Title: Systematic insights into cell density-dependent transcriptional responses upon medium replacements

Understanding the molecular mechanisms governing transfection efficiency and particle secretion in high cell density cultures is critical to overcome the cell density effect upon transient gene expression. The effect of different conditioned media in HEK293 transcriptome at low and high cell density is explored. A systematic pair-wise comparative study was performed to shed light on the effect on previous phenotypical characteristics of different media conditions: fresh, exhausted and media depleted from extracellular vesicles (EVs) as well as associated proteins and RNAs. The obtained results suggest that restorative effects observed in transfection efficiency when employing EV-depleted media may arise predominantly from physicochemical alterations rather than cellular processes. A significant downregulation of genes associated with nucleocytoplasmic transport for the conditions involving the use of exhausted or EV-depleted media was observed. Moreover, upregulation of histone-related genes in EV-depleted media suggest a role for histone signaling in response to cellular stress or growth limitations, thereby highlighting the potential of manipulating histone levels as a promising strategy to enhance transient transfection. It was also corroborated that the accumulation of extracellular matrix proteins upon cell growth may inhibit transfection by an already-known competitive effect between cell membrane-bound and free proteoglycans. Proteomic characterization of EV-depleted media further unveiled enrichment of pathways associated with infection response and double-strand DNA breaks, suggesting that HEK293 cells undergo an induced infection-like state that disrupts cellular processes. Importantly, the study reveals that EV-depleted media stimulates virion release pathways underscoring the complex interplay between extracellular vesicles and viral budding.