Airway epithelial cells present as one of the first

Airway epithelial cells, present as one of the first lines of defense for inhaled particulate matter, represent a major determinant in the interaction of a foreign body with other major body compartments. In addition, epithelial eletriptan cost are involved with the formation and maintenance of tight junctions between neighboring epithelial cells, only permitting polarized secretory functions (such as ion transport) and routine cellular trafficking, while preventing access to xenobiotics and pathogens [1,28,32,34]. Moreover, these tight junctions assist in keeping cytokines, toxins, and pathogens from infiltrating the epithelial layer. Indeed, these dynamic protein structures govern the paracellular permeability of the epithelium, permitting only that which is necessary under normal circumstances [13,26]. Consequently, pulmonary barrier function plays a pivotal role in controlling penetration of inhaled nanoparticles into the interstitium, which can lead to rapid interstitial fibrosis [19,35]. For example, dispersed single- or multi-walled CNTs rapidly enter the alveolar interstitial space and induce a progressive interstitial fibrotic response with minimal lung inflammation [19,23,35]. However, due to a lack of studies evaluating the effects of CNT exposure on lung epithelial barriers, a key determinant of pulmonary toxicity due to ENMs, the pathogenic mechanisms underlying these effects have not been fully elucidated. This is due, in part, to a lack of effective, consistent methods to reliably predict in vivo outcomes in an in vitro setting. Further compounding this issue is the notion that unique physicochemical characteristics of ENMs, such as particle size, shape, and surface modification, may contribute to progressive, toxic responses, including heightened lung epithelial barrier permeability. To date, it remains inadequately understood how each of these fundamental features distinctly affects the airway epithelium. In addition, due to the escalating costs and limitations of evaluating individual cell type-specific outcomes when animals are exposed to ENMs in vivo, more in vitro approaches are being explored as alternatives. Epithelial cells in culture form tight junction complexes, thereby making in vitro models of lung epithelium a favorable choice to mimic and predict the respiratory behavior in vivo upon exposure to ENMs such as CNT materials [30]. Several studies have sought to explore the effects of various ENMs on epithelial function in vitro using the Calu-3 small airway epithelial cell line [21,28,38]; however, inconsistent methods to do so limit comparisons and conclusions. Hence, our current study utilized the Calu-3 cell line that demonstrates the characteristics of differentiated, functional human epithelia [9,33] with the primary objective to provide the technical details and characterization of an in vitro pulmonary barrier model to consistently screen for the pathogenicity of various nanoparticles on lung epithelium. Trans-epithelial electrical resistance (TEER) is a commonly used endpoint to assess integrity and permeability of epithelial monolayers, as it is an instantaneous measure of ion flux [32] and an indirect measurement of the formation of tight junctions [36]. Thus, we have employed this parameter for use as a biosensor for epithelial monolayer formation and integrity. In addition, the peculiar effects of cell number and serum constituents have been examined in an effort to reliably study the effect of exposed particles on lung epithelial barrier permeability in vitro. Providing the results of our various testing strategies to optimize these conditions will not only provide consistent methodologies that can be implemented by others, but will also help to elucidate potential differences in past studies that have used alternate culture conditions with similar model systems, particularly Calu-3 cells. Additionally, as a means to substantiate this model, we have utilized the optimized conditions outlined in this study with an exposure to CNTs. We hypothesized that the physicochemical properties of CNTs play a key role in determining the effects that these particles have on epithelial barrier integrity, thereby affecting penetration of CNTs into the interstitium. Such high-throughput in vitro cell models of the airway epithelium could be advantageous in predicting the interaction of other nanoparticles with lung epithelial barriers to mimic the respiratory behavior in vivo.