Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications

Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma.

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

Pharmaceuticals ◽

10.3390/ph14070618 ◽

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.

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Serial Coupling of Ion-Exchange and Size-Exclusion Chromatography to Determine Aggregation Levels in mAbs in The Presence of a Proteinaceous Excipient, Recombinant Human Serum Albumin

Journal of Pharmaceutical Sciences ◽

10.1002/jps.24275 ◽

2015 ◽

Vol 104 (2) ◽

pp. 548-556 ◽

Cited By ~ 4

Author(s):

Paul Luigi Gargani Weisbjerg ◽

Mikael Bjerg Caspersen ◽

Ken Cook ◽

Marco Van De Weert

Keyword(s):

Human Serum Albumin ◽

Ion Exchange ◽

Serum Albumin ◽

Human Serum ◽

Size Exclusion Chromatography ◽

Size Exclusion ◽

Recombinant Human Serum Albumin ◽

Exclusion Chromatography

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

Biosensors ◽

10.3390/bios10110173 ◽

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.

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

Scientific Reports ◽

10.1038/s41598-020-78422-y ◽

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.

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Tuberous Sclerosis Complex Axis Controls Renal Extracellular Vesicle Production and Protein Content

International Journal of Molecular Sciences ◽

10.3390/ijms21051729 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1729 ◽

Cited By ~ 2

Author(s):

Fahad Zadjali ◽

Prashant Kumar ◽

Ying Yao ◽

Daniel Johnson ◽

Aristotelis Astrinidis ◽

...

Keyword(s):

Protein Content ◽

Tuberous Sclerosis ◽

Tuberous Sclerosis Complex ◽

Growth Regulation ◽

Primary Cilia ◽

Collecting Duct ◽

Extracellular Vesicle ◽

Size Exclusion ◽

Chromatography Method ◽

Significant Difference

The tuberous sclerosis complex (Tsc) proteins regulate the conserved mTORC1 growth regulation pathway. We identified that loss of the Tsc2 gene in mouse inner medullary collecting duct (mIMCD) cells induced a greater than two-fold increase in extracellular vesicle (EV) production compared to the same cells having an intact Tsc axis. We optimized EV isolation using a well-established size exclusion chromatography method to produce high purity EVs. Electron microscopy confirmed the purity and spherical shape of EVs. Both tunable resistive pulse sensing (TRPS) and dynamic light scattering (DLS) demonstrated that the isolated EVs possessed a heterogenous size distribution. Approximately 90% of the EVs were in the 100–250 nm size range, while approximately 10% had a size greater than 250 nm. Western blot analysis using proteins isolated from the EVs revealed the cellular proteins Alix and TSG101, the transmembrane proteins CD63, CD81, and CD9, and the primary cilia Hedgehog signaling-related protein Arl13b. Proteomic analysis of EVs identified a significant difference between the Tsc2-intact and Tsc2-deleted cell that correlated well with the increased production. The EVs may be involved in tissue homeostasis and cause disease by overproduction and altered protein content. The EVs released by renal cyst epithelia in TSC complex may serve as a tool to discover the mechanism of TSC cystogenesis and in developing potential therapeutic strategies.

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Size Exclusion Chromatography and Related Separation Techniques

Analytical Chemistry ◽

10.1021/a1980015t ◽

1998 ◽

Vol 70 (12) ◽

pp. 251-278 ◽

Cited By ~ 102

Author(s):

Howard G. Barth ◽

Barry E. Boyes ◽

Christian Jackson

Keyword(s):

Size Exclusion Chromatography ◽

Size Exclusion ◽

Separation Techniques ◽

Exclusion Chromatography

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

10.1101/2021.04.01.437817 ◽

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.

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

Cellular and Molecular Life Sciences ◽

10.1007/s00018-019-03071-y ◽

2019 ◽

Vol 76 (12) ◽

pp. 2369-2382 ◽

Cited By ~ 36

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

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

Cancers ◽

10.3390/cancers12113156 ◽

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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