scholarly journals Comparison of Extracellular Vesicle Isolation Methods from Human Biofluids using Single Molecule Arrays

2020 ◽  
Author(s):  
Dmitry Ter-Ovanesyan ◽  
Maia Norman ◽  
Wendy Trieu ◽  
Ju-Hyun Lee ◽  
Roey Lazarovits ◽  
...  
Keyword(s):  
Single Molecule ◽  
Relative Efficiency ◽  
Transmembrane Proteins ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Great Promise ◽  
Disease Biomarkers ◽  
Isolation Methods ◽  
Exclusion Chromatography ◽  
Protein Contamination

AbstractExtracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs and using them for diagnostics is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive assays to quantify EVs by immuno-isolating and detecting EV transmembrane proteins in microwell arrays. We developed single molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays to rapidly optimize EV isolation using SEC from plasma and CSF. Our results highlight the utility of quantifying EVs using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.

scholarly journals Framework for rapid comparison of extracellular vesicle isolation methods

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Dmitry Ter-Ovanesyan ◽  
Maia Norman ◽  
Roey Lazarovits ◽  
Wendy Trieu ◽  
Ju-Hyun Lee ◽  
...  
Keyword(s):  
Single Molecule ◽  
Relative Efficiency ◽  
Transmembrane Proteins ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Great Promise ◽  
Disease Biomarkers ◽  
Isolation Methods ◽  
Exclusion Chromatography ◽  
Protein Contamination

Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive, single molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays, all on one platform, to improve SEC isolation from plasma and CSF. Our results highlight the utility of quantifying EV proteins using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.

Extracellular vesicle isolation methods: rising impact of size-exclusion chromatography

2019 ◽  
Vol 76 (12) ◽  
pp. 2369-2382 ◽  
Author(s):  
Marta Monguió-Tortajada ◽  
Carolina Gálvez-Montón ◽  
Antoni Bayes-Genis ◽  
Santiago Roura ◽  
Francesc E. Borràs
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Isolation Methods ◽  
Exclusion Chromatography

scholarly journals Serum Extracellular Vesicle-Derived circHIPK3 and circSMARCA5 Are Two Novel Diagnostic Biomarkers for Glioblastoma Multiforme

2021 ◽  
Vol 14 (7) ◽  
pp. 618
Author(s):  
Michele Stella ◽  
Luca Falzone ◽  
Angela Caponnetto ◽  
Giuseppe Gattuso ◽  
Cristina Barbagallo ◽  
...  
Keyword(s):  
Glioblastoma Multiforme ◽  
Roc Analysis ◽  
Characteristic Curve ◽  
Area Under The Curve ◽  
Digital Pcr ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Circular Rnas ◽  
Diagnostic Biomarkers ◽  
Exclusion Chromatography

Glioblastoma multiforme (GBM) is the most frequent and deadly human brain cancer. Early diagnosis through non-invasive biomarkers may render GBM more easily treatable, improving the prognosis of this currently incurable disease. We suggest the use of serum extracellular vesicle (sEV)-derived circular RNAs (circRNAs) as highly stable minimally invasive diagnostic biomarkers for GBM diagnosis. EVs were isolated by size exclusion chromatography from sera of 23 GBM and 5 grade 3 glioma (GIII) patients, and 10 unaffected controls (UC). The expression of two candidate circRNAs (circSMARCA5 and circHIPK3) was assayed by droplet digital PCR. CircSMARCA5 and circHIPK3 were significantly less abundant in sEVs from GBM patients with respect to UC (fold-change (FC) of −2.15 and −1.92, respectively) and GIII (FC of −1.75 and −1.4, respectively). Receiver operating characteristic curve (ROC) analysis, based on the expression of sEV-derived circSMARCA5 and circHIPK3, allowed us to distinguish GBM from UC (area under the curve (AUC) 0.823 (0.667–0.979) and 0.855 (0.704 to 1.000), with a 95% confidence interval (CI), respectively). Multivariable ROC analysis, performed by combining the expression of sEV-derived circSMARCA5 and circHIPK3 with preoperative neutrophil to lymphocyte (NLR), platelet to lymphocyte (PLR) and lymphocyte to monocyte (LMR) ratios, three known diagnostic and prognostic GBM markers, allowed an improvement in the GBM diagnostic accuracy (AUC 0.901 (0.7912 to 1.000), 95% CI). Our data suggest sEV-derived circSMARCA5 and circHIPK3 as good diagnostic biomarkers for GBM, especially when associated with preoperative NLR, PLR and LMR.

scholarly journals Electromagnetic Piezoelectric Acoustic Sensor Detection of Extracellular Vesicles through Interaction with Detached Vesicle Proteins

