Evaluation of the Storage Stability of Extracellular Vesicles

Richter, Maximilian; Fuhrmann, Kathrin; Fuhrmann, Gregor

doi:10.3791/59584

Cited by 20 publications

(19 citation statements)

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“…Moreover, it is possible that Lipo‐NPs could be lyophilized to aid in storage for clinical applications. Indeed, studies have shown that EVs retain function after lyophilization 81,82. Although less clinically feasible, Lipo‐NPs could alternatively be stored at −80 °C (for up to two years), as studies have shown that both EVs and lipoproteins remain stable under such conditions 83,84…”

Section: Resultsmentioning

confidence: 99%

Adipose‐Derived Biogenic Nanoparticles for Suppression of Inflammation

Tian

Ticer

Wang

et al. 2020

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Extracellular vesicles secreted from adipose‐derived mesenchymal stem cells (ADSCs) have therapeutic effects in inflammatory diseases. However, production of extracellular vesicles (EVs) from ADSCs is costly, inefficient, and time consuming. The anti‐inflammatory properties of adipose tissue‐derived EVs and other biogenic nanoparticles have not been explored. In this study, biogenic nanoparticles are obtained directly from lipoaspirate, an easily accessible and abundant source of biological material. Compared to ADSC‐EVs, lipoaspirate nanoparticles (Lipo‐NPs) take less time to process (hours compared to months) and cost less to produce (clinical‐grade cell culture facilities are not required). The physicochemical characteristics and anti‐inflammatory properties of Lipo‐NPs are evaluated and compared to those of patient‐matched ADSC‐EVs. Moreover, guanabenz loading in Lipo‐NPs is evaluated for enhanced anti‐inflammatory effects. Apolipoprotein E and glycerolipids are enriched in Lipo‐NPs compared to ADSC‐EVs. Additionally, the uptake of Lipo‐NPs in hepatocytes and macrophages is higher. Lipo‐NPs and ADSC‐EVs have comparable protective and anti‐inflammatory effects. Specifically, Lipo‐NPs reduce toll‐like receptor 4‐induced secretion of inflammatory cytokines in macrophages. Guanabenz‐loaded Lipo‐NPs further suppress inflammatory pathways, suggesting that this combination therapy can have promising applications for inflammatory diseases.

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Section: Resultsmentioning

confidence: 99%

Adipose‐Derived Biogenic Nanoparticles for Suppression of Inflammation

Tian

Ticer

Wang

et al. 2020

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“…The resuspended pellet was purified by size-exclusion chromatography (SEC) column loaded with Sepharose CL-2B (GE Life Science, UK) to separate MVs from soluble protein impurities. Eluted fractions of 1 mL were collected in polypropylene tubes (Axygen, Corning, Germany) and then stored at 4 • C for no longer than a week (27).…”

Section: Purificationmentioning

confidence: 99%

Streptococcal Extracellular Membrane Vesicles Are Rapidly Internalized by Immune Cells and Alter Their Cytokine Release

et al. 2020

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Extracellular vesicles are membranous structures shed by almost every living cell. Bacterial gram-negative outer membrane vesicles (OMVs) and gram-positive membrane vesicles (MVs) play important roles in adaptation to the surrounding environment, cellular components' exchange, transfer of antigens and virulence factors, and infection propagation. Streptococcus pneumoniae is considered one of the priority pathogens, with a global health impact due to the increase in infection burden and growing antibiotic resistance. We isolated MVs produced from the S. pneumoniae reference strain (R6) and purified them via size exclusion chromatography (SEC) to remove soluble protein impurities. We characterized the isolated MVs by nanoparticle tracking analysis (NTA) and measured their particle size distribution and concentration. Isolated MVs showed a mean particle size range of 130-160 nm and a particle yield of around 10 12 particles per milliliter. Cryogenic transmission electron microscopy (cryo-TEM) images revealed a very heterogeneous nature of isolated MVs with a broad size range and various morphologies, arrangements, and contents. We incubated streptococcal MVs with several mammalian somatic cells, namely, human lung epithelial A549 and human keratinocytes HaCaT cell lines, and immune cells including differentiated macrophage-like dTHP-1 and murine dendritic DC2.4 cell lines. All cell lines displayed excellent viability profile and negligible cytotoxicity after 24-h incubation with MVs at concentrations reaching 10 6 MVs per cell (somatic cells) and 10 5 MVs per cell (immune cells). We evaluated the uptake of fluorescently labeled MVs into these four cell lines, using flow cytometry and confocal microscopy. Dendritic cells demonstrated prompt uptake after 30-min incubation, whereas other cell lines showed increasing uptake after 2-h incubation and almost complete colocalization/internalization of MVs after only 4-h incubation. We assessed the influence of streptococcal MVs on antigen-presenting cells, e.g., dendritic cells, using enzyme-linked immunosorbent assay (ELISA) and observed enhanced release of tumor necrosis factor (TNF)-α, a slight increase of interleukin (IL)-10 Mehanny et al. Streptococcal Membrane Vesicles secretion, and no detectable effect on IL-12. Our study provides a better understanding of gram-positive streptococcal MVs and shows their potential to elicit a protective immune response. Therefore, they could offer an innovative avenue for safe and effective cell-free vaccination against pneumococcal infections.

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“…Several studies were dedicated specifically to EVs stability at +4˚C or room temperature or contain large sections devoted to this topic [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. Additional pieces of information could be found in research papers, where the EVs stability has been checked [29][30][31][32][33][34][35][36][37] in line with the general topic.…”

Section: Introductionmentioning

confidence: 99%

Adsorption of extracellular vesicles onto the tube walls during storage in solution

et al. 2020

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Short term storage of extracellular vesicle (EV) solutions at +4°C is a common practice, but the stability of EVs during this procedure has not been fully understood yet. Using nanoparticle tracking analysis, we have shown that EVs isolated from the conditioned medium of HT-29 cells exhibit a pronounced concentration decrease when stored in PBS in ordinary polypropylene tubes within the range of (0.5–2.1) × 1010 particles/ml. EV losses reach 51±3% for 0.5 ml of EVs in Eppendorf 2 ml tube at 48 hours of storage at +4°C. Around 2/3 of the observed losses have been attributed to the adsorption of vesicles onto tube walls. This result shows that the lower part (up to at least 2 × 1010 particles/ml) of the practically relevant concentration range for purified EVs is prone to adsorption losses at +4°C. Total particle losses could be reduced to 18–21% at 48 hours by using either Eppendorf Protein LoBind tubes or ordinary tubes with the surface blocked with bovine serum albumin or EVs. Reduction of losses to 15% has been shown for isolated EVs dissolved in the supernatant after 100 000 g centrifugation as a model of conditioned medium. Also, a previously unknown feature of diffusion-controlled adsorption was revealed for EVs. In addition to the decrease in particle count, this process causes the predominant losses of smaller particles.

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Evaluation of the Storage Stability of Extracellular Vesicles

Cited by 20 publications

References 0 publications

Adipose‐Derived Biogenic Nanoparticles for Suppression of Inflammation

Adipose‐Derived Biogenic Nanoparticles for Suppression of Inflammation

Streptococcal Extracellular Membrane Vesicles Are Rapidly Internalized by Immune Cells and Alter Their Cytokine Release

Adsorption of extracellular vesicles onto the tube walls during storage in solution

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