Polymer-based Precipitation Research Articles

Protective effects of exosomes derived from lyophilized porcine liver against acetaminophen damage on HepG2 cells

BackgroundRecently, extracellular vesicles have come to the fore following their emerging role in cell communication, thanks to their ability to reach cells into the human body without dissipating their cargo, transferring biological active molecules, such as proteins, nucleic acids, lipids, etc. They appear as a promising tool in medicine, because of their capability to modulate cellular response in recipient cells. Moreover, a considerable number of publications suggests that exosome uptake is selective but not specific, and it can cross species and cell-type boundaries. This study aims to explore the potential role of porcine liver derived extracellular vesicles, exosomes in particular, to protect human cells from acute damage induced by acetaminophen.MethodsExtracellular vesicles were isolated from porcine lyophilized liver using polymer-based precipitation and a further enrichment was performed using affinity beads. The effects of obtained fractions, total extracellular vesicles and enriched extracellular vesicles, were assessed on human liver derived HepG2 cells. Cell growth and survival were tested, with MTT and area coverage analysis designed by us, as well as protein expression, with immunofluorescence and Western blot. Oxidative stress in live cells was also measured with fluorogenic probes.ResultsAfter proving that porcine extracellular vesicles did not have a toxic effect on HepG2, quite the contrary total extracellular vesicle fraction improved cell growth, we investigated their protective capability with a preconditioning strategy in APAP-induced damage. EVs displayed not only the ability to strongly modulate cell survival responses, but they also were able to boost cell cycle progression.ConclusionsExtracellular vesicles derived from farm animal food derivatives are able to modulate human hepatic cell metabolism, also improving cell survival in a damaged context.

Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed.

The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific strategies are still required to enrich less abundant proteins and get rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins. One of the alternatives that has become more promising is to characterize circulating extracellular vesicles from serum samples that have great interest in biomedicine. In the present work, we enriched the extracellular vesicles fraction from human serum by applying different techniques, including ultracentrifugation, size-exclusion chromatography, and two commercial precipitation methods based on different mechanisms of action. To improve the performance and efficacy of the techniques to promote purity of the preparations, we have employed a small volume of serum samples (<100 mL). The comparative proteomic profiling of the enriched preparations shows that ultracentrifugation procedure yielded a larger and completely different set of proteins than other techniques, including mitochondrial and ribosome related proteins. The results showed that size exclusion chromatography carries over lipoprotein associated proteins, while a polymer-based precipitation kit has more affinity for proteins associated with granules of platelets. The precipitation kit that targets glycosylation molecules enriches differentially protein harboring glycosylation sites, including immunoglobulins and proteins of the membrane attack complex.

Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography.

The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer‐based precipitation and size exclusion chromatography (Pre‐SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early‐ and late‐eluting EV fractions, we performed a quantitative proteomic analysis of MDA‐MB‐468‐derived EVs. We identified 286 exclusive proteins in early‐eluting fractions and 148 proteins with a differential concentration between early‐ and late‐eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies.

Isolation, Characterization, and MicroRNA Analysis of Extracellular Vesicles from Bovine Oviduct and Uterine Fluids.

Intercellular communication can be carried out by circulating systemic and/or locally released extracellular vesicles (EVs), produced by nearly every cell type and tissue, and are involved in physiological and pathological processes. In recent years, EVs have been identified in reproductive tissues, such as oviduct and uterus, and have been shown to be related to several events important for reproductive success. The understanding of their functions in reproduction has important implications for assisted reproductive technologies, for the treatment of infertility in humans and improvement of reproduction efficiency in animals. To study such EVs, it is necessary to isolate and concentrate them from fluid samples, which in the case of reproductive tissues, are usually of limited volume. Several methods for EV isolation are available such as chromatography, ultracentrifugation, polymer-based precipitation, and immunoaffinity.Outcomes can be variable in terms of the amount and quality of isolated EVs, due to the type of isolation method. The choice of method, or a different combination of methods, may depend on the type of sample and scientific question to be addressed in a given study. In this chapter, we describe a method for isolation of EVs from bovine oviductal and uterine fluids for use in functional studies. The method combines size exclusion chromatography and ultracentrifugation. We also describe the different protocols for characterization of isolated EVs (transmission electron microscopy, nanoparticle tracking analysis, and western blot), as well as the isolation of RNA content in EVs, and their miRNAs profiling for functional studies.