Biosensors ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 173
Author(s):  
Loránd Románszki ◽  
Zoltán Varga ◽  
Judith Mihály ◽  
Zsófia Keresztes ◽  
Michael Thompson
Keyword(s):  
Storage Period ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Frequency Change ◽  
Acoustic Sensor ◽  
C Storage ◽  
Thaw Cycle ◽  
Signal Change ◽  
Freeze Thaw Cycle ◽  
Exclusion Chromatography

An electromagnetic piezoelectric acoustic sensor (EMPAS) was used to study the non-specific adsorption of human red blood cell-derived extracellular vesicle preparations. Vesicle storage history (temperature and duration) highly affected the obtained results: The signal change, namely the frequency decrease of the crystal measured at 20 °C, was negligibly small (<1 s−2) when the vesicle solutions had previously been stored at 4 °C, and was in the order of 10 s−2 when the vesicle solutions had been stored at −30 °C. Moreover, the rate of frequency decrease increased exponentially with the storage time at −30 °C. Upon a 4 °C storage period following the −30 °C storage period of the same sample, the measured frequency decrease dropped, suggesting a partial relaxation of the system. The results are explained by the disintegration of the vesicles triggered by the freeze–thaw cycle, likely due to the detachment of proteins from the vesicle surface as was proved by size-exclusion chromatography. Surface modification of the sensor crystal provided the possibility of signal enhancement, as the maximum rate of the frequency change for the same vesicle concentrations was higher on hydrophobic, octadecyl trichlorosilane–modified quartz than on hydrophilic, bare quartz. The EMPAS signal has been associated with the amount of detached proteins, which in turn is proportional to the originating vesicle concentration.

scholarly journals Enrichment of plasma extracellular vesicles for reliable quantification of their size and concentration for biomarker discovery

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Marija Holcar ◽  
Jana Ferdin ◽  
Simona Sitar ◽  
Magda Tušek-Žnidarič ◽  
Vita Dolžan ◽  
...  
Keyword(s):  
Biomarker Discovery ◽  
Plasma Source ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enrichment Method ◽  
Transmission Electron ◽  
Relative Standard ◽  
Exclusion Chromatography ◽  
Reliable Quantification

AbstractHuman plasma is a complex fluid, increasingly used for extracellular vesicle (EV) biomarker studies. Our aim was to find a simple EV-enrichment method for reliable quantification of EVs in plasma to be used as biomarker of disease. Plasma of ten healthy subjects was processed using sedimentation rate- (sucrose cushion ultracentrifugation—sUC) and size- (size exclusion chromatography—SEC) based methods. According to nanoparticle tracking analysis (NTA), asymmetrical flow field-flow fractionation coupled to detectors (AF4-UV-MALS), miRNA quantification, transmission electron microscopy and enzyme-linked immunosorbent assay, enrichment of EVs from plasma with sUC method lead to high purity of EVs in the samples. High nanoparticle concentrations after SEC resulted from substantial contamination with lipoproteins and other aggregates of EV-like sizes that importantly affect downstream EV quantification. Additionally, sUC EV-enrichment method linked to quantification with NTA or AF4-UV-MALS is repeatable, as the relative standard deviation of EV size measured in independently processed samples from the same plasma source was 5.4% and 2.1% when analyzed by NTA or AF4-UV-MALS, respectively. In conclusion, the sUC EV-enrichment method is compatible with reliable measurement of concentration and size of EVs from plasma and should in the future be tested on larger cohorts in relation to different diseases. This is one of the first studies using AF4-UV-MALS to quantify EVs in blood plasma, which opens new possible clinical utility for the technique.

scholarly journals Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques

2021 ◽  
Author(s):  
Marvin Droste ◽  
Tobias Tertel ◽  
Stefanie Jeruschke ◽  
Robin Dittrich ◽  
Evangelia Kontopoulou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Method Comparison ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Western Blots ◽  
Preparation Methods ◽  
Imaging Flow Cytometry ◽  
Particle Yield ◽  
Exclusion Chromatography ◽  
Ultra Filtration

Extracellular vesicles (EVs) from several body fluids, including urine, appear as promising biomarkers. Within the last decade, numerous groups have compared the efficacy of EV preparation protocols. Frequently, the efficacy of EV preparation methods is judged by the recovery of particles as estimated by conventional nanoparticle tracking analysis (NTA) or other particle quantification devices. Here, at the example of different urinary EV (uEV) preparation methods, we determined the particle yield in obtained samples with conventional NTA, analyzed their EV content by imaging flow cytometry (IFCM) and quantified the intensity of TSG101 and the contaminant protein uromodulin (UMOD) in Western blots. Our results demonstrate a correlation among CD9-positive objects detected by IFCM and TSG101 Western blot intensities, while particle numbers as determined by NTA correlated with the amount of UMOD. Consequently, our results question the reliability of conventional NTA analyses for identifying the optimal EV preparation method. Here, in our method comparison, a combination of size exclusion chromatography followed by ultra-filtration showed the highest CD9-positive object and TSG101 protein recovery, and in relation to the number of CD9-positive objects, the lowest amount of UMOD contamination.

scholarly journals Single molecule fingerprinting reveals different amplification properties of α-synuclein oligomers and preformed fibrils in seeding assay.

