isolating mouse lens epithelial cells to study lens-related disorders

Dyrkning og karakterisering af 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.

Forfatter Spotlight: Lås op for grå stærs hemmeligheder - Undersøgelse af redoxreparationsenzymer i linsepitelceller

Optimering af musens primære linseepitelcellekultur: En omfattende guide til 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 af muselinseepitelceller for at studere linserelaterede lidelser

Til at begynde med skal du placere den aflivede mus på den kirurgiske platform. Brug kirurgisk saks til at fjerne øjenlågene forsigtigt. Derefter påføres forsigtigt tryk på modsatte sider af øjenhulen ved hjælp af buede pincet, hvilket får øjet til at stikke udad.Brug grå stær kniv til at gøre et omhyggeligt snit på hornhinden og anvende buede pincet til forsigtigt at trække linsen. Brug derefter buede pincet med stumpe spidser til at overføre linserne til en 60 millimeter plastkulturskål indeholdende fem ml steril DPBS og Gentamicin. Skyl forsigtigt linserne med DPBS-opløsning indeholdende Gentamicin.Når skylningsprocessen er afsluttet, skal du placere linsen på et stykke filterpapir og lade det tørre. Når linsen er tilstrækkelig tør, skal du overføre den forsigtigt til dækslet på en petriskål for at forberede fjernelse af linsekapslen. Drej derefter linsen opad, og sørg for, at det forreste segment vender opad.Mens du holder den forreste kapsel med pincet, skal du bruge capsulorhexis tang i den dominerende hånd for at skabe en lille tåre. Træk forsigtigt de to værktøjer i modsatte retninger for at fjerne kapslen. Placer kapslen i DPBS, indtil alle linsedissektioner er afsluttet.Derefter overføres linsekapslen forsigtigt til en plade med seks brønde. Tilsæt en milliliter 0.05%Trypsinopløsning til hver brønd for at starte den enzymatiske fordøjelsesproces. Omrør forsigtigt Trypsin-opløsningen for at sikre jævn gennemtrængning.Placer pladen i en cellekulturinkubator og lad kapslen fordøje i otte til 10 minutter ved 37 grader Celsius. Brug derefter dissekersaks til omhyggeligt at hakke den fordøjede linsekapsel for at fremme celleseparation. Tilsæt 0.5 ml af kulturmediet indeholdende 10% FBS for at slukke trypsinet.Vævsprøverne overføres til et centrifugeringsrør og centrifugeres ved 1000 G i fem minutter. Supernatanten fjernes forsigtigt uden at forstyrre cellepellet. Brug en milliliter dyrkningsmedium til at resuspendere cellerne og frø cellerne i en 24-brøndplade.Det fasede kontrastbillede af de dyrkede linseepitelceller viste, at cellerne mellem den tredje og femte dag indledte deres proliferationsfase. Hurtig vækst og logaritmiske spredningsfaser kunne observeres mellem den syvende og 10. dag.

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, ...