scholarly journals Methods for Separation and Characterization of Extracellular Vesicles: Results of a Worldwide Survey Performed by the ISEV Rigor and Standardization Subcommittee

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1955 ◽  
Author(s):  
Felix Royo ◽  
Clotilde Théry ◽  
Juan M. Falcón-Pérez ◽  
Rienk Nieuwland ◽  
Kenneth W. Witwer
Keyword(s):  
Extracellular Vesicles ◽  
Initial Response ◽  
Size Exclusion ◽  
Disease Biomarkers ◽  
Quality Controls ◽  
Isolation And Characterization ◽  
Measurement Results ◽  
Exclusion Chromatography ◽  
Important Objective

Research on extracellular vesicles (EVs) is growing exponentially due to an increasing appreciation of EVs as disease biomarkers and therapeutics, an expanding number of EV-containing materials under study, and application of new preparation, detection, and cargo analysis methods. Diversity of both sources and methodologies imposes challenges on the comparison of measurement results between studies and laboratories. While reference guidelines and minimal requirements for EV research have achieved the important objective of assembling community consensus, it is also essential to understand which methodologies and quality controls are currently being applied, and how usage trends are evolving. As an initial response to this need, the International Society for Extracellular Vesicles (ISEV) performed a worldwide survey in 2015 on “Techniques used for the isolation and characterization of extracellular vesicles” and published the results from this survey in 2016. In 2019, a new survey was performed to assess the changing state of the field. The questionnaire received more than 600 full or partial responses, and the present manuscript summarizes the results of this second worldwide survey. The results emphasize that separation methods such as ultracentrifugation and density gradients are still the most commonly used methods, the use of size exclusion chromatography has increased, and techniques based on tangential flow and microfluidics are now being used by more than 10% of respondents. The survey also reveals that most EV researchers still do not perform sample quality controls before or after isolation of EVs. Finally, the majority of EV researchers emphasize that separation and characterization of EVs should receive more attention.

scholarly journals The Past, the Present, and the Future of the Size Exclusion Chromatography in Extracellular Vesicles Separation

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2272
Author(s):  
Hussein Kaddour ◽  
Malik Tranquille ◽  
Chioma M. Okeoma
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Cell Types ◽  
Size Exclusion ◽  
Target Cells ◽  
Purification And Characterization ◽  
Extracellular Milieu ◽  
Exclusion Chromatography ◽  
Single Vesicle

Extracellular vesicles (EVs) are cell-derived membranous particles secreted by all cell types (including virus infected and uninfected cells) into the extracellular milieu. EVs carry, protect, and transport a wide array of bioactive cargoes to recipient/target cells. EVs regulate physiological and pathophysiological processes in recipient cells and are important in therapeutics/drug delivery. Despite these great attributes of EVs, an efficient protocol for EV separation from biofluids is lacking. Numerous techniques have been adapted for the separation of EVs with size exclusion chromatography (SEC)-based methods being the most promising. Here, we review the SEC protocols used for EV separation, and discuss opportunities for significant improvements, such as the development of novel particle purification liquid chromatography (PPLC) system capable of tandem purification and characterization of biological and synthetic particles with near-single vesicle resolution. Finally, we identify future perspectives and current issues to make PPLC a tool capable of providing a unified, automated, adaptable, yet simple and affordable particle separation resource.

scholarly journals Isolation and Characterization of Equine Uterine Extracellular Vesicles: A Comparative Methodological Study

2021 ◽  
Vol 22 (2) ◽  
pp. 979
Author(s):  
Carmen Almiñana ◽  
Alba Rudolf Vegas ◽  
Muhittin Tekin ◽  
Mubbashar Hassan ◽  
Rustem Uzbekov ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Optimal Method ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Uterine Fluid ◽  
Exclusion Chromatography ◽  
Protein Classes ◽  
Uterine Lavage ◽  
Phosphate Buffered Saline

Extracellular vesicles (EVs) have been identified in the uterine fluid in different species and have been pointed as key players in the embryo-maternal dialogue, maternal recognition of pregnancy and establishment of pregnancy. However, little is known about the uterine EVs in the mare. Therefore, the present study aimed at characterizing EVs from uterine lavage of cyclic mares by comparing five EVs isolation methods and the combination of them: (1) ultracentrifugation (UC); (2) concentration of lavage volume by Centricon ultrafiltration (CE); (3) the use of CE with different washing steps (phosphate-buffered saline with or without trehalose); (4) size-exclusion chromatography with iZON-qEV columns, and (5) a combination of the methods with best results based on EVs yield, purity, and protein cargo profiles. Transmission electron microscopy and Western blotting confirmed the isolation of EVs by all methods but with quantitative and qualitative differences. Mass spectrometry provided differences in protein profiles between methods, number of identified proteins, and protein classes. Our results indicate that the combination of CE/trehalose/iZON/UC is an optimal method to isolate equine uterine EVs with good yield and purity that can be applied in future studies to determine the role of equine uterine EVs in embryo-maternal interactions.