Circulating exosomes express α4β7 integrin and compete with CD4+ T cells for the binding to Vedolizumab.

Vedolizumab (VDZ) is a therapeutic monoclonal antibody approved for the treatment of inflammatory bowel diseases (IBD). VDZ selectively binds to the α4β7 integrin and blocks trafficking of a specific subset of gastrointestinal-homing T-lymphocytes to inflamed tissue. Although VDZ has shown promising results in numerous clinical studies a subgroup of patients do not respond adequately. Mechanistic insights and prognostic biomarkers able to predict which patients might benefit from VDZ therapy are currently lacking. Circulating exosomes were isolated from serum of blood donors and VDZ-treated patients by polymer-based precipitation. The surface expression of α4β7 integrin was evaluated by flow cytometry and the levels of exosome-bound VDZ were investigated by Promonitor-VDZ ELISA kit. The capacity of exosomes to interfere with the adhesion of VDZ-treated CD4+ T cells was assessed by adhesion assay. In this study, we showed that serum exosomes isolated from both blood donor and ulcerative colitis patients express on their surface the VDZ target α4β7 integrin. We observed an increased exosomal sequestration of VDZ in anti-TNF exposed patients compared to anti- TNFα naïve patients, according to a greater expression of α4β7 integrin on vesicles surface. Circulating exosomes could compete for VDZ binding with CD4+ T cells since we found that the amount of VDZ bound to T cells was impaired in the presence of exosomes. In addition, we demonstrated that exosomes bind VDZ, which consequently becomes unable to block MadCAM-1-mediated adhesion of lymphocytes. Circulating exosomes might contribute to drug sequestration, possibly affecting the therapeutic efficacy of VDZ in IBD patients. Our data suggest that previous biologic therapy may have altered the sequestration capacity of circulating exosomes, thus reducing the efficacy of VDZ in patients who failed anti-TNF agents.

Deep dive on the proteome of salivary extracellular vesicles: comparison between ultracentrifugation and polymer-based precipitation isolation.

Salivary extracellular vesicles (EVs), as novel functional carriers and potential biomarkers, are usually obtained by ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation methods. However, salivary EVs obtained by these two methods have not been systematically compared. Here, we perform an in-depth analysis on EVs isolated by these two methods using proteomics. Both methods obtain EVs ranging from 40 to 210nm, with the PEG method resulting in a wider size distribution. PEG-separated products were irregularly shaped and aggregated, while UC-separated ones were monodispersed and teacup-shaped. Additionally, the expression of EV-specific markers was higher in UC-separated EVs. Using tandem mass spectrometry proteomics, we identified and quantified 1217 kinds of saliva exosomal proteins and 361 kinds of differential proteins, showing that UC can isolate more EV-related proteins. These results offer some guidance for EV separating and provide potential direction for the use of EVs in non-invasive diagnosis.

Seminal exosomal miR-210-3p as a potential marker of Sertoli cell damage in Varicocele.

Varicocoele-associated stressors, such as hypoxia and heat, can damage cell function and viability, and some exosomal biomarkers released from impaired cells may reflect the cell status in testis. To find if seminal exosomal microRNAs can reflect the Sertoli cell function in varicocoele. Experimental left varicocoele rat model was established (n=24), and patients with different grades of varicocoele (n=104) were enrolled. Primary rat Sertoli cells were isolated with enzymatic hydrolysis. Exosomes were isolated from primary rat Sertoli cells, rat epididymis tissue, and human seminal plasma with polymer-based precipitation method. Exosomal microRNAs were quantified with qPCR. Inhibin-B was detected with enzyme immunoassay. The correlation analysis between microRNA and inhibin-B was evaluated with Spearman's correlation. We screened 12 previously reported hypoxia-responsive microRNAs in the primary rat Sertoli cells and found that 4 exosomal microRNAs increased significantly in response to in vitro hypoxia treatment (P<.05). Of the 4 microRNAs, only miR-210-3p was upregulated in the rats with experimental varicocoele (P<.01). In the patients with varicocoele, we found that seminal exosomal miR-210-3p significantly increased in patients with grade II and III varicocoele (P<.01), and miR-210-3p negatively correlated with sperm count (P<.01) and seminal inhibin-B expression (r=-0.39, P<.01). For the 30 patients with microsurgical varicocelectomy, the operation notably decreased miR-210-3p (P<.01). Seminal exosomal miR-210-3p may be a novel, sensitive, and non-invasive biomarker of Sertoli cell damage in varicocoele.

