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    • ghrelin receptor Consistent with our findings a previous stu

    2018-11-01

    Consistent with our findings, a previous study by Rotoli et al. demonstrated a significant decrease in the TEER measurements of Calu-3 monolayers with relatively low doses of MWCNTs (5–10μg/mL), and a striking TEER decrease with exposure of 100μg/mL MWCNTs or SWCNTs [28]. Rotoli et al. also observed that the ghrelin receptor in monolayer permeability was accompanied by an increase in the paracellular permeability of mannitol, and that there was no significant alteration in the cell monolayer viability. As mentioned, our data is in agreement with these results using occupationally relevant concentrations of CNTs, in the range of 0.2 – 6μg/cm2, equivalent to approximately 0.33μg/mL and 9.9μg/mL, respectively. Interestingly, we observed a much more dramatic difference in TEER values between the control and MWCNTs at approximately 10μg/mL. The study by Rotoli et al. utilizes a similar method to that reported herein, with the exception that the authors employed a cell seeding density of 7.5×104cells/well. When considering the results reported in Fig. 1, it is plausible that the increased seeding density resulted in higher baseline TEER values, and thus, required larger concentrations of CNTs to induce a greater decrease in TEER. These results highlight the importance of implementing more standardized methods for the assessment of the effects of ENMs on epithelial cells, and underscore the utility of the results reported here to allow for comparisons between studies. Previous reports have demonstrated that the physicochemical parameters of CNTs, such as surface modification, can greatly influence monolayer formation and barrier integrity. During their interaction with biological tissues, surface chemistry of ENMs plays a key role in determining potential toxic pulmonary responses, including airway perturbation. It is believed that surface functional groups can affect the stability and dispersion of the MWCNT by altering the surface charge and reactivity of the particle, thus influencing the interaction of the particles with cells. Consistently, we observed an ENM- dose dependent effect on barrier function while studying f-MWCNTs compared to non-functionalized MWCNTs. The f-MWCNTs resulted in permeability that was consistent with controls; in contrast, their pristine counterparts greatly altered epithelial integrity. This observation is consistent with the recent report by Sager et al. in which it was demonstrated that f-MWCNTs, the same CNTs that we have used in the current study, were less bioactive and showed reduced signs of inflammation in vivo as compared to non-functionalized MWCNTs [29]. Our findings also suggest that the type of CNT that the epithelium is exposed to plays a prominent role in influencing the extent of airway barrier changes. For instance, we observed that pristine MWCNT caused a large decrease in TEER as compared to controls, whereas the SWCNTs had significantly smaller effects. MWCNTs have been shown to have a lower surface/volume ratio as compared to SWCNTs, and such structural differences may contribute to the greater perturbations of barrier function induced by MWCNTs [28]. This may be related to the morphology of the MWCNTs compared to SWCNTs, as MWCNTs tend to be observed as more rigid fibers that may be capable of poking through the epithelium [20,40], while SWCNTs can often be found as more flexible, rounded particles [39,40]. In addition, length-based impairment of barrier function has been reported using SWCNTs and MWCNTs of different lengths, where longer SWCNTs and MWCNTs induced a larger reduction in TEER values, indicating that fiber length led to a greater amount of epithelial dysfunction [27].
    Conclusions In conclusion, this study demonstrated that the Calu-3 epithelial cell line is a highly effective in vitro epithelial barrier model that can be implemented to test the effects of ENM on the epithelium. Cell density and serum concentration are major factors affecting the formation of monolayer complexes, and should be carefully considered and reported. We have found that 5.0×104cells/well in the Transwell cell culture system is optimal for assessing epithelial cell barrier function, and that 10% serum concentrations in culture media should be maintained. Furthermore, SWCNT and MWCNTs were found to induce a dose- and time-dependent change in monolayer integrity of Calu-3 cells in vitro, and the physicochemical characteristics of CNTs, such as COOH-functionalization and the type of CNT, can influence monolayer formation and permeability. Overall, this work demonstrates a well-characterized model of in vitro epithelial barrier function by providing the experimental conditions required for achieving tight junction complexes and relevant CNT exposure doses, which further aid in predicting and comparing the interaction of numerous novel nanoparticles with the biological system and mimic the respiratory behavior in vivo.