scholarly journals Isolation and characterization of BanLec-I, a mannoside-binding lectin from Musa paradisiac (banana)

1990 ◽  
Vol 272 (3) ◽  
pp. 721-726 ◽  
Author(s):  
V L Koshte ◽  
W van Dijk ◽  
M E van der Stelt ◽  
R C Aalberse
Keyword(s):  
Cell Proliferation ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Size Exclusion ◽  
T Cell Proliferation ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Binding Lectin

A lectin (BanLec-I) from banana (Musa paradisiac) with a binding specificity for oligomannosidic glycans of size classes higher than (Man)6GlcNAc was isolated and purified by affinity chromatography on a Sephadex G-75 column. It did not agglutinate untreated human or sheep erythrocytes, but it did agglutinate rabbit erythrocytes. BanLec-I stimulated T-cell proliferation. On size-exclusion chromatography, BanLec-I has a molecular mass of approx. 27 kDa, and on SDS/PAGE the molecular mass is approx. 13 kDa. The isoelectric point is 7.2-7.5. BanLec-I was found to be very effective as a probe in detecting glycoproteins, e.g. on nitrocellulose blots.

scholarly journals Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues

2020 ◽  
Vol 21 (15) ◽  
pp. 5586 ◽  
Author(s):  
Marie Bordas ◽  
Géraldine Genard ◽  
Sibylle Ohl ◽  
Michelle Nessling ◽  
Karsten Richter ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Cell Communication ◽  
Size Exclusion ◽  
Lymphoid Tissues ◽  
Premetastatic Niche ◽  
Diagnosis And Prognosis ◽  
Isolation And Characterization ◽  
Optimized Protocol ◽  
Exclusion Chromatography

Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches—(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols.

The structure of the extracellular teichoic acids from the allergy-protective bacterium Lactococcus lactis G121

2012 ◽  
Vol 393 (8) ◽  
pp. 749-755
Author(s):  
Kathleen Fischer ◽  
Evgueny Vinogradov ◽  
Buko Lindner ◽  
Holger Heine ◽  
Otto Holst
Keyword(s):  
Lactococcus Lactis ◽  
Molecular Mechanisms ◽  
Cell Envelope ◽  
Teichoic Acid ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
Protective Properties ◽  
Isolation And Characterization ◽  
Exclusion Chromatography

Abstract The Gram-positive bacterium Lactococcus lactis G121 is a farm isolate that protects mice from ovalbumin-induced asthma. To understand the molecular mechanisms of such allergy-protective properties, the isolation and characterization of cell envelope constituents is crucial. Here, structural analyses of the extracellular teichoic acid (EC TA) from L. lactis G121 are presented. Extraction with 0.9% saline afforded a crude TA fraction. Consecutive size exclusion chromatography on Biogel P60 and P10 matrix was performed to purify the sample. Chemical component analyses, high-resolution electrospray ionization Fourier-transformed ion cyclotron mass spectrometry, and nuclear magnetic resonance spectroscopy were conducted for structural elucidation. The EC TA was a poly(glycosylglycerol phosphate) molecule with a repeating unit of -6)-[β-d-Glcp-(1→3)-][α-d-GlcpNAc-(1→4)-]α-d-GalpNAc-(1→3)-β-d-GlcpNAc-(1→2)-glycerol-(1-P-).

scholarly journals SVF-derived extracellular vesicles carry characteristic miRNAs in lipedema

2019 ◽  
Author(s):  
Eleni Priglinger ◽  
Karin Strohmeier ◽  
Moritz Weigl ◽  
Carolin Lindner ◽  
Martin Barsch ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Extracellular Vesicles ◽  
Stromal Vascular Fraction ◽  
Size Exclusion ◽  
Significant Difference ◽  
Chronic Progressive Disease ◽  
Exclusion Chromatography ◽  
Median Particle