Cytokine profiling of extracellular vesicles isolated from plasma in myalgic encephalomyelitis/chronic fatigue syndrome: a pilot study

BackgroundMyalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease of unknown etiology lasting for a minimum of 6 months but usually for many years, with features including fatigue, cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Dysregulation of cytokine signaling could give rise to many of these symptoms. Cytokines are present in both plasma and extracellular vesicles, but little investigation of EVs in ME/CFS has been reported. Therefore, we aimed to characterize the content of extracellular vesicles (EVs) isolated from plasma (including circulating cytokine/chemokine profiling) from individuals with ME/CFS and healthy controls.MethodsWe included 35 ME/CFS patients and 35 controls matched for age, sex and BMI. EVs were enriched from plasma by using a polymer-based precipitation method and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM) and immunoblotting. A 45-plex immunoassay was used to determine cytokine levels in both plasma and isolated EVs from a subset of 19 patients and controls. Linear regression, principal component analysis and inter-cytokine correlations were analyzed.ResultsME/CFS individuals had significantly higher levels of EVs that ranged from 30 to 130 nm in size as compared to controls, but the mean size for total extracellular vesicles did not differ between groups. The enrichment of typical EV markers CD63, CD81, TSG101 and HSP70 was confirmed by Western blot analysis and the morphology assessed by TEM showed a homogeneous population of vesicles in both groups. Comparison of cytokine concentrations in plasma and isolated EVs of cases and controls yielded no significant differences. Cytokine-cytokine correlations in plasma revealed a significant higher number of interactions in ME/CFS cases along with 13 inverse correlations that were mainly driven by the Interferon gamma-induced protein 10 (IP-10), whereas in the plasma of controls, no inverse relationships were found across any of the cytokines. Network analysis in EVs from controls showed 2.5 times more significant inter-cytokine interactions than in the ME/CFS group, and both groups presented a unique negative association.ConclusionsElevated levels of 30-130 nm EVs were found in plasma from ME/CFS patients and inter-cytokine correlations revealed unusual regulatory relationships among cytokines in the ME/CFS group that were different from the control group in both plasma and EVs. These disturbances in cytokine networks are further evidence of immune dysregulation in ME/CFS.

Optimized method for extraction of exosomes from human primary muscle cells

Skeletal muscle is increasingly considered an endocrine organ secreting myokines and extracellular vesicles (exosomes and microvesicles), which can affect physiological changes with an impact on different pathological conditions, including regenerative processes, aging, and myopathies. Primary human myoblasts are an essential tool to study the muscle vesicle secretome. Since their differentiation in conditioned media does not induce any signs of cell death or cell stress, artefactual effects from those processes are unlikely. However, adult human primary myoblasts senesce in long-term tissue culture, so a major technical challenge is posed by the need to avoid artefactual effects resulting from pre-senescent changes. Since these cells should be studied within a strictly controlled pre-senescent division count (<21 divisions), and yields of myoblasts per muscle biopsy are low, it is difficult or impossible to amplify sufficiently large cell numbers (some 250 × 106 myoblasts) to obtain sufficient conditioned medium for the standard ultracentrifugation approach to exosome isolation.Thus, an optimized strategy to extract and study secretory muscle vesicles is needed. In this study, conditions are optimized for the in vitro cultivation of human myoblasts, and the quality and yield of exosomes extracted using an ultracentrifugation protocol are compared with a modified polymer-based precipitation strategy combined with extra washing steps. Both vesicle extraction methods successfully enriched exosomes, as vesicles were positive for CD63, CD82, CD81, floated at identical density (1.15-1.27 g.ml−1), and exhibited similar size and cup-shape using electron microscopy and NanoSight tracking. However, the modified polymer-based precipitation was a more efficient strategy to extract exosomes, allowing their extraction in sufficient quantities to explore their content or to isolate a specific subpopulation, while requiring >30 times fewer differentiated myoblasts than what is required for the ultracentrifugation method. In addition, exosomes could still be integrated into recipient cells such as human myotubes or iPSC-derived motor neurons.Modified polymer-based precipitation combined with extra washing steps optimizes exosome yield from a lower number of differentiated myoblasts and less conditioned medium, avoiding senescence and allowing the execution of multiple experiments without exhausting the proliferative capacity of the myoblasts.

