Isolation of native EVs from primary biofluids—Free‐flow electrophoresis as a novel approach to purify ascites‐derived EVs

Abstract

Although extracellular vesicles (EVs) have been extensively characterized, efficient purification methods, especially from primary biofluids, remain challenging. Here we introduce free‐flow electrophoresis (FFE) as a novel approach for purifying EVs from primary biofluids, in particular from the peritoneal fluid (ascites) of ovarian cancer patients. FFE represents a versatile, fast, matrix‐free approach for separating different analytes with inherent differences in charge density and/or isoelectric point (pI). Using a series of buffered media with different pH values allowed us to collect 96 fractions of ascites samples. To characterize the composition of the individual fractions, we used state‐of‐the‐art methods such as nanoflow and imaging flow cytometry (nFCM and iFCM) in addition to classical approaches. Of note, tetraspanin‐positive events measured using nFCM were enriched in a small number of distinct fractions. This observation was corroborated by Western blot analysis and electron microscopy, demonstrating only minor contamination with soluble proteins and lipid particles. In addition, these gently purified EVs remain functional. Thus, FFE represents a new, efficient and fast method for separating native and highly purified EVs from complicated primary samples.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

“…Fluid flow‐based techniques separate EVs and other particles based on one or more particle properties, but without relying on a ‘matrix’ or stationary phase. The two main categories of flow‐based techniques currently used in EV studies are field‐flow fractionation (FFF, Giddings et al., 1976 ) and free‐flow electrophoresis (FFE, Preußer et al., 2022 ). These techniques can be applied to highly heterogeneous input materials, and the absence of a solid phase allows high particle recovery.…”

“…AF4 is also not a preparative technique. In contrast, FFE combines flow with electrophoresis, adding separation by, for example, isoelectric point (Preußer et al., 2022 ). Introducing separation buffers with different pH or other characteristics across the separation channel allows high‐resolution separation of different EV and other EP populations.…”