Isolation and Characterization of Milk Extracellular Vesicles

Extracellular vesicles from milk were isolated by a combination of ultracentrifugation and size exclusion chromatography [24 (link)–26 (link)]. Briefly, 50 μl of fat separated milk was centrifuged at 2,000×g for 10 min at 4°C and then at 10,000×g for 30 min at 4°C. The supernatant was then ultracentrifuged at 100,000×g for 3 hours (TL-100 benchtop ultracentrifuge, Beckman-Coulter). The obtained crude EV pellet was washed in PBS once at 100,000×g for 3 hours. The washed pellet was resuspended in 1 ml of PBS and EVs were purified by mini-size exclusion chromatography (mini-SEC) using 1.5 cm x 12 cm mini-columns (Bio-Rad, Hercules, CA, USA; Econo-Pac columns) packed with 10 mL of Sepharose 2B (Millipore-Sigma, St. Louis, MO). Crude EVs (1.0 ml) obtained from ultracentrifugation were loaded onto the column and five 1 ml fractions corresponding to the void volume peak were collected in PBS. Fraction four was collected and used for subsequent experiments as the ‘EV’ fraction. Given that SEC is a size-dependent assay, we anticipate that the EVs obtained using this approach contain a heterogeneous mixture of exomeres, exosomes, and microvesicles in the size range of 30–200 nm [27 (link)]. The EVs were characterized by nanoparticle tracking analysis (NTA) and Western blotting as per MISEV2018 guidelines [28 (link)]. Antibodies used for Western blot experiments are described in Table 1.