A novel approach for enumeration of extracellular vesicles from crude and purified cell culture samples

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

Cytotherapy ◽

10.1016/s1465324921004643 ◽

2021 ◽

Vol 23 (5) ◽

pp. S118

Author(s):

S. Staubach ◽

T. Tertel ◽

V. Börger ◽

C. Grätz ◽

M. Pfaffl ◽

...

Keyword(s):

Cell Culture ◽

Human Plasma ◽

Extracellular Vesicles ◽

Free Flow ◽

Culture Supernatants

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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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NANOmetric BIO-Banked MSC-Derived Exosome (NANOBIOME) as a Novel Approach to Regenerative Medicine

Journal of Clinical Medicine ◽

10.3390/jcm7100357 ◽

2018 ◽

Vol 7 (10) ◽

pp. 357 ◽

Cited By ~ 7

Author(s):

Bruna Codispoti ◽

Massimo Marrelli ◽

Francesco Paduano ◽

Marco Tatullo

Keyword(s):

Plasma Membrane ◽

Regenerative Medicine ◽

Extracellular Vesicles ◽

Biological Fluids ◽

Cell Types ◽

Endogenous Factors ◽

Cell Lineages ◽

Novel Approach ◽

Therapeutic Uses ◽

Technical Issues

Mesenchymal stem cells (MSCs) are well known for their great potential in clinical applications. In fact, MSCs can differentiate into several cell lineages and show paracrine behavior by releasing endogenous factors that stimulate tissue repair and modulate local immune response. Each MSC type is affected by specific biobanking issues—technical issues as well as regulatory and ethical concerns—thus making it quite tricky to safely and commonly use MSC banking for swift regenerative applications. Extracellular vesicles (EVs) include a group of 150–1000 nm vesicles that are released by budding from the plasma membrane into biological fluids and/or in the culture medium from varied and heterogenic cell types. EVs consist of various vesicle types that are defined with different nomenclature such as exosomes, shedding vesicles, nanoparticles, microvesicles and apoptotic bodies. Ectosomes, micro- and nanoparticles generally refer to the direct release of single vesicles from the plasma membrane. While many studies describe exosomes as deriving from multivesicular bodies, solid evidence about the origin of EVs is often lacking. Extracellular vesicles represent an important portion of the cell secretome. Their numerous properties can be used for diagnostic, prognostic, and therapeutic uses, so EVs are considered to be innovative and smart theranostic tools. The aim of this review is to investigate the usefulness of exosomes as carriers of the whole information panel characterizing the use of MSCs in regenerative medicine. Our purpose is to make a step forward in the development of the NANOmetric BIO-banked MSC-derived Exosome (NANOBIOME).

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