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

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.

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Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

10.26434/chemrxiv.12234683 ◽

2020 ◽

Author(s):

Dario Brambilla ◽

Laura Sola ◽

Elisa Chiodi ◽

Natasa Zarovni ◽

Diogo Fortunato ◽

...

Keyword(s):

Cell Culture ◽

Extracellular Vesicles ◽

Culture Media ◽

Biological Fluids ◽

Imaging Techniques ◽

Cell Communication ◽

Disease Diagnosis ◽

Cell Culture Supernatant ◽

Transmission Electron ◽

Downstream Analysis

Extracellular vesicles (EVs) have attracted great interest among researchers due to their role in cell-cell communication, disease diagnosis, and drug delivery. In spite of their potential in the medical field, there is no consensus on the best method for separating microvesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation is made complex by the fact that blood and cell culture media, contain a large number of nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles requires harsh conditions that hinder their use in certain types of downstream analysis. Herein, a novel capture and release approach for small extracellular vesicles (sEVs), based on DNAdirected immobilization of antiCD63 antibody is presented. The flexible DNAlinker increases the capture efficiency and allows releasing of EVs by exploiting the endonucleasic activity of DNAse I. This separation protocol works under mild conditions, enabling the release of intact vesicles that can be successfully analyzed by imaging techniques. In this article sEVs recovered from plasma were characterized by established techniques for EVs analysis including nanoparticle tracking and transmission electron microscopy.<br>

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Isolation and characterization of multipotent mesenchymal stem cells in nasal polyps

Experimental Biology and Medicine ◽

10.1177/1535370214553898 ◽

2014 ◽

Vol 240 (2) ◽

pp. 185-193 ◽

Cited By ~ 12

Author(s):

Jung-Sun Cho ◽

Joo-Hoo Park ◽

Ju-Hyung Kang ◽

Sung Eun Kim ◽

Il-Ho Park ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Nasal Polyps ◽

Isolation And Characterization ◽

Multipotent Mesenchymal Stem Cells

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Orally Administered 5-aminolevulinic Acid for Isolation and Characterization of Circulating Tumor-Derived Extracellular Vesicles in Glioblastoma Patients

Cancers ◽

10.3390/cancers12113297 ◽

2020 ◽

Vol 12 (11) ◽

pp. 3297

Author(s):

Sybren L. N. Maas ◽

Thomas S. van Solinge ◽

Rosalie Schnoor ◽

Anudeep Yekula ◽

Joeky T. Senders ◽

...

Keyword(s):

Extracellular Vesicles ◽

Aminolevulinic Acid ◽

Culture Media ◽

Protoporphyrin Ix ◽

Liquid Biopsies ◽

Guided Surgery ◽

Tumor Visualization ◽

Isolation And Characterization ◽

Droplet Pcr

Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.

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Isolation and characterization of extracellular vesicles

Trillium Exctracellular Vesicles ◽

10.47184/tev.2019.01.02 ◽

2019 ◽

Vol 1 (1) ◽

pp. 18-26

Author(s):

Fabia Fricke ◽

Dominik Buschmann ◽

Michael W. Pfaffl

Keyword(s):

Cell Culture ◽

Extracellular Vesicles ◽

Culture Media ◽

Body Fluids ◽

Cell Culture Media ◽

Advantages And Disadvantages ◽

The Past ◽

Isolation And Characterization ◽

Basic Biology

Research into extracellular vesicles (EVs) gained significant traction in the past decade and EVs have been investigated in a wide variety of studies ranging from basic biology to diagnostic and therapeutic applications. Since EVs are secreted by most, if not all, eukaryotic and prokaryotic cells, they have been detected in body fluids as diverse as blood, urine and saliva as well as in cell culture media. In this chapter, we will provide an overview of EV isolation and characterization strategies and highlight their advantages and disadvantages.

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Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

10.26434/chemrxiv.12234683.v1 ◽

2020 ◽

Author(s):

Dario Brambilla ◽

Laura Sola ◽

Elisa Chiodi ◽

Natasa Zarovni ◽

Diogo Fortunato ◽

...

Keyword(s):

Cell Culture ◽

Extracellular Vesicles ◽

Culture Media ◽

Biological Fluids ◽

Imaging Techniques ◽

Cell Communication ◽

Disease Diagnosis ◽

Cell Culture Supernatant ◽

Transmission Electron ◽

Downstream Analysis

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79 MicroRNAs of extracellular vesicles secreted by embryos as an early biomarker of competence

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab79 ◽

2020 ◽

Vol 32 (2) ◽

pp. 166

Author(s):

