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 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 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.

scholarly journals An isolation system to collect high quality and purify extracellular vesicles from serum

2021 ◽  
Author(s):  
Jian Yang ◽  
Li Liao ◽  
Xin Gao ◽  
Xiaotao Xing ◽  
Haisen Huang ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Size Exclusion ◽  
High Quality ◽  
Time Saving ◽  
Isolation System ◽  
Transmission Electron ◽  
Isolation Methods ◽  
Exclusion Chromatography ◽  
Sepharose 4B

Abstract Background: Extracellular vesicles (EVs) are membrane encapsulated nanoparticles that function as carriers and play a role in intercellular communication. There are a large number of EVs in the blood and serve as an indicator of pathophysiological conditions. Studies on the basics and application of EVs are hampered by the limitations of current protocols to isolate EVs from blood. However, current isolation methods are difficult to achieve a balance between yield and purity.Results: Firstly, we use Sepharose-4B to build a self-made size exclusion chromatography (SEC) column and perform separation and identification. Then we use the SEC column to systematically compare the efficiency with the most common EV isolation methods: ultracentrifugation (UC) and total exosomes isolation commercial kit (TEI). The EVs isolated through different methods were characterized the yield and size of EVs, analyzed their protein profiles, the morphology and purity were observed under the transmission electron microscope. To further improve the quality and purity, we combined SEC and UC methods and established a two-steps method to isolated EVs from serum.Conclusion: Our study presents the combination of size-exclusion chromatography and ultracentrifugation as a feasible and time-saving method to isolate high quality and purity extracellular vesicles from serum.

The size of extracellular vesicles secreted by different types of stem cells

2018 ◽  
pp. 38-42
Author(s):  
И.Б. Алчинова ◽  
М.В. Полякова ◽  
И.Н. Сабурина ◽  
М.Ю. Карганов
Keyword(s):  
Stem Cells ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Living Organism ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Study Results ◽  
Multipotent Mesenchymal Stem Cells ◽  
Rat Adipose Tissue ◽  
Almost All

Механизм терапевтического действия мультипотентных мезенхимных стволовых клеток (ММСК) на облученный организм в последнее время вызывает повышенный интерес исследователей. В качестве активного участника паракринного механизма реализации этого эффекта предлагают рассматривать внеклеточные везикулы, секретируемые практически всеми клетками живого организма. Цель работы: выделить и охарактеризовать внеклеточные везикулы, продуцируемые стволовыми клетками различной природы. Материалы и методы. Суспензии внеклеточных везикул, выделенных по модифицированному протоколу дифференциального центрифугирования из культуральных жидкостей от культур ММСК костного мозга человека 2-го пассажа и ММСК жировой ткани крысы 4-го пассажа, были проанализированы методом просвечивающей электронной микроскопии и методом анализа траекторий наночастиц. Результаты. Исследование показало наличие в обоих образцах микрочастиц размерами до и около 100 нм, однако процентное содержание частиц разных размеров в суспензии различалось для двух анализируемых типов клеток. Заключение. Полученные результаты могут свидетельствовать о специфике секреции, обусловленной клеточным типом. A mechanism of the therapeutic effect of multipotent mesenchymal stem cells (MMSC) on irradiated body has recently arisen much interest of researchers. Extracellular vesicles (EVs) secreted by almost all cells of a living organism were suggested to actively contribute to the paracrine mechanism of this effect. The aim of the study was isolation and characterization of extracellular vesicles produced by various types of stem cells. Materials and methods. Suspensions of EVs were isolated from culture media of passage 2 human bone marrow-derived MMSC and passage 4 rat adipose tissue-derived MMSC using a modified protocol of differential centrifugation and then studied using transmission electron microscopy and nanoparticle tracking analysis. Results. The study showed the presence of microparticles with a size of >100 nm in the examined samples. However, the percent content of particles with different sizes in the suspension was different in two analyzed types of cell culture. Conclusion. The study results might reflect a specificity of secretion determined by the cell type.

scholarly journals A Bacterially-Expressed Recombinant Envelope Protein from Usutu Virus Induces Neutralizing Antibodies in Rabbits

Vaccines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 157
Author(s):  
Kinga Böszörményi ◽  
Janet Hirsch ◽  
Gwendoline Kiemenyi Kayere ◽  
Zahra Fagrouch ◽  
Nicole Heijmans ◽  
...  
Keyword(s):  
Envelope Protein ◽  
Neutralizing Antibodies ◽  
Size Exclusion ◽  
Vitro Assay ◽  
Usutu Virus ◽  
Transmission Electron ◽  
Exclusion Chromatography ◽  
Efficacious Vaccine ◽  
Human Infections

Background: Recently, an emerging flavivirus, Usutu virus (USUV), has caused an epidemic among birds in Europe, resulting in a massive die-off in Eurasian blackbirds. Currently found only in Europe and Africa, it can be envisioned that Usutu virus will follow the path of other flaviviruses, like West Nile virus and Zika virus, and will spread via its mosquito vectors and bird hosts to other parts of the world. Several cases of human infections by Usutu virus have already been published. Anticipating this spread, development of an efficacious vaccine would be highly desirable. Method: This study describes the production in E. coli, purification, and refolding of a partial USUV envelope protein. Prior to immunization, the protein was characterized using size exclusion chromatography, transmission electron microscopy and dynamic light scattering, showing the limited presence of virus-like structures, indicating that the protein solution is probably a mixture of mono and multimeric envelope proteins. Results: Immunizations of two rabbits with the refolded E-protein fraction, mixed with a strong adjuvant, resulted in the generation of neutralizing antibodies, as evidenced in an in vitro assay. Discussion: The way forward towards a subunit vaccine against Usutu virus infection is discussed.

scholarly journals Kinetics and Topology of DNA Associated with Circulating Extracellular Vesicles Released during Exercise

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 522
Author(s):  
Elmo W. I. Neuberger ◽  
Barlo Hillen ◽  
Katharina Mayr ◽  
Perikles Simon ◽  
Eva-Maria Krämer-Albers ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Affinity Capture ◽  
Healthy Humans ◽  
Dnase Digestion ◽  
And Topology ◽  
Single Bout ◽  
Exclusion Chromatography ◽  
Free Circulating Dna ◽  
Line Elements

Although it is widely accepted that cancer-derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9+, CD63+, and CD81+ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12% of the total cfDNA occurs in association with CD9+/CD63+/CD81+EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20% are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV-associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen.

Microfluidic Size Exclusion Chromatography (μSEC) for Extracellular Vesicles and Plasma Protein Separation

Small ◽  
2022 ◽  
pp. 2104470
Author(s):  
Sheng Yuan Leong ◽  
Hong Boon Ong ◽  
Hui Min Tay ◽  
Fang Kong ◽  
Megha Upadya ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Plasma Protein ◽  
Protein Separation ◽  
Size Exclusion ◽  
Exclusion Chromatography

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.

Sign in / Sign up

Export Citation Format

Share Document