A novel approach for large-scale manufacturing of small extracellular vesicles from bone marrow-derived mesenchymal stromal cells using a hollow fiber bioreactor
Mesenchymal stromal cells (MSCs) are promising therapeutic candidates in a variety of diseases due to having immunomodulatory and pro-regenerative properties. In recent years, MSC-derived small extracellular vesicles (sEVs) have attracted increasing interest as a possible alternative to conventional...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-01-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1107055/full |
| _version_ | 1797944946515247104 |
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| author | Viktoria Jakl Melanie Ehmele Martina Winkelmann Simon Ehrenberg Tim Eiseler Benedikt Friemert Markus Thomas Rojewski Markus Thomas Rojewski Hubert Schrezenmeier Hubert Schrezenmeier |
| author_facet | Viktoria Jakl Melanie Ehmele Martina Winkelmann Simon Ehrenberg Tim Eiseler Benedikt Friemert Markus Thomas Rojewski Markus Thomas Rojewski Hubert Schrezenmeier Hubert Schrezenmeier |
| author_sort | Viktoria Jakl |
| collection | DOAJ |
| description | Mesenchymal stromal cells (MSCs) are promising therapeutic candidates in a variety of diseases due to having immunomodulatory and pro-regenerative properties. In recent years, MSC-derived small extracellular vesicles (sEVs) have attracted increasing interest as a possible alternative to conventional cell therapy. However, translational processes of sEVs for clinical applications are still impeded by inconsistencies regarding isolation procedures and culture conditions. We systematically compared different methods for sEV isolation from conditioned media of ex vivo expanded bone marrow-derived MSCs and demonstrated considerable variability of quantity, purity, and characteristics of sEV preparations obtained by these methods. The combination of cross flow filtration with ultracentrifugation for sEV isolation resulted in sEVs with similar properties as compared to isolation by differential centrifugation combined with ultracentrifugation, the latter is still considered as gold standard for sEV isolation. In contrast, sEV isolation by a combination of precipitation with polyethylene glycol and ultracentrifugation as well as cross flow filtration and size exclusion chromatography resulted in sEVs with different characteristics, as shown by surface antigen expression patterns. The MSC culture requires a growth-promoting supplement, such as platelet lysate, which contains sEVs itself. We demonstrated that MSC culture with EV-depleted platelet lysate does not alter MSC characteristics, and conditioned media of such MSC cultures provide sEV preparations enriched for MSC-derived sEVs. The results from the systematic stepwise evaluation of various aspects were combined with culture of MSCs in a hollow fiber bioreactor. This resulted in a strategy using cross flow filtration with subsequent ultracentrifugation for sEV isolation. In conclusion, this workflow provides a semi-automated, efficient, large-scale-applicable, and good manufacturing practice (GMP)-grade approach for the generation of sEVs for clinical use. The use of EV-depleted platelet lysate is an option to further increase the purity of MSC-derived sEVs. |
| first_indexed | 2024-04-10T20:47:37Z |
| format | Article |
| id | doaj.art-0dd0984c8c6741dfbd3819d911228179 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-10T20:47:37Z |
| publishDate | 2023-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-0dd0984c8c6741dfbd3819d9112281792023-01-24T04:53:14ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-01-011110.3389/fbioe.2023.11070551107055A novel approach for large-scale manufacturing of small extracellular vesicles from bone marrow-derived mesenchymal stromal cells using a hollow fiber bioreactorViktoria Jakl0Melanie Ehmele1Martina Winkelmann2Simon Ehrenberg3Tim Eiseler4Benedikt Friemert5Markus Thomas Rojewski6Markus Thomas Rojewski7Hubert Schrezenmeier8Hubert Schrezenmeier9Institute for Transfusion Medicine, University Hospital Ulm, Ulm, GermanyInstitute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Donation Service Baden-Württemberg—Hessia and University Hospital Ulm, Ulm, GermanyInstitute for Transfusion Medicine, University Hospital Ulm, Ulm, GermanyInstitute for Transfusion Medicine, University Hospital Ulm, Ulm, GermanyClinic of Internal Medicine I, University Hospital Ulm, Ulm, GermanyClinic for Trauma Surgery and Orthopedics, Army Hospital Ulm, Ulm, GermanyInstitute for Transfusion Medicine, University Hospital