B. Melo-Baez ◽

Y. S. Wong ◽

J. Cabezas ◽

C. J. Aguilera ◽

F. O. Castro ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Extracellular Vesicles ◽

Culture Media ◽

Blastocyst Stage ◽

Fold Change ◽

Target Cells ◽

Bovine Embryos ◽

Log2 Fold Change ◽

Transmission Electron

Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted by different cell types and participate in cellular communication by carrying molecules as microRNAs (miRNAs) that can interfere with gene expression of target cells. Extracellular vesicles have become relevant as a mechanism of embryo-maternal communication. The aim of this study was to evaluate miRNA content in EVs secreted after embryonic genome activation, by bovine embryos with different developmental potential. Bovine embryos were produced invitro and cultured in group until Day 3.5 in synthetic oviductal fluid (SOF) medium. Only 8-16-cell embryos were cultured individually in EVs-depleted SOF until Day 5. The SOF was EV depleted by ultrafiltration. Culture media (CM) were collected at Day 5 and embryos continued in culture until Day 7 with fresh SOF. Collected media were conserved individually and identified with the corresponding embryo. Then, CM were classified according to capacity of its embryo to reach blastocyst stage at Day 7: G1-CM (blocked embryos in 8-16 cell) and G2-CM (embryos that reach blastocyst stage). The EV isolation was carried out using the protocol described by Mellisho et al. (2017). Recovered EVs were evaluated by nanoparticle tracking analysis (NTA), Transmission electron microscopy and the presence of surface markers (CD9, CD63, CD81, and CD40L). After NTA, individual CM were pooled to organise 3 replicates of 10CM each, for G1 and G2. The whole miRNA isolation, library preparation, and sequencing was performed by Norgen Biotek facilities (Canada). The quality of libraries was analysed using the FastQC program platform followed by Trimmomatic to remove remnant adapters. For the miRNA library it accepted reads with value above 30 Phreads and 22 to 30bp length. The reads were mapped against the reference genome ARS-UCD1.2 using Bowtie2 software and miRDeep2 mapper, and the gene counts were calculated using HTSeq. Differential expression analysis was performed in EdgeR package. To expand this information, principal component analysis, Heatmap, and Volcano plot were plotted and pathway enrichment analysis was conducted. The NTA, transmission electron microscopy, and flow cytometry confirmed the presence of exosomes and microvesicles in isolated EVs. According to NTA, the mean size of EVs was 102.1-176.2nm and concentration of 8.4×107-8.6×108 particlesmL−1 in G1 and G2, respectively. We identified 96 miRNAs significantly expressed across the samples. Only eight miRNAs in EVs were differentially expressed between groups (G2 vs. G1). The bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were up-regulated (Log2 fold-change>1), whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were down-regulated (Log2 fold-change<1). The more significant (P-value<0.01) up-regulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The EVs secreted by embryos to culture environment carry miRNAs that can reflect the molecular state of their parental cell. This lets us suggest culture media derived-EVs and their miRNA cargo as early biomarkers to select more competent bovine embryos. This research was supported by FONDECYT, Chile (1170310).

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Exosomes of Multipotent Mesenchymal Stem Cells: Prospects for Clinical Application

10.33920/med-03-2002-05 ◽

2020 ◽

pp. 34-51

Author(s):

N. Meshcheryakova ◽

V. Kuzmenko ◽

Ya. Stanishevskiy

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Clinical Application ◽

Extracellular Vesicles ◽

Clinical Use ◽

Multipotent Mesenchymal Stem Cells

Exosomes are considered to be a population of extracellular vesicles, multipotent mesenchymal stem cells as a population of stem cells. Studies on the clinical use of exosomes of multipotent mesenchymal stem cells were analyzed. Transplantation of exosomes of multipotent mesenchymal stem cells is promising in the treatment of various diseases.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22020979 ◽

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.

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Extracellular Vesicles: New Tools for Early Diagnosis of Breast and Genitourinary Cancers

International Journal of Molecular Sciences ◽

10.3390/ijms22168430 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8430

Author(s):

Anna Testa ◽

Emilio Venturelli ◽

Maria Felice Brizzi

Keyword(s):

Early Diagnosis ◽

Extracellular Vesicles ◽

Cell Types ◽

Mortality Rates ◽

Potential Contribution ◽

Breast Cancers ◽

Characterization Methods ◽

Non Invasive ◽

Isolation And Characterization ◽

Almost All

Breast cancers and cancers of the genitourinary tract are the most common malignancies among men and women and are still characterized by high mortality rates. In order to improve the outcomes, early diagnosis is crucial, ideally by applying non-invasive and specific biomarkers. A key role in this field is played by extracellular vesicles (EVs), lipid bilayer-delimited structures shed from the surface of almost all cell types, including cancer cells. Subcellular structures contained in EVs such as nucleic acids, proteins, and lipids can be isolated and exploited as biomarkers, since they directly stem from parental cells. Furthermore, it is becoming even more evident that different body fluids can also serve as sources of EVs for diagnostic purposes. In this review, EV isolation and characterization methods are described. Moreover, the potential contribution of EV cargo for diagnostic discovery purposes is described for each tumor.

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