uma permeação uniforme.Coloque a placa em uma incubadora de cultura celular e deixe a cápsula digerir por oito a 10 minutos a 37 graus Celsius. Em seguida, use uma tesoura dissecante para picar cuidadosamente a cápsula da lente digerida para promover a separação celular. Adicionar 0,5 mililitros do meio de cultura contendo 10%FBS para extinguir a tripsina.Transfira as amostras de tecido para um tubo de centrifugação e centrifuga a 1000 G durante cinco minutos. Remova cuidadosamente o sobrenadante sem perturbar o pellet celular. Use um mililitro de meio de cultura para ressuspender as células e semear as células em uma placa de 24 poços.A imagem de contraste faseado das células epiteliais do cristalino cultivadas mostrou que, entre o terceiro e o quinto dia, as células iniciaram sua fase de proliferação. Fases de crescimento rápido e proliferação logarítmica puderam ser observadas entre o sétimo e o 10º dia.

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, ...