Polymeric Precipitation Inhibitor–Based Solid Supersaturable SMEDD Formulation of Canagliflozin: Improved Bioavailability and Anti-diabetic Activity

The current investigation explores the enhanced biopharmaceutical attributes of solid supersaturable self-microemulsifying drug delivery system (Solid SS SMEDDS) for poorly water soluble drug canagliflozin (CFZ). Solid SS SMEDD formulation was prepared by spray drying using Neusilin US2 as an adsorbent. The developed SS SMEDDS were characterized for physicochemical and solid state characterizations. Theoretical molecular simulations were performed by in silico docking. BET measurements were done for evaluating surface area topography. In vitro dissolution, ex vivo permeation, and pharmacokinetics studies were performed to determine release rate, permeability, and absorption behavior, respectively. Anti-diabetic studies and histopathology were done as per standard protocols. The solid SS SMEDD formulation containing 320 mg Lauroglycol FCC, 1200 mg Tween 80, 480 mg Transcutol-P, and 2.24% w/v Poloxamer 188 as polymer-based precipitation inhibitor (PPI) was optimized. The solid state attributes of CFZ in solid SS SMEDDS revealed its molecularly dispersed state. The prepared formulation demonstrated uniform flow properties, nanoparticulate size distribution, and acceptable drug content. Theoretical simulations revealed strong intermolecular hydrogen bonding with a docking score of − 5.184 kJ/mol. In vitro dissolution studies depicted enhanced release of CFZ from solid SS SMEDDS in various dissolution media. Remarkable improvement in ex vivo permeability was observed across the jejunum segment of rat intestine. These studies revealed enhanced absorption of CFZ in rats following oral administration of solid SS SMEDDS, as compared with that of pure drug and marketed product. Pharmacological evaluation of optimized solid SS SMEDDS also revealed enhanced anti-diabetic activity, as evident from urinary glucose excretion studies and SGLT II protein expression in rat kidneys. Accelerated stability study confirmed the stability of the formulation up to 6 months storage period. The designed delivery system indicated promising results in developing an effective formulation of CFZ for diabetes mellitus.

Polymer-Based Precipitation of Extracellular Vesicular miRNAs from Serum Improve Gastric Cancer miRNA Biomarker Performance

Circulating miRNAs are promising liquid biopsy biomarkers for noninvasive cancer detection. However, detection of subtle, but meaningful differences in circulating miRNA quantities between diseased and healthy samples remains a key challenge in clinical settings because biomarker signal/noise ratios are often low. Because extracellular vesicles (EVs) are key sources of circulating miRNAs in serum, it was hypothesized that isolating EVs would enrich miRNA biomarkers, leading to enhanced diagnostic ability and improved biomarker performance. This research assessed the performance of EV-miRNAs against serum miRNAs as biomarkers for gastric cancer (GC). It was first determined that polymer-based precipitation (PBP) gave the highest EV-miRNA recovery when compared with ultracentrifugation, column affinity, peptide affinity, and immunobead affinity EV purification. Four PBP reagents were used to isolate EV-miRNAs from 15 GC and 15 healthy controls and 133 GC-related miRNAs were profiled from EV fractions and total serum using real-time quantitative PCR. A PBP reagent that generated the most EV-miRNA biomarkers was selected and used to validate 11 EV-miRNAs in an independent set of 20 GC and 20 controls. Eight of these EV-miRNA biomarkers were found to give better GC detection accuracy (area under the curve, approximately 0.8). Overall, data suggest that EV miRNAs can improve GC detection performance compared with serum miRNAs and led to the identification of eight EV-miRNAs as potential noninvasive biomarkers for GC.

