Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200438, P. R. China.
Department of Urology, The Second Affiliated Hospital of Dalian Medical University, Dalian 116023, P. R. China.
J Proteome Res. 2020 Jun 5;19(6):2217-2225. doi: 10.1021/acs.jproteome.9b00693. Epub 2020 Apr 28.
Exosomes, a subtype of extracellular vesicles secreted by mammalian cells with a typical size range of 30-150 nm, have been implicated in many biological processes as intercellular communication carriers. The isolation of exosomes is an essential and challenging step before subsequent analysis and functional studies, due to the complexity of body fluids, as well as the small size and low density of exosomes. Ultracentrifugation (UC) and size exclusion chromatography (SEC) are two methods that have been extensively used for exosomes isolation in biological studies in recent years. In this work, we compared the characteristics of urinary exosomes extracted with SEC and UC methods in detail. Results showed that the SEC isolation method was superior to UC in the recovery of exosomal particles and proteins. The results of proteomics analysis showed that more purified exosomes were extracted with the SEC method. We also observed that parts of exosomes were ruptured and precipitated insufficiently during UC isolations. It not only led to a low recovery of exosome proteins but also resulted in a considerable loss of exosomal particles. Moreover, the exosomal rupture and particle loss in UC could not be avoided by resuspension of the exosomal particles. Our results also showed that exosomes from SEC purifications possessed a high internalization capability from 4 to 6 h when incubated with EA.hy926 and HCV29 cell lines.
外泌体是哺乳动物细胞分泌的一种细胞外囊泡亚类，其典型大小范围为 30-150nm，作为细胞间通讯载体参与许多生物学过程。由于体液的复杂性以及外泌体的体积小、密度低，因此外泌体的分离是后续分析和功能研究的重要且具有挑战性的步骤。超速离心（UC）和尺寸排阻色谱（SEC）是近年来在生物学研究中外泌体分离中广泛使用的两种方法。在这项工作中，我们详细比较了 SEC 和 UC 方法提取的尿外泌体的特性。结果表明，SEC 分离方法在外泌体颗粒和蛋白质的回收方面优于 UC。蛋白质组学分析的结果表明，SEC 方法提取的外泌体更纯净。我们还观察到 UC 分离过程中外泌体部分破裂和沉淀不足，不仅导致外泌体蛋白回收率低，而且导致外泌体颗粒大量损失。此外，通过重悬外泌体颗粒不能避免 UC 中的外泌体破裂和颗粒损失。我们的结果还表明，从 SEC 纯化中提取的外泌体在与 EA.hy926 和 HCV29 细胞系孵育 4 至 6 小时后具有较高的内化能力。
