ABSTRACT

Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellular communication, and their presence in biofluids can be indicative for (patho)physiological conditions. Studies aiming to resolve functionalities of EV or to discover EV-associated biomarkers for disease in liquid biopsies are hampered by limitations of current protocols to isolate EV from biofluids or cell culture medium. EV isolation is complicated by the >10⁵-fold numerical excess of other types of particles, including lipoproteins and protein complexes. In addition to persisting contaminants, currently available EV isolation methods may suffer from inefficient EV recovery, bias for EV subtypes, interference with the integrity of EV membranes, and loss of EV functionality. In this study, we established a novel three-step non-selective method to isolate EV from blood or cell culture media with both high yield and purity, resulting in 71% recovery and near to complete elimination of unrelated (lipo)proteins. This EV isolation procedure is independent of ill-defined commercial kits, and apart from an ultracentrifuge, does not require specialised expensive equipment.

 抽象的

细胞外囊泡（EV）是膜封装的纳米颗粒，可以在细胞间通讯中发挥作用，它们在生物体液中的存在可以指示（病理）生理状况。旨在解决 EV 功能或在液体活检中发现与 EV 相关的疾病生物标志物的研究受到当前从生物流体或细胞培养基中分离 EV 方案的限制的阻碍。 EV 分离由于其他类型颗粒（包括脂蛋白和蛋白质复合物）的 >10 ⁵倍过量而变得复杂。除了持续存在的污染物之外，目前可用的 EV 隔离方法还可能面临 EV 回收效率低下、EV 亚型偏差、EV 膜完整性干扰以及 EV 功能丧失等问题。在这项研究中，我们建立了一种新颖的三步非选择性方法，以高产量和高纯度从血液或细胞培养基中分离 EV，回收率达到 71%，并且几乎完全消除了不相关的（脂）蛋白。这种 EV 分离程序独立于定义不明确的商业套件，并且除了超速离心机之外，不需要专门的昂贵设备。