Growth media conditions influence the secretion route and release levels of engineered extracellular vesicles

Extracellular vesicles (EVs) are nanosized cell-derived vesicles found in all bodily fluids which provide a route of intercellular communication by transmitting biological cargo. While EVs offer promise as therapeutic agents, the molecular mechanisms of EV biogenesis are not yet fully elucidated, in part due to the concurrence of numerous interwoven pathways which give rise to heterogenous EV populations in vitro. The effects of conditions in the cellular environment on EV production are of particular interest. In this study, we utilize a quantifiable EV-engineering approach to investigate how various cell media conditions alter EV production. The presence of serum, exogenous EVs, and other signaling factors in cell media alters EV production on physical, molecular, and transcriptional levels. Further, we demonstrate that exosome biogenesis pathways are the major factors contributing to EV production under optimized conditions. Our findings suggest a novel understanding to the mechanisms underlying EV production in cell culture which can be applied to develop advanced EV production methods. Overall design: Sequencing of HEK293 cells in different media conditions