title: Comparative study of culture conditions for maintaining CYP3A4 and ATP-binding cassette transporters activity in primary cultured human hepatocytes.
abstract: The aim of this study was to determine suitable culture conditions for maintaining the activity of cytochrome p450 (CYP) 3A4 and drug transporters in primary cultured human hepatocytes. Human hepatocytes were isolated using the two-step collagenase perfusion technique and were cultured with four different media, serum-free William's E medium (serum-free WEM), WEM containing fetal calf serum (FCS-WEM), WEM with human serum (HS-WEM), and Lanford's medium. The albumin levels were maintained for 7 days in hepatocytes. Although CYP3A4 mRNA levels gradually decreased from 3 days, CYP3A4 and hepatocyte nuclear factor-4α alpha protein levels and activities were maintained for 7 days in hepatocytes cultured with serum-free WEM and Lanford's but not in those with FCS-WEM and HS-WEM. Furthermore, CYP3A4 protein levels were significantly increased by the addition of rifampicin and dexamethasone to the culture media, indicating that the induction potential was maintained. The protein levels of P-glycoprotein, multi-drug-resistance-2, and breast cancer-resistance protein were maintained for 7 days in all media. Serum-free WEM and Lanford's also maintained protein levels of CYP2C19, CYP2D6, and organic anion transporter polypeptide in the hepatocytes. Serum-free WEM and Lanford's may be appropriate culture media for maintaining CYP3A4 and drug transporter protein levels in primary cultured hepatocytes.
authors: Department of Pharmacology, St. Marianna University School of Medicine, Kawasaski, Kanagawa 216-8511, Japan. takebay@marianna-u.ac.jp
authors: Yuko
authors: Y
authors: Takeba
authors: Naoki
authors: N
authors: Matsumoto
authors: Sachiko
authors: S
authors: Takenoshita-Nakaya
authors: Yoshie
authors: Y
authors: Harimoto
authors: Toshio
authors: T
authors: Kumai
authors: Yuichi
authors: Y
authors: Kinoshita
authors: Hiroshi
authors: H
authors: Nakano
authors: Takehito
authors: T
authors: Ohtsubo
authors: Shinichi
authors: S
authors: Kobayashi
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D003470:Culture Media; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D051557:Hepatocyte Nuclear Factor 4 / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D012293:Rifampin / Q000494:pharmacology
chemical_list: D018528:ATP-Binding Cassette Transporters; D003470:Culture Media; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D012293:Rifampin