A long-term culture system based on a collagen vitrigel membrane chamber that supports liver-specific functions of hepatocytes isolated from mice with humanized livers

Abstract

During drug discovery, in vitro models are used to predict the in vivo pharmacokinetic and toxicological properties of drug candidates in humans. However, the conventional method of culturing human hepatocytes as monolayers does not necessarily replicate biologic reactions and does not support liver-specific functions, such as cytochrome P450 (CYP) activities, for prolonged periods. To remedy these problems and thus increase and prolong hepatic functions, we developed a culture system comprising a collagen vitrigel membrane (CVM) chamber and PXB-cells®, fresh hepatocytes isolated from liver-humanized chimeric mice (PXB-mice®). To quantitatively assess our new system, we evaluated the activities of 5 major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A), albumin secretion, and urea synthesis. First, between Days 14 and 21, the activities of all CYP isoforms tested in vitrigel culture were equal to or higher than in conventional monolayer culture system. Second, the activities of CYP3A, CYP2C9, and CYP2C19 during Days 10 through 17 were higher in vitrigel culture than in suspended PXB-cells prepared on Day 0 (suspension assay). Third, albumin secretion and urea synthesis were higher in vitrigel culture than in conventional monolayer culture. Fourth, the vitrigel-cultured PXB-cells showed the characteristic morphology of parenchymal hepatocytes and were almost all alive in monolayer. These results indicate that our vitrigel culture method is superior to the conventional monolayer method in terms of diverse liver-specific functions, including CYP activity. Our findings suggest that the vitrigel culture method could be a powerful in vitro tool for predicting the pharmacokinetic and toxicological properties of drug candidates in humans.

INTRODUCTION

During drug discovery, preclinical studies are important to predict the pharmacokinetic and toxicological properties of candidate compounds in humans. For example, cell-based assays using cryopreserved or fresh human hepatocytes are used to predict the metabolism and hepatotoxicity of drugs. However, current in vitro hepatocyte models using conventional monolayer culture systems do not always replicate in vivo biological functions. In particular: 1) freshly isolated or cryopreserved human hepatocytes lose their cytochrome P450 (CYP) activities within several days (Rodríguez-Antona et al., 2002; Guguen-Guillouzo and Guillouzo, 2010); 2) CYP activities are lower in conventional adherent monolayers than in hepatocytes placed in suspension cultures immediately after their isolation (Boess et al., 2003); and 3) long-term maintenance of hepatocytes in the conventional monolayer method is difficult (den Braver-Sewradj et al., 2016; Griffin and Houston, 2005). Furthermore, the reactive metabolites (RMs) produced by CYP isoforms reportedly reflect the hepatotoxicity of test compounds (Thompson et al., 2016), and high CYP activities are required to assess RM-induced hepatotoxicity. Therefore, the selection of appropriate cells as well as the development of a long-term culture method is central to increasing the accuracy of predictions regarding drug metabolism and toxicity in humans.

To assess the metabolism and toxicity of drug candidates, an “on demand” supply of fresh human hepatocytes, which have high CYP activities, from the same donor is ideal but not possible. As an alternative, PXB-mice®, chimeric mice in which more than 70% of the liver is replaced with normal human hepatocytes, are a useful animal model for predicting drug metabolism (Kitamura and Sugihara, 2014; Sanoh et al., 2015; Sanoh and Ohta, 2014). In addition, PXB-mice® are a ready source of suitable replacement cells for fresh human hepatocytes. Therefore, we isolated chimeric hepatocytes from PXB-mice® (PXB-cells) and assessed several representative liver-specific functions in the current study.

It is difficult to maintain liver functions including CYP activities over the long term in conventional monolayers of cultured hepatocytes. Therefore, the conventional monolayer culture method is inadequate for hepatotoxicity assays that examine the effects of long-term exposure to test compounds, and new methods of culturing hepatocytes that maintain their functional properties, including CYP activities, for prolonged periods are needed urgently. To address the difficulty regarding persistent functionality, three-dimensional cultures such as spheroid (Bell et al., 2016), a micropatterned hepatocyte-fibroblast co-culture system (Khetani and Bhatia, 2008), and a hepatic co-culture system (Hultman et al., 2016) have been developed and characterized. Although these culture systems are expected to facilitate drug discovery ultimately, they are complicated culture methods to require the other kind of cells such as feeder cells. Therefore, we designed a new culture system that is based on a collagen vitrigel membrane (CVM) chamber, which was developed for activating the hepatic functions of HepG2