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Mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) are considered a promising therapeutic tool in cell therapy due to their immunomodulatory, regenerative and angiogenic capabilities. However, there is a lack of process knowledge, particularly for a large-scale production of MSC-EV using fully controlled stirred tank bioreactor (STR) systems. For the establishment of a STR-based process, we investigated dynamic process set-ups in spinner flasks, using three different microcarriers, as well as in shaking flasks, using microcarrier-free spheroids. An immortalized cell line (hMSC-TERT) and a particle-free chemically defined medium was used for all approaches. Cell characteristics (e.g., growth, metabolism, cell-specific particle production rates), MSC-EV epitope markers and MSC-EV potency in migration assays were analyzed. We showed that the transfer to a dynamic system (non-porous microcarrier, spinner flask) significantly increased the cell-specific particle production rate (6-fold) and the expression of EV-specific markers. Moreover, MSC proliferation and, most importantly, the therapeutic potency of MSC-derived particles including EVs was maintained. We demonstrated that high cell-specific particle production rates were associated with an increased glucose consumption rate rather than cell growth, which can be utilized for future process development. Furthermore, we showed that dynamic conditions of a controlled 1 L STR significantly increased the cell-specific particle production rate (24-fold) as well as the final concentration (3-fold) of potent MSC-derived particles including EVs. This indicates that fully controlled STRs are an efficient production system for MSC-derived particles including EVs that may open and facilitate the path for clinical applications. [ABSTRACT FROM AUTHOR]