nanoparticle tracking analysis
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2022 ◽  
Vol 13 (1) ◽  
Ramana Vaka ◽  
Saad Khan ◽  
Bin Ye ◽  
Yousef Risha ◽  
Sandrine Parent ◽  

Abstract Background Although 90% of infections with the novel coronavirus 2 (COVID-19) are mild, many patients progress to acute respiratory distress syndrome (ARDS) which carries a high risk of mortality. Given that this dysregulated immune response plays a key role in the pathology of COVID-19, several clinical trials are underway to evaluate the effect of immunomodulatory cell therapy on disease progression. However, little is known about the effect of ARDS associated pro-inflammatory mediators on transplanted stem cell function and survival, and any deleterious effects could undermine therapeutic efficacy. As such, we assessed the impact of inflammatory cytokines on the viability, and paracrine profile (extracellular vesicles) of bone marrow-derived mesenchymal stromal cells, heart-derived cells, and umbilical cord-derived mesenchymal stromal cells. Methods All cell products were manufactured and characterized to established clinical release standards by an accredited clinical cell manufacturing facility. Cytokines and Extracellular vesicles in the cell conditioned media were profiled using proteomic array and nanoparticle tracking analysis. Using a survey of the clinical literature, 6 cytotoxic cytokines implicated in the progression of COVID-19 ARDS. Flow cytometry was employed to determine receptor expression of these 6 cytokines in three cell products. Based on clinical survey and flow cytometry data, a cytokine cocktail that mimics cytokine storm seen in COVID-19 ARDS patients was designed and the impact on cytokine cocktail on viability and paracrine secretory ability of cell products were assessed using cell viability and nanoparticle tracking analysis. Results Flow cytometry revealed the presence of receptors for all cytokines but IL-6, which was subsequently excluded from further experimentation. Despite this widespread expression, exposure of each cell type to individual cytokines at doses tenfold greater than observed clinically or in combination at doses associated with severe ARDS did not alter cell viability or extracellular vesicle character/production in any of the 3 cell products. Conclusions The paracrine production and viability of the three leading cell products under clinical evaluation for the treatment of severe COVID-19 ARDS are not altered by inflammatory mediators implicated in disease progression.

2021 ◽  
Xiao-Dong Yang ◽  
Qingfeng Yang ◽  
Zhou Limin ◽  
Li-Juan Zhang ◽  
Jun Hu

Abstract The anoxia of coastal water has already been a serious problem all over the word. Nanobubbles are proved to have great applications in water remediation because they could effectively increase the oxygen content and degrade organic matters in water. But the existing methods to produce nanobubbles are complicated and high cost to operate, especially in deep sea. In this paper, we presented a low-cost method, hydraulic air compression (HAC), to produce a large number of nanobubbles and proved that nanoscale gas bubbles could be produced by HAC for the first time. Nanoparticle tracking analysis was used to measure the size and concentration of produced nanobubbles. It indicated that the concentration of nanobubbles would increase as the downpipe height increases. Degassed measurements proved that produced “nanoparticles” are gas nanobubbles indeed. More dissolved oxygen in water would provide the source for larger number of nanobubble formation. Those results are expected to be very helpful for water remediation in ocean in the future.

2021 ◽  
Vol 14 (12) ◽  
pp. 1820-1827
Guang-Hui He ◽  
Meng Dong ◽  
Song Chen ◽  
Yu-Chuan Wang ◽  

AIM: To determine the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSCs) on the expression of vascular endothelial growth factor A (VEGF-A) in human retinal vascular endothelial cells (HRECs). METHODS: Exosomes were isolated from hUCMSCs using cryogenic ultracentrifugation and characterized by transmission electron microscopy, Western blotting and nanoparticle tracking analysis. HRECs were randomly divided into a normal control group (group A), a high glucose model group (group B), a high glucose group with 25 μg/mL (group C), 50 μg/mL (group D), and 100 μg/mL exosomes (group E). Twenty-four hours after coculture, the cell proliferation rate was detected using flow cytometry, and the VEGF-A level was detected using immunofluorescence. After coculture 8, 16, and 24h, the expression levels of VEGF-A in each group were detected using PCR and Western blots. RESULTS: The characteristic morphology (membrane structured vesicles) and size (diameter between 50 and 200 nm) were observed under transmission electron microscopy. The average diameter of 122.7 nm was discovered by nanoparticle tracking analysis (NTA). The exosomal markers CD9, CD63, and HSP70 were strongly detected. The proliferation rate of the cells in group B increased after 24h of coculture. Immunofluorescence analyses revealed that the upregulation of VEGF-A expression in HRECs stimulated by high glucose could be downregulated by cocultured hUCMSC-derived exosomes (F=39.03, P<0.01). The upregulation of VEGF-A protein (group C: F=7.96; group D: F=17.29; group E: F=11.89; 8h: F=9.45; 16h: F=12.86; 24h: F=42.28, P<0.05) and mRNA (group C: F=4.137; group D: F=13.64; group E: F=22.19; 8h: F=7.253; 16h: F=16.98; 24h: F=22.62, P<0.05) in HRECs stimulated by high glucose was downregulated by cocultured hUCMSC-derived exosomes (P<0.05). CONCLUSION: hUCMSC-derived exosomes downregulate VEGF-A expression in HRECs stimulated by high glucose in time and concentration dependent manner.

