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Methods for Investigating Corneal Cell Interactions and Extracellular Vesicles In Vitro

机译：研究角膜细胞相互作用和体外细胞外囊泡的方法

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摘要

Science and medicine have become increasingly “human‐centric” over the years. A growing shift away from the use of animals in basic research has led to the development of sophisticated in vitro models of various tissues utilizing human‐derived cells to study physiology and disease. The human cornea has likewise been modeled in vitro using primary cells derived from corneas obtained from cadavers or post‐transplantation. By utilizing a cell's intrinsic ability to maintain its tissue phenotype ina pre‐designed microenvironment containing the required growth factors, physiological temperature, and humidity, tissue‐engineered corneas can be grown and maintained in culture for relatively long periods of time on the scale of weeks to months. Due to its transparency and avascularity, the cornea is an optimal tissue for studies of extracellular matrix and cell‐cell interactions, toxicology and permeability of drugs, and underlying mechanisms of scarring and tissue regeneration. This paper describes methods for the cultivation of corneal keratocytes, fibroblasts, epithelial, and endothelial cells for in vitro applications. We also provide detailed, step‐by‐step protocols for assembling and culturing 3D constructs of the corneal stroma, epithelial‐ and endothelial‐stromal co‐cultures and isolation of extracellular vesicles.
机译：多年来，科学和医学越来越“以人为本”。远离基础研究中的动物的越来越多的转变导致了利用人源细胞研究生理学和疾病的各种组织的复杂体外模型的复杂体外模型的发展。人体角膜同样使用衍生自尸体或移植后的玉米体的主要细胞进行模拟。通过利用细胞的内在能力维持其组织表型INA预先设计的微环境，含有所需的生长因子，生理温度和湿度，可以在几周的规模上生长和维持在培养上的培养和维持培养到几个月。由于其透明度和缺血性，角膜是用于研究细胞外基质和细胞 - 细胞相互作用，毒理学和药物渗透性的最佳组织，以及瘢痕和组织再生的基础机制。本文介绍了用于体外应用的角膜角膜织物，成纤维细胞，上皮和内皮细胞的方法。我们还提供了用于组装和培养角膜基质，上皮和内皮 - 基质共培养物的3D构建体和细胞外囊泡的3D构建方案的详细的逐步方案。

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  • 来源
    《Current Protocols in Cell Biology》 |2020年第1期|共20页
  • 作者

    Tina B. McKay; Xiaoqing Guo; Audrey E. K. Hutcheon;

  • 作者单位

    Schepens Eye Research Institute of Massachusetts Eye and Ear and Department of Ophthalmology Harvard Medical School Boston Massachusetts;

    Schepens Eye Research Institute of Massachusetts Eye and Ear and Department of Ophthalmology Harvard MedicalSchool Boston Massachusetts;

    Schepens Eye Research Institute of Massachusetts Eye and Ear and Department of Ophthalmology Harvard Medical School Boston Massachusetts;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 细胞生物学;
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