Transitioning from static to suspension culture system for large‐scale production of xeno‐free extracellular vesicles derived from mesenchymal stromal cells
Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters...
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| Veröffentlicht in: | Biotechnology progress 2024-05, Vol.40 (3), p.e3419-n/a |
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| creator | Santos, Natália Cristine Dias Bruzadelle‐Vieira, Paula Cássia Noronha, Nádia Mizukami‐Martins, Amanda Orellana, Maristela Delgado Bentley, Maria Vitória L. B. Covas, Dimas Tadeu Swiech, Kamilla Malmegrim, Kelen Cristina Ribeiro |
| description | Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters monitoring. In this study, we reported the establishment of a scalable bioprocess to produce MSC‐EV in suspension cultures using spinner flasks and human collagen‐coated microcarriers (3D culture system). We compared the EV production in this 3D culture system with the standard static culture using T‐flasks (2D culture system). The EV produced in both systems were characterized and quantify by western blotting and nanoparticle tracking analysis. The presence of the typical protein markers CD9, CD63, and CD81 was confirmed by western blotting analyses for EV produced in both culture systems. The cell fold‐increase was 5.7‐fold for the 3D culture system and 4.6‐fold for the 2D culture system, signifying a fold‐change of 1.2 (calculated as the ratio of fold‐increase 3D to fold‐increase 2D). Furthermore, it should be noted that the total cell production in the spinner flask cultures was 4.8 times higher than that in T‐flask cultures. The total cell production in the spinner flask cultures was 5.2‐fold higher than that in T‐flask cultures. While the EV specific production (particles/cell) in T‐flask cultures (4.40 ± 1.21 × 108 particles/mL, p |
| doi_str_mv | 10.1002/btpr.3419 |
| format | Article |
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B. ; Covas, Dimas Tadeu ; Swiech, Kamilla ; Malmegrim, Kelen Cristina Ribeiro</creator><creatorcontrib>Santos, Natália Cristine Dias ; Bruzadelle‐Vieira, Paula ; Cássia Noronha, Nádia ; Mizukami‐Martins, Amanda ; Orellana, Maristela Delgado ; Bentley, Maria Vitória L. B. ; Covas, Dimas Tadeu ; Swiech, Kamilla ; Malmegrim, Kelen Cristina Ribeiro</creatorcontrib><description>Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters monitoring. In this study, we reported the establishment of a scalable bioprocess to produce MSC‐EV in suspension cultures using spinner flasks and human collagen‐coated microcarriers (3D culture system). We compared the EV production in this 3D culture system with the standard static culture using T‐flasks (2D culture system). The EV produced in both systems were characterized and quantify by western blotting and nanoparticle tracking analysis. The presence of the typical protein markers CD9, CD63, and CD81 was confirmed by western blotting analyses for EV produced in both culture systems. The cell fold‐increase was 5.7‐fold for the 3D culture system and 4.6‐fold for the 2D culture system, signifying a fold‐change of 1.2 (calculated as the ratio of fold‐increase 3D to fold‐increase 2D). Furthermore, it should be noted that the total cell production in the spinner flask cultures was 4.8 times higher than that in T‐flask cultures. The total cell production in the spinner flask cultures was 5.2‐fold higher than that in T‐flask cultures. While the EV specific production (particles/cell) in T‐flask cultures (4.40 ± 1.21 × 108 particles/mL, p < 0.05) was higher compared to spinner flask cultures (2.10 ± 0.04 × 108 particles/mL, p < 0.05), the spinner flask culture system offers scalability, making it capable of producing enough MSC‐EV at a large scale for clinical applications. Therefore, we concluded that 3D culture system evaluated here serves as an efficient transitional platform that enables the scaling up of MSC‐EV production for therapeutic purposes by utilizing stirred tank bioreactors and maintaining xeno‐free conditions.