Division of Pulmonary and Critical Care Medicine, Department of Medicine, Boston University Medical Campus, Boston, Massachusetts.
Department of Biology and Chemistry, Changwon National University, Changwon, Korea.
Am J Physiol Lung Cell Mol Physiol. 2021 Apr 1;320(4):L522-L529. doi: 10.1152/ajplung.00318.2020. Epub 2021 Jan 13.
Extracellular vesicles (EVs) in bodily fluids play an essential role in cell-cell cross talk and potentially serve as novel biomarkers in "liquid biopsy." It is crucial to have a consistent, efficient, and reliable method to separate EVs from bodily fluids. Currently, there is no universally accepted, "best" method to separate EVs. Besides differential ultracentrifugation (UC), polyethylene glycol (PEG) is among the commonly used methods for EV separation from bodily fluids. However, the optimal concentration of PEG to be used remains inadequately addressed. We initially observed that the concentration of PEG has a significant impact on the amount of separated EVs and EV-cargos, which are recovered from bronchoalveolar lavage fluid (BALF). To determine the optimal PEG concentration to be used in EV separation from BALF, we first separated the BALF and serum from wild-type C57BL/6 mice. Next, various concentrations of PEG (5%, 10%, and 15% PEG), a commercial kit, and UC were used to obtain EVs from BALF and serum. EVs were characterized, and EV-cargo protein, RNA, and miRNA levels were determined. We found that high concentration of PEG (10% and 15%) altered various EV parameters that are frequently used in EV studies, including EV yield, purity, and morphology. Using miR-15a, miR-142, and miR-223 as examples, we found that 10% and 15% PEG robustly reduced the detected levels of EV-cargo miRNAs compared with those in the EVs separated using UC or 5% PEG. Collectively, low concentration of PEG facilitates the optimal BALF EV separation.
体液中的细胞外囊泡 (EVs) 在细胞间通讯中发挥着重要作用，并可能作为“液体活检”中的新型生物标志物。拥有一种一致、高效和可靠的方法来从体液中分离 EVs 至关重要。目前，还没有一种被普遍接受的、“最佳”的方法来分离 EVs。除了差速超速离心 (UC) 之外，聚乙二醇 (PEG) 也是从体液中分离 EV 的常用方法之一。然而，PEG 的最佳浓度仍未得到充分解决。我们最初观察到，PEG 的浓度对从支气管肺泡灌洗液 (BALF) 中分离出的 EV 和 EV cargos 的数量有显著影响。为了确定从 BALF 中分离 EV 时使用的最佳 PEG 浓度，我们首先从野生型 C57BL/6 小鼠中分离出 BALF 和血清。接下来，我们使用不同浓度的 PEG（5%、10%和 15%PEG）、商业试剂盒和 UC 从 BALF 和血清中获得 EVs。对 EVs 进行了表征，并测定了 EV cargos 蛋白、RNA 和 miRNA 水平。我们发现，高浓度的 PEG（10%和 15%）改变了 EV 研究中常用的各种 EV 参数，包括 EV 产量、纯度和形态。以 miR-15a、miR-142 和 miR-223 为例，我们发现与使用 UC 或 5%PEG 分离的 EV 相比，10%和 15%PEG 显著降低了 EV cargos miRNA 的检测水平。总的来说，低浓度的 PEG 有利于最佳的 BALF EV 分离。
