Abstract
Milk is a highly complex, heterogeneous biological fluid that contains non-nutritive, bioactive extracellular vesicles called exosomes. Characterization of milk-derived exosomes (MDEs) is challenging due to the lack of standardized methods that are currently being used for milk pre-processing, storage, and exosome isolation. In this study, we tested: 1) three pre-processing methods to remove cream, fat, cellular debris, and casein proteins from bovine milk to determine whether pre-processing of whole milk prior to long-term storage improves MDE isolations, 2) the suitability of two standard exosome isolation methods for MDE fractionation, and 3) four extraction protocols for obtaining high quality RNA from bovine and human MDEs. MDEs were characterized via Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western immunoblotting for CD9, CD63, and Calnexin protein markers. We also present an optimized method of TEM sample preparation for MDEs. Our results indicate that: 1) Removal of cream and fat globules from unpasteurized bovine milk, prior to long-term storage, improves the MDE yield but not purity, 2) Differential ultracentrifugation (DUC) combined with serial filtration is better suited for bovine MDE isolation compared to ExoQuick (EQ) combined with serial filtration, however both methods were comparable for human milk, and 3) TRIzol LS is better suited for RNA extraction from bovine MDEs isolated by EQ and DUC methods. 4) TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for RNA extractions from human MDEs isolated by EQ, yet the TRIzol LS method is better suited for human MDEs isolated by DUC. The QIAzol + miRNeasy Mini Kit produced the lowest RNA yield for bovine and human MDEs.
Highlights
Maternal milk is the primary nutritional source of newborn mammals
In terms of the exosome purity across G1-G3 samples isolated via Differential Ultracentrifugation (DUC), CD9 and CD63 exosome markers generated single protein bands at 28 kDa and 53 kDa, respectively, and Calnexin failed to cross react at 68 kDa (Fig 5C) in the milk-derived exosomes (MDEs) isolations yet generated a strong band in the human microglia sample
We found that the EQ and DUC methods when combined with serial filtration and processing steps to remove fat, cream, and casein proteins, were efficient in isolating MDEs from 1.5 mL of unpasteurized bovine colostrum (Figs 4 and 5), where the pellets contained more intact MDEs compared to their respective supernatants
Summary
Maternal milk is the primary nutritional source of newborn mammals. Mammalian milk is a highly complex and heterogeneous biofluid that contains protein, lipids, carbohydrates, minerals, vitamins, active enzymes, hormones, immune factors, and microbiota [1,2,3,4]. Mammalian milk is biologically customized to fit the physiological, neurodevelopmental, and immune requirements of offspring as they age [3,4,5,6]. Maternal milk was found to contain functional microRNAs (miRNAs) encapsulated within protective lipid droplets, referred to as milk-derived exosomes (MDEs) [9,10,11,12,13,14,15,16,17]. MDEs are exclusively secreted from mammary gland epithelial cells (MECs), can travel across offspring’s intestinal endothelium post-ingestion into circulation, and are taken up by surrounding tissues [13]
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