Disentangling the Impact of Growth Conditions and Isolation Methods on Extracellular Particle Yield and Molecular Profiles

Introduction Extracellular particle (EP) populations—including large extracellular vesicles (lEVs), small extracellular vesicles (sEVs), and non-enveloped extracellular particles (NVEPs)—exhibit distinct molecular profiles and play key roles in intercellular communication. The ability to isolate specific EP subtypes is critical for detailed molecular and functional analyses in both research and clinical contexts. Nutrient composition in the culture environment have also been reported to influence transcriptome of breast cancer cells1 . These factors point to the need for better insight into how media and methods impact EP research output. Aim Breast cancer cells display different growth patterns depending on culture conditions—forming monolayers in DMEM and clusters in EMEM. This study aims to separate EP subpopulations from cells cultured under these conditions using ultracentrifugation (UC) and tangential flow filtration (TFF). By comparing particle size distributions, yields, and molecular profiles, the goal is to identify the most effective isolation strategy and evaluate how culture media influence EP characteristics. Methods MCF7 were cultured in DMEM and EMEM. EPs were isolated using UC and TFF, then analyzed by nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), protein quantification, and RNA analysis. Yield, size distribution, and molecular cargo were compared across methods and media conditions. Results TFF yielded more EPs compared to UC, especially enriching small EPs (<50 nm) with higher RNA content. Both methods showed EPs <100nm enriched in RNA, consistent with prior findings2,3. MCF7 release more sEVs in EMEM, but more lEVs in DMEM. Conclusions This study highlights the advantages of TFF over UC for EP separation, offering a more efficient and scalable approach. Additionally, culture media selection significantly impacts EP characteristics, emphasizing the need for standardized conditions in EP research. These findings contribute to optimizing separation workflows, ensuring reproducibility and efficiency in extracellular particle studies.