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.


| INTRODUC TI ON
Drug metabolism occurs mainly in the liver. Drugs undergo structural alterations catalyzed by the metabolic enzyme cytochrome P450 (CYP), which is expressed in the intestine and in hepatocytes.
CYP3A4 metabolizes approximately 50% of marketed drugs. 1,2 Therefore, drug discovery and development require evaluation of the cytotoxicity and drug-drug interactions of candidate compounds before and after absorption in the human body using human hepatocytes. A high-content screening (HCS)-based assay for many candidates is necessary to select nontoxic and effective compounds, as it would improve cost and time effectiveness in the early stages of drug discovery and development. HCS evaluates multiparameter images acquired with a motor-driven microscope and extracts quantitative data. Therefore, HCS requires a large amount of adult primary human hepatocytes (PHHs) expressing CYP3A4. However, PHHs are limited in number because of their poor proliferative ability, and individual and lot differences negatively affect data reproducibility. 2,3 Therefore, it is indispensable to develop a model of adult PHHs that can be stably supplied, are scalable and qualitatively uniform and express CYP3A4. Moreover, certain chemicals can alter the susceptibility to a drug by promoting or inhibiting the pharmacological actions of coadministered drugs. [4][5][6][7][8] To predict the clinical effect of drug-drug interactions, it is also necessary to evaluate the possible transcription-inducing function of CYP3A4.
HepaRG cells have characteristics similar to those of human fetal liver cells, hepatoblasts (HBs), and addition of 1-2% dimethylsulphoxide (DMSO) to the culture medium induces their differentiation into adult-type HLCs. 9,10 HepaRG HLCs are the only cultured cells expressing CYP3A4 at high levels similar to those expressed by adult PHHs. 11,12 In previous work by our group, we generated transgenic HepaRG cells, and determined the presence of HB-like cells expressing fetal-type CYP3A7 and adult-type HLCs expressing CYP3A4 by detecting the expression of DsRed and EGFP, respectively, before and after cell differentiation. 13,14 The transgenic CYP3A4G/7R HepaRG provides an easily applicable and inexpensive system to determine the degree of cell differentiation according to the color shift from red fluorescence to green fluorescence. 14,15 Compounds that induce CYP3A4 transcription can also be conveniently evaluated based on the fold increase of normalized green fluorescence intensity. When cell numbers seeded in each well are strictly controlled, and enough number of wells are analyzed for each condition, total green fluorescence intensity per area can represent relative CYP3A4 transcriptional levels among wells. Moreover, cell death induced by hepatotoxic compounds can be better monitored by the reduction of total green fluorescence intensity per area, as both of transcription and cell numbers are reduced. In previous work by our group, we generated transgenic mice for the CYP3A7-DsRed BAC reporter gene, which contains the complete transcriptional regulatory units of CYP3A7 and CYP3A4 and the gene body of CYP3A7 was replaced with DsRed. Then we found that 5% carbon tetrachloride (CCl 4 ) injection induces red fluorescence reactivation in the liver of adult transgenic mice via transient HB expansion during liver regeneration. 16 Therefore, red fluorescence recovery could be used as a tool to identify hepatotoxic compounds. In both cases, achieving automatic scanning with a confocal microscope and limiting light leakage from adjacent wells require the use of multiwell plates with a flat and thin bottom and a black wall for HLC culture.
In recent years, multiwell plates suitable for 3D cell culture were developed and are commercially available. It is believed that these are necessary because hepatocytes that form microspheroids tend to exhibit high enzyme activities comparable to those of hepatocytes under biological conditions. 17 Therefore, the combination of CYP3A4-expressing HLCs, 3D culture of HLCs, and a multiwell plate with a flat surface is essential for reliable screening. However, an optimal system remains to be developed; in particular, a multiwell plate suitable for live-cell imaging is lacking.
In this study, we aimed to establish a multiwell cell culture system for HepaRG-derived HLCs showing high levels of CYP3A4 enzyme activity. We first prepared a sufficient amount of homogenously differentiated HepaRG-derived HLCs. Specific numbers of HLCs were seeded in each well of five 96-well plates and one 24-well plate, to compare re-colonization status and subsequent re-maturation efficiency.
The relative expression levels of CYP3A4 were evaluated by measuring total green fluorescence intensity and EGFP and CYP3A4 mRNA expression levels using confocal microscopy and reverse transcription-quantitative PCR (RT-qPCR), respectively. The enzyme activity of CYP3A4 was assessed by measuring the amount of 1'-hydroxymidazolam, a probe for measuring CYP3A4-mediated metabolism using liquid chromatography tandem-mass spectrometry (LC-MS/MS). To identify the cell culture system capable of providing the most sensitive evaluation of CYP3A4 transcription induction, induction rates were compared in HLCs cultured on six plates and treated for 48 hours with rifampicin (RIF).

