ISEV2025 Abstract Book

Introduction: Extracellular particle (EP) populations of varying sizes, such as large extracellular vesicles (lEVs), small extracellular vesicles (sEVs), and non-enveloped extracellular particles(NVEPs), have distinct molecular profiles and roles in intercellular communication. Isolating specific EP populations enables a more detailed molecular and functional characterization in research and clinical applications. It has been observed that cells grow in a monolayer in DMEM media and exhibit cluster growth in EMEM, while variations in molecular composition under different media conditions have also been reported. This study aims to sort EP subpopulations from differently cultured breast cancer cells using ultracentrifugation and filtration-based techniques. By comparing the size and yield of populations, it aims to identify the most effective sorting method while assessing how nutritional conditions shape the EP profile. Methods: MCF7 cells were grown in EMEM + insulin or DMEM(4× more nutrients than EMEM) supplemented with 10% FBS.EPs were isolated from 2-day serum-free MCF7 conditioned media using differential ultracentrifugation and 3-step dead-end or tangential flow filtration (TFF). EP fractions were characterized using dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA).Protein content in each fraction was quantified using a BCA assay, while TRIzol purified RNA using absorbance. Results: All three methods successfully fractionated EPs to varying degrees, as confirmed by DLS, NTA, and TRPS. Filtration-based methods achieved higher particle recovery than ultracentrifugation, with TFF providing the purest fraction (based on the protein-to-particle ratio) and the highest lEV enrichment relative to smaller EPs. Across the three isolation methods tested, DMEM-cultured cells released more lEVs, whereas EMEM-cultured cells released more NVEPs. RNA quantification revealed that the NVEP fraction had a higher RNA content than sEVs or lEVs. Summary/Conclusion: The observed differences suggest the potential to modulate EP production through tailored culture media, offering a strategy to selectively enhance specific EP subpopulations for targeted applications. The enrichment of RNA in the < 50 nm EP fraction aligns with prior findings. This study also highlights the efficiency of filtration-based fractionation, particularly TFF, which enables higher EP yields and improved molecular cargo recovery in less time compared to ultracentrifugation.