AbstractLipedema is a chronic, progressive disease of adipose tissue with lack of consistent diagnostic criteria. The aim of this study was a thorough comparative characterization of extracellular microRNAs from the stromal vascular fraction (SVF) of healthy and lipedema adipose tissue. For this, we analyzed 187 extracellular microRNAs in concentrated conditioned media (cCM) and specifically in small extracellular vesicles (sEVs) enriched thereof by size exclusion chromatography. No significant difference in median particle size and concentration was observed between sEV fractions in healthy and lipedema. We found the majority of miRNAs located predominantly in cCM compared to sEV enriched fraction. Surprisingly, hierarchical clustering of the most variant miRNAs showed that only sEV miRNA profiles – but not cCM miRNAs – were impacted by lipedema. Seven sEV miRNAs (miR–16-5p, miR-29a-3p, miR-24-3p, miR-454-p, miR–144-5p, miR-130a-3p, let-7c-5p) were differently regulated in lipedema and healthy, whereas only one cCM miRNA (miR-188-5p) was significantly downregulated in lipedema. Comparing SVF from healthy and lipedema patients, we identified sEVs as the lipedema relevant miRNA fraction. This study contributes to identify the potential role of SVF secreted miRNAs in lipedema.

scholarly journals The Past, the Present, and the Future of the Size Exclusion Chromatography in Extracellular Vesicles Separation

2021 ◽  
Author(s):  
Hussein Kaddour ◽  
Malik Tranquille ◽  
Chioma M. Okeoma
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Cell Types ◽  
Size Exclusion ◽  
Target Cells ◽  
Purification And Characterization ◽  
Extracellular Milieu ◽  
Exclusion Chromatography ◽  
Single Vesicle

Extracellular vesicles (EVs) are cell-derived membranous particles secreted by all cell types into the extracellular milieu. EVs carry, protect, and transport a wide array of bioactive cargoes to recipient/target cells. EVs regulate physiological and pathophysiological processes in recipient cells and are important in therapeutics/drug delivery. Despite these great attributes of EVs, an efficient protocol for EV separation from biofluids is lacking. Numerous techniques have been adapted for the separation of EVs with size exclusion chromatography (SEC)-based methods being the most promising. Here, we review the SEC protocols used for EV separation, and discuss opportunities for significant improvements, such as the development of novel particle purification liquid chromatography (PPLC) system capable of tandem purification and characterization of biological and synthetic particles with near-single vesicle resolution. Finally, we identify future perspectives and current issues to make PPLC a tool capable of providing a unified, automated, adaptable, yet simple and affordable particle separation resource.

Isolation and characterization of lignins from Eucalyptus grandis Hill ex Maiden and Eucalyptus globulus Labill. by enzymatic mild acidolysis (EMAL)

Holzforschung ◽  
2008 ◽  
Vol 62 (1) ◽  
pp. 24-30 ◽  
Author(s):  
Anderson Guerra ◽  
Lucian A. Lucia ◽  
Dimitris S. Argyropoulos
Keyword(s):  
Eucalyptus Globulus ◽  
Eucalyptus Grandis ◽  
Raw Material ◽  
Size Exclusion ◽  
Hydroxyl Groups ◽  
Aryl Ether ◽  
Isolation And Characterization ◽  
White Fir ◽  
Exclusion Chromatography

Abstract Despite the growing importance of Eucalyptus wood as raw material for pulp and paper, there is a lack of knowledge on the chemistry of their macromolecular components. The present paper addresses this issue by applying the recently developed protocol for isolating enzymatic mild acidolysis lignins (EMAL) from Eucalyptus grandis, Eucalyptus globulus and the softwood species Douglas fir and white fir, which were used for comparative purposes. The structures of EMALs were investigated by quantitative 31P NMR, DFRC/31P NMR (derivatization followed by reductive cleavage followed by quantitative 31P NMR) and size exclusion chromatography (SEC). Overall, the yields of EMALs isolated from Eucalyptus were higher than those from the softwoods examined. Lignin from E. globulus was found to contain higher contents of arylglycerol-β-aryl ether structures, free phenolic hydroxyl groups and syringyl-type units than lignin from E. grandis. New insights provided by the DFRC/31P NMR revealed that up to 62.2% of arylglycerol-β-aryl ether structures in E. globulus are uncondensed, while in E. grandis the amount of such uncondensed structures was found to be lower than 48%. SEC analyses showed that lignins from E. grandis and softwoods associate in greater extension than lignin from E. globulus.

scholarly journals 109 Isolation and characterization of myometrium and decidual cell-derived extracellular vesicles

2021 ◽  
Vol 224 (2) ◽  
pp. S75-S76
Author(s):  
Megan Shepherd ◽  
Enkhtuya Radnaa ◽  
Rheanna Urrabaz-Garza ◽  
Talar Kechichian ◽  
Ourlad Alzeus G. Tantengco ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Decidual Cell ◽  
Isolation And Characterization
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