Scalability of spheroid-derived small extracellular vesicles production in stirred systems

Thibaud Dauphin¹Laurence De Beaurepaire¹Apolline Salama¹Quentin Pruvost¹Clémentine Claire^1,2Karine Haurogné¹Sophie Sourice¹Aurélien Dupont³Jean-Marie Bach^1,2Julie Hervé¹Eric Olmos⁴Steffi Bosch¹Blandine Lieubeau¹Mathilde Mosser^1,2*

• ¹Oniris, INRAE, IECM, Nantes, France
• ²Oniris, B-FHIT, Nantes, France
• ³University of Rennes, CNRS, Biosit, MRic, Rennes, France
• ⁴University of Lorraine, CNRS, LRGP, Nancy, France

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 anchoragedependent cells. Using a human β cell line, we highlight that the generation of spheroids in a chemically defined medium allows the culture of highly viable cells in suspension in stirred systems. We demonstrate that spheroid size depends 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 a 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 cultures and scale-up in stirred systems for production of sEV-based therapies.