Workflow for the Isolation and Characterisation of Human Milk Extracellular Vesicles (HMEVs) and Their Inflammatory Biomarker Profile.

Human milk extracellular vesicles (HMEVs), secreted by mammary epithelial cells, are enriched in bioactive molecules that support intestinal epithelial integrity. Among these, oxylipins, that is, lipid mediators derived from polyunsaturated fatty acids, are gaining interest for their immunomodulatory and neuroprotective functions in breastfed infants. However, current workflows for oxylipin profiling in HMEVs often lack sensitivity or breadth, limiting mechanistic insights. This study presents an optimised workflow for comprehensive oxylipin profiling in HMEVs. HMEVs were isolated via size-exclusion chromatography and ultracentrifugation, followed by characterisation using attenuated total reflectance-Fourier transform infrared spectroscopy, Western blotting, Exoview immunocapture, tunable resistive pulse sensing and transmission electron microscopy. The influence of different pre-analytical protocols on HMEV recovery was assessed. Cryolysis with liquid nitrogen was employed for vesicle lysis before targeted oxylipin quantification using ultra-performance liquid chromatography-tandem mass spectrometry. The analysis of 10 human milk samples revealed 9,10-DiHOME, 12,13-DiHOME and 11,12-EET as the most abundant oxylipins, with concentrations ranging from 0.5 to 3.7, 0.8 to 4.5 and 0.1 to 0.3 nM, respectively. This refined pipeline enables in-depth oxylipin profiling in HMEVs and serves as a robust platform for future in vitro and in vivo investigations into EV-mediated lipid signalling.