2021 ◽  
Author(s):  
Derrick Lau ◽  
Chloe Magnan ◽  
Kathryn Hill ◽  
Antony Cooper ◽  
Yann Gambin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Amyloid Fibrils ◽  
Size Exclusion ◽  
Single Molecule Detection ◽  
Single Molecule Spectroscopy ◽  
Parkinsons Disease ◽  
Exclusion Chromatography ◽  
Particle Level ◽  
Amplification Assay ◽  
Multiple Species

The quantification of α-synuclein (α-syn) aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinsons disease. However, multiple species such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein and these species are likely to co-exist in biological samples and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify which species can be detected in seeding assays, recombinant oligomers and preformed fibrils were produced and purified to characterise their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent but our results reveal that their seeding behaviour is very different compared to preformed fibrils in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples are likely to dominate the response in seeding assays.

scholarly journals Isolation and Characterization of Urine Microvesicles from Prostate Cancer Patients: Different Approaches, Different Visions

2021 ◽  
Author(s):  
María García-Flores ◽  
Christian M. Sánchez-López ◽  
Marta Ramírez-Calvo ◽  
Antonio Fernández-Serra ◽  
Antonio Marcilla ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Clinical Decision Making ◽  
Clinical Decision ◽  
Protein Quantification ◽  
Size Exclusion ◽  
Biologically Relevant ◽  
Isolation And Characterization ◽  
Isolation Methods ◽  
Exclusion Chromatography ◽  
Significant Enrichment

Abstract BackgroundBecause of their specific and biologically relevant cargo, urine extracellular vesicles (EVs) constitute a valuable source of potential non-invasive biomarkers that could support the clinical decision-making to improve the management of prostate cancer (PCa) patients. Different EV isolation methods differ in terms of complexity and yield, conditioning, as consequence, the analytical result. MethodsThe aim of this study was to compare three different isolation methods for urine EVs: ultracentrifugation (UC), size exclusion chromatography (SEC), and a commercial kit (Exolute® Urine Kit). Urine samples were collected from 6 PCa patients and 4 healthy donors (HDs). After filtered through 0.22 µm filters, urine was divided in 3 equal volumes to perform EVs isolation with each of the three approaches. Isolated EVs were characterized by spectrophotometric protein quantification, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), AlphaScreen Technology, and whole miRNA Transcriptome. ResultsOur results showed that UC and SEC provided better results in terms of EVs yield and purity than Exolute®, non-significant differences were observed in terms of EV-size. Interestingly, luminescent AlphaScreen assay demonstrated a significant enrichment of CD9 and CD63 positive microvesicles in SEC and UC methods compared with Exolute®. This heterogeneity was also demonstrated in terms of miRNA content indicating that the best correlation was observed between UC and SEC. ConclusionsOur study highlights the importance of standardizing the urine EV isolation methods to guaranty the analytical reproducibility necessary for their implementation in a clinical setting.

scholarly journals Size-Exclusion Chromatography as a Technique for the Investigation of Novel Extracellular Vesicles in Cancer

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3156
Author(s):  
Daniel S. K. Liu ◽  
Flora M. Upton ◽  
Eleanor Rees ◽  
Christopher Limb ◽  
Long R. Jiao ◽  
...  
Keyword(s):  
Systematic Review ◽  
Cancer Cells ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Biomarker Discovery ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Isolation Method ◽  
Isolation And Purification ◽  
Exclusion Chromatography

Cancer cells release extracellular vesicles, which are a rich target for biomarker discovery and provide a promising mechanism for liquid biopsy. Size-exclusion chromatography (SEC) is an increasingly popular technique, which has been rediscovered for the purposes of extracellular vesicle (EV) isolation and purification from diverse biofluids. A systematic review was undertaken to identify all papers that described size exclusion as their primary EV isolation method in cancer research. In all, 37 papers were identified and discussed, which showcases the breadth of applications in which EVs can be utilised, from proteomics, to RNA, and through to functionality. A range of different methods are highlighted, with Sepharose-based techniques predominating. EVs isolated using SEC are able to identify cancer cells, highlight active pathways in tumourigenesis, clinically distinguish cohorts, and remain functionally active for further experiments.

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