Spent Medium Inhibits rVSV Infection.

The cell density effect, defined as reduced cell-specific productivity above a critical cell density, remains a major limitation in virus manufacturing processes. While medium exchange prior to infection has been reported to mitigate this effect, the role of spent medium during the early phase of infection is poorly understood. Here, we show that spent medium conditioned by high-density HEK293 cultures inhibits infection with recombinant vesicular stomatitis virus (rVSV), even when infection is performed at low cell density. The strength of inhibition increased with the density and conditioning time of the donor culture and resulted in slower replication kinetics, thereby delaying the optimal harvest time and potentially reducing overall yield. Notably, the inhibitory effect was reversible when the virus was added to cells maintained in fresh medium, indicating that inhibition is mediated by the medium rather than intrinsic changes in the cells. We excluded pH effects within 7.1-8.0, nutrient depletion, and lactate/ammonium accumulation as primary causes. Removal of cell debris and extracellular vesicles by filtration (down to 0.02 µm) and size-based retention down to 3 kDa did not restore infection, and AUC indicated no major differences in particle distributions between fresh and conditioned media. Together, our data suggest an unidentified <3 kDa inhibitor in spent medium that partially suppresses rVSV infection and slows replication kinetics.