</description><identifier>ISSN: 8756-7938</identifier><identifier>ISSN: 1520-6033</identifier><identifier>EISSN: 1520-6033</identifier><identifier>DOI: 10.1002/btpr.3419</identifier><identifier>PMID: 38247123</identifier><language>eng</language><publisher>Hoboken, USA: John Wiley & Sons, Inc</publisher><subject>bioprocess ; Bioreactors ; CD63 antigen ; CD81 antigen ; CD9 antigen ; Cell culture ; Culture ; Extracellular vesicles ; Flasks ; Mesenchymal stem cells ; mesenchymal stromal cells ; Nanoparticles ; Protozoa ; Scaling up ; static culture ; Stromal cells ; Suspension culture ; Therapeutic applications ; Vesicles ; Western blotting</subject><ispartof>Biotechnology progress, 2024-05, Vol.40 (3), p.e3419-n/a</ispartof><rights>2024 American Institute of Chemical Engineers.</rights><rights>2024 American Institute of Chemical Engineers</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c3139-47888ec71659fcbaf7cd408d0cf8957bc190d31d9dc1a9786b1c1d1cf45d30183</cites><orcidid>0000-0002-9693-6289 ; 0000-0002-9303-0086 ; 0000-0003-2149-0101</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fbtpr.3419$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fbtpr.3419$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,27901,27902,45550,45551</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/38247123$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Santos, Natália Cristine Dias</creatorcontrib><creatorcontrib>Bruzadelle‐Vieira, Paula</creatorcontrib><creatorcontrib>Cássia Noronha, Nádia</creatorcontrib><creatorcontrib>Mizukami‐Martins, Amanda</creatorcontrib><creatorcontrib>Orellana, Maristela Delgado</creatorcontrib><creatorcontrib>Bentley, Maria Vitória L. B.</creatorcontrib><creatorcontrib>Covas, Dimas Tadeu</creatorcontrib><creatorcontrib>Swiech, Kamilla</creatorcontrib><creatorcontrib>Malmegrim, Kelen Cristina Ribeiro</creatorcontrib><title>Transitioning from static to suspension culture system for large‐scale production of xeno‐free extracellular vesicles derived from mesenchymal stromal cells</title><title>Biotechnology progress</title><addtitle>Biotechnol Prog</addtitle><description>Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters monitoring. In this study, we reported the establishment of a scalable bioprocess to produce MSC‐EV in suspension cultures using spinner flasks and human collagen‐coated microcarriers (3D culture system). We compared the EV production in this 3D culture system with the standard static culture using T‐flasks (2D culture system). The EV produced in both systems were characterized and quantify by western blotting and nanoparticle tracking analysis. The presence of the typical protein markers CD9, CD63, and CD81 was confirmed by western blotting analyses for EV produced in both culture systems. The cell fold‐increase was 5.7‐fold for the 3D culture system and 4.6‐fold for the 2D culture system, signifying a fold‐change of 1.2 (calculated as the ratio of fold‐increase 3D to fold‐increase 2D). Furthermore, it should be noted that the total cell production in the spinner flask cultures was 4.8 times higher than that in T‐flask cultures. The total cell production in the spinner flask cultures was 5.2‐fold higher than that in T‐flask cultures. While the EV specific production (particles/cell) in T‐flask cultures (4.40 ± 1.21 × 108 particles/mL, p < 0.05) was higher compared to spinner flask cultures (2.10 ± 0.04 × 108 particles/mL, p < 0.05), the spinner flask culture system offers scalability, making it capable of producing enough MSC‐EV at a large scale for clinical applications. Therefore, we concluded that 3D culture system evaluated here serves as an efficient transitional platform that enables the scaling up of MSC‐EV production for therapeutic purposes by utilizing stirred tank bioreactors and maintaining xeno‐free conditions.