Free flow electrophoresis allows quick preparation of extracellular vesicles from cell culture supernatants and human plasma

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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Extracellular Vesicles from PC-3 Cell Culture Supernatant

Food Science Faculty Data Sets ◽

10.36837/chapman.000204 ◽

2020 ◽

Author(s):

John Miklavcic

Keyword(s):

Cell Culture ◽

Extracellular Vesicles ◽

Culture Supernatant ◽

Cell Culture Supernatant

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Extracellular Vesicles from Human Plasma Show a Distinctive Proteome and miRNome Profile in Patients with Severe Cutaneous Adverse Reactions

Chemical Research in Toxicology ◽

10.1021/acs.chemrestox.1c00047 ◽

2021 ◽

Author(s):

Orlando Salinas-Jaramillo ◽

Alejandra Monroy-Arreola ◽

Sebastian Herrera-Noreña ◽

Ana L. Guzmán-Ortiz ◽

Abrahan Hernández-Hernández ◽

...

Keyword(s):

Human Plasma ◽

Extracellular Vesicles ◽

Adverse Reactions ◽

Severe Cutaneous Adverse Reactions ◽

Cutaneous Adverse Reactions

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Importance of stem cell culture conditions for their derived extracellular vesicles therapeutic effect

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2021.03.028 ◽

2021 ◽

Author(s):

Cristina Mas-Bargues ◽

Consuelo Borrás

Keyword(s):

Cell Culture ◽

Stem Cell ◽

Therapeutic Effect ◽

Extracellular Vesicles ◽

Culture Conditions ◽

Stem Cell Culture ◽

Cell Culture Conditions

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Delivery of functional exogenous proteins by plant-derived vesicles to human cells in vitro

Scientific Reports ◽

10.1038/s41598-021-85833-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Luiza Garaeva ◽

Roman Kamyshinsky ◽

Yury Kil ◽

Elena Varfolomeeva ◽

Nikolai Verlov ◽

...

Keyword(s):

Cell Culture ◽

Colon Cancer ◽

Extracellular Vesicles ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Human Cells ◽

Bioactive Molecules ◽

Native Plant ◽

Peripheral Blood Mononuclear

AbstractPlant-derived extracellular vesicles (EVs) gain more and more attention as promising carriers of exogenous bioactive molecules to the human cells. Derived from various edible sources, these EVs are remarkably biocompatible, biodegradable and highly abundant from plants. In this work, EVs from grapefruit juice were isolated by differential centrifugation followed by characterization of their size, quantity and morphology by nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy and cryo-electron microscopy (Cryo-EM). In Cryo-EM experiments, we visualized grapefruit EVs with the average size of 41 ± 13 nm, confirmed their round-shaped morphology and estimated the thickness of their lipid bilayer as 5.3 ± 0.8 nm. Further, using cell culture models, we have successfully demonstrated that native grapefruit-derived extracellular vesicles (GF-EVs) are highly efficient carriers for the delivery of the exogenous Alexa Fluor 647 labeled bovine serum albumin (BSA) and heat shock protein 70 (HSP70) into both human peripheral blood mononuclear cells and colon cancer cells. Interestingly, loading to plant EVs significantly ameliorated the uptake of exogenous proteins by human cells compared to the same proteins without EVs. Most importantly, we have confirmed the functional activity of human recombinant HSP70 in the colon cancer cell culture upon delivery by GF-EVs. Analysis of the biodistribution of GF-EVs loaded with 125I-labeled BSA in mice demonstrated a significant uptake of the grapefruit-derived extracellular vesicles by the majority of organs. The results of our study indicate that native plant EVs might be safe and effective carriers of exogenous proteins into human cells.

