Extracellular Vesicle Depletion Protocols of Foetal Bovine Serum Influence Umbilical Cord Mesenchymal Stromal Cell Phenotype, Immunomodulation, and Particle Release
The immunomodulatory properties of MSCs can be recreated using their extracellular vesicles (EVs). Yet, the true capabilities of the MSC EVs cannot be distinguished from contaminating bovine EVs and protein derived from supplemental foetal bovine serum (FBS). FBS EV depletion protocols can minimise...
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MDPI AG
2023-05-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/11/9242 |
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| author | Rebecca Davies Shannen Allen Claire Mennan Mark Platt Karina Wright Oksana Kehoe |
| author_facet | Rebecca Davies Shannen Allen Claire Mennan Mark Platt Karina Wright Oksana Kehoe |
| author_sort | Rebecca Davies |
| collection | DOAJ |
| description | The immunomodulatory properties of MSCs can be recreated using their extracellular vesicles (EVs). Yet, the true capabilities of the MSC EVs cannot be distinguished from contaminating bovine EVs and protein derived from supplemental foetal bovine serum (FBS). FBS EV depletion protocols can minimise this, but vary in terms of depletion efficiency, which can negatively impact the cell phenotype. We explore the impact of FBS EV depletion strategies, including ultracentrifugation, ultrafiltration, and serum-free, on umbilical cord MSC characteristics. Whilst a greater depletion efficiency, seen in the ultrafiltration and serum-free strategies, did not impact the MSC markers or viability, the MSCs did become more fibroblastic, had slower proliferation, and showed inferior immunomodulatory capabilities. Upon MSC EV enrichment, more particles, with a greater particle/protein ratio, were isolated upon increasing the FBS depletion efficiency, except for serum-free, which showed a decreased particle number. Whilst all conditions showed the presence of EV-associated markers (CD9, CD63, and CD81), serum-free was shown to represent a higher proportion of these markers when normalised by total protein. Thus, we caution MSC EV researchers on the use of highly efficient EV depletion protocols, showing that it can impact the MSC phenotype, including their immunomodulatory properties, and stress the importance of testing in consideration to downstream objectives. |
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| format | Article |
| id | doaj.art-6077b5b4c1d345e89836a32376f9197f |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T03:06:33Z |
| publishDate | 2023-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-6077b5b4c1d345e89836a32376f9197f2023-11-18T07:56:20ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-05-012411924210.3390/ijms24119242Extracellular Vesicle Depletion Protocols of Foetal Bovine Serum Influence Umbilical Cord Mesenchymal Stromal Cell Phenotype, Immunomodulation, and Particle ReleaseRebecca Davies0Shannen Allen1Claire Mennan2Mark Platt3Karina Wright4Oksana Kehoe5Centre for Regenerative Medicine Research, School of Medicine (Keele University), RJAH Orthopaedic Hospital, Shropshire SY10 7AG, UKCentre for Regenerative Medicine Research, School of Medicine (Keele University), RJAH Orthopaedic Hospital, Shropshire SY10 7AG, UKCentre for Regenerative Medicine Research, School of Pharmacy and Bioengineering (Keele University), RJAH Orthopaedic Hospital, Shropshire SY10 7AG, UKDepartment of Chemistry, Centre for Analytical Science, Loughborough University, Loughborough LE11 3TU, UKCentre for Regenerative Medicine Research, School of Pharmacy and Bioengineering (Keele University), RJAH Orthopaedic Hospital, Shropshire SY10 7AG, UKCentre for Regenerative Medicine Research, School of Medicine (Keele University), RJAH Orthopaedic Hospital, Shropshire SY10 7AG, UKThe immunomodulatory properties of MSCs can be recreated using their extracellular vesicles (EVs). Yet, the true capabilities of the MSC EVs cannot be distinguished from contaminating bovine EVs and protein derived from supplemental foetal bovine serum (FBS). FBS EV depletion protocols can minimise this, but vary in terms of depletion efficiency, which can negatively impact the cell phenotype. We explore the impact of FBS EV depletion strategies, including ultracentrifugation, ultrafiltration, and serum-free, on umbilical cord MSC characteristics. Whilst a greater depletion efficiency, seen in the ultrafiltration and serum-free strategies, did not impact the MSC markers or viability, the MSCs did become more fibroblastic, had slower proliferation, and showed inferior immunomodulatory capabilities. Upon MSC EV enrichment, more particles, with a greater particle/protein ratio, were isolated upon increasing the FBS depletion efficiency, except for serum-free, which showed a decreased particle number. Whilst all conditions showed the presence of EV-associated markers (CD9, CD63, and CD81), serum-free was shown to represent a higher proportion of these markers when normalised by total protein. Thus, we caution MSC EV researchers on the use of highly efficient EV depletion protocols, showing that it can impact the MSC phenotype, including their immunomodulatory properties, and stress the importance of testing in consideration to downstream objectives.https://www.mdpi.com/1422-0067/24/11/9242mesenchymal stromal cellsextracellular vesiclesvesicle depletion protocolsimmunomodulation |
| spellingShingle | Rebecca Davies Shannen Allen Claire Mennan Mark Platt Karina Wright Oksana Kehoe Extracellular Vesicle Depletion Protocols of Foetal Bovine Serum Influence Umbilical Cord Mesenchymal Stromal Cell Phenotype, Immunomodulation, and Particle Release International Journal of Molecular Sciences mesenchymal stromal cells extracellular vesicles vesicle depletion protocols immunomodulation |
| title | Extracellular Vesicle Depletion Protocols of Foetal Bovine Serum Influence Umbilical Cord Mesenchymal Stromal Cell Phenotype, Immunomodulation, and Particle Release |
| title_full | Extracellular Vesicle Depletion Protocols of Foetal Bovine Serum Influence Umbilical Cord Mesenchymal Stromal Cell Phenotype, Immunomodulation, and Particle Release |
| title_fullStr | Extracellular Vesicle Depletion Protocols of Foetal Bovine Serum Influence Umbilical Cord Mesenchymal Stromal Cell Phenotype, Immunomodulation, and Particle Release |
| title_full_unstemmed | Extracellular Vesicle Depletion Protocols of Foetal Bovine Serum Influence Umbilical Cord Mesenchymal Stromal Cell Phenotype, Immunomodulation, and Particle Release |
| title_short | Extracellular Vesicle Depletion Protocols of Foetal Bovine Serum Influence Umbilical Cord Mesenchymal Stromal Cell Phenotype, Immunomodulation, and Particle Release |
| title_sort | extracellular vesicle depletion protocols of foetal bovine serum influence umbilical cord mesenchymal stromal cell phenotype immunomodulation and particle release |
| topic | mesenchymal stromal cells extracellular vesicles vesicle depletion protocols immunomodulation |
| url | https://www.mdpi.com/1422-0067/24/11/9242 |
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