| Cell culture
All media used for HepaRG cell culture were purchased from Biopredic International, Rennes, France. Cell culture media used in this study were 710, 720, and 640, and MIL502. The contents of these media are not disclosed by the manufacturer, but they differ in serum and DMSO concentration and are specified for cell proliferation, cell differentiation, transcription induction assays, and mass spectrometry.
Transgenic and wild-type (WT) HepaRG cells at the passage (P)11 and P19 were used, respectively. Cells were seeded at 1.5 to two times the recommended number of cells to maintain differentiation potency. After 2-week (2 W) cell culture in medium 710 at 37°C and 5% CO 2 , the DMSO concentration in the medium was gradually increased from 0.1% to 0.4% from 3 to 4 days before subculture. To produce HLCs, cells were seeded in fifteen 25-cm 2 cell culture flasks at a density of 0.5 × 10 6 cells/cm 2 and cultured in medium 710 for 2 W. The cells were then cultured in the 0.4% DMSO medium for 3 days, the 1% DMSO medium for 2 days, and finally in the 1.7% DMSO medium for 9 days. Therefore, the initial cell differentiation of HepaRG cells to HLCs was completed in 4 weeks (4 W).
The dissociation of tight adhesion among HLCs induces cell dedifferentiation from HLCs to growing HB-LCs, so mildly dissociated cell aggregates were transferred to multiwell plates. An aliquot of the 4-W differentiated cell mixture collected from the 15 flasks was placed into each well of a multiwell plate (7.2 × 10 4 cells/well). All plates used are commercially available as listed in Table 1 (Cosmo Bio or Thermo Fisher Scientific). Additional cell cultures on assay plates were achieved as described in the Results section. Immediately prior to use, some of plates were coated with 2-20% Cellmatrix Type I-A (Nitta Gelatin).

| Fluorescence microscopy and image analysis
Fluorescence microscopic images were captured with an A1 confocal microscope (Nikon, Tokyo, Japan). The mean and standard de-

| RNA extraction and RT-qPCR analyses
Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands), and cDNA was prepared from 0.5 µg of total RNA using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO). cDNA from 10 ng of RNA was amplified in 25 µl reactions using KOD SYBR® qPCR Master Mix (TOYOBO) and a 7500 Real-Time PCR System (Applied Biosystems). ACTB was used as an internal control. Primer sets used for RT-qPCR are listed in Table 2.

| LC-MS/MS analyses
The medium MIL502 supplemented with 50 μmol L −1 midazolam was prepared before use. An aliquot (0.5 × 10 6 ) of the original 4-W HLCs was incubated for 1 hours at 37°C in a 500 μl midazolam mixture. The

| Statistics
Data are presented as the mean ± SD. The Z test Excel formula was used to evaluate the statistical significance of differences between two samples. Significance was determined using equal-variance Z values on both sides. The correlation coefficient (R) was calculated using the software in Microsoft Excel. Values of P < .01 were considered significant. *P < .01; **P < .001.

| Effect of culture plate type on colony morphology of HepaRG-derived HLCs
In this study, five 96-well plates with different characteristics were used to compare the cell maturation abilities of CYP3A4-expressing HLCs (Table 1)   Because the BAC reporter gene CYP3A4G/7R contains a complete transcriptional element, EGFP transcription can be enhanced by the CYP3A4 transcriptional inducer RIF ( Figure 1A). Therefore, we also attempted to select plates suitable for identifying compounds that enhance CYP3A4 transcription via nuclear receptors. On D8, a medium containing 0.1% DMSO or 10 µmol L −1 RIF was added to three wells per plate (n = 3) and further cultured for 3 days under comparable conditions ( Figure 1B) HAG plate (HAG) also has a PDMS membrane bottom, which promotes cell growth more efficiently than the G plate ( Figure 1D,E).
The properties of the Preset VECELL® (V) plate were previously reported using hepatoma HepG2 cells. 19 Cells were seeded in a 24well V plate at a comparable density to that in a 96-well plate. Cell

| Effect of plate type on maturation into HLCs
In CYP3A4G/7R HepaRG cells, EGFP expression is regulated by the  Figure 2D).
To determine whether D10 cells that were cultured on the O plate reliably differentiated into HLCs, the cells were subjected to an ICC analysis for representative markers of mature human PHHs.