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3473
Magdalena Dlugolecka ◽  
Jacek Szymanski ◽  
Lukasz Zareba ◽  
Zuzanna Homoncik ◽  
Joanna Domagala-Kulawik ◽  

The current lack of reliable methods for quantifying extracellular vesicles (EVs) isolated from complex biofluids significantly hinders translational applications in EV research. The recently developed fluorescence nanoparticle tracking analysis (FL-NTA) allows for the detection of EV-associated proteins, enabling EV content determination. In this study, we present the first comprehensive phenotyping of bronchopulmonary lavage fluid (BALF)-derived EVs from non-small cell lung cancer (NSCLC) patients using classical EV-characterization methods as well as the FL-NTA method. We found that EV immunolabeling for the specific EV marker combined with the use of the fluorescent mode NTA analysis can provide the concentration, size, distribution, and surface phenotype of EVs in a heterogeneous solution. However, by performing FL-NTA analysis of BALF-derived EVs in comparison to plasma-derived EVs, we reveal the limitations of this method, which is suitable only for relatively pure EV isolates. For more complex fluids such as plasma, this method appears to not be sensitive enough and the measurements can be compromised. Our parallel presentation of NTA-based phenotyping of plasma and BALF EVs emphasizes the great impact of sample composition and purity on FL-NTA analysis that has to be taken into account in the further development of FL-NTA toward the detection of EV-associated cancer biomarkers.

2021 ◽  
Vol 22 (22) ◽  
pp. 12436
Marvin Droste ◽  
Tobias Tertel ◽  
Stefanie Jeruschke ◽  
Robin Dittrich ◽  
Evangelia Kontopoulou ◽  

Small extracellular vesicles isolated from urine (uEVs) are increasingly recognized as potential biomarkers. Meanwhile, different uEV preparation strategies exist. Conventionally, the performance of EV preparation methods is evaluated by single particle quantification, Western blot, and electron microscopy. Recently, we introduced imaging flow cytometry (IFCM) as a next-generation single EV analysis technology. Here, we analyzed uEV samples obtained with different preparation procedures using nanoparticle tracking analysis (NTA), semiquantitative Western blot, and IFCM. IFCM analyses demonstrated that urine contains a predominant CD9+ sEV population, which exceeds CD63+ and CD81+ sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9+ sEVs. Although overall reduced, the highest CD9+ sEV recovery was obtained from urine samples stored at −80 °C and the lowest from those stored at −20 °C. Upon comparing the yield of the different uEV preparations, incongruencies between NTA and IFCM data became apparent. Results obtained by both NTA and IFCM were consistent with Western blot analyses for EV marker proteins; however, NTA results correlated with the amount of the impurity marker uromodulin. Despite demonstrating that the combination of ultrafiltration and size exclusion chromatography appears as a reliable uEV preparation technique, our data challenge the soundness of traditional NTA for the evaluation of different EV preparation methods.

2021 ◽  
Vol 11 (1) ◽  
Jeong Il Lee ◽  
Han Sol Huh ◽  
Joong Yull Park ◽  
Jung-Geun Han ◽  
Jong-Min Kim

AbstractIn recent years, minuscule gas bubbles called bulk nanobubbles (BNBs) have drawn increasing attention due to their unique properties and broad applicability in various technological fields, such as biomedical engineering, water treatment, and nanomaterials. However, questions remain regarding the stability and behavior of BNBs. In the present work, BNBs were generated in water using a gas–liquid mixing method. NB analysis was performed using a nanoparticle tracking analysis (NTA) method to investigate the coarsening behavior of BNBs in water over time. The diameters of the BNBs increased, and their cubic radii increased linearly (r3 ~ t) over time. While the concentration of BNBs decreased, the total volume of BNBs remained the same. The size distribution of the BNBs broadened, and the concentration of larger BNBs increased over time. These results indicate that relatively small BNBs disappeared due to dissolution and larger BNBs grew through mass transfer between BNBs instead of coalescence. In other words, BNBs underwent Ostwald ripening: gas molecules from smaller BNBs diffused through the continuous phase to be absorbed into larger BNBs.

2021 ◽  
Vol 22 (19) ◽  
pp. 10343
Yuanfei Zhou ◽  
Mao Xia ◽  
Chenbin Cui ◽  
Hongkui Wei ◽  
Siwen Jiang ◽  

Maternal obesity disrupts both placental angiogenesis and fetus development. However, the links between adipocytes and endothelial cells in maternal obesity are not fully understood. The aim of this study was to characterize exosome-enriched miRNA from obese sow’s adipose tissue and evaluate the effect on angiogenesis of endothelial cells. Plasma exosomes were isolated and analyzed by nanoparticle tracking analysis (NTA), electron morphological analysis, and protein marker expression. The number of exosomes was increased as the gestation of the sows progressed. In addition, we found that exosomes derived from obese sows inhibited endothelial cell migration and angiogenesis. miRNA detection showed that miR-221, one of the miRNAs, was significantly enriched in exosomes from obese sows. Further study demonstrated that exosomal miR-221 inhibited the proliferation and angiogenesis of endothelial cells through repressing the expression of Angptl2 by targeting its 3′ untranslated region. In summary, miR-221 was a key component of the adipocyte-secreted exosomal vesicles that mediate angiogenesis. Our study may be a novel mechanism showing the secretion of “harmful” exosomes from obesity adipose tissues causes placental dysplasia during gestation.

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