Methods for Investigating Corneal Cell Interactions and Extracellular Vesicles In Vitro
Q3 Biochemistry, Genetics and Molecular Biology
Tina B. McKay, Xiaoqing Guo, Audrey E. K. Hutcheon, Dimitrios Karamichos, Joseph B. Ciolino
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Abstract
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 in a 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. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : Isolating and culturing human corneal keratocytes and fibroblasts
Basic Protocol 2 : Isolating and culturing human corneal epithelial cells
Basic Protocol 3 : Isolating and culturing human corneal endothelial cells
Basic Protocol 4 : 3D corneal stromal construct assembly
Basic Protocol 5 : 3D corneal epithelial-stromal construct assembly
Basic Protocol 6 : 3D corneal endothelial-stromal construct assembly
Basic Protocol 7 : Isolating extracellular vesicles from corneal cell conditioned medium
Support Protocol : Cryopreserving human corneal fibroblasts, corneal epithelial cells, and corneal endothelial cells
角膜细胞相互作用和细胞外囊泡的体外研究方法
多年来,科学和医学变得越来越“以人为中心”。在基础研究中越来越少地使用动物,这导致了利用人类来源的细胞研究生理和疾病的各种组织的复杂体外模型的发展。人类角膜也同样在体外建立了模型,使用的原代细胞来源于尸体或移植后的角膜。通过利用细胞在预先设计的微环境中维持其组织表型的内在能力,该微环境包含所需的生长因子、生理温度和湿度,组织工程角膜可以在培养中生长和维持相对较长的时间(数周至数月)。由于其透明和无血管性,角膜是研究细胞外基质和细胞间相互作用、毒理学和药物渗透性、瘢痕形成和组织再生的潜在机制的最佳组织。本文描述了用于体外应用的角膜角质细胞、成纤维细胞、上皮细胞和内皮细胞的培养方法。我们还提供了详细的,一步一步的协议组装和培养角膜基质,上皮和内皮基质共培养和分离细胞外囊泡的3D结构。©2020 Wiley期刊有限公司基本方案1:分离和培养人角膜角质细胞和成纤维细胞基本方案2:分离和培养人角膜上皮细胞基本方案3:分离和培养人角膜内皮细胞基本方案4:3D角膜基质构建组装基本方案5:3D角膜上皮-基质构建组装基本方案6:3D角膜内皮-基质构建组装基本方案7:从角膜细胞条件培养基中分离细胞外囊泡支持方案:冷冻保存人角膜成纤维细胞、角膜上皮细胞和角膜内皮细胞
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