Department of Microbiology and Immunology, Eulji University School of Medicine, Daejeon, South Korea.
PLoS One. 2020 Jul 7;15(7):e0235793. doi: 10.1371/journal.pone.0235793. eCollection 2020.
Extracellular vesicles (EVs) are small vesicles secreted from cells. They have crucial biological functions in intercellular communications and may even be biomarkers for cancer. The various methods used to isolate EVs from body fluid and cell culture supernatant have been compared in prior studies, which determined that the component yield and physical properties of isolated EVs depend largely on the isolation method used. Several novel and combined methods have been recently developed, which have not yet been compared to the established methods. Therefore, the purpose of this study is to compare the physical and functional differences in EVs isolated using a differential centrifugation method, the precipitation-based Invitrogen kit, the ExoLutE kit, and the Exodisc, of which the latter two were recently developed. We investigated the properties of EVs isolated from non-infected and Kaposi's sarcoma-associated herpesvirus-infected human umbilical vein endothelial cells using each method and determined the yields of DNA, RNA, and proteins using quantitative polymerase chain reaction and bicinchoninic acid assays. Additionally, we determined whether the biological activity of EVs correlated with the quantity or physical properties of the EVs isolated using different methods. We found that Exodisc was the most suitable method for obtaining large quantities of EVs, which might be useful for biomarker investigations, and that the EVs separated using Exodisc exhibited the highest complement activation activity. However, we also found that the functional properties of EVs were best maintained when differential centrifugation was used. Effective isolation is necessary to study EVs as tools for diagnosing cancer and our findings may have relevant implications in the field of oncology by providing researchers with data to assist their selection of a suitable isolation method.
细胞外囊泡 (EVs) 是细胞分泌的小囊泡。它们在细胞间通讯中具有重要的生物学功能，甚至可能是癌症的生物标志物。先前的研究比较了从体液和细胞培养上清液中分离 EV 的各种方法，这些方法确定了分离的 EV 的成分产量和物理性质在很大程度上取决于所使用的分离方法。最近开发了几种新的和组合的方法，但尚未与已建立的方法进行比较。因此，本研究的目的是比较使用差速离心法、基于沉淀的 Invitrogen 试剂盒、ExoLutE 试剂盒和 Exodisc 分离 EV 时的物理和功能差异，其中后两种方法是最近开发的。我们使用每种方法研究了从未感染和卡波西肉瘤相关疱疹病毒感染的人脐静脉内皮细胞中分离的 EV 的特性，并使用定量聚合酶链反应和双缩脲法测定 DNA、RNA 和蛋白质的产量。此外，我们还确定了 EV 分离的生物活性是否与使用不同方法分离的 EV 的数量或物理性质相关。我们发现，Exodisc 是获得大量 EV 的最适合方法，这可能对生物标志物研究有用，并且使用 Exodisc 分离的 EV 表现出最高的补体激活活性。然而，我们还发现，当使用差速离心时，EV 的功能特性得到了最好的保持。为了将 EV 作为诊断癌症的工具进行研究，有效的分离是必要的，我们的发现可能为肿瘤学领域提供相关意义，为研究人员提供数据以协助他们选择合适的分离方法。
