Heinemann Mitja L, Vykoukal Jody
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103, Leipzig, Germany.
McCombs Institute for the Early Detection and Treatment of Cancer, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030, USA.
Methods Mol Biol. 2017;1660:33-41. doi: 10.1007/978-1-4939-7253-1_4.
A prevalent challenge in isolating extracellular vesicles (EVs) from biological fluids is the reliable depletion of abundant contaminants-including free proteins and biomolecules, as well as nontarget vesicle subpopulations and other nanoparticulates-from the sample matrix while maximizing recovery. Sequential Filtration is a recently published approach for the size-based isolation of exosomes that is ideally suited for large-volume biofluid samples such as ascites , urine , lavage fluid, or cell-conditioned media. We describe a straightforward, three-step protocol comprising back-to-back steps of dead-end (normal) filtration, tangential-flow filtration, and track-etched membrane filtration that can be applied to yield a homogeneous population of exosome-sized extracellular vesicles. The approach is scalable and employs relatively gentle manipulation forces to fractionate and concentrate extracellular vesicles with good purity and functional integrity.
从生物流体中分离细胞外囊泡（EVs）时，一个普遍存在的挑战是如何可靠地去除样本基质中大量的污染物，包括游离蛋白质和生物分子，以及非靶标囊泡亚群和其他纳米颗粒，同时实现回收率最大化。连续过滤是最近发表的一种基于尺寸分离外泌体的方法，非常适合用于处理大量生物流体样本，如腹水、尿液、灌洗液或细胞条件培养基。我们描述了一种简单的三步方案，包括死端（常规）过滤、切向流过滤和径迹蚀刻膜过滤的背靠背步骤，该方案可用于产生均一的外泌体大小的细胞外囊泡群体。该方法具有可扩展性，采用相对温和的操作力对外细胞囊泡进行分级和浓缩，具有良好的纯度和功能完整性。
