Dynamic cultivation of human mesenchymal stem/stromal cells for the production of extracellular vesicles in a 3D bioreactor system

verfasst von

Ciarra Almeria, René Weiss, Maike Keck, Viktoria Weber, Cornelia Kasper, Dominik Egger

Abstract

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 × 10

⁹ ± 1.5 × 10

⁹ and 9.7 × 10

⁹ ± 3.1 × 10

⁹ particles/mL) compared to static 2D culture (4.2 × 10

⁹ ± 7.5 × 10

⁸ and 3.9 × 10

⁹ ± 3.0 × 10

⁸ 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

⁷ ± 1.1 × 10

⁷ particles/µg protein in 2D to 1.6 × 10

⁸ ± 8.3 × 10

⁶ 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.

Organisationseinheit(en)

Institut für Zellbiologie und Biophysik
Biofabrikation für Wirkstofftestung

Externe Organisation(en)

Universität für Bodenkultur Wien (BOKU)
Universität für Weiterbildung Krems
Agaplesion Diakonieklinikum Hamburg

Typ

Artikel

Journal

Biotechnology letters

Band

Seiten

279–293

Anzahl der Seiten

ISSN

0141-5492

Publikationsdatum

04.2024

Publikationsstatus

Veröffentlicht

Peer-reviewed

ASJC Scopus Sachgebiete

Angewandte Mikrobiologie und Biotechnologie, Bioengineering, Biotechnologie

Elektronische Version(en)

https://doi.org/10.1007/s10529-024-03465-4 (Zugang: Offen)

BibTeX

@article{552ef9182e994ee795335fe6641f4542,
title = "Dynamic cultivation of human mesenchymal stem/stromal cells for the production of extracellular vesicles in a 3D bioreactor system",
abstract = "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{\textregistered} 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{\textregistered} bioreactor system demonstrated a higher concentration of MSC-EVs from the 3D bioreactor (9.1 × 10 9 ± 1.5 × 10 9 and 9.7 × 10 9 ± 3.1 × 10 9 particles/mL) compared to static 2D culture (4.2 × 10 9 ± 7.5 × 10 8 and 3.9 × 10 9 ± 3.0 × 10 8 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 7 ± 1.1 × 10 7 particles/µg protein in 2D to 1.6 × 10 8 ± 8.3 × 10 6 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.",
keywords = "3D cell culture, Bioreactors, Extracellular vesicles, Hypoxia, Mesenchymal stem cells",
author = "Ciarra Almeria and Ren{\'e} Weiss and Maike Keck and Viktoria Weber and Cornelia Kasper and Dominik Egger",
note = "Funding Open Access funding enabled and organized by Projekt DEAL. The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.",
year = "2024",
month = apr,
doi = "10.1007/s10529-024-03465-4",
language = "English",
volume = "46",
pages = "279–293",
journal = "Biotechnology letters",
issn = "0141-5492",
publisher = "Springer Netherlands",
number = "2",
}