Separation of small extracellular vesicles (sEV) from human blood by Superose 6 size exclusion chromatography
Abstract Extracellular vesicles (EVs) are valuable targets for liquid biopsy. However, attempts to introduce EV‐based biomarkers into clinical practice have not been successful to the extent expected. One of the reasons for this failure is the lack of reliable methods for EV baseline purification fr...
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| Format: | Article |
| Language: | English |
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Wiley
2024-10-01
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| Series: | Journal of Extracellular Vesicles |
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| Online Access: | https://doi.org/10.1002/jev2.70008 |
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| author | Jerome Nouvel Gonzalo Bustos‐Quevedo Tony Prinz Ramsha Masood George Daaboul Tanja Gainey‐Schleicher Uwe Wittel Sophia Chikhladze Bence Melykuti Martin Helmstaedter Karl Winkler Irina Nazarenko Gerhard Pütz |
| author_facet | Jerome Nouvel Gonzalo Bustos‐Quevedo Tony Prinz Ramsha Masood George Daaboul Tanja Gainey‐Schleicher Uwe Wittel Sophia Chikhladze Bence Melykuti Martin Helmstaedter Karl Winkler Irina Nazarenko Gerhard Pütz |
| author_sort | Jerome Nouvel |
| collection | DOAJ |
| description | Abstract Extracellular vesicles (EVs) are valuable targets for liquid biopsy. However, attempts to introduce EV‐based biomarkers into clinical practice have not been successful to the extent expected. One of the reasons for this failure is the lack of reliable methods for EV baseline purification from complex biofluids, such as cell‐free plasma or serum. Because available one‐step approaches for EV isolation are insufficient to purify EVs, the majority of studies on clinical samples were performed either on a mixture of EVs and lipoproteins, whilst the real number of EVs and their individual specific biomarker content remained elusive, or on a low number of samples of sufficient volume to allow elaborate 2‐step EV separation by size and density, resulting in a high purity but utmost low recovery. Here we introduce Fast Protein Liquid Chromatography (FPLC) using Superose 6 as a matrix to obtain small EVs from biofluids that are almost free of soluble proteins and lipoproteins. Along with the estimation of a realistic number of small EVs in human samples, we show temporal resolution of the effect of the duration of postprandial phase on the proportion of lipoproteins in purified EVs, suggesting acceptable time frames additionally to the recommendation to use fasting samples for human studies. Furthermore, we assessed a potential value of pure EVs for liquid biopsy, exemplarily examining EV‐ and tumour‐biomarkers in pure FPLC‐derived fractions isolated from the serum of patients with pancreatic cancer. Consistent among different techniques, showed the presence of diseases‐associated biomarkers in pure EVs, supporting the feasibility of using single‐vesicle analysis for liquid biopsy. |
| format | Article |
| id | doaj-art-588507e5d9a54be8a0d1eefab47b6573 |
| institution | Kabale University |
| issn | 2001-3078 |
| language | English |
| publishDate | 2024-10-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Vesicles |
| spelling | doaj-art-588507e5d9a54be8a0d1eefab47b65732024-12-18T07:26:42ZengWileyJournal of Extracellular Vesicles2001-30782024-10-011310n/an/a10.1002/jev2.70008Separation of small extracellular vesicles (sEV) from human blood by Superose 6 size exclusion chromatographyJerome Nouvel0Gonzalo Bustos‐Quevedo1Tony Prinz2Ramsha Masood3George Daaboul4Tanja Gainey‐Schleicher5Uwe Wittel6Sophia Chikhladze7Bence Melykuti8Martin Helmstaedter9Karl Winkler10Irina Nazarenko11Gerhard Pütz12Institute for Infection Prevention and Hospital Epidemiology Freiburg GermanyInstitute for Infection Prevention and Hospital Epidemiology Freiburg GermanyInstitute for Infection Prevention and Hospital Epidemiology Freiburg GermanyInstitute for Infection Prevention and Hospital Epidemiology Freiburg GermanyNanoView Biosciences Boston Massachusetts