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Abstract P469: Scale-up And Characterization Of Therapeutic Mesenchymal Stromal Cell-derived Extracellular Vesicles

Circulation Research ◽

10.1161/res.129.suppl_1.p469 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Selen Uman ◽

Jason A Burdick

Keyword(s):

Cell Culture ◽

Extracellular Vesicles ◽

Animal Studies ◽

Scale Up ◽

Spinner Flask ◽

Perfusion Bioreactor ◽

Large Animal ◽

Culture Methods ◽

Therapeutic Benefits ◽

Cell Culture Methods

Introduction: Early studies have shown therapeutic benefits of mesenchymal stromal cells (MSCs) in cardioprotection due to their angiogenic, proliferative, anti-apoptotic and anti-inflammatory properties, which are now attributed to secreted factors such as extracellular vesicles (EVs). While MSC-EVs have shown promise in small animals for cardiovascular therapies, large animal studies are required to evaluate the therapeutic benefit of MSC-EVs for clinical translation. One of the biggest challenges for large animal studies is the need to generate clinically-relevant quality and quantity of EVs without batch-to-batch variations that could compromise efficacy. This study aims to explore three different cell culture methods (traditionally-used tissue culture plates (TCP), 3-D printed bioscaffolds in a perfusion system (P), and microcarriers in dynamic spinner flask conditions (M)) to scale-up the production of MSC-EVs across four different biological donors and rigorously investigate EV yield, size, shape, and content. Methods: MSCs were isolated from the iliac crest of four different Yucatan minipigs using heparinized syringes, and cells were expanded to passage four, at which point they were seeded onto the respective cell culture methods. EVs were collected from conditioned medium (CM) via differential ultracentrifugation. EV size, distribution, yield, and protein concentration were studied using Nanoparticle Tracking Analysis (NTA) and microBCA assays. Results: Both perfusion bioreactor and spinner flask systems enabled sustained maintenance of large numbers of cells. Across biological donors and fabrication methods, modes remained within 50-150 nm and were not statistically different. Microcarrier-based spinner flasks and perfusion bioreactor set-ups both improved EV yield, up to 6 times in efficiency. Ongoing research focuses on examining differences in EV content across biological donors using RNA-sequencing and proteomics.

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Enrichment of Exosome-Like Extracellular Vesicles from Plasma Suitable for Clinical Vesicular miRNA Biomarker Research

Journal of Clinical Medicine ◽

10.3390/jcm8111995 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1995 ◽

Cited By ~ 5

Author(s):

Moon ◽

Shin ◽

Kim ◽

Lee ◽

Mankhong ◽

...

Keyword(s):

Human Plasma ◽

Extracellular Vesicles ◽

Proteinase K ◽

High Yield ◽

Clinical Samples ◽

Diagnostic Biomarkers ◽

Micro Rnas ◽

Biomarker Research ◽

Protein Rich ◽

Yield And Purity

Exosome-like extracellular vesicles (ELVs) contain biomolecules that have potential as diagnostic biomarkers, such as proteins, micro-RNAs (miRNAs), and lipids. However, it is difficult to enrich ELVs consistently with high yield and purity from clinical samples, which hampers the development of ELV biomarkers. This is particularly true for miRNAs in protein-rich plasma. Hence, we modified ELV isolation protocols of three commercially available polymer-precipitation-based kits using proteinase K (PK) treatment to quantify ELV-associated miRNAs in human plasma. We compared the yield, purity, and characteristics of enriched plasma ELVs, and measured the relative quantity of three selected miRNAs (miR-30c, miR-126, and miR-192) in ELVs using six human plasma samples. Compared with the original protocols, we demonstrated that ELVs can be isolated with PK treatment with high purity (i.e., lack of non-exosomal proteins and homogeneous size of vesicles) and yield (i.e., abundancy of exosomal markers), which were dependent on kits. Using the kit with the highest purity and yield with PK treatment, we successfully quantified ELV miRNAs (levels of 45%–65% in total plasma) with acceptable variability. Collectively, ELV enrichment using the modified easy-to-use method appears suitable for the analysis of miRNAs, although its clinical applicability needs to be confirmed in larger clinical studies.

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