Isolation and characterization of extracellular vesicles from human milk for potential use as a dietary supplement in clinical research with preterm infants

Background/objectives Human milk (HM) is the gold standard for neonatal nutrition, providing essential macronutrients and bioactive compounds that promote immune and gastrointestinal development. Among these components, HM-derived extracellular vesicles (HMEVs) are emerging as key mediators of intestinal maturation and protection against necrotizing enterocolitis (NEC). HMEVs carry miRNAs, proteins, and bioactive lipids that resist digestion and modulate critical signaling pathways in the immature gut, making them particularly relevant for preterm infants. Methods This study describes the development and adaptation of a robust, scalable workflow for isolating HMEVs from donor HM for potential use as a nutritional supplement. An initial laboratory-scale isolation protocol was successfully scaled up to a sterile, clinically compatible process. Key modifications included increasing ultracentrifugation speed, eliminating filtration, and replacing phosphate-buffered saline (PBS) with a resuspension medium suitable for nutritional applications. Results The scaled procedure increased the processed milk volume from 25 mL to 63 mL while reducing the HMEVs isolation time from 14 hs to 7.7 h. Tunable Resistive Pulse Sensing (TRPS) and ExoView Ⓡ analysis showed that these modifications led to an approximate 2-fold increase in EV recovery following centrifugation speed optimization. In addition, omission of filtration yielded a 4.3-fold increase in HMEV recovery. The isolated HMEVs exhibited high purity, reaching up to 3.98 × 10 13 particles/g protein. The final product was resuspended in 5% glucose solution, chosen for its physiological compatibility, favorable osmolarity (< 380 mOsmol/kg), and ability to preserve EV stability for up to 30 days at −80 °C. The HMEV preparation was produced under sterile conditions, and endotoxin testing returned negative results. Conclusions This optimized and scalable method enables the safe and efficient isolation of HMEVs for use in neonatal nutritional supplements. These findings establish methodological groundwork for future translational studies of milk-derived EVs aimed at supporting intestinal development and immune protection, and potentially reducing the risk of NEC in preterm infants.