title: Culture medium type affects endocytosis of multi-walled carbon nanotubes in BEAS-2B cells and subsequent biological response.
abstract: We examined the cytotoxicity of multi-walled carbon nanotubes (MWCNTs) and the resulting cytokine secretion in BEAS-2B cells or normal human bronchial epithelial cells (HBEpCs) in two types of culture media (Ham's F12 containing 10% FBS [Ham's F12] and serum-free growth medium [SFGM]). Cellular uptake of MWCNT was observed by fluorescent microscopy and analyzed using flow cytometry. Moreover, we evaluated whether MWCNT uptake was suppressed by 2 types of endocytosis inhibitors. We found that BEAS-2B cells cultured in Ham's F12 and HBEpCs cultured in SFGM showed similar biological responses, but BEAS-2B cells cultured in SFGM did not internalize MWCNTs, and the 50% inhibitory concentration value, i.e., the cytotoxicity, was increased by more than 10-fold. MWCNT uptake was suppressed by a clathrin-mediated endocytosis inhibitor and a caveolae-mediated endocytosis inhibitor in BEAS-2B cells cultured in Ham's F12 and HBEpCs cultured in SFGM. In conclusion, we suggest that BEAS-2B cells cultured in a medium containing serum should be used for the safety evaluation of nanomaterials as a model of normal human bronchial epithelial cells. However, the culture medium composition may affect the proteins that are expressed on the cytoplasmic membrane, which may influence the biological response to MWCNTs.
authors: Department of Orthopaedic Surgery, Shinshu University School of Medicine, Matsumoto, Nagano, Japan. hhaniu@shinshu-u.ac.jp
authors: Hisao
authors: H
authors: Haniu
authors: Naoto
authors: N
authors: Saito
authors: Yoshikazu
authors: Y
authors: Matsuda
authors: Tamotsu
authors: T
authors: Tsukahara
authors: Kayo
authors: K
authors: Maruyama
authors: Yuki
authors: Y
authors: Usui
authors: Kaoru
authors: K
authors: Aoki
authors: Seiji
authors: S
authors: Takanashi
authors: Shinsuke
authors: S
authors: Kobayashi
authors: Hiroki
authors: H
authors: Nomura
authors: Masanori
authors: M
authors: Okamoto
authors: Masayuki
authors: M
authors: Shimizu
authors: Hiroyuki
authors: H
authors: Kato
mesh_terms: D001980:Bronchi / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D016207:Cytokines / Q000378:metabolism; D004705:Endocytosis; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D037742:Nanotubes, Carbon / Q000633:toxicity*; D044967:Serum
chemical_list: D003470:Culture Media; D016207:Cytokines; D037742:Nanotubes, Carbon
keywords: AB; Alamar blue; BEAS-2B cells; CNT; Cytotoxicity; DIC; Dulbecco’s-PBS containing 10% FBS; Endocytosis; F-DPBS; FCM; H33342; HBEpC; Ham’s F12; Ham’s F12 containing 10% fetal bovine serum; IL; MWCNT; Multi-walled carbon nanotube; Normal human bronchial epithelial cells; PBS; SE; SFGM; SSC; bisbenzimide H33342 fluorochrome trihydrochloride; carbon nanotube; differential interference contrast; flow cytometry; human bronchial epithelial cell; interleukin; multi-walled carbon nanotube; phosphate buffered saline; serum-free growth medium; side scatter; standard error