Comprehensive evaluation of methods for small extracellularvesicles separation from human plasma, urine and cell culturemedium

Dong L, Zieren RC, Horie K, Kim CJ, Mallick E, Jing Y, Feng M, Kuczler MD, Green J, Amend SR, Witwer KW, de Reijke TM, Cho YK, Pienta KJ, Xue W. Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium. J Extracell Vesicles. 2020 Dec;10(2):e12044. doi: 10.1002/jev2.12044. Epub 2021 Jan 15. PMID: 33489012; PMCID: PMC7810129.

Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) researchfield is the lack of a consensus method for EV separation. This may also explain thediversity of the experimental results, as co-separated soluble proteins and lipoproteinsmay impede the interpretation of experimental findings. In this study, we compre-hensively evaluated the EV yields and sample purities of three most popular EV sep-aration methods, ultracentrifugation, precipitation and size exclusion chromatogra-phy combined with ultrafiltration, along with a microfluidic tangential flow filtrationdevice, Exodisc, in three commonly used biological samples, cell culture medium,human urine and plasma. Single EV phenotyping and density-gradient ultracentrifu-gation 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 con-taminants when the non-EV particle levels are high in input materials. We found no100% pure EV preparations due to the overlap of their size and density with manynon-EV particles in biofluids. Precipitation has the lowest sample purity, regardlessof sample type. The purities of the other techniques may vary in different sampletypes and are largely dependent on their working principles and the intrinsic compo-sition of the input sample. Researchers should choose the proper separation methodaccording to the sample type, downstream analysis and their working scenarios.

Serum extracellular vesicles trace COVID-19 progression and immune responses

Yim KHW, Borgoni S, Chahwan R. Serum extracellular vesicles profiling is associated with COVID-19 progression and immune responses. J Extracell Biol. 2022 Apr;1(4):e37. doi: 10.1002/jex2.37. Epub 2022 Apr 20. PMID: 35574251; PMCID: PMC9088353.
Abstract
Coronavirus disease 2019 (COVID-19) has transformed very quickly into a world pandemic with severe and unexpected consequences on human health. Concerted efforts to generate better diagnostic and prognostic tools have been ongoing. Research, thus far, has primarily focused on the virus itself or the direct immune response to it. Here, we propose extracellular vesicles (EVs) from serum liquid biopsies as a new and unique modality to unify diagnostic and prognostic tools for COVID-19 analyses. EVs are a novel player in intercellular signaling particularly influencing immune responses. We herein show that innate and adaptive immune EVs profiling, together with SARS-CoV-2 Spike S1 EVs provide a novel signature for COVID-19 infection. It also provides a unique ability to trace the co-existence of viral and host cell signatures to monitor affected tissues and severity of the disease progression. And provide a phenotypic insight into COVID-associated EVs.⁺

EV研究の最先端を目指す世界最大の細胞外小胞学会の年次総会「ISEV2023」が2023年5月17日から21日までの4日間、アメリカ合衆国シアトルで開催されました。この会議ではEV研究における基礎、臨床、診断から治療までのあらゆる側面が網羅されており、世界のEV研究コミュニティの中心的存在です。
このISEV2023のプログラムにて、NanoFCMに関連した2つの講演とポスターで最新の研究紹介が行われました。
■Novel fluorescent labelling of internal protein targets in ARMMs as a model extracellular vesicle by Ben Peacock (talk)
■Sensitive and Quantitative Detection of EV Subpopulations Using Dedicated Nano Flow Cytometry by Dr. Clayton Deighan (talk)
■Investigation into commercially available EV membrane dyes to improve EV purity analysis in complex biofluids (poster)
NanoFCMはEVの全範囲（40~1,000 nm）における単一粒子のハイスループットなマルチパラメーター解析を可能にするナノフローサイトメーターです。これまで解明されなかったEVの様々な情報をこの装置で明らかにすることができます。