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Development of an Effective Isolation Method for Plasma Extracellular Vesicles Excluding Lipoproteins by Precipitation With Polyethylene Glycol

Kosuke Otani,Yusei Fujioka,M. Okada,H. Yamawaki

Published 2025 in Journal of Extracellular Biology

ABSTRACT

ABSTRACT Various molecules in plasma extracellular vesicles (EVs) are expected to be applied to minimally invasive diagnosis; however, the high concentration of lipoproteins in plasma, which are similar in size, density and content to EVs, hampers analysis on plasma EVs. To overcome this, we explored an effective isolation method for plasma EVs that excludes lipoproteins by applying precipitation methods that are conventionally used to separate lipoproteins. Human plasma was mixed with heparin and MnCl2, phosphotungstic acid and MgCl2, or polyethylene glycol (PEG), and the expression level of CD9, Apo B and Apo A‐I in both the supernatant and pellet was measured by enzyme‐linked immunosorbent assay. Morphology was observed by transmission electron microscopy to assess EV yield and lipoprotein contamination. The combination of heparin and MnCl2, or phosphotungstic acid and MgCl2, could not separate plasma EVs and lipoproteins. PEG precipitated EVs and lipoproteins differently, and EVs were specifically precipitated by PEG (3%) to some extent. In comparison with differential ultracentrifugation (UC), size‐exclusion chromatography, density gradient centrifugation and precipitation with PEG (8%) followed by UC, PEG (3%) was not inferior in efficiency but was superior in terms of time and cost. The precipitation method using PEG (3%) may contribute to the application of plasma EVs in disease diagnosis.

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