br Materials and methods br Results br

Materials and methods
Results
Discussion This study revealed the optimal conditions required to investigate in vitro epithelial barrier formation using Calu-3 cells, to be used as a screening model for ENM exposure to mimic in vivo conditions. Details on the specific cell line, cell number, serum concentration, and duration required to achieve stable monolayer formation have been described herein to establish a consistent, reliable in vitro method for further implementation within the field. Additionally, CNT exposure, including dose range and various types of CNTs, was used to test this model for its utility in examining the potential health hazards of ENM exposure in an in vitro manner. TEER is commonly used as an indirect parameter of tight junction formation and as a measure of epithelial monolayer integrity and permeability [32,36]. Thus, this technique offers fast and efficient analysis of the integrity of epithelial barriers, and we have employed this tool as a biosensor in our model in an effort to standardize more high-throughput in vitro screening of ENMs for potential health hazards. Our study has also utilized the Calu-3 cell line, which is commonly used for tracheobronchial epithelial cell studies given their ability to form tight junction complexes in vitro. They display several of the primary culture properties and exhibit characteristics of differentiated, functional human airway epithelia [9,33]. Previous studies have demonstrated that Calu-3 palmitic acid develop a high TEER and exhibit a similar pattern of expression of epithelial markers when compared to primary cells [2]. More importantly, Calu-3 cells are immortalized and of human origin, thereby overcoming the constraints of using primary differentiated cell cultures, including cost and limited passage number. Our results indicate that both cell seeding density, as well as FBS concentration can influence monolayer formation, as indicated by the changes in TEER values under the various conditions. Many extracellular stimuli, including nutrients, cytokines, and immune cells, have been shown to regulate or alter the barrier function of tight junctions, while serum proteins may open the already established tight junctions or inhibit their formation, thus influencing changes in TEER [22]. Accordingly, we found that high serum concentrations (15% and 10%) altered monolayer integrity, as indicated by low TEER measurements when compared with TEER values for lower serum concentrations at the same time point. Calu-3 cells cultured with serum concentrations as low as 2% displayed considerably increased TEER values, which corresponds with increased tight junction formation. Importantly, the serum effects were found to be dose-dependent and the barrier-weakening effect was not mediated by cell density since the same cell number was utilized for all serum concentrations. Our results also agree with earlier studies showing the inhibitory effect of serum on tight junctions in the blood brain barrier [5,6]. Thus, we suggest that using a concentration of 10% FBS in culture media provides satisfactory growth conditions while minimizing the effect of serum on TEER values. It is also noteworthy that the observed TEER values for this concentration of FBS in culture medium resulted in the most consistent TEER values at days 10 through 14 in culture compared with the other concentrations tested. The increased use and demand for ENMs in a wide range of industries has fostered the need for more in-depth analysis on the health hazards following exposure. The potential exposure to CNTs of workers in small-scale production facilities has been reported by field studies to be airborne in trace amounts up to a few thousandμg/m3[10,17]. Consistently, a recent study determined the average inhalable elemental carbon mass concentration at 8 facilities within the United States to be approximately 10μg/m3[8]. Hence, correlating exposure levels in cultured cells with the aerosolized levels likely to occur in the workspace is essential, owing to the probable biological effects on respiratory barrier function and mucocilliary clearance. To achieve this goal, our study made use of the occupationally relevant concentration range of 0.2–6μg/cm2 of CNTs [19,24,37,40]. As evident in Fig. 5, MWCNTs caused dose- and time-dependent reductions in epithelial barrier integrity using these relevant concentrations. At low doses of 0.2 and 0.6μg/cm2, MWCNTs had no effect on TEER values; however, at higher doses of 2 and 6μg/cm2, MWCNTs induced a significant decrease in the resistance compared to controls after only 24h of treatment (day 10 in culture). Conversely, f-MWCNTs and SWCNTs at the same high doses did not elicit significant changes in monolayer resistance, demonstrating a particle type-dependent alteration of airway barrier function.