Cost-effective Strategies for Depletion of Endogenous Extracellular Vesicles from Fetal Bovine Serum

Despite the prominent therapeutic potentials of stem cells, their use in cell therapy has been challenged with some unreproducible and inconsistent outcomes in addition to the risk of rejection and tumorigenesis. Gaining novel insights to the importance of the conditioned medium, secretory factors and extracellular vesicles as the functional components of the cultured stem cells, suggested the idea of substituting the cells with their cell-free counterparts. Biological properties of these products are influenced by the cues received from their microenvironment. Hence, providing optimal and fully defined culture conditions is essential for their preparation. Fetal bovine serum (FBS), one of the most routine supplements of cell culture, is enriched by endogenous extracellular vesicles (EVs). These EVs will affect the yield, purity and functional features of the cell-free products. Here, we endeavored to examine and compare three different methods including ultrasonication, ultrafiltration and polymer-based precipitation, to deplete EVs from FBS. We chose easy to perform and fast methods with the capacity for high-throughput applications. Based on our observations, although all examined methods were able to deplete EVs from FBS to some extent, polymer-based precipitation could be considered as the method of choice with minimal consequences on the biological requirements of FBS to support cell growth and characteristics. Due to similarities between FBS and some other biological solutions, this strategy would be suitable for EV-depletion from other liquids with high concentrations of proteins and nutrients. Moreover, it could be applied for preparation of optimal culture conditions for nanoparticle applications.

Isolation of Small Extracellular Vesicles From Human Serum Using a Combination of Ultracentrifugation With Polymer-Based Precipitation.

Methods for reproducibly isolating and enriching small extracellular vesicles (EVs) from blood are essential for clinical utilization of small EVs in cancer patients. We combined ultracentrifugation (UC) with polymer-based precipitation (ExoQuick [EQ] or Total Exosome Isolation [TEI] kit) to isolate small EVs (diameter, 30–150 nm) from the serum of breast cancer patients. We compared the performance of four cycles of UC (UC4x) with that of two cycles of UC followed by enrichment using the EQ (UC2x→EQ) or TEI (UC2x→TEI) kits. The mean concentration of small EVs isolated from 1 mL of serum using UC2x→EQ (139.0±29.1 µg) and UC2x→TEI (140.4±5.0 µg) did not differ from that obtained using UC4x (141.8±26.9 µg). The mean number of EV particles obtained using UC4x was 29.2±9.9×109 per mL of serum, whereas UC2x→EQ and UC2x→TEI yielded higher numbers of EVs (50.7±17.0×109 and 59.3±20.6×109, respectively). Concentrations of EV microRNAs, including miR-21 and miR-155, did not differ between the three methods. In conclusion, performing UC prior to the use of polymer-based precipitation kits could be feasible for isolating small EVs from human serum in large sample-based translational researches.

Proteomics comparison of exosomes from serum and plasma between ultracentrifugation and polymer-based precipitation kit methods.

Exosomes are vesicles with sizes ranging from 30 to 150nm. The analysis and detection of blood exosomes offers an effective route for cancer diagnosis, prognosis assessment, and therapeutic evaluation of diseases. Due to the difference in separation procedure, collection method and the usage of anticoagulants, serum and plasma samples show diversity test results. In order to evaluate the isolation effect of exosomes in serum and plasma samples, two commonly used exosomal isolation methods, ultracentrifugation and polymer-based precipitation kit, were used, respectively. And the isolation effects were evaluated by comparing the composition and abundant of proteins from isolated exosomes based on MS-based proteomics analysis. The results showed that the plasma exosomes extracted by ultracentrifugation identified more exosome biomarkers, and the concentrations of these biomarkers were higher than others. And plasma exosomes could be a better sample for blood-based proteomics research of exosomes. It would be more useful for future targeted biomarker discovery.

Serum overexpression of miR-301a and miR-23a in patients with colorectal cancer.

