Comparative Study of Culture Conditions for Maintaining CYP3A4 and ATP-Binding Cassette Transporters Activity in Primary Cultured Human Hepatocytes

Abstract

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.

Anti-CD28 Monoclonal Antibody–Stimulated Cytokines Released from Blood Suppress CYP1A2, CYP2B6, and CYP3A4 in Human Hepatocytes In Vitro

“…at ASPET Journals on May 10, 2018 dmd.aspetjournals.org the levels of immunoreactive albumin, HNF-4a, CYP2C enzymes, or CYP2D6 (Takeba et al, 2011). In our study, although the two control plasma samples had similar effects on CYP1A2, their effects on CYP3A4 mRNA differed (Fig.…”

“…The effects of saline plasma were comparable to those of naïve plasma (data not shown). Inclusion of isotype-specific IgG control for evaluation of ANC28.1 The effects of plasma during an extended culture of human hepatocytes on DMEs have not been reported in the literature, but the effects of human serum have been described by Takeba et al (2011). Compared with medium alone, human serum (10% in Williams' E medium) reduced the levels of immunoreactive CYP3A enzymes in fresh-plated human hepatocytes cultured for up to 7 days.…”

Increased Levels of Fatty Acids Contributed to Induction of Hepatic CYP3A4 Activity Induced by Diabetes — In Vitro Evidence From HepG2 Cell and Fa2N-4 Cell Lines

“…Primary human hepatocytes are considered to be the gold standard model for xenobiotic metabolism, but large interindividual donor variability, rapid loss of drug metabolizing enzyme expression, poor availability, and high cost have limited its application (17,18). Several human hepatic cell lines (e.g., HepG2, LS180, Fa2N4, and HepaRG) have been introduced to overcome the previously mentioned drawbacks despite the lower constitutive levels of drugmetabolizing enzymes in these cell lines relative to human hepatocytes.…”

Functional expression and regulation of drug transporters in monolayer- and sandwich-cultured mouse hepatocytes

“…By contrast, sandwich-cultured rat hepatocytes exhibit functional canalicular networks (LeCluyse et al, 1994;Liu et al, 1999b), even if they also display reduced expression of sinusoidal influx transporters with time in culture when compared to freshly isolated hepatocytes (Borlak and Klutcka, 2004;Kotani et al, 2011;Tchaparian et al, 2011). With regard to human hepatocytes cultured either in monolayer or sandwich conditions, expression of drug transporters appears to be much better preserved with time in culture when compared to rat 4 counterparts (Hoffmaster et al, 2004;Jigorel et al, 2005;Kotani et al, 2011;Li et al, 2009;Schaefer et al, 2012;Takeba et al, 2011).…”