Elfar Miran Yousri, Brown Helen Louise, Clayton Aled, Stephens Phil
School of Dentistry, Cardiff University, Health Park Campus, Cardiff, CF14 4XY, UK.
School of Biosciences, Cardiff University, Sir Martin Evans Building, Cardiff, CF10 3AX, UK.
Sci Rep. 2025 Aug 3;15(1):28310. doi: 10.1038/s41598-025-14186-7.
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.
慢性伤口常常滋生如金黄色葡萄球菌等病原体，这引发了人们对开发新的抗菌和伤口愈合策略的兴趣，包括利用细胞外囊泡（EVs）。虽然最近在EV领域一直强调要确保表征和分离技术的标准化，但对于用于收集含囊泡条件培养基（CM）的上游组织培养方法的关注较少。因此，本研究调查了用于EV富集的CM的抗菌特性。CM对青霉素敏感的金黄色葡萄球菌NCTC 6571具有抑菌作用，但对青霉素耐药的金黄色葡萄球菌1061 A没有作用。进一步分析表明，抗菌活性是由于残留抗生素而非细胞分泌因子，具体是青霉素在组织培养塑料表面的残留和释放。预先洗涤细胞并尽量减少基础培养基中的抗生素浓度可降低这种残留效应。这些发现强调了在组织培养中控制抗生素使用的重要性，以避免对CM或EVs的抗菌潜力得出误导性结论。研究人员在方法开发过程中应仔细考虑培养基的选择和补充，因为准确确定任何CM的抗菌机制对于验证未来基于细胞的治疗应用至关重要。
