Dataset Information

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

ABSTRACT: 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, we 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. We 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, our 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.

SUBMITTER: Blans K

PROVIDER: S-EPMC5373680 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

Blans Kristine K Hansen Maria S MS Sørensen Laila V LV Hvam Michael L ML Howard Kenneth A KA Möller Arne A Wiking Lars L Larsen Lotte B LB Rasmussen Jan T JT

Journal of extracellular vesicles 20170315 1

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 whe ...[more]

PMID: 28386391

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Project description:Extracellular vesicles (EVs) are a heterogeneous population of biological particles released by cells. They represent an attractive source of potential biomarkers for early detection of diseases such as cancer. However, it is critical that sufficient amounts of EVs can be isolated and purified in a robust and reproducible manner. Several isolation methods that seem to produce distinct populations of vesicles exist, making data comparability difficult. While some methods induce cellular stress that may affect both the quantity and function of the EVs produced, others involve expensive reagents or equipment unavailable for many laboratories. Thus, there is a need for a standardized, feasible and cost-effective method for isolation of EVs from cell culture supernatants. Here we present the most common obstacles in the production and isolation of small EVs, and we suggest a combination of relatively simple strategies to avoid these. Three distinct cell lines were used (human oral squamous cell carcinoma (PE/CA-PJ49/E10)), pancreatic adenocarcinoma (BxPC3), and a human melanoma brain metastasis (H3). The addition of 1% exosome-depleted FBS to Advanced culture media enabled for reduced presence of contaminating bovine EVs while still ensuring an acceptable cell proliferation and low cellular stress. Cells were gradually adapted to these new media. Furthermore, using the Integra CELLine AD1000 culture flask we increased the number of cells and thereby EVs in 3D-culture. A combination of ultrafiltration with different molecular weight cut-offs and size-exclusion chromatography was further used for the isolation of a heterogeneous population of small EVs with low protein contamination. The EVs were characterized by nanoparticle tracking analysis, immunoaffinity capture, flow cytometry, Western blot and transmission electron microscopy. We successfully isolated a significant amount of small EVs compatible with exosomes from three distinct cell lines in order to demonstrate reproducibility with cell lines of different origin. The EVs were characterized as CD9 positive with a size between 60-140 nm. We conclude that this new combination of methods is a robust and improved strategy for the isolation of EVs, and in particular small EVs compatible with exosomes, from cell culture media without the use of specialized equipment such as an ultracentrifuge.

Project description:Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque. Effects of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with macaque, suggesting opportunities for optimization. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

Dataset Information

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

Publications

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

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