An improvised one-step sucrose cushion ultracentrifugation method for exosome isolation from culture supernatants of mesenchymal stem cells.
An improvised one-step sucrose cushion ultracentrifugation method for exosome isolation from culture supernatants of mesenchymal stem cells.
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DOI：
10.1186/s13287-018-0923-0
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发表时间：
2018-07-04
影响因子：
7.5
通讯作者：
Mohanty S
中科院分区：
文献类型：
--
作者：
Gupta S;Rawat S;Arora V;Kottarath SK;Dinda AK;Vaishnav PK;Nayak B;Mohanty S
外泌体是内体起源的纳米囊泡（30 - 120nm）。这些外泌体含有各种功能蛋白和rna，可用于治疗目的。目前，有一种标准的方法来分离外泌体，保持其生物特性，提高产量和纯度是一个主要的挑战。最常用的方法是差示超离心，但它有自己的缺点，包括高耗时，由于破坏外泌体完整性而导致产量低，以及高蛋白质污染物。在这项研究中，我们已经确定了一种改进的方法来解决这些问题，使用超离心分离外泌体，因为它是经济有效的，并在世界范围内使用。我们比较了差分超离心法和改良的一步蔗糖缓冲超离心法分离外泌体。收集培养48 h的人间充质干细胞条件无血清培养基，分离外泌体。300g离心10 min去除细胞碎片，10000 g离心30 min去除微囊泡。采用等量预处理条件培养基直接超离心和一步蔗糖缓冲超离心分离外泌体。用这些方法分离的外泌体的大小、形态、浓度和表面标记蛋白的表达都被表征。通过纳米颗粒跟踪分析和电镜观察发现，一步蔗糖垫超离心获得的杯状外泌体回收率较高。Western blotting和流式细胞术证实了这些结果。结果表明，该方法可用于不同起始物质的外泌体分离，是一种有效的、可重复性好的潜在标准方法。我们相信这种方法将在细胞外囊泡研究领域有广泛的应用，而高产量和高纯度的外泌体分离是必不可少的一步。组织特异性人间充质干细胞UC和SUC外泌体分离方法的比较示意图。SUC分离方法可获得数量更多的杯状外泌体，其群体相对均匀，可大规模生产外泌体用于下游分析。缩写：SUC一步蔗糖缓冲超离心，UC直接超离心。本文的在线版本（10.1186/s13287-018-0923-0）包含补充材料，授权用户可使用。
Exosomes are nanovesicles (30–120 nm) of endosomal origin. These exosomes contain various functional proteins and RNAs that could be used for therapeutic purposes. Currently, having a standard method for exosome isolation retaining its biological properties with increased yield and purity is a major challenge. The most commonly used method is differential ultracentrifugation but it has its own disadvantages, which include high time consumption, low yield due to disruption of exosome integrity, and high protein contaminants. In this study, we have identified an improved method addressing these problems for exosome isolation using ultracentrifugation since it is cost-effective and used worldwide. We have compared differential ultracentrifugation with the modified method called one-step sucrose cushion ultracentrifugation for exosome isolation. The conditioned serum-free media from human mesenchymal stem cells cultured for 48 h was collected for exosome isolation. The cellular debris was removed by centrifugation at 300g for 10 min, followed by centrifugation at 10,000g for 30 min to remove microvesicles. Equal volumes of pre-processed conditioned media were used for exosome isolation by direct ultracentrifugation and one-step sucrose cushion ultracentrifugation. The exosomes isolated using these methods were characterized for their size, morphology, concentration, and surface marker protein expression. It was observed that the recovery of exosomes with cup-shaped morphology from one-step sucrose cushion ultracentrifugation was comparatively high as estimated by nanoparticle tracking analysis and electron microscopy. These results were confirmed by Western blotting and flow cytometry. We conclude that this one-step sucrose cushion ultracentrifugation method provides an effective and reproducible potential standard method which could be used for various starting materials for isolating exosomes. We believe that this method will have a wide application in the field of extracellular vesicle research where exosome isolation with high yield and purity is an imperative step. Schematic representation of comparison of UC and SUC exosome isolation methods for tissue-specific human mesenchymal stem cells. The SUC isolation method yields a greater number of cup-shaped exosomes with a relatively homogenous population for mass-scale production of exosomes for downstream analysis. Abbreviations: SUC One-step sucrose cushion ultracentrifugation, UC Direct ultracentrifugation. The online version of this article (10.1186/s13287-018-0923-0) contains supplementary material, which is available to authorized users.
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影响因子：
4.6
作者：
Katsuda, Takeshi;Tsuchiya, Reiko;Kosaka, Nobuyoshi;Yoshioka, Yusuke;Takagaki, Kentaro;Oki, Katsuyuki;Takeshita, Fumitaka;Sakai, Yasuyuki;Kuroda, Masahiko;Ochiya, Takahiro
通讯作者：
Ochiya, Takahiro
影响因子：
14
作者：
Watson DC;Bayik D;Srivatsan A;Bergamaschi C;Valentin A;Niu G;Bear J;Monninger M;Sun M;Morales-Kastresana A;Jones JC;Felber BK;Chen X;Gursel I;Pavlakis GN
通讯作者：
Pavlakis GN
影响因子：
16
作者：
Lobb RJ;Becker M;Wen SW;Wong CS;Wiegmans AP;Leimgruber A;Möller A
通讯作者：
Möller A
影响因子：
1.9
作者：
Bhasin, Ashu;Srivastava, M. V. Padma;Bose, Sushmita
通讯作者：
Bose, Sushmita
DOI：
10.1007/978-1-4939-2550-6_15
发表时间：
2015-01-01
期刊：
PROTEOMIC PROFILING: METHODS AND PROTOCOLS
影响因子：
--
作者：
Greening, David W.;Xu, Rong;Simpson, Richard J.
通讯作者：
Simpson, Richard J.