Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies

Benedikter, Birke J.; Bouwman, Freek G.; Vajen, Tanja; Heinzmann, Alexandra C.A.; Grauls, Gert; Mariman, E.C.M.; Wouters, Emiel F.M.; Savelkoul, Paul H. M.; López‐Iglesias, Carmen; Koenen, Rory R.; Rohde, Gernot; Stassen, Frank R. M.

doi:10.1038/s41598-017-15717-7

Cited by 246 publications

(197 citation statements)

References 26 publications

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“…Benedikter et al (2017) found that EV from CSEstimulated pulmonary epithelial cells did not significantly modify IL-8 secretion by naïve cells. The same CSE-EV were less potent inducers of monocyte adhesion to endothelial cells than EV secreted by unexposed cells , questioning a role of CSE-induced EV in immune cell recruitment.…”

Section: Conflicting Datamentioning

confidence: 83%

Extracellular vesicles released in response to respiratory exposures: implications for chronic disease

Benedikter

Wouters

Savelkoul

et al. 2018

Journal of Toxicology and Environmental Health, Part B

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Extracellular vesicles (EV) are secreted signaling entities that enhance various pathological processes when released in response to cellular stresses. Respiratory exposures such as cigarette smoke and air pollution exert cellular stresses and are associated with an increased risk of several chronic diseases. The aim of this review was to examine the evidence that modifications in EV contribute to respiratory exposure-associated diseases. Publications were searched using PubMed and Google Scholar with the search terms (cigarette smoke OR tobacco smoke OR air pollution OR particulate matter) AND (extracellular vesicles OR exosomes OR microvesicles OR microparticles OR ectosomes). All original research articles were included and reviewed. Fifty articles were identified, most of which investigated the effect of respiratory exposures on EV release in vitro (25) and/or on circulating EV in human plasma (24). The majority of studies based their main observations on the relatively insensitive scatter-based flow cytometry of EV (29). EV induced by respiratory exposures were found to modulate inflammation (19), thrombosis (13), endothelial dysfunction (11), tissue remodeling (6), and angiogenesis (3). By influencing these processes, EV may play a key role in the development of cardiovascular diseases and chronic obstructive pulmonary disease and possibly lung cancer and allergic asthma. The current findings warrant additional research with improved methodologies to evaluate the contribution of respiratory exposure-induced EV to disease etiology, as well as their potential as biomarkers of exposure or risk and as novel targets for preventive or therapeutic strategies.

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Section: Conflicting Datamentioning

confidence: 83%

Extracellular vesicles released in response to respiratory exposures: implications for chronic disease

Benedikter

Wouters

Savelkoul

et al. 2018

Journal of Toxicology and Environmental Health, Part B

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“…Therefore, we filtered the supernatant through a 0.22 μm filter to remove larger particles. Recently it was reported that ultrafiltration, rather than ultracentrifugation, results in less damage to vesicles and a higher recovery rate 24 . While most ultrafiltration substrates will bind EVs, regenerated cellulose does not 25 , so we decanted and concentrated the 0.22 μm filtrate using a 10 kD mwco regenerated cellulose filter to the final volume of 1 ml at 4C (MilliporeSigma, Burlington MA, USA; Cat # UFC901008) (FIG 1B).…”

Section: Fractionationmentioning

confidence: 99%

Isolation and characterization of extracellular vesicles from Caenorhabditis elegans for multi-omic analysis

et al. 2018

Preprint

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Cells from bacteria to human release vesicles into their extracellular environment. These extracellular vesicles (EVs) contain multiple classes of molecules, including nucleic acids, proteins, and lipids. The isolation and analysis of EV cargos from mammalian cell culture and liquid biopsy samples has become a powerful approach for uncovering the messages that are packaged into these organelles. However, this approach has not been tenable in invertebrate model systems due to lack of sufficient amounts of pure EVs. Here we report a robust and reproducible procedure to isolate EVs from Caenorhabditis elegans with yields similar to those obtained from human cell culture. Through nanoparticle tracking, transmission electron microscopy, flow cytometry, mass spectrometry, RNAseq, and immunoaffinity analysis we provide the first ever detailed characterization of C.elegans EV composition and demonstrate that C. elegans EVs share fundamentally similar properties with their mammalian counterparts. These include vesicle size, enrichment for lipid rafts, and similar types of RNA and protein cargos. This ability of isolate pure EVs on a scale amenable to multiple types of downstream analyses permits, multi-omics characterization of EV cargos in an invertebrate model system. BACKGROUNDThe cellular secretion of small membrane-bound extracellular vesicles (EVs) into the external environment is an ancient capacity conserved throughout evolution 1,2,3 . EVs range in size from 30-1000 nm in diameter and can be internalized into recipient cells via endocytosis or membrane fusion. There is growing recognition that EVs may play important roles in facilitating intercellular communication through transferring protein, lipid, and genetic cargos 4,5 . The content of EVs are influenced by the physiological state of the cells and are thought to play critical roles in diverse cellular processes as well as multiple types of pathological conditions including cancer, immunity, and neurodegenerative diseases.Many studies have characterized the composition of mammalian EVs. Such EVs are highly enriched in the lipid raft species cholesterol, and sphingomyelin, and in proteins that associate with lipid rafts, including glycosylphosphatidylinositol-anchored (GPI) proteins 6,7,8 . The two main types of EVs studied so far are exosomes, which release from the endosomal network and microvesicles which bud directly from the plasma membrane 9,10 . Mammalian exosomes commonly contain membrane proteins such as CD9, CD63, and CD81, as well as lysosomal and endosomal-marking proteins, and various amounts of extracellular matrix proteins while are largely free of nuclear proteins 11 . Microvesicles have less defined protein markers but may contain proteins of mitochondria and endoplasmic reticulum origin 11 . However, the methods utilized to purify EVs do not separate these types of vesicles, so it is currently unclear how to definitively distinguish the cargos from different types of EVs 12 . EVs from diverse species, including humans, are known to carry RNA ...

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“…EV isolation was performed by a combination of 10 kDa ultrafiltration and sepharose CL-4B SEC as described previously [13]. In brief, 60 ml of conditioned media were concentrated to 500 μl on an Amicon Ultra-15 Centrifugal Filter Unit with Ultracel 10 membrane (10 kDa MWCO, Merck Millipore), followed by separation of EVs and free protein by SEC on a 10 ml sepharose CL4B column into 24 fractions of 0.5 ml.…”

Section: Ev Isolationmentioning

confidence: 99%

“…Although the airway epithelium forms the first line of contact with inhaled cigarette smoke, studies on the functions of EVs released by smoke-exposed airway epithelial cells are scarce. Previously, we have shown that airway epithelial cells secrete small EVs (mode size 110 nm) expressing the tetraspanins CD63, CD81 and CD9, at control conditions and when exposed to CSE [5,13]. In this study, we aimed to predict the functions of these EVs.…”

Section: Introductionmentioning

confidence: 99%

Proteomic analysis reveals procoagulant properties of cigarette smoke‐induced extracellular vesicles

Benedikter

Bouwman

Heinzmann

et al. 2019

J of Extracellular Vesicle

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Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers. ARTICLE HISTORY

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Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies

Cited by 246 publications

References 26 publications

Extracellular vesicles released in response to respiratory exposures: implications for chronic disease

Extracellular vesicles released in response to respiratory exposures: implications for chronic disease

Isolation and characterization of extracellular vesicles from Caenorhabditis elegans for multi-omic analysis

Proteomic analysis reveals procoagulant properties of cigarette smoke‐induced extracellular vesicles

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