{"title":"在三维生物反应器系统中动态培养人类间充质干细胞/基质细胞以生产细胞外囊泡。","authors":"Ciarra Almeria, René Weiss, Maike Keck, Viktoria Weber, Cornelia Kasper, Dominik Egger","doi":"10.1007/s10529-024-03465-4","DOIUrl":null,"url":null,"abstract":"<p><strong>Purpose: </strong>3D cell culture and hypoxia have been demonstrated to increase the therapeutic effects of mesenchymal stem/stromal cells (MSCs)-derived extracellular vesicles (EVs). In this study, a process for the production of MSC-EVs in a novel 3D bioreactor system under normoxic and hypoxic conditions was established and the resulting EVs were characterized.</p><p><strong>Methods: </strong>Human adipose-derived MSCs were seeded and cultured on a 3D membrane in the VITVO® bioreactor system for 7 days. Afterwards, MSC-EVs were isolated and characterized via fluorescence nanoparticle tracking analysis, flow cytometry with staining against annexin V (Anx5) as a marker for EVs exposing phosphatidylserine, as well as CD73 and CD90 as MSC surface markers.</p><p><strong>Results: </strong>Cultivation of MSC in the VITVO® bioreactor system demonstrated a higher concentration of MSC-EVs from the 3D bioreactor (9.1 × 10<sup>9</sup> ± 1.5 × 10<sup>9</sup> and 9.7 × 10<sup>9</sup> ± 3.1 × 10<sup>9</sup> particles/mL) compared to static 2D culture (4.2 × 10<sup>9</sup> ± 7.5 × 10<sup>8</sup> and 3.9 × 10<sup>9</sup> ± 3.0 × 10<sup>8</sup> particles/mL) under normoxic and hypoxic conditions, respectively. Also, the particle-to-protein ratio as a measure for the purity of EVs increased from 3.3 × 10<sup>7</sup> ± 1.1 × 10<sup>7</sup> particles/µg protein in 2D to 1.6 × 10<sup>8</sup> ± 8.3 × 10<sup>6</sup> particles/µg protein in 3D. Total MSC-EVs as well as CD73<sup>-</sup>CD90<sup>+</sup> MSC-EVs were elevated in 2D normoxic conditions. The EV concentration and size did not differ significantly between normoxic and hypoxic conditions.</p><p><strong>Conclusion: </strong>The production of MSC-EVs in a 3D bioreactor system under hypoxic conditions resulted in increased EV concentration and purity. This system could be especially useful in screening culture conditions for the production of 3D-derived MSC-EVs.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":" ","pages":"279-293"},"PeriodicalIF":2.0000,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10902030/pdf/","citationCount":"0","resultStr":"{\"title\":\"Dynamic cultivation of human mesenchymal stem/stromal cells for the production of extracellular vesicles in a 3D bioreactor system.\",\"authors\":\"Ciarra Almeria, René Weiss, Maike Keck, Viktoria Weber, Cornelia Kasper, Dominik Egger\",\"doi\":\"10.1007/s10529-024-03465-4\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Purpose: </strong>3D cell culture and hypoxia have been demonstrated to increase the therapeutic effects of mesenchymal stem/stromal cells (MSCs)-derived extracellular vesicles (EVs). In this study, a process for the production of MSC-EVs in a novel 3D bioreactor system under normoxic and hypoxic conditions was established and the resulting EVs were characterized.</p><p><strong>Methods: </strong>Human adipose-derived MSCs were seeded and cultured on a 3D membrane in the VITVO® bioreactor system for 7 days. Afterwards, MSC-EVs were isolated and characterized via fluorescence nanoparticle tracking analysis, flow cytometry with staining against annexin V (Anx5) as a marker for EVs exposing phosphatidylserine, as well as CD73 and CD90 as MSC surface markers.</p><p><strong>Results: </strong>Cultivation of MSC in the VITVO® bioreactor system demonstrated a higher concentration of MSC-EVs from the 3D bioreactor (9.1 × 10<sup>9</sup> ± 1.5 × 10<sup>9</sup> and 9.7 × 10<sup>9</sup> ± 3.1 × 10<sup>9</sup> particles/mL) compared to static 2D culture (4.2 × 10<sup>9</sup> ± 7.5 × 10<sup>8</sup> and 3.9 × 10<sup>9</sup> ± 3.0 × 10<sup>8</sup> particles/mL) under normoxic and hypoxic conditions, respectively. Also, the particle-to-protein ratio as a measure for the purity of EVs increased from 3.3 × 10<sup>7</sup> ± 1.1 × 10<sup>7</sup> particles/µg protein in 2D to 1.6 × 10<sup>8</sup> ± 8.3 × 10<sup>6</sup> particles/µg protein in 3D. Total MSC-EVs as well as CD73<sup>-</sup>CD90<sup>+</sup> MSC-EVs were elevated in 2D normoxic conditions. The EV concentration and size did not differ significantly between normoxic and hypoxic conditions.</p><p><strong>Conclusion: </strong>The production of MSC-EVs in a 3D bioreactor system under hypoxic conditions resulted in increased EV concentration and purity. This system could be especially useful in screening culture conditions for the production of 3D-derived MSC-EVs.</p>\",\"PeriodicalId\":8929,\"journal\":{\"name\":\"Biotechnology Letters\",\"volume\":\" \",\"pages\":\"279-293\"},\"PeriodicalIF\":2.0000,\"publicationDate\":\"2024-04-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10902030/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biotechnology Letters\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1007/s10529-024-03465-4\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/2/13 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q3\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology Letters","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1007/s10529-024-03465-4","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/2/13 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Dynamic cultivation of human mesenchymal stem/stromal cells for the production of extracellular vesicles in a 3D bioreactor system.
Purpose: 3D cell culture and hypoxia have been demonstrated to increase the therapeutic effects of mesenchymal stem/stromal cells (MSCs)-derived extracellular vesicles (EVs). In this study, a process for the production of MSC-EVs in a novel 3D bioreactor system under normoxic and hypoxic conditions was established and the resulting EVs were characterized.
Methods: Human adipose-derived MSCs were seeded and cultured on a 3D membrane in the VITVO® bioreactor system for 7 days. Afterwards, MSC-EVs were isolated and characterized via fluorescence nanoparticle tracking analysis, flow cytometry with staining against annexin V (Anx5) as a marker for EVs exposing phosphatidylserine, as well as CD73 and CD90 as MSC surface markers.
Results: Cultivation of MSC in the VITVO® bioreactor system demonstrated a higher concentration of MSC-EVs from the 3D bioreactor (9.1 × 109 ± 1.5 × 109 and 9.7 × 109 ± 3.1 × 109 particles/mL) compared to static 2D culture (4.2 × 109 ± 7.5 × 108 and 3.9 × 109 ± 3.0 × 108 particles/mL) under normoxic and hypoxic conditions, respectively. Also, the particle-to-protein ratio as a measure for the purity of EVs increased from 3.3 × 107 ± 1.1 × 107 particles/µg protein in 2D to 1.6 × 108 ± 8.3 × 106 particles/µg protein in 3D. Total MSC-EVs as well as CD73-CD90+ MSC-EVs were elevated in 2D normoxic conditions. The EV concentration and size did not differ significantly between normoxic and hypoxic conditions.
Conclusion: The production of MSC-EVs in a 3D bioreactor system under hypoxic conditions resulted in increased EV concentration and purity. This system could be especially useful in screening culture conditions for the production of 3D-derived MSC-EVs.
期刊介绍：
Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them.
All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included.
Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields.
The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories.
Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.
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