Ulm, Ulm, GermanyInstitute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Donation Service Baden-Württemberg—Hessia and University Hospital Ulm, Ulm, GermanyInstitute for Transfusion Medicine, University Hospital Ulm, Ulm, GermanyInstitute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Donation Service Baden-Württemberg—Hessia and University Hospital Ulm, Ulm, GermanyMesenchymal stromal cells (MSCs) are promising therapeutic candidates in a variety of diseases due to having immunomodulatory and pro-regenerative properties. In recent years, MSC-derived small extracellular vesicles (sEVs) have attracted increasing interest as a possible alternative to conventional cell therapy. However, translational processes of sEVs for clinical applications are still impeded by inconsistencies regarding isolation procedures and culture conditions. We systematically compared different methods for sEV isolation from conditioned media of ex vivo expanded bone marrow-derived MSCs and demonstrated considerable variability of quantity, purity, and characteristics of sEV preparations obtained by these methods. The combination of cross flow filtration with ultracentrifugation for sEV isolation resulted in sEVs with similar properties as compared to isolation by differential centrifugation combined with ultracentrifugation, the latter is still considered as gold standard for sEV isolation. In contrast, sEV isolation by a combination of precipitation with polyethylene glycol and ultracentrifugation as well as cross flow filtration and size exclusion chromatography resulted in sEVs with different characteristics, as shown by surface antigen expression patterns. The MSC culture requires a growth-promoting supplement, such as platelet lysate, which contains sEVs itself. We demonstrated that MSC culture with EV-depleted platelet lysate does not alter MSC characteristics, and conditioned media of such MSC cultures provide sEV preparations enriched for MSC-derived sEVs. The results from the systematic stepwise evaluation of various aspects were combined with culture of MSCs in a hollow fiber bioreactor. This resulted in a strategy using cross flow filtration with subsequent ultracentrifugation for sEV isolation. In conclusion, this workflow provides a semi-automated, efficient, large-scale-applicable, and good manufacturing practice (GMP)-grade approach for the generation of sEVs for clinical use. The use of EV-depleted platelet lysate is an option to further increase the purity of MSC-derived sEVs.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1107055/fullmesenchymal stromal cellsmesenchymal stem cellsplatelet lysatehollow fiber bioreactorexosomessmall extracellular vesicles |
| spellingShingle | Viktoria Jakl Melanie Ehmele Martina Winkelmann Simon Ehrenberg Tim Eiseler Benedikt Friemert Markus Thomas Rojewski Markus Thomas Rojewski Hubert Schrezenmeier Hubert Schrezenmeier A novel approach for large-scale manufacturing of small extracellular vesicles from bone marrow-derived mesenchymal stromal cells using a hollow fiber bioreactor Frontiers in Bioengineering and Biotechnology mesenchymal stromal cells mesenchymal stem cells platelet lysate hollow fiber bioreactor exosomes small extracellular vesicles |
| title | A novel approach for large-scale manufacturing of small extracellular vesicles from bone marrow-derived mesenchymal stromal cells using a hollow fiber bioreactor |
| title_full | A novel approach for large-scale manufacturing of small extracellular vesicles from bone marrow-derived mesenchymal stromal cells using a hollow fiber bioreactor |
| title_fullStr | A novel approach for large-scale manufacturing of small extracellular vesicles from bone marrow-derived mesenchymal stromal cells using a hollow fiber bioreactor |
| title_full_unstemmed | A novel approach for large-scale manufacturing of small extracellular vesicles from bone marrow-derived mesenchymal stromal cells using a hollow fiber bioreactor |
| title_short | A novel approach for large-scale manufacturing of small extracellular vesicles from bone marrow-derived mesenchymal stromal cells using a hollow fiber bioreactor |
| title_sort | novel approach for large scale manufacturing of small extracellular vesicles from bone marrow derived mesenchymal stromal cells using a hollow fiber bioreactor |
| topic | mesenchymal stromal cells mesenchymal stem cells platelet lysate hollow fiber bioreactor exosomes small extracellular vesicles |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1107055/full |
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