, 2006, Sayes et al., 2006, Herzog R428 clinical trial et al., 2007, Wick et al., 2007, Donaldson and Poland, 2009, Shvedova et al., 2009, Kolosnjaj-Tabi et al., 2010, Nagai et al., 2011 and Haniu et al., 2012b). We recently reported that the cell type also plays a critical role in the biological response to CNTs (Haniu et al., 2011b). BEAS-2B human bronchial epithelial cells, MESO-1 malignant pleural mesothelioma cells, and THP-1 cells differentiated

to macrophage-like cells that, when exposed to MWCNTs, showed cell growth inhibition and increased cytokine secretion. These cells had the potential to internalize MWCNTs into the cytoplasm. Moreover, we showed that the cellular concentration of MWCNTs correlates with cytotoxicity in BEAS-2B and MESO-1 cells (Haniu et al., 2011a). BEAS-2B is the most popular cell line for the evaluation of the respiratory safety of nanomaterials (Herzog et al., 2007, Park et al., 2008 and Eom and Choi, 2009), and it is used in the safety assessment of CNTs (Lindberg et al., 2009, Hirano et al., 2010, He et al., 2011, Tsukahara and Haniu, 2011 and Wang et al., 2011). However, even when the different types of CNTs BIRB 796 datasheet studied are accounted for, the concentrations of CNTs that show cytotoxicity vary greatly. This variability may be caused by the cell culture medium, because cytotoxicity at low CNT concentrations was observed when the cells were cultured in a medium containing

serum, whereas cytotoxicity was only observed at very high CNT concentrations when serum was not present in the medium. In this study, we determined the influence of serum on the cellular responses to MWCNTs and compared

the biological response between BEAS-2B cells and HBEpCs. Moreover, we confirmed the effect of endocytosis of MWCNTs. MWCNTs manufactured by a chemical vapor deposition method were provided by Hodogaya Chemical (MWNT-7; Tokyo, Japan). The properties of these MWCNTs were obtained from Hodogaya Chemicals (Table 1). Autoclave sterilization conditions were 121 °C for 15 min. MWNT-7 was dispersed with 0.1% gelatin (Nippi, Tokyo, Japan) in phosphate-buffered saline (PBS) Montelukast Sodium and sonicated for 30 min by using a water-bath sonicator. The BEAS-2B human bronchial epithelial cell line was purchased from American Type Culture Collection (Manassas, VA, USA). Normal HBEpCs were purchased from Cell Application (San Diego, CA, USA). BEAS-2B cells were cultured in Ham’s nutrient mixture F-12 (Nacalai, Tokyo, Japan) with 10% fetal bovine serum (Ham’s F12) and passaged twice a week, or cultured in bronchial/tracheal epithelial cell serum-free growth medium kit with 0.1 μg/ml retinoic acid (SFGM; Cell Application) and passaged every 4 days in SFGM, with the medium exchanged every other day. HBEpCs were cultured in SFGM and passaged every 4 days, with the medium exchanged every other day. HBEpCs were used by passage 4.