The Impact of Different Isolation Methods on Extracellular Vesicle Yield and Composition in human Breast Milk: A Pilot Study

Extracellular vesicles (EVs) play a key role in intercellular communication and are present in diverse physiological fluids, including breast milk (BM). Evidence has suggested that BM-derived EVs (BMEVs) impact the health and well-being of infants. To gain insights into the biochemical composition and functionality of BMEVs, effective isolation with minimal contamination is essential. Various methods have been utilised for BMEV isolation, each with inherent advantages and limitations. This pilot study compares three isolation methods-ultracentrifugation (UC), size exclusion chromatography (SEC), and multimodal chromatography with a charged core bead Capto Core column (CC)-to determine the most efficient approach for enriching BMEVs from small BM sample volumes.

BMEVs were isolated from samples collected from three donors using UC, SEC, and CC. BMEV yields and cargo were evaluated using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), BCA protein quantification, western blotting, proteomics, and small RNA sequencing.

NTA showed similar particle concentrations and size distributions across all three isolation methods, and TEM validated the presence of EVs. Particle-to-protein ratios differed significantly across isolation methods, with SEC showing a significantly higher ratio than UC. Western blot and proteomics analyses showed greater enrichment of EV-associated proteins in SEC and CC compared to UC, while CC showed the lowest abundance of BMEV contaminants (alpha-S1-casein, lactoferrin and α-lactalbumin). UC captured a distinct miRNA profile including higher enrichment of hsa-miR-16-5p compared to other methods.

Based on our findings, the CC isolation method may serve as a viable approach for isolating an EV-enriched fraction from BM. CC was more effective at reducing co-isolated contaminants compared to UC or SEC, while maintaining enrichment of EV-associated proteins. Additionally, CC-based isolation is cost-effective, user friendly, and highly scalable, thus making it a potential practical and efficient solution for both routine research and large-scale clinical applications.