Zheng Yaochao, Streleckis Aiden Charles, Chen Hongyu, Yao Yao
Regenerative Bioscience Center, Department of Animal and Dairy Science, College of Agricultural and Environmental Science, University of Georgia.
Regenerative Bioscience Center, Department of Animal and Dairy Science, College of Agricultural and Environmental Science, University of Georgia;
J Vis Exp. 2025 Feb 7(216). doi: 10.3791/67439.
Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells to transport bioactive cargo, such as proteins, RNAs, and DNAs, for intercellular communication. Investigating EV-mediated crosstalk among cells in muscle homeostasis and diseases offers significant potential to enhance our understanding of muscle development, regeneration, and atrophy. However, current protocols for isolating skeletal muscle-derived EVs (SkM-EVs) face challenges in achieving high purity and yield, primarily due to difficulties in releasing EVs from muscle tissues without compromising cellular membranes. This article presents an efficient protocol for SkM-EV isolation, comprising mechanical detachment, enzymatic dissociation, filtration, and ultracentrifugation. These steps are optimized to enhance EV release from muscle tissues, yielding high-purity SkM-EVs. Subsequently, nano-flow cytometry, BCA assay, and Western blot assay are performed to characterize the quantity and quality of the isolated SkM-EVs. This protocol holds promise for establishing a reliable platform to obtain tissue-derived EVs for advancing basic research, disease diagnosis, and drug delivery.
细胞外囊泡（EVs）是由细胞释放的脂质双层包裹的纳米颗粒，用于运输生物活性物质，如蛋白质、RNA和DNA，以进行细胞间通讯。研究EV介导的肌肉稳态和疾病中细胞间的串扰，对于增进我们对肌肉发育、再生和萎缩的理解具有巨大潜力。然而，目前分离骨骼肌来源的EVs（SkM-EVs）的方法在实现高纯度和高产量方面面临挑战，主要原因是难以在不破坏细胞膜的情况下从肌肉组织中释放出EVs。本文介绍了一种高效的SkM-EV分离方法，包括机械分离、酶解、过滤和超速离心。这些步骤经过优化，以增强EVs从肌肉组织中的释放，从而获得高纯度的SkM-EVs。随后，进行纳米流式细胞术、BCA测定和蛋白质免疫印迹测定，以表征分离出的SkM-EVs的数量和质量。该方法有望建立一个可靠的平台，以获得组织来源的EVs，用于推进基础研究、疾病诊断和药物递送。
