Heparin affinity purification of extracellular vesicles.
Heparin affinity purification of extracellular vesicles.
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DOI：
10.1038/srep10266
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发表时间：
2015-05-19
影响因子：
4.6
通讯作者：
Maguire CA
中科院分区：
文献类型：
--
作者：
Balaj L;Atai NA;Chen W;Mu D;Tannous BA;Breakefield XO;Skog J;Maguire CA
细胞外囊泡（EV）是由细胞释放的脂质膜囊泡。它们携带活性生物分子，包括DNA，RNA和蛋白质，可以转移到受体细胞。从细胞培养基和生物流体中分离和纯化EV仍然是一个重大挑战。最广泛使用的隔离方法是超浓缩（UC），它需要昂贵的设备，只能部分净化电动汽车。以前我们已经表明，肝素阻断EV在细胞中的摄取，支持直接EV-肝素相互作用。在这里，我们表明，可以使用超滤（UF），然后肝素亲和珠从细胞培养基和人血浆中纯化EV。来自细胞培养物的UF/肝素纯化的EV显示EV标志物阿利克斯，含有不同的RNA谱，具有较低水平的蛋白质污染，并且在结合和摄取到细胞中时具有功能。通过UC分离的EV的RNA产率相似。我们能够在血浆样品中检测到与UC样品水平相当的mRNA。总之，我们已经发现了一种简单、可扩展且有效的方法来利用EV的肝素亲和力纯化EV。
Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity.
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