PhD Scientific Days 2024

Pharmaceutical Sciences and Health Technologies II.

Scalibity of spheroid-derived extracellular vesicles production in stirred system

Text of the abstract

Introduction: Extracellular vesicles (EV)-based therapies have gained widespread interest, but efforts remain to ensure standardisation and high-scale production. Implementing processes in stirred-tank bioreactors (STR) is crucial to finely control the cell environment and scaling-up production, but it remains a significant challenge for adherent cells. Here, we investigate the impact of culturing a pancreatic cell line as spheroids in stirred systems on cell fate and EV release. In order to fix the agitation rate for large-scale production, we assessed whether the power input (P/V) or impeller tip speed should be keep constant.
Aims: We aim to establish a standardised process for spheroid-derived EV production and evaluate the influence of culture mode on EV properties.
Methods: 1.4E7 cells were cultured as spheroids in spinner flasks (SpF) or ambr®250 STR. After 24h of culture in serum-free medium, EV were isolated by tangential flow filtration and size exclusion chromatography. EV size and concentration were determined by nanoparticle tracking analysis of tetraspanin-labelled particles. Protein expression was detected by western blot. EV immune properties were assessed through mixed lymphocytes reaction (MLR).
Results: Culturing 1.4E7 cells in suspension reduced their specific growth rate but allowed the formation of homogeneous and viable spheroids. Spheroid formation is dependent of the P/V, and maintaining this criterion constant across scales proved to be the optimal scale-up strategy. However, the process transfer to STR altered spheroid fate due to impeller design and its impact on flow regime. Compared to a monolayer process, EV yield decreased (2-fold) in SpF but increased in STR (2-fold), and EV exhibited distinct immune responses in MLR.
Conclusion: As EV are the mirror of their parental cells, it is crucial to understand and master the culture environment and to fine tune each process parameter during set-up and scale-up to warrant EV final quality and quantity attributes. We present a comprehensive framework addressing key challenges encountered in developing scalable EV-based therapy production from anchorage-dependent cells.
Funding: it is supported by the French National Research Agency (ANR), the Ministry of Agriculture and Food Sovereignty, and the "Pays de la Loire" region.