At present, no effective therapies are available for traumatic spinal cord injury (SCI). Following the initial trauma, persistent inflammation exacerbates neuronal and functional losses. Our recent findings highlight the potential of acute treatment with MSC-derived extracellular vesicles (MSC-EVs) in mitigating inflammation and its consequential damages after SCI. However, with numerous protocols available for EV preparation from MSC-derived secretomes, we sought to identify the most efficacious method for generating EV preparations capable of reducing the pro-inflammatory polarization of microglia.We assessed the potential and biological activity of EV fractions produced with several protocols involving various pore sizes for tangential flow filtration (TFF), in optional combination with size exclusion chromatography (SEC), or ultracentrifugation, and supplementation of the culture medium with human platelet lysate. EV fractions were applied in vitro for 24 hours on LPS-treated primary microglia or BV-2 microglia. Thereafter cells and medium were harvested for analysis.We observed that the production of nitric oxide was comparably reduced by all EV fractions, regardless of the preparation protocol used. Nevertheless, EVs fractions enriched via small pore size (100 kDa) TFF led to higher anti-inflammatory activities.We are currently conducting a transcriptomic analysis comparing samples obtained from naive microglia with those from LPS-activated microglia, which were treated with either the most efficacious or the least efficacious EV fractions, or left untreated.Elucidating the specific signaling pathways activated by potent anti-inflammatory EV fractions, will provide valuable insights to further refine EV preparation protocols and identify novel molecular targets for combating inflammatory conditions.