Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated.
In this study, researchers from Aarhus University present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. The researchers show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, these data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.
Summary of the MEV isolation approaches
Raw human or bovine milk was skimmed by centrifugation to remove the milk fat globules (MFG) in the cream layer, and to sediment somatic milk cells. Two parallel MEV (milk extracellular vesicle) isolation approaches, based on differential centrifugation and size–exclusion chromatography (SEC), were employed on human and bovine skim milk. (1) Ultracentrifugation of the skim milk (39 ml per tube) results in a solid pellet (340K pellet) containing most of the skim milk casein, a supernatant of milk serum (340K supernatant), and a viscous phospholipid rich soluble concentrate positioned adjacent to the casein pellet (see photo in Sup. Figure S1). The phospholipid concentrate obtained by ultracentrifugation of skim milk is traditionally called the “fluff layer”. (2) A lower g-force centrifugation of human and bovine skim milk (27 ml per tube) results in a casein pellet, and a supernatant of milk serum. Naturally, the casein pellet of human skim milk is smaller compared to bovine skim milk, with the latter depicted in the figure. Subsequently, MEV present in the fluff layer or milk serum can be isolated from remaining soluble casein and whey proteins by SEC. MEV isolation and analysis from the fluff layer is highlighted in red and milk serum in green.