Isolation of native EVs from primary biofluids—Free‐flow electrophoresis as a novel approach to purify ascites‐derived EVs. Issue 12 (25th December 2022)

• Record Type: Journal Article
• Authors: Preußer, Christian; Stelter, Kathrin; Tertel, Tobias; Linder, Manuel; Helmprobst, Frederik; Szymanski, Witold; Graumann, Johannes; Giebel, Bernd; Reinartz, Silke; Müller, Rolf; Weber, Gerhard; von Strandmann, Elke Pogge
• Is Part Of: Journal of extracellular biology. Volume 1:Issue 12(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-12-25
• Subjects: ascites -- extracellular vesicles -- free‐flow electrophoresis -- microvesicles; Coated vesicles -- Periodicals; Extracellular matrix -- Periodicals; Cytology -- Periodicals; Molecular biology -- Periodicals; Extracellular Vesicles; Extracellular Matrix; Coated vesicles; Cytology; Extracellular matrix; Periodical; Periodicals; 571.65
• DOI: 10.1002/jex2.71
• Languages: English
• ISSNs: 2768-2811

Abstract

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.