Scalability of spheroid-derived small extracellular vesicles production in stirred systems
Year: 2025
Type: article
Abstract: Introduction: Small extracellular vesicle (sEV)-based therapies have gained widespread interest, but challenges persist to ensure standardization and high-scale production. Implementing upstream processes in a chemically defined media in stirred-tank bioreactors (STBr) is mandatory to closely control the cell environment, and to scale-up production, but it remains a significant challenge for anchorage-dependent cells. Methods: We used a human β cell line, grown as monolayer or in suspension as spheroid in stirred systems. We assessed the consequences of culturing these cells in 3D with, or without fetal bovine serum in a chemically defined medium, for cell growth, viability and metabolism. We next explored how different scale-up strategies might influence cell and spheroid formation in spinner flask, with the aim to transfer the process in instrumented Ambr®250 STBr. Lastly, we analyzed and characterized sEV production in monolayer, spinner flask and STBr. Results and discussion: Generation of spheroids in a chemically defined medium allowed the culture of highly viable cells in suspension in stirred systems. Spheroid size depended on the system's volumetric power input (P/V), and maintaining this parameter constant during scale-up proved to be the optimal strategy for standardizing the process. However, transferring the spinner flask (SpF) process to the Ambr®250 STBr at constant P/V modified spheroid size, due to important geometric differences and impeller design. Compared to a monolayer reference process, sEV yield decreased two-fold in SpF, but increased two-fold in STBr. Additionally, a lower expression of the CD63 tetraspanin was observed in sEV produced in both stirred systems, suggesting a reduced release of exosomes compared to ectosomes. This study addresses the main issues encountered in spheroid culture scale-up in stirred systems, rather conducive for the production of ectosomes.
Institutions: Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement, Oniris, Centre National de la Recherche Scientifique, Inserm, Structure Fédérative de Recherche en Biologie et Santé de Rennes, Université de Rennes, Laboratoire Réactions et Génie des Procédés, Université de Lorraine
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,801
OpenAlex ID: W4409953034