</description><subject>bioprocess</subject><subject>Bioreactors</subject><subject>CD63 antigen</subject><subject>CD81 antigen</subject><subject>CD9 antigen</subject><subject>Cell culture</subject><subject>Culture</subject><subject>Extracellular vesicles</subject><subject>Flasks</subject><subject>Mesenchymal stem cells</subject><subject>mesenchymal stromal cells</subject><subject>Nanoparticles</subject><subject>Protozoa</subject><subject>Scaling up</subject><subject>static culture</subject><subject>Stromal cells</subject><subject>Suspension culture</subject><subject>Therapeutic applications</subject><subject>Vesicles</subject><subject>Western blotting</subject><issn>8756-7938</issn><issn>1520-6033</issn><issn>1520-6033</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2024</creationdate><recordtype>article</recordtype><recordid>eNp1kU9u1TAQhy0Eoo_CggsgS2xgkdYTJ7G9hIp_UiUQeqwjxx6XVEn88CSlb8cROELP1pPg8AoLJFYjeb75ZqwfY09BnIAQ5Wk379KJrMDcYxuoS1E0Qsr7bKNV3RTKSH3EHhFdCiG0aMqH7EjqslJQyg272SY7UT_3ceqnCx5SHDnNdu4dnyOnhXaY23HibhnmJSGnPc048hATH2y6wNsfP8nZAfkuRb-4VcRj4Nc4xdwKCZHj9Zysw2FY8gS_QurdgMQ9pv4K_WHniIST-7of7ZD355dc1xF6zB4EOxA-uavH7MvbN9uz98X5x3cfzl6dF06CNEWltNboFDS1Ca6zQTlfCe2FC9rUqnNghJfgjXdgjdJNBw48uFDVXgrQ8pi9OHjzP74tSHM79rReYCeMC7WlAVXXlRJVRp__g17GJU35ulYKBRWAMk2mXh4olyJRwtDuUj_atG9BtGts7Rpbu8aW2Wd3xqUb0f8l_-SUgdMD8L0fcP9_U_t6--nzb-UvNVmosw</recordid><startdate>202405</startdate><enddate>202405</enddate><creator>Santos, Natália Cristine Dias</creator><creator>Bruzadelle‐Vieira, Paula</creator><creator>Cássia Noronha, Nádia</creator><creator>Mizukami‐Martins, Amanda</creator><creator>Orellana, Maristela Delgado</creator><creator>Bentley, Maria Vitória L. B.</creator><creator>Covas, Dimas Tadeu</creator><creator>Swiech, Kamilla</creator><creator>Malmegrim, Kelen Cristina Ribeiro</creator><general>John Wiley & Sons, Inc</general><general>Wiley Subscription Services, Inc</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>7T7</scope><scope>7U7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>M7N</scope><scope>P64</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-9693-6289</orcidid><orcidid>https://orcid.org/0000-0002-9303-0086</orcidid><orcidid>https://orcid.org/0000-0003-2149-0101</orcidid></search><sort><creationdate>202405</creationdate><title>Transitioning from static to suspension culture system for large‐scale production of xeno‐free extracellular vesicles derived from mesenchymal stromal cells</title><author>Santos, Natália Cristine Dias ; Bruzadelle‐Vieira, Paula ; Cássia Noronha, Nádia ; Mizukami‐Martins, Amanda ; Orellana, Maristela Delgado ; Bentley, Maria Vitória L. B. ; Covas, Dimas Tadeu ; Swiech, Kamilla ; Malmegrim, Kelen Cristina Ribeiro</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3139-47888ec71659fcbaf7cd408d0cf8957bc190d31d9dc1a9786b1c1d1cf45d30183</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2024</creationdate><topic>bioprocess</topic><topic>Bioreactors</topic><topic>CD63 antigen</topic><topic>CD81 antigen</topic><topic>CD9 antigen</topic><topic>Cell culture</topic><topic>Culture</topic><topic>Extracellular vesicles</topic><topic>Flasks</topic><topic>Mesenchymal stem cells</topic><topic>mesenchymal stromal cells</topic><topic>Nanoparticles</topic><topic>Protozoa</topic><topic>Scaling up</topic><topic>static culture</topic><topic>Stromal cells</topic><topic>Suspension culture</topic><topic>Therapeutic applications</topic><topic>Vesicles</topic><topic>Western blotting</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Santos, Natália Cristine Dias</creatorcontrib><creatorcontrib>Bruzadelle‐Vieira, Paula</creatorcontrib><creatorcontrib>Cássia Noronha, Nádia</creatorcontrib><creatorcontrib>Mizukami‐Martins, Amanda</creatorcontrib><creatorcontrib>Orellana, Maristela Delgado</creatorcontrib><creatorcontrib>Bentley, Maria Vitória L. B.