@article{d575999d8fd14df498df7f7935b41724,
title = "Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium",
abstract = "One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co-separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density-gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co-separate contaminants when the non-EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non-EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios.",
keywords = "EV yield, methods comparison, sample purity, single particle phenotyping, small extracellular vesicle separation",
author = "Liang Dong and Zieren, {Richard C.} and Kengo Horie and Kim, {Chi Ju} and Emily Mallick and Yuezhou Jing and Mingxiao Feng and Kuczler, {Morgan D.} and Jordan Green and Amend, {Sarah R.} and Witwer, {Kenneth W.} and {de Reijke}, {Theo M.} and Cho, {Yoon Kyoung} and Pienta, {Kenneth J.} and Wei Xue",
note = "Funding Information: The authors thank Barbara Smith, microscopy specialist of the Microscope Facility at Johns Hopkins University School of Medicine, for acquiring TEM images. RZ was supported by the Stichting Cure for Cancer foundation, Amsterdam, the Netherlands. KH was supported by Japan Society for the Promotion of Science (KAKENHI: 19K18555). CJK was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI19C1122). CJK and YKC were supported by a grant (IBS-R020-D1) from Institute for Basic Science (IBS), Ulsan, Republic of Korea. JG was supported by the NIH grant (R01CA228133). WX was supported by Program of Shanghai Subject Chief Scientist (19XD1402300) and Program for Outstanding Medical Academic Leader (2019LJ11). SRA was supported by the Patrick C. Walsh Prostate Cancer Research Fund and the Prostate Cancer Foundation. KJP was supported by NCI grants U54CA143803, CA163124, CA093900, and CA143055, and the Prostate Cancer Foundation. Funding Information: The authors thank Barbara Smith, microscopy specialist of the Microscope Facility at Johns Hopkins University School of Medicine, for acquiring TEM images. RZ was supported by the Stichting Cure for Cancer foundation, Amsterdam, the Netherlands. KH was supported by Japan Society for the Promotion of Science (KAKENHI: 19K18555). CJK was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI19C1122). CJK and YKC were supported by a grant (IBS‐R020‐D1) from Institute for Basic Science (IBS), Ulsan, Republic of Korea. JG was supported by the NIH grant (R01CA228133). WX was supported by Program of Shanghai Subject Chief Scientist (19XD1402300) and Program for Outstanding Medical Academic Leader (2019LJ11). SRA was supported by the Patrick C. Walsh Prostate Cancer Research Fund and the Prostate Cancer Foundation. KJP was supported by NCI grants U54CA143803, CA163124, CA093900, and CA143055, and the Prostate Cancer Foundation. Publisher Copyright: {\textcopyright} 2020 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles",
year = "2020",
month = dec,
doi = "10.1002/jev2.12044",
language = "English (US)",
volume = "10",
journal = "Journal of Extracellular Vesicles",
issn = "2001-3078",
publisher = "Taylor and Francis Ltd.",
number = "2",
}