Framework for rapid comparison of extracellular vesicle isolation methods

eLife

Dmitry Ter-Ovanesyan¹  Wendy Trieu¹  Roey Lazarovits¹  Ju-Hyun Lee¹  David R Walt²  George M Church³  Maia Norman⁴ 

[1] Wyss Institute for Biologically Inspired Engineering, Boston, United States;Wyss Institute for Biologically Inspired Engineering, Boston, United States;Department of Pathology, Brigham and Women’s Hospital, Boston, United States;Harvard Medical School, Boston, United States;Wyss Institute for Biologically Inspired Engineering, Boston, United States;Harvard Medical School, Boston, United States;Wyss Institute for Biologically Inspired Engineering, Boston, United States;Tufts University School of Medicine, Boston, United States;Department of Pathology, Brigham and Women’s Hospital, Boston, United States;

关键词: extracellular vesicles;    exosomes;    diagnostics;    biomarkers;    size exclusion chromatography;    Human;   

DOI  :  10.7554/eLife.70725

来源: eLife Sciences Publications, Ltd

【 摘 要 】

Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive, single-molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays, all on one platform, to improve SEC isolation from plasma and CSF. Our results highlight the utility of quantifying EV proteins using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.

【 授权许可】

CC BY