</creatorcontrib><creatorcontrib>Covas, Dimas Tadeu</creatorcontrib><creatorcontrib>Swiech, Kamilla</creatorcontrib><creatorcontrib>Malmegrim, Kelen Cristina Ribeiro</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Toxicology Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biotechnology progress</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Santos, Natália Cristine Dias</au><au>Bruzadelle‐Vieira, Paula</au><au>Cássia Noronha, Nádia</au><au>Mizukami‐Martins, Amanda</au><au>Orellana, Maristela Delgado</au><au>Bentley, Maria Vitória L. B.</au><au>Covas, Dimas Tadeu</au><au>Swiech, Kamilla</au><au>Malmegrim, Kelen Cristina Ribeiro</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Transitioning from static to suspension culture system for large‐scale production of xeno‐free extracellular vesicles derived from mesenchymal stromal cells</atitle><jtitle>Biotechnology progress</jtitle><addtitle>Biotechnol Prog</addtitle><date>2024-05</date><risdate>2024</risdate><volume>40</volume><issue>3</issue><spage>e3419</spage><epage>n/a</epage><pages>e3419-n/a</pages><issn>8756-7938</issn><issn>1520-6033</issn><eissn>1520-6033</eissn><abstract>Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters monitoring. In this study, we reported the establishment of a scalable bioprocess to produce MSC‐EV in suspension cultures using spinner flasks and human collagen‐coated microcarriers (3D culture system). We compared the EV production in this 3D culture system with the standard static culture using T‐flasks (2D culture system). The EV produced in both systems were characterized and quantify by western blotting and nanoparticle tracking analysis. The presence of the typical protein markers CD9, CD63, and CD81 was confirmed by western blotting analyses for EV produced in both culture systems. The cell fold‐increase was 5.7‐fold for the 3D culture system and 4.6‐fold for the 2D culture system, signifying a fold‐change of 1.2 (calculated as the ratio of fold‐increase 3D to fold‐increase 2D). Furthermore, it should be noted that the total cell production in the spinner flask cultures was 4.8 times higher than that in T‐flask cultures. The total cell production in the spinner flask cultures was 5.2‐fold higher than that in T‐flask cultures. While the EV specific production (particles/cell) in T‐flask cultures (4.40 ± 1.21 × 108 particles/mL, p < 0.05) was higher compared to spinner flask cultures (2.10 ± 0.04 × 108 particles/mL, p < 0.05), the spinner flask culture system offers scalability, making it capable of producing enough MSC‐EV at a large scale for clinical applications. Therefore, we concluded that 3D culture system evaluated here serves as an efficient transitional platform that enables the scaling up of MSC‐EV production for therapeutic purposes by utilizing stirred tank bioreactors and maintaining xeno‐free conditions.</abstract><cop>Hoboken, USA</cop><pub>John Wiley & Sons, Inc</pub><pmid>38247123</pmid><doi>10.1002/btpr.3419</doi><tpages>13</tpages><orcidid>https://orcid.org/0000-0002-9693-6289</orcidid><orcidid>https://orcid.org/0000-0002-9303-0086</orcidid><orcidid>https://orcid.org/0000-0003-2149-0101</orcidid></addata></record> |
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| source | Wiley Online Library Journals Frontfile Complete |
| subjects | bioprocess Bioreactors CD63 antigen CD81 antigen CD9 antigen Cell culture Culture Extracellular vesicles Flasks Mesenchymal stem cells mesenchymal stromal cells Nanoparticles Protozoa Scaling up static culture Stromal cells Suspension culture Therapeutic applications Vesicles Western blotting |
| title | Transitioning from static to suspension culture system for large‐scale production of xeno‐free extracellular vesicles derived from mesenchymal stromal cells |
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