Health Unit, Flemish Institute for Technological Research, Mol, Belgium.
Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium.
PLoS One. 2021 Feb 4;16(2):e0245835. doi: 10.1371/journal.pone.0245835. eCollection 2021.
Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day.
细胞外囊泡 (EVs) 在广泛的生物医学应用中引起了关注。EV 临床应用的一个主要限制是缺乏快速且可重复的分离方法以及随后从生物体液中检测 EV 亚群的方法，以及它们的储存方法。为了推进这一应用领域，具有单 EV 分辨率的基于荧光的表征技术，如高灵敏度流式细胞术 (HS-FCM)，非常强大，可以评估 EV 分组方法和储存条件。此外，由于具有高通量、多重分析的潜力，HS-FCM 和 EV 亚群的荧光标记的使用正在扩大，但需要进一步的方法开发来提高测量的重现性。在这项研究中，我们应用 HS-FCM 测量技术以及标准的 EV 表征技术，包括纳米颗粒跟踪分析，来比较两种 EV 分离方法，即差速离心后超速离心和 exoEasy 膜亲和柱纯化，从脂多糖刺激的单核细胞 THP-1 细胞中获得的 EV 级分的产量和纯度。我们观察到 EV 产量和纯度的差异。此外，我们还研究了 EV 在 4°C 或 -80°C 下储存长达一个月对 EV 浓度和 EV 相关荧光标记物稳定性的影响。体外细胞衍生的 EV 级分的浓度在测试的储存条件下显示保持稳定，然而，在 4°C 下储存的标记 EV 的荧光强度在一天内开始下降。
