Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles
Abstract Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stabilit...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2022-09-01
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| Series: | Journal of Extracellular Vesicles |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/jev2.12266 |
| _version_ | 1811208854024749056 |
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| author | Kekoolani S. Visan Richard J. Lobb Sunyoung Ham Luize G. Lima Carlos Palma Chai Pei Zhi Edna Li‐Ying Wu Harsha Gowda Keshava K. Datta Gunter Hartel Carlos Salomon Andreas Möller |
| author_facet | Kekoolani S. Visan Richard J. Lobb Sunyoung Ham Luize G. Lima Carlos Palma Chai Pei Zhi Edna Li‐Ying Wu Harsha Gowda Keshava K. Datta Gunter Hartel Carlos Salomon Andreas Möller |
| author_sort | Kekoolani S. Visan |
| collection | DOAJ |
| description | Abstract Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum‐containing media. The majority of researchers use serum‐containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non‐sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA‐MB‐231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano‐flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large‐scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations. |
| first_indexed | 2024-04-12T04:28:48Z |
| format | Article |
| id | doaj.art-47a5770b4d0c49f1bafcb6ca8e551f66 |
| institution | Directory Open Access Journal |
| issn | 2001-3078 |
| language | English |
| last_indexed | 2024-04-12T04:28:48Z |
| publishDate | 2022-09-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Vesicles |
| spelling | doaj.art-47a5770b4d0c49f1bafcb6ca8e551f662022-12-22T03:47:59ZengWileyJournal of Extracellular Vesicles2001-30782022-09-01119n/an/a10.1002/jev2.12266Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesiclesKekoolani S. Visan0Richard J. Lobb1Sunyoung Ham2Luize G. Lima3Carlos Palma4Chai Pei Zhi Edna5Li‐Ying Wu6Harsha Gowda7Keshava K. Datta8Gunter Hartel9Carlos Salomon10Andreas Möller11Tumour Microenvironment Laboratory QIMR Berghofer Medical Research Institute Herston QLD AustraliaTumour Microenvironment Laboratory QIMR Berghofer Medical Research Institute Herston QLD AustraliaTumour Microenvironment Laboratory QIMR Berghofer Medical Research Institute Herston QLD AustraliaTumour Microenvironment Laboratory QIMR Berghofer Medical Research Institute Herston QLD AustraliaExosome Biology Laboratory Faculty of Medicine and Biomedical Sciences Centre for Clinical Diagnostics University of Queensland Centre for Clinical Research Royal Brisbane and Women's Hospital The University of Queensland Brisbane QLD AustraliaTumour Microenvironment Laboratory QIMR Berghofer Medical Research Institute Herston QLD AustraliaTumour Microenvironment Laboratory QIMR Berghofer Medical Research Institute Herston QLD AustraliaCancer Precision Medicine Laboratory QIMR Berghofer Medical Research Institute Herston QLD AustraliaCancer Precision Medicine Laboratory QIMR Berghofer Medical Research Institute Herston QLD AustraliaStatistics Unit QIMR Berghofer Medical Research Institute Herston QLD AustraliaExosome Biology Laboratory Faculty of Medicine and Biomedical Sciences Centre for Clinical Diagnostics University of Queensland Centre for Clinical Research Royal Brisbane and Women's Hospital The University of Queensland Brisbane QLD AustraliaTumour Microenvironment Laboratory QIMR Berghofer Medical Research Institute Herston QLD AustraliaAbstract Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum‐containing media. The majority of researchers use serum‐containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non‐sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA‐MB‐231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano‐flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large‐scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.https://doi.org/10.1002/jev2.12266cell cultureextracellular vesiclesisolationtangential flow filtration |
| spellingShingle | Kekoolani S. Visan Richard J. Lobb Sunyoung Ham Luize G. Lima Carlos Palma Chai Pei Zhi Edna Li‐Ying Wu Harsha Gowda Keshava K. Datta Gunter Hartel Carlos Salomon Andreas Möller Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles Journal of Extracellular Vesicles cell culture extracellular vesicles isolation tangential flow filtration |
| title | Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles |
| title_full | Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles |
| title_fullStr | Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles |
| title_full_unstemmed | Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles |
| title_short | Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles |
| title_sort | comparative analysis of tangential flow filtration and ultracentrifugation both combined with subsequent size exclusion chromatography for the isolation of small extracellular vesicles |
| topic | cell culture extracellular vesicles isolation tangential flow filtration |
| url | https://doi.org/10.1002/jev2.12266 |
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