Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany.
Martin Luther University Halle-Wittenberg, Biocenter, Halle (Saale), Germany.
Sci Rep. 2021 Jun 2;11(1):11585. doi: 10.1038/s41598-021-91129-y.
Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples.
细胞外囊泡（EVs）因其能够从液体活检中提供诊断信息而引起了人们的兴趣。细胞不断释放大小、内容和特征各不相同的囊泡，这取决于它们的生物发生、起源和功能。这种异质性在试图分离和表征 EVs 时增加了一层复杂性，导致了各种方案的出现。EVs 在所有体液中的高丰度及其稳定的来源依赖性生物标志物使其成为生物标志物发现和诊断的有力工具。然而，应用受到样品定义质量的限制。在这里，我们比较了常用的分离技术：超速离心、密度梯度离心、超滤和尺寸排阻色谱。然后，我们旨在使用免疫磁珠从细胞培养上清液中进行组织特异性分离前列腺衍生的 EVs。通过纳米颗粒跟踪分析、western blot 和电子显微镜确认了 EVs 的质量和数量。此外，还开发了点样抗体微阵列来表征 EV 亚群。在一个微阵列上对 16 个样本进行 6 种不同 EV 表面标志物的 3 次重复分析，可以轻松扩展，从而提供更快、更经济的方法来表征样本。
