Assessment of isolation strategies to remove caseins for high-quality milk-derived extracellular vesicles

Increasing studies have highlighted the significance of milk-derived extracellular vesicles (MEV) in mother-newborn integration, as well as their application as novel drug delivery systems and diagnostic biomarkers. However, conventional ultracentrifugation (UC) often results in the co-precipitation of casein micelles in MEV pellets. In this study, we compared methods with different principles to screen the optimal pretreatment in caseins removal, and found that isoelectric precipitation by hydrochloric acid (HA) could most effectively remove caseins in porcine milk. We further characterized MEV populations isolated by UC and HA/UC from diverse aspects, including particle methodology via nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), RNA and protein contents, and purity analysis. Importantly, the proliferative and anti-inflammatory effects of MEV were evaluated in vitro, showing the superiority of MEV via HA/UC in functionality compared with UC. Our results suggest that HA pretreatment before ultracentrifugation could effectively remove caseins and other protein complexes, making MEV with higher purity and more significant effects in vitro. This study provides valuable insights for the advancement of MEV isolation techniques across different species and accurate function analysis of MEV.