USAInstitute for Infection Prevention and Hospital Epidemiology Freiburg GermanyMedical Center, Faculty of Medicine, University of Freiburg University of Freiburg Freiburg GermanyMedical Center, Faculty of Medicine, University of Freiburg University of Freiburg Freiburg GermanyInstitute for Infection Prevention and Hospital Epidemiology Freiburg GermanyMedical Center, Faculty of Medicine, University of Freiburg University of Freiburg Freiburg GermanyMedical Center, Faculty of Medicine, University of Freiburg University of Freiburg Freiburg GermanyInstitute for Infection Prevention and Hospital Epidemiology Freiburg GermanyMedical Center, Faculty of Medicine, University of Freiburg University of Freiburg Freiburg GermanyAbstract Extracellular vesicles (EVs) are valuable targets for liquid biopsy. However, attempts to introduce EV‐based biomarkers into clinical practice have not been successful to the extent expected. One of the reasons for this failure is the lack of reliable methods for EV baseline purification from complex biofluids, such as cell‐free plasma or serum. Because available one‐step approaches for EV isolation are insufficient to purify EVs, the majority of studies on clinical samples were performed either on a mixture of EVs and lipoproteins, whilst the real number of EVs and their individual specific biomarker content remained elusive, or on a low number of samples of sufficient volume to allow elaborate 2‐step EV separation by size and density, resulting in a high purity but utmost low recovery. Here we introduce Fast Protein Liquid Chromatography (FPLC) using Superose 6 as a matrix to obtain small EVs from biofluids that are almost free of soluble proteins and lipoproteins. Along with the estimation of a realistic number of small EVs in human samples, we show temporal resolution of the effect of the duration of postprandial phase on the proportion of lipoproteins in purified EVs, suggesting acceptable time frames additionally to the recommendation to use fasting samples for human studies. Furthermore, we assessed a potential value of pure EVs for liquid biopsy, exemplarily examining EV‐ and tumour‐biomarkers in pure FPLC‐derived fractions isolated from the serum of patients with pancreatic cancer. Consistent among different techniques, showed the presence of diseases‐associated biomarkers in pure EVs, supporting the feasibility of using single‐vesicle analysis for liquid biopsy.https://doi.org/10.1002/jev2.70008biomarkerextracellular vesiclesfast protein liquid chromatographyliquid biopsypancreatic cancersingle particle interferometric imaging |
| spellingShingle | Jerome Nouvel Gonzalo Bustos‐Quevedo Tony Prinz Ramsha Masood George Daaboul Tanja Gainey‐Schleicher Uwe Wittel Sophia Chikhladze Bence Melykuti Martin Helmstaedter Karl Winkler Irina Nazarenko Gerhard Pütz Separation of small extracellular vesicles (sEV) from human blood by Superose 6 size exclusion chromatography Journal of Extracellular Vesicles biomarker extracellular vesicles fast protein liquid chromatography liquid biopsy pancreatic cancer single particle interferometric imaging |
| title | Separation of small extracellular vesicles (sEV) from human blood by Superose 6 size exclusion chromatography |
| title_full | Separation of small extracellular vesicles (sEV) from human blood by Superose 6 size exclusion chromatography |
| title_fullStr | Separation of small extracellular vesicles (sEV) from human blood by Superose 6 size exclusion chromatography |
| title_full_unstemmed | Separation of small extracellular vesicles (sEV) from human blood by Superose 6 size exclusion chromatography |
| title_short | Separation of small extracellular vesicles (sEV) from human blood by Superose 6 size exclusion chromatography |
| title_sort | separation of small extracellular vesicles sev from human blood by superose 6 size exclusion chromatography |
| topic | biomarker extracellular vesicles fast protein liquid chromatography liquid biopsy pancreatic cancer single particle interferometric imaging |
| url | https://doi.org/10.1002/jev2.70008 |
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