isolating mouse lens epithelial cells to study lens-related disorders

수정체 상피세포의 배양 및 특성화(Cultivation and Characterization of Lens Epithelial Cells)

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.

저자 스포트라이트: 백내장의 비밀을 밝히다 – 수정체 상피 세포의 산화 환원 복구 효소 조사

Mouse Primary Lens 상피 세포 배양 최적화: 트립신화에 대한 종합 가이드

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.

수정체 관련 질환을 연구하기 위한 마우스 수정체 상피 세포 분리

시작하려면 안락사된 쥐를 수술 플랫폼에 놓습니다. 수술용 가위를 사용하여 눈꺼풀을 섬세하게 제거합니다. 그런 다음 구부러진 핀셋을 사용하여 눈구멍의 반대쪽에 부드러운 압력을 가하여 눈이 바깥쪽으로 튀어나오도록 합니다.백내장 칼을 사용하여 각막을 조심스럽게 절개하고 구부러진 핀셋을 사용하여 수정체를 조심스럽게 빼냅니다. 그런 다음 끝이 뭉툭한 곡선형 핀셋을 사용하여 5ml의 멸균 DPBS와 겐타마이신이 들어 있는 60mm 플라스틱 배양 접시에 렌즈를 옮깁니다. 겐타마이신이 함유된 DPBS 용액으로 렌즈를 부드럽게 헹굽니다.헹굼 과정이 끝나면 렌즈를 여과지 위에 놓고 건조시킵니다. 렌즈가 충분히 건조되면 렌즈 캡슐을 제거할 준비를 하기 위해 페트리 접시의 덮개에 조심스럽게 옮깁니다. 그런 다음 렌즈를 위쪽으로 돌려 앞쪽 세그먼트가 위쪽을 향하도록 합니다.핀셋으로 전방 캡슐을 잡고 주로 사용하는 손에 캡슐 헥시스 겸자를 사용하여 작은 찢어짐을 만듭니다. 두 도구를 반대 방향으로 부드럽게 당겨 캡슐을 제거합니다. 모든 렌즈 박리가 완료될 때까지 캡슐을 DPBS에 넣습니다.그런 다음 렌즈 캡슐을 6웰 플레이트에 조심스럽게 옮깁니다. 각 웰에 0.05%트립신 용액 1밀리리터를 첨가하여 효소 분해 과정을 시작합니다. 트립신 용액을 부드럽게 저어 균일한 침투를 보장합니다.플레이트를 세포 배양 인큐베이터에 넣고 캡슐을 섭씨 37도에서 8-10분 동안 소화시킵니다. 그런 다음 해부 가위를 사용하여 소화된 수정체 캡슐을 조심스럽게 다져 세포 분리를 촉진합니다. 트립신을 담금질하기 위해 10% FBS가 함유된 배양 배지 0.5mL를 추가합니다.조직 샘플을 원심분리 튜브로 옮기고 1000G에서 5분 동안 원심분리합니다. 세포 펠릿을 방해하지 않고 상층액을 조심스럽게 제거하십시오. 1m리터의 배양 배지를 사용하여 세포를 재현탁시키고 24웰 플레이트에 세포를 파종합니다.배양된 수정체 상피 세포의 위상 대비 이미지는 3일에서 5일 사이에 세포가 증식 단계를 시작했음을 보여주었습니다. 급속한 성장과 대수적 증식 단계는 7일에서 10일 사이에 관찰될 수 있습니다.

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, ...