Extracellular vesicles (EVs) are a heterogeneous group of membrane-bound vesicles with complex cargoes including proteins, lipids, and nucleic acids. EVs have received significant attention due to their specific features including stability under harsh conditions and involvement in cell-to-cell communication. Circulating EVs and the molecules associated with them are important in the diagnosis and prognosis of cancers. MicroRNAs (miRNAs) are a group of small noncoding RNAs that have a role in regulating gene expression. Current literature shows that circulating miRNAs can be used as noninvasive biomarkers for early detection of cancers. The present study was set to investigate the potential role of serum exosomal miRNA expression levels in colorectal cancer (CRC) patients and evaluate their correlation with clinicopathologic features. Exosome-enriched fractions were isolated from the serum of 25 CRC patients and 13 age- and sex-matched healthy controls using a polymer-based precipitation method. During the pilot phase, real-time polymerase chain reaction (RT-PCR) was carried out on 12 CRC patients and eight healthy participants to evaluate the expression difference of 11 candidate miRNAs between CRC patients and tumor free subjects. Finally, the results were validated in a separate group, which was similar in size to the pilot group. The clinicopathologic data were also collected and the relationship between aberrant miRNA expression and clinicopathological parameters were investigated. There were high expressions of exosomal miR-23a and miR-301a in serum samples of CRC patients compared to normal controls in training and validation phases; these differences were not significantly correlated with clinicopathologic features. Receiver operating characteristic curve analysis showed that miR-301a and miR-23a were able to discriminate CRC patients from normal subjects. The findings provide evidence on the roles of miR-301a and miR-23a in CRC development and their potential roles as noninvasive biomarkers for early detection of CRC.

Remote ischaemic preconditioning increases serum extracellular vesicle concentrations with altered micro-RNA signature in CABG patients.

Remote ischaemic preconditioning (RIPC) can attenuate myocardial ischaemia/reperfusion injury but its underlying mechanisms remain largely unknown. Recently, extracellular vesicles (EVs) containing microRNAs (miRNAs) were shown to mediate distant intercellular communication that may be involved in cardioprotection. We tested the hypothesis that RIPC in anaesthetized patients undergoing coronary artery bypass (CABG) surgery results in the release of EVs from the ischaemic/reperfused arm into the blood stream harbouring cardioprotective miRNAs. In 58 patients randomised to RIPC (three 5/5minutes episodes of left arm ischaemia/reperfusion by suprasystolic blood pressure cuff inflations/deflations) or Sham, a subprotocol comprising of parallel right radial artery and regional (left subclavian) venous blood sampling before (awake) and 5 and 60minutes after RIPC/Sham during isoflurane/sufentanil anaesthesia could be completed. EVs were extracted by polymer-based precipitation methods, their concentrations measured, and their miRNA signature analysed. Five minutes after RIPC, regional venous EV concentrations downstream from the cuff increased and arterial concentrations increased after 60minutes (fold change [fc]: RIPC: 1.33±0.5, Sham: 0.91±0.31; P=0.003 for interaction). Already 5minutes after RIPC, expression of 26 miRNAs (threshold fc: 3.0, P<0.05) isolated from EVs including the cardioprotective miR-21 had increased. RIPC also decreased postoperative Troponin I concentrations (AUC RIPC: 336ng/mL×72hours±306 vs Sham: 713±1013; P = 0.041). Remote ischaemic preconditioning increases serum EV concentrations, most likely by early EV release from the patients' left (RIPC) arm, alters their miRNA signature, and is associated with myocardial protection. Thus, an increased EV concentration with an altered miR-signature may mediate the RIPC effect.

Multiparametric Analysis of Circulating Exosomes and Other Small Extracellular Vesicles by Advanced Imaging Flow Cytometry.

Extracellular vesicles (EVs) are responsible for a multitude of physiological functions, including immunomodulation. A heterogenous mixture of small EV (sEV) subsets, including putative exosomes, is derived when commonly used “exosome” isolation techniques are employed. Subset diversity relates in part to their different intracellular origins, and can be associated with distinct functional properties. Recent progress in the EV field has enabled the categorization of such subsets based on their surface composition. For the first time, we combine such emerging subset-specific markers with advanced imaging flow cytometry (iFCM) to perform high-throughput, multiparametric, vesicle-by-vesicle characterization, and functional assessment of specific small EV subsets, and exosomes in particular. The approach allows researchers to address three important applications. First, it is known that different isolation techniques result in the divergent recovery of particular vesicle subsets. Taking three commonly used “exosome” isolation techniques as test cases (ultracentrifugation, size-exclusion chromatography, and polymer-based precipitation), the capacity for convenient and accurate isolate compositional analysis by iFCM is demonstrated. The approach was able to corroborate and to quantify the known skewing of subtype recovery among different isolation approaches. Second, exosomes are a particularly widely studied EV subset. Applying exosome-specific markers to samples collected from an optimal clinical transplantation model, we verify the capacity for iFCM to detect exosomes in circulation, to establish their tissue of origin, and to provide insights as to their functional immunological potential. Finally, we describe a technique for establishing whether the transfer of a molecule of interest to a target cell is exosomally mediated. In so doing, we highlight the approach’s utility in assessing the functional impact of circulating exosomes and in identifying their targets. In conclusion, we set out a new methodological approach by which small extracellular vesicle subsets, exosomes in particular, can be conveniently and comprehensively investigated, thereby offering novel phenotypic and functional insights.

