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 fro...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2022-12-01
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| Series: | Journal of Extracellular Biology |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/jex2.71 |
| _version_ | 1827888357420564480 |
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| author | Christian Preußer Kathrin Stelter Tobias Tertel Manuel Linder Frederik Helmprobst Witold Szymanski Johannes Graumann Bernd Giebel Silke Reinartz Rolf Müller Gerhard Weber Elke Pogge vonStrandmann |
| author_facet | Christian Preußer Kathrin Stelter Tobias Tertel Manuel Linder Frederik Helmprobst Witold Szymanski Johannes Graumann Bernd Giebel Silke Reinartz Rolf Müller Gerhard Weber Elke Pogge vonStrandmann |
| author_sort | Christian Preußer |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-12T20:33:29Z |
| format | Article |
| id | doaj.art-aa869d584cef4b56a97195f8d97e47aa |
| institution | Directory Open Access Journal |
| issn | 2768-2811 |
| language | English |
| last_indexed | 2024-03-12T20:33:29Z |
| publishDate | 2022-12-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Biology |
| spelling | doaj.art-aa869d584cef4b56a97195f8d97e47aa2023-08-01T18:55:38ZengWileyJournal of Extracellular Biology2768-28112022-12-01112n/an/a10.1002/jex2.71Isolation of native EVs from primary biofluids—Free‐flow electrophoresis as a novel approach to purify ascites‐derived EVsChristian Preußer0Kathrin Stelter1Tobias Tertel2Manuel Linder3Frederik Helmprobst4Witold Szymanski5Johannes Graumann6Bernd Giebel7Silke Reinartz8Rolf Müller9Gerhard Weber10Elke Pogge vonStrandmann11Institute for Tumor Immunology, Center for Tumor Biology and Immunology Philipps University Marburg MarburgGermanyInstitute for Tumor Immunology, Center for Tumor Biology and Immunology Philipps University Marburg MarburgGermanyInstitute for Transfusion Medicine University Hospital Essen University of Duisburg‐Essen Essen GermanyInstitute for Tumor Immunology, Center for Tumor Biology and Immunology Philipps University Marburg MarburgGermanyCore Facility for Mouse Pathology and Electron Microscopy, Institute of Neuropathology Philipps University Marburg Marburg GermanyInstitute of Translational Proteomics Philipps University of Marburg Marburg GermanyInstitute of Translational Proteomics Philipps University of Marburg Marburg GermanyInstitute for Transfusion Medicine University Hospital Essen University of Duisburg‐Essen Essen GermanyTranslational Oncology Group, Center for Tumor Biology and Immunology Philipps University Marburg Marburg GermanyTranslational Oncology Group, Center for Tumor Biology and Immunology Philipps University Marburg Marburg GermanyFFE Service GmbH Feldkirchen GermanyInstitute for Tumor Immunology, Center for Tumor Biology and Immunology Philipps University Marburg MarburgGermanyAbstract 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.https://doi.org/10.1002/jex2.71ascitesextracellular vesiclesfree‐flow electrophoresismicrovesicles |
| spellingShingle | Christian Preußer Kathrin Stelter Tobias Tertel Manuel Linder Frederik Helmprobst Witold Szymanski Johannes Graumann Bernd Giebel Silke Reinartz Rolf Müller Gerhard Weber Elke Pogge vonStrandmann Isolation of native EVs from primary biofluids—Free‐flow electrophoresis as a novel approach to purify ascites‐derived EVs Journal of Extracellular Biology ascites extracellular vesicles free‐flow electrophoresis microvesicles |
| title | Isolation of native EVs from primary biofluids—Free‐flow electrophoresis as a novel approach to purify ascites‐derived EVs |
| title_full | Isolation of native EVs from primary biofluids—Free‐flow electrophoresis as a novel approach to purify ascites‐derived EVs |
| title_fullStr | Isolation of native EVs from primary biofluids—Free‐flow electrophoresis as a novel approach to purify ascites‐derived EVs |
| title_full_unstemmed | Isolation of native EVs from primary biofluids—Free‐flow electrophoresis as a novel approach to purify ascites‐derived EVs |
| title_short | Isolation of native EVs from primary biofluids—Free‐flow electrophoresis as a novel approach to purify ascites‐derived EVs |
| title_sort | isolation of native evs from primary biofluids free flow electrophoresis as a novel approach to purify ascites derived evs |
| topic | ascites extracellular vesicles free‐flow electrophoresis microvesicles |
| url | https://doi.org/10.1002/jex2.71 |
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