Enhanced bioprocess control to advance the manufacture of mesenchymal stromal cell‐derived extracellular vesicles in stirred‐tank bioreactors

• Published in: Biotechnology and bioengineering Vol. 120; no. 9; pp. 2725 - 2741
• Main Authors: Costa, Marta H. G., Costa, Margarida S., Painho, Beatriz, Sousa, Carolina D., Carrondo, Inês, Oltra, Enrique, Pelacho, Beatriz, Prosper, Felipe, Isidro, Inês A., Alves, Paula, Serra, Margarida
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.09.2023
• Subjects: Angiogenesis; bioprocess analytics; Bioprocessing; Bioreactors; Cell culture; Extracellular vesicles; extracellular vesicles (EVs); Glucose; mesenchymal stem/stromal cells (MSCs); metabolic preconditioning; Metabolites; Quality management; Raman spectroscopy; Size exclusion chromatography; stirred‐tank bioreactors; Stromal cells; Ultracentrifugation; Vesicles; stirred-tank bioreactors; bioprocess analytics; extracellular vesicles (EVs); mesenchymal stem/stromal cells (MSCs); metabolic preconditioning; Raman spectroscopy
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.28378

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) act as signaling mediators of cellular responses. However, despite representing a promising alternative to cell‐based therapies, clinical translation of EVs is currently limited by their lack of scalability and standardized bioprocessing. Herein, we integrated scalable downstream processing protocols with standardized expansion of large numbers of viable cells in stirred‐tank bioreactors to improve EV production. Higher EV yields were linked to EV isolation by tangential flow filtration followed by size exclusion chromatography, rendering 5 times higher number of EVs comparatively to density gradient ultracentrifugation protocols. Additionally, when compared to static culture, EV manufacture in bioreactors resulted in 2.2 higher yields. Highlighting the role of operating under optimal cell culture conditions to maximize the number of EVs secreted per cell, MSCs cultured at lower glucose concentration favored EV secretion. While offline measurements of metabolites concentration can be performed, in this work, Raman spectroscopy was also applied to continuously track glucose levels in stirred‐tank bioreactors, contributing to streamline the selection of optimal EV collection timepoints. Importantly, MSC‐derived EVs retained their quality attributes and were able to stimulate angiogenesis in vitro, therefore highlighting their promising therapeutic potential.