Cushioned-Density Gradient Ultracentrifugation (C-DGUC) improves the isolation efficiency of extracellular vesicles
Cushioned-Density Gradient Ultracentrifugation (C-DGUC) improves the isolation efficiency of extracellular vesicles
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DOI：
10.1371/journal.pone.0215324
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发表时间：
2019-04-11
期刊：
影响因子：
3.7
通讯作者：
Raffai, Robert L.
中科院分区：
文献类型：
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作者：
Duong, Phat;Chung, Allen;Raffai, Robert L.
超离心（UC）被认为是用于分离细胞外囊泡（EV）的稳健方法。然而，最近的研究强调了UC的局限性，包括回收效率低和可能影响下游功能分析的EV聚集。我们测试了在UC期间使用碘克沙醇液体垫以解决这些缺点的益处。在这项研究中，我们比较了通过常规UC和纯化UC（C-UC）从J774A.1巨噬细胞条件培养基中分离的EV的产率和纯度。我们扩展了我们的研究，包括其他两种常见的EV分离方法：超滤（UF）和聚乙二醇（PEG）沉降。在使用这四种方法浓缩EV后，通过使用OptiPrep密度梯度超离心（DGUC）对浓缩物进行进一步纯化。我们的数据表明，C-DGUC提供了一个两倍的改善EV恢复比传统的UC-DGUC。我们还发现，UF-DGUC保留的蛋白质是C-DGUC的十倍，而PEG-DGUC在纳米颗粒和蛋白质回收方面的性能与C-DGUC相似。关于通过纳米颗粒与蛋白质比率评估的纯度，我们的数据显示，通过UC-DGUC分离的EV达到最高纯度，而C-DGUC和PEG-DGUC产生类似的纯制剂。总的来说，我们证明了与常规UC相比，在初始浓缩步骤期间使用高密度碘克沙醇垫提高了源自细胞培养基的EV的产率。这种提高的产率而没有蛋白质污染物的大量保留，并且没有暴露于引起聚集的力，为分离随后可用于功能研究的EV提供了新的机会。
Ultracentrifugation (UC) is recognized as a robust approach for the isolation of extracellular vesicles (EVs). However, recent studies have highlighted limitations of UC including low recovery efficiencies and aggregation of EVs that could impact downstream functional analyses. We tested the benefit of using a liquid cushion of iodixanol during UC to address such shortcomings. In this study, we compared the yield and purity of EVs isolated from J774A.1 macrophage conditioned media by conventional UC and cushioned-UC (C-UC). We extended our study to include two other common EV isolation approaches: ultrafiltration (UF) and polyethylene glycol (PEG) sedimentation. After concentrating EVs using these four methods, the concentrates underwent further purification by using OptiPrep density gradient ultracentrifugation (DGUC). Our data show that C-DGUC provides a two-fold improvement in EV recovery over conventional UC-DGUC. We also found that UF-DGUC retained ten-fold more protein while PEG-DGUC achieved similar performance in nanoparticle and protein recovery compared to C-DGUC. Regarding purity as assessed by nanoparticle to protein ratio, our data show that EVs isolated by UC-DGUC achieved the highest purity while C-DGUC and PEG-DGUC led to similarly pure preparations. Collectively, we demonstrate that the use of a high-density iodixanol cushion during the initial concentration step improves the yield of EVs derived from cell culture media compared to conventional UC. This enhanced yield without substantial retention of protein contaminants and without exposure to forces causing aggregation offers new opportunities for the isolation of EVs that can subsequently be used for functional studies.