We reported the enhanced liver-specific function and structure of HepG2 cells by the oxygenation culture via a collagen vitrigel membrane (CVM). The cells were conditioned in our laboratory for a long period, so their characteristics may change from the original HepG2 cells registered in RIKEN cell bank (RCB) with the number of 1648 (HepG2-RCB1648 cells). We named the conditioned HepG2-RCB1648 cells in our laboratory as HepG2-NIAS cells. Here, we clarified the features of HepG2 cells with three

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... different culture histories by analyzing their morphology and viability, CYP3A4 activity, the potential to form bile canaliculus-like structures, and the expression of drug transporters. On plastic, HepG2-NIAS cells grew as a monolayer without the formation of large aggregates involving dead cells that were observed in HepG2-RCB1648 cells and HepG2-RCB1886 cells. In the oxygenation culture via a CVM, the CYP3A4 activity of HepG2-NIAS cells increased to almost half level in direct comparison to that of differentiated HepaRG cells cultured on a collagen-coated plate; however, that of HepG2-RCB1648 cells and HepG2-RCB1886 cells was almost not detected. HepG2-NIAS cells formed bile canaliculus-like networks in which fluorescein was accumulated after the exposure of fluorescein diacetate, although HepG2-RCB1648 cells and HepG2-RCB1886 cells did not possess the potential. Also, immunohistological observations revealed that HepG2-NIAS cells remarkably enhanced the expression of drug transporters, NTCP, OATP1B1, OATP1B3, BSEP, MDR1, MRP2, and BCRP. These results suggest that HepG2-NIAS cells are a new subline of HepG2 cells useful for drug development studies. HepG2-NIAS cells were registered in RCB with the number of 4679.