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 and collection. Here, we fully characterised the EVs released by MDA-MB-231 or HEK-293T cells cultured in 10% EV-DEP DMEM or a commonly used serum-free synthetic medium, Opti-MEM. Cells cultured in Opti-MEM released a significantly higher yield of CD9- and CD63-positive EVs as compared to cells grown in EV-DEP DMEM. In MDA-MB-231 cells the increased EV release was likely driven by nutrient deprivation (decreased phospho-S6) and increased cell stress (increased phospho-AKT). MDA-MB-231 cells grown in EV-DEP DMEM relied upon actin cytoskeleton dynamics for up to 50% of EVs released, while cells in Opti-MEM did not use ROCK kinases for EV biogenesis. Mass spectrometry analysis showed that the EV proteomes from both conditions were largely unchanged other than EV-DEP DMEM EVs contained more histones and bovine proteins. Addition of 2.5 - 10% serum or 2.5 mg/mL albumin to Opti-MEM medium partially rescued some cell stress phenotypes and returned EV release rates to that of cells in EV-DEP DMEM. The choice of media is an important consideration when designing EV studies and their downstream applications. While Opti-MEM increased cell stress, it achieved a high yield of EVs that were depleted of histones and contaminating bovine proteins - useful for therapeutic applications. Alternatively, EV-DEP DMEM produced a decreased yield of EVs in lower stress conditions, however, the EVs were histone and bovine protein rich which may have important implications for use for use in therapeutic or immunological experiments.