| Effect of plate type on the fluorometric CYP3A4 transcriptional induction test
After adding RIF or 0.1% DMSO, fluorescence images were captured at 24, 48, and 72 hours ( Figure 4A). Image J was used to calculate total fluorescence per area at each time point in five plates.

| Effect of plate type on the metabolic activity of CYP3A4
Midazolam was added to the medium of the six plate types to determine which plate would provide high-quality HLCs that had

| Optimization of 2D culture conditions for the formation of mini-liver-like structures containing HLCs and biliary epithelium on O plates
To optimize the cell culture conditions of HepaRG-derived HLCs, O plates were collagen-coated with 0%, 2%, 5%, 10%, 15%, or 20% Cellmatrix Type I-A (n = 4). In this experiment, D10 HLCs from WT HepaRG cells were subjected to ICC analysis using anti-human CYP3A4 and human anti-human CK19 antibodies, which are representative markers of mature HLCs and bile duct epithelium, respectively. ICC images were acquired using a confocal microscope ( Figure 7A,B), and the mean relative fluorescence intensity normalized by DNA staining intensity for CYP3A4 and CK19 was calculated to evaluate the effects of collagen on HLC maturation ( Figure 7C). In addition, cell growth, evaluated by the relative intensities of DNA staining, was 1.4 times higher in untreated plates than in 10% collagen-coated wells.
However, CYP3A4 and CK19 protein expression levels in uncoated plates were equivalent to those in 10% collagen-coated plates. The maximum values for CYP3A4 and CK19 were obtained in 2% collagencoated O plates, which were approximately 1.3-fold higher than those obtained in 10% collagen-coated wells (n = 4, P < .01). These results indicated that O plates coated with 2% Cellmatrix Type I-A were the most effective at promoting the maturation of HepaRG cells. Further investigation is necessary to determine whether this phenomenon is HepaRG specific or if it occurs in human PHHs. In addition to HepaRG, attempts have been made to produce functional adult-type

| D ISCUSS I ON
HLCs from human-induced pluripotent stem cells (iPSCs), although mature HLCs remain to be produced from human iPSCs. 21,22 Therefore, the establishment of an in vitro system capable of promoting HLC maturation would be favorable. The 2D culture system presented here may also be effective for the maturation of human iPSC-derived HLCs.
The use of CYP3A4G/7R HepaRG-derived HLCs cultured in the 96-well glass-bottom plates selected in this study enables semi-quantitative evaluations of compounds based not only on fluorescence, but also on high levels of enzyme activity. This system would enable additional analyses such as cell death caused by hepatotoxicity and metabolite evaluation.

ACK N OWLED G M ENTS
This work was supported by a grant awarded to MT, the Naito Taishun Science and Technology Foundation 2017. We sincerely thank Dr C.
Guguen-Guillouzo and Dr C. Chesne (Biopredic International, France) for expert advice on HepaRG cells. We also thank Dr K. Nagata and Dr Y. Nakayama (Research Center for Bioscience and Technology, Tottori University, Japan) for expert advice on LC-MS/MS analysis.

D I SCLOS U R E
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Masako Tada, the corresponding author, designed the research, involved in final proofreading before submission, collected the data, and contributed to data analysis. Keiko Ooeda, Mao Yamashita, and Shota Okuyama conducted the experiments, performed data analysis, and contributed to the writing of the manuscript. Musashi Kubiura-Ichimaru, Akari Mine, Saori Tsuji, and Takafumi Ueyama conducted experiments and performed data analysis. Fumihiko Kawamura contributed new reagents or analytic tools.

E TH I C A L S TATEM ENT
The authors declare that this study was performed in accordance with the research policy of Toho University.

O PEN R E S E A RCH BA D G E
This article has earned an Open Data and Open material badges for making publicly available the digitally-shareable data necessary to reproduce the reported results. All data and materials are available in the article.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data discussed in this publication is included in this manuscript.
Further information and requests for data and reagents should be requested to the corresponding author, Masako Tada. Please contact masako.tada@sci.toho-u.ac.jp.