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

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>

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

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>

scholarly journals Isolation of small extracellular vesicles from cell culture supernatant

2020 ◽  
Author(s):  
Eun-Ju Im ◽  
Chan-Hyeong Lee ◽  
Moon-Chang Baek
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Supernatant ◽  
Culture Media ◽  
Cell Culture Supernatant ◽  
Cell Culture Media

Abstract This protocol describes how to isolate small extracellular vesicles (sEV) from cell culture media using an ultracentrifuge and analyze the number of sEV by nanoparticle tracking analyzer.

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 Isolation of Extracellular Vesicles from Cell Culture Media by Differential Ultracentrifugation  v1 (protocols.io.bnr3md8n)

protocols.io ◽  
2020 ◽  
Author(s):  
Dima Ter ◽  
Wendy Trieu ◽  
Maia Norman ◽  
Roey Lazarovits ◽  
George Church ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Cell Culture Media

scholarly journals CSIG-09. PROTEOMIC ANALYSIS OF MENINGIOMA CELL-DERIVED EXTRACELLULAR VESICLES: FIRST OF A KIND

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi45-vi46
Author(s):  
Franz Ricklefs ◽  
Manka Fuh ◽  
Cecile Maire ◽  
Mareike Holz ◽  
Katharina Kolbe ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Extracellular Vesicles ◽  
Cell Cultures ◽  
Cell Communication ◽  
Benign Tumors ◽  
Short Term ◽  
Meningioma Cell ◽  
Particle Counts ◽  
Mass Spectometry

Abstract BACKGROUND Extracellular vesicles (EVs) play an important role in cell-cell communication in different types of tumors, carrying multiple layers of biological functional molecules, including proteins, RNA, DNA and lipids. Their implication as biomarkers in tumor disease is under current investigation. We previously showed that EVs in glioblastoma reflect the tumor subtype and that glioblastoma patients have elevated circulating particle counts. Regarding to meningioma, it is not known to what extent these usually benign tumors secrete EVs and how these EVs reflect the tumor. Here we report the first study that analyzed meningioma cell-derived EVs. METHODS Meningioma tissue, short-term cell cultures and cell culture-derived EVs (menEVs), (n=4) were analyzed by global mass-spectromety, immunoblotting and imaging flow cytometry and compared to EVs from glioblastoma short-term cell cultures (gEVs), (n=4). Plasma EVs from meningioma patients (n = 12) were analyzed for their tetraspanin marker expression (CD9, CD81 and CD63). EVs were further analyzed by nanoparticle analysis (NTA) and electron microscopy. RESULTS menEVs were 110-140nm in size and exhibited vesicular structures by electron microscopy. We identified 269 proteins in menEVs through mass spectometry. 45 proteins were upregulated in menEVs compared to short-term cell culture and original tumor tissue. 99 proteins were exclusively found in menEVs compared to gEVs, with osteopontin being the top highly expressed protein within the mEV fraction. Both meningioma and glioblastoma patients have elevated circulating plasma EV counts (p< 0.01), as measured by NTA. CONCLUSION The increase in circulating plasma EVs in meningioma patients suggests that tumor cell-derived EVs augment the pool of circulating EVs and could be utilized to obtain information on the tumor by liquid biopsy. Osteopontin is known to be expressed at high levels in meningiomas and its association with menEVs may facilitate isolation of circulating meningioma-specific EVs for analysis.

scholarly journals Factors influencing the measurement of the secretion rate of extracellular vesicles

The Analyst ◽  
2020 ◽  
Vol 145 (17) ◽  
pp. 5870-5877
Author(s):  
Yi Wen ◽  
Yundi Chen ◽  
Guosheng Wang ◽  
Komal Abhange ◽  
Fei Xue ◽  
...  
Keyword(s):  
Cell Culture ◽  
Direct Measurement ◽  
Extracellular Vesicles ◽  
Culture Supernatant ◽  
Extracellular Vesicle ◽  
Secretion Rate ◽  
Cell Culture Supernatant ◽  
Factors Influencing

Direct measurement of the small extracellular vesicle in the cell culture supernatant.

scholarly journals Immuno-Acoustic Sorting of Disease-Specific Extracellular Vesicles by Acoustophoretic Force

Micromachines ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1534
Author(s):  
Junyuan Liu ◽  
Yuxin Qu ◽  
Han Wang
Keyword(s):  
Extracellular Vesicles ◽  
Processing Time ◽  
Life Science ◽  
Biological Fluids ◽  
Label Free ◽  
Her2 Positive ◽  
Diagnosis And Prognosis ◽  
Efficient Recovery ◽  
Downstream Analysis ◽  
Disease Specific

Methods for the isolation and analysis of extracellular vesicles (EVs) have been extensively explored in the field of life science and in clinical diagnosis in recent years. The separation and efficient recovery of high-purity target EVs from biological samples are important prerequisites in the study of EVs. So far, commonly used methods of EV separation include ultracentrifugation, filtration, solvent precipitation and immunoaffinity capturing. However, these methods suffer from long processing time, EV damage and low enrichment efficiency. The use of acoustophoretic force facilitates the non-contact label-free manipulation of cells based on their size and compressibility but lacks specificity. Additionally, the acoustophoretic force exerted on sub-micron substances is normally weak and insufficient for separation. Here we present a novel immuno-acoustic sorting technology, where biological substances such as EVs, viruses, and biomolecules, can be specifically captured by antibody/receptor coated microparticles through immunoaffinity, and manipulated by an acoustophoretic force exerted on the microparticles. Using immuno-acoustic sorting technology, we successfully separated and purified HER2-positive EVs for further downstream analysis. This method holds great potential in isolating and purifying specific targets such as disease-related EVs from biological fluids and opens new possibilities for the EV-based early diagnosis and prognosis of diseases.

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