Extracellular vesicles from oral squamous carcinoma cells display pro- and anti-angiogenic properties.

A new intercellular communication mode established by neoplastic cells and tumor microenvironment components is based on extracellular vesicles (EVs). However, the biological effects of the EVs released by tumor cells on angiogenesis are not completely understood. Here, we aimed to understand the biological effects of EVs isolated from two cell lines of oral squamous cell carcinoma (OSCC) (SCC15 and HSC3) on endothelial cell tubulogenesis. OSCC-derived EVs were isolated with a polymer-based precipitation method, quantified using nanoparticle tracking analysis and verified for EV markers by dot blot. Functional assays were performed to assess the angiogenic potential of the OSCC-derived EVs. The results showed that EVs derived from both cell lines displayed typical spherical-shaped morphology and expressed the EV markers CD63 and Annexin II. Although the average particle concentration and size were quite similar, SCC15-derived EVs promoted a pronounced tubular formation associated with significant migration and apoptosis rates of the endothelial cells, whereas EVs derived from HSC3 cells inhibited significantly endothelial cell tubulogenesis and proliferation. The findings of this study reveal that EVs derived from different OSCC cell lines by a polymer-based precipitation method promote pro- or anti-angiogenic effects.

Polymer-based precipitation preserves biological activities of extracellular vesicles from an endometrial cell line.

Extracellular vesicles (EVs) are membrane-bound vesicles released by cells and act as media for transfer of proteins, small RNAs and mRNAs to distant sites. They can be isolated by different methods. However, the biological activities of the purified EVs have seldom been studied. In this study, we compared the use of ultracentrifugation (UC), ultra-filtration (UF), polymer-based precipitation (PBP), and PBP with size-based purification (PBP+SP) for isolation of EVs from human endometrial cells and mouse uterine luminal fluid (ULF). Electron microscopy revealed that the diameters of the isolated EVs were similar among the tested methods. UF recovered the highest number of EVs followed by PBP, while UC and PBP+SP were significantly less efficient (P<0.05). Based on the number of EVs-to-protein ratios, PBP had the least protein contamination, significantly better than the other methods (P<0.05). All the isolated EVs expressed exosome-enriched proteins CD63, TSG101 and HSP70. Incubation of the trophoblast JEG-3 cells with an equal amount of the fluorescence-labelled EVs isolated by the studied methods showed that many of the PBP-EVs treated cells were fluorescence positive but only a few cells were labelled in the UC- and UF-EVs treated groups. Moreover, the PBP-EVs could transfer significantly more miRNA to the recipient cells than the other 3 methods (P<0.05). The PBP method could isolate EVs from mouse ULF; the diameter of the isolated EVs was 62±19 nm and expressed CD63, TSG101 and HSP70 proteins. In conclusion, PBP could best preserve the activities of the isolated EVs among the 4 methods studied and was able to isolate EVs from a small volume of sample. The simple setup and low equipment demands makes PBP the most suitable method for rapid EV assessment and isolation of EVs in clinical and basic research settings.

Assessment of Extracellular Vesicles Purity Using Proteomic Standards.

The increasing interest in extracellular vesicles (EVs) research is fueled by reports indicating their unique role in intercellular communication and potential connection to the development of common human diseases. The unique role assumes unique protein and nucleic acid cargo. Unfortunately, accurate analysis of EVs cargo faces a challenge of EVs isolation. Generally used isolation techniques do not separate different subtypes of EVs and even more, poorly separate EVs from non-EVs contaminants. Further development of EVs isolation protocols urgently needs a quantitative method of EVs purity assessment. We report here that multiple reaction monitoring assay using internal standards carrying peptides for quantification of EVs and non-EVs proteins is a suitable approach to assess purity of EVs preparations. As a first step in potential standardization of EVs isolation, we have evaluated polymer-based precipitation techniques and compared them to traditional ultracentrifugation protocol.