Antibiotic carry over is a confounding factor for cell-based antimicrobial research applications.

Chronic wounds often host pathogens like Staphylococcus aureus, prompting interest in developing new antimicrobial and wound healing strategies, including the utilisation of extracellular vesicles (EVs). Whilst there has been a recent emphasis within the EV community to ensure standardization of characterization and isolation techniques, there has been less focus placed on the upstream tissue culture methodologies used for collection of vesicle-containing conditioned medium (CM). Hence, this study investigated the antimicrobial properties of the CM used for EV enrichment. CM exhibited bacteriostatic effects against penicillin-sensitive S. aureus NCTC 6571, but not penicillin-resistant S. aureus 1061 A. Further analysis revealed that the antimicrobial activity was due to residual antibiotics rather than cell-secreted factors, specifically the retention and release of penicillin to tissue culture plastic surfaces. Pre-washing cells and minimizing antibiotic concentrations in basal medium reduced this carry-over effect. These findings emphasize the importance of controlling antibiotic use in tissue culture to avoid misleading conclusions about the antimicrobial potential of CM or EVs. Researchers should carefully consider medium selection and supplementation during method development as accurately determining the antimicrobial mechanisms of any CM is essential for validating future cell-based therapeutic applications.