Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales.
Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales.
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DOI：
10.1080/20013078.2018.1528109
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发表时间：
2018
影响因子：
16
通讯作者：
Giebel B
中科院分区：
文献类型：
--
作者：
Ludwig AK;De Miroschedji K;Doeppner TR;Börger V;Ruesing J;Rebmann V;Durst S;Jansen S;Bremer M;Behrmann E;Singer BB;Jastrow H;Kuhlmann JD;El Magraoui F;Meyer HE;Hermann DM;Opalka B;Raunser S;Epple M;Horn PA;Giebel B
细胞外囊泡（EVs）在不同器官内部和器官之间的局部和远处的细胞间提供了一种复杂的细胞间信号传递手段。根据其细胞类型特异性特征，ev可以作为多种疾病的一类新的生物标志物，并可以用作药物递送载体。此外，来自某些细胞类型的ev在再生医学和免疫调节中发挥有益作用。有几种技术可用于从各种体液或细胞培养上清中收获ev。传统的方法包括差速离心、密度梯度离心、尺寸排除层析和基于免疫捕获的方法来从含有ev的液体中收获ev。由于这些方法的可扩展性有限，我们设计并优化了一种基于聚乙二醇（PEG）的沉淀方法，从细胞培养上清中富集ev。我们证明了该方法的可重复性和可扩展性，并将其与更经典的ev收获方法进行了比较。研究表明，对聚乙二醇颗粒进行洗涤和超离心再沉淀可以去除大量聚乙二醇共沉淀分子，如牛血清白蛋白（BSA）。然而，尺寸排除色谱的结果显示，获得的EV样品的每毫克蛋白质的颗粒纯度更高，peg制备的EV样品很可能仍然含有一定比例的其他非EV相关分子。由于peg富集的EV在缺血性卒中模型中显示出与相应细胞相同的治疗活性，因此这种共纯化分子不太可能对获得的EV样品的功能特性产生负面影响。综上所述，如果要对纯EV样品进行分子分析，优化后的PEG协议可能不是首选的纯化方法，但它是一种可扩展且可重复的方法，可以很容易地被配备超离心机的实验室采用，以富集功能性活性EV。
Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.
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DOI：
10.1007/s00018-018-2773-4
发表时间：
2018-08
期刊：
Cellular and molecular life sciences : CMLS
影响因子：
--
作者：
Karimi N;Cvjetkovic A;Jang SC;Crescitelli R;Hosseinpour Feizi MA;Nieuwland R;Lötvall J;Lässer C
通讯作者：
Lässer C
DOI：
10.1073/pnas.1521230113
发表时间：
2016-02-23
影响因子：
11.1
作者：
Kowal, Joanna;Arras, Guillaume;Thery, Clotilde
通讯作者：
Thery, Clotilde
影响因子：
2.9
作者：
KURFURST, MM
通讯作者：
KURFURST, MM
影响因子：
6
作者：
Doeppner, Thorsten R.;Herz, Josephine;Hermann, Dirk M.
通讯作者：
Hermann, Dirk M.
DOI：
10.1128/aem.30.3.464-471.1975
发表时间：
1975-01-01
期刊：
APPLIED MICROBIOLOGY
影响因子：
--
作者：
BRONSON, DL;ELLIOTT, AY;RITZI, D
通讯作者：
RITZI, D