DMEM, or Opti-MEM, that is the Question: An Important Consideration for Extracellular Vesicle Isolation and their Downstream Applications.

Serum-free synthetic media are frequently used as an alternative to extracellular vesicle-depleted serum containing media (EV-DEP) for EV production. However, the impact of this medium on EV biogenesis, release, and composition remains poorly understood. Here, we comprehensively characterised EV release by MDA-MB-231 or HEK-293T cells cultured in EV-DEP DMEM versus a serum-free synthetic medium, Opti-MEM. In Opti-MEM culture, cells released significantly more CD9- and CD63-positive EVs compared to EV-DEP DMEM. Proteomic analysis revealed that EVs from EV-DEP DMEM contained more histones and bovine proteins. Cells cultured in Opti-MEM released a substantially higher proportion of small-EVs derived from the sphingomyelinase-MVB pathway (60%) compared to EV-DEP DMEM (30%). Conversely, cells cultured in EV-DEP DMEM were more reliant upon the ROCK kinase pathway to release ectosomes (45 %) compared to OptiMEM (15 %). Where cells cultured in EV-DEP DMEM employed RabGTPase-dependent mechanisms for MVB-derived EV release (Rab3d, Rab27a, and Rab27b), cells cultured in Opti-MEM did not. Given that cells cultured in Opti-MEM vs EV-DEP DMEM produce EVs with different protein signatures from distinct molecular pathways, the choice of medium should be carefully considered when designing EV studies - particularly if they are to be used in therapeutic or immunological experiments.