Unlocking the Secrets of Human Milk: Isolation and Characterization of Extracellular Vesicles

Abstract

ABSTRACT Extracellular vesicles from human milk (HMEVs) are crucial for neonatal development, immune modulation, and protection against pathogens. However, the lack of standardized isolation and characterization protocols poses significant challenges. This review aims to evaluate and compare various methods for the isolation and characterization of HMEVs, highlighting their effectiveness and potential applications. Preliminary purification steps, including the removal of cells, fat globules, and casein micelles, enhance the purity of isolated HMEVs. We categorized isolation methods into density-based, size-based, and affinity-based techniques. Density-based methods include differential and density gradient ultracentrifugation. Size-based methods encompass polymer precipitation, membrane filtration, electrophoretic filtration, size exclusion chromatography, and microfluidics. Affinity-based methods involve immunoisolation using antibodies specific to HMEV surface proteins. Characterization techniques discussed include flow cytometry, dynamic light scattering, nanoparticle tracking analysis, tunable resistive pulse sensing, electron microscopy, atomic force microscopy, confocal microscopy, western blotting, ELISA, and lateral flow immunoassay systems. Differential ultracentrifugation, considered the “gold standard,” provides high purity but is time-consuming. Density gradient ultracentrifugation offers precise separation. Size-based methods like polyethylene glycol precipitation and membrane filtration are simple and fast. Electrophoretic filtration and microfluidics provide precise control of sample flow. Affinitybased methods are highly specific but costly. Advanced characterization techniques provide comprehensive insights into HMEV properties and functions. Standardizing isolation protocols and employing advanced characterization techniques are essential for advancing HMEV research. Future studies should focus on understanding the molecular mechanisms of HMEVs, exploring the impact of maternal health, and developing targeted delivery technologies. These efforts will enhance the therapeutic potential of HMEVs in neonatal care and contribute to personalized nutritional interventions. Keywords: extracellular vesicles, ultracentrifugation, flow cytometry, exosomes, human milk, breast milk