A two-dimensional multiwell cell culture method for the production of CYP3A4-expressing hepatocyte-like cells from HepaRG cells

Abstract

Cytochrome P450 enzymes (CYP) function in drug metabolism in the liver. To evaluate numerous drug candidates, a high-content screening (HCS) system with hepatocyte-like cells (HLCs) that can replace adult human hepatocytes is required. Human hepatocellular carcinoma HepaRG is the only cell line capable of providing HLCs with high CYP3A4 expression comparable to that in adult hepatocytes after cell differentiation. The aim of this study was to design an ideal multiwell culture system for HLCs using transgenic HepaRG cells expressing the EGFP coding an enhanced green fluorescent protein under CYP3A4 transcriptional regulation. HLCs were matured on five different types of 96-well black plates. Culturing HLCs on glass-bottom Optical CVG plates significantly promoted cell maturation and increased metabolic activity by twofold under two-dimensional (2D) culture conditions, and these features were enhanced by 2% collagen coating. Three plates for three-dimensional (3D) cell cultures with a gas-exchangeable fabric or dimethylpolysiloxane membrane bottom formed multiple round colonies, whereas they were ineffective for CYP3A4 expression. Under optimized conditions presented here, HLCs lost responsiveness to nuclear receptor-mediated transcriptional induction of CYP3A4, suggesting that CYP3A4 transcription has already been fully upregulated. Therefore, HepaRG-derived HLCs will provide an alternative to human hepatocytes with high levels of CYP3A4 enzyme activity even under 2D culture conditions. This will improve a variety of drug screening methods.

Keywords: CYP3A4; CYP3A7; HepaRG; a dual-color reporter; cytochrome P450 enzymes; hepatocyte-like cells; high-content screening.

FIGURE 1

Effect of cell culture plate type on cell proliferation and colony formation of hepatocyte‐like cells (HLCs) differentiated from transgenic CYP3A4G/7R HepaRG. A, Structure of the CYP3A4/7R BAC reporter. EGFP and DsRed were used as transcriptional reporter genes for CYP3A4 and CYP3A7, respectively. B, Schematic showing the cell culture schedule. C, Morphology and fluorescent images of differentiated CYP3A4G/7R HepaRG cells. D2, 2 days; 4 W, 4 weeks; PhC, phase contrast image. D, Illustrations showing the characteristics of a well in each plate. E, Colony morphology of D10 HLCs after replating

FIGURE 2

Effect of cell culture plate type on maturation into hepatocyte‐like cells (HLCs) expressing EGFP fluorescence. A, EGFP‐positive cell images in each well of 96‐well plates captured by confocal microscopy (upper). Enlarged view of the center of the well (bottom). B, Semi‐quantitative fluorometric evaluation of EGFP‐positive HLC frequencies. Relative values were calculated according to the average value obtained from D9 cells cultured on O plates in the 1.7% DMSO medium taken as 100 (n = 3). Black circles, cells were cultured in the 1.7% DMSO medium; open circles, DMSO concentration reduced from 1.7% to 0.1% on the last 3 days of the cell culture period. C, Comparison of relative fluorescence intensity of EGFP in D10 HLCs between five plates (n = 3). D, Relative cell numbers were estimated based on the fluorescence intensity of DNA staining with Hoechst 33 258

FIGURE 3

Protein expression for general hepatocyte‐specific markers in D10 HepaRG cells cultured on the O plate. A, CYP3A4 and EGFP; B, HNF4A, ALB, and CK8/18. a–d, An enlarged view of the center of the well is shown in A and B

FIGURE 4

Effect of culture plate type on fluorometric CYP3A4 transcriptional induction tests. A, Images of EGFP‐positive cells were captured on D10 after culturing in medium containing 0.1% DMSO (upper, untreated control) or 10 µmol L⁻¹ rifampicin (RIF) (bottom, treated sample) for 48 hours. B, Semi‐quantitative fluorometric evaluation of EGFP‐positive hepatocyte‐like cells (HLCs). Each value was normalized to the mean value obtained in D9 cells cultured in O plates in the 1.7% DMSO medium as 100 (n = 3). Black circles, cells cultured in medium containing 10 µmol L⁻¹ RIF; open circles, cells cultured in the 0.1% DMSO medium during the last 3 days of the cell culture period

FIGURE 5

Effect of plate type on CYP3A4 mRNA transcription assessed by RT‐qPCR. A, Enhanced expression of CYP3A4 and EGFP mRNA evaluated by RT‐qPCR (n = 3, triplicate measurements). B, CYP3A4 and EGFP transcriptional induction rates estimated according to mRNA levels. Graphs show the mean ± SD of the fold change with the 0.1% DMSO control considered as 1

FIGURE 6

Effect of plate type on the metabolic activity of CYP3A4 assessed by LC‐MS/MS. A, Comparison of the amounts of the CYP3A4‐mediated metabolite midazolam (mixture from four wells). B, Induction rate of CYP3A4 transcription estimated according to the amount of CYP3A4 metabolite after treatment with 10 μmol L⁻¹ RIF for 48 hours

FIGURE 7

Optimized 2D culture system for the formation of hepatocyte‐like cells (HLCs) from HepaRG cells and biliary epithelium on O plates coated with 2% Cellmatrix Type I‐A. A, Images of CYP3A4‐positive cells (green) and CK19‐positive cells (red) generated by confocal microscopy (upper). An enlarged view of the center of the well is shown in B. C, Semi‐quantitative fluorometric evaluation of HLCs and the biliary epithelium according to the ICC signal intensity for CYP3A4 and CK19, respectively. Cell growth under the indicated conditions was estimated based on the fluorescence intensity of DNA staining with Hoechst 33 258 (cells). Normalized relative values were calculated according to the average value obtained from D10 cells cultured on noncoated O plates taken as 100 (n = 4)