Human HepaRG Cells can be Cultured in Hanging-drop Plates for Cytochrome P450 Induction and Function Assays

Title:Human HepaRG Cells can be Cultured in Hanging-drop Plates for Cytochrome P450 Induction and Function Assays

Volume: 9 Issue: 1

Author(s): Norie Murayama, Takashi Usui, Nicky Slawny, Christophe Chesne and Hiroshi Yamazaki

Affiliation:

Keywords: CYP1A2, CYP2B6, CYP3A4, drug interaction, hanging-drop, HepaRG, human hepatocytes.

Abstract: Recent guidance/guidelines for industry recommend that cytochrome P450 induction can be assessed using human hepatocyte enzyme activity and/or mRNA levels to evaluate potential drug– drug interactions. To evaluate time-dependent cytochrome P450 induction precisely, induction of CYP1A2, CYP2B6, and CYP3A4 mRNA was confirmed (> 2-fold) by the treatment with omeprazole, phenobarbital, and rifampicin, respectively, for 24 or 48 h on day 3 from the start of culture. After 24 h, the fold induction of CYP1A2 with 3.6 and 1.8 x 10⁴ HepaRG cells per well was lower than that for 7.2 x 10⁴ cells. CYP1A2 induction levels at 24 h were higher than those after 48 h. In contrast, higher CYP2B6 inductions were confirmed after 48 h exposure than after 24 h, independent of the number of cells per well. To help reduce the use of human cryopreserved hepatocytes, typical P450-dependent enzyme activities were investigated in human HepaRG cells cultured in commercial hanging-drop plates. Newly designed 96-well hanging-drop plates were capable of maintaining human CYP3A-dependent midazolam hydroxylation activities for up to 4 days using only 10% of the recommended initial 7.2 x 10⁴ cells per well. Favorable HepaRG function using hanging-drop plates was confirmed by detecting 1′- hydroxymidazolam O-glucuronide on day 3, suggesting an improvement over traditional control plates in which this metabolite can be detected for 24-well plates. These results suggest that the catalytic function and/or induction of CYP1A2, CYP2B6, and CYP3A4 can be readily assessed with reduced numbers of starting HepaRG cells cultured in three-dimensional cultures in drops prepared with hanging-drop plates.

Cite this article as:

Murayama Norie, Usui Takashi, Slawny Nicky, Chesne Christophe and Yamazaki Hiroshi, Human HepaRG Cells can be Cultured in Hanging-drop Plates for Cytochrome P450 Induction and Function Assays, Drug Metabolism Letters 2015; 9 (1) . https://dx.doi.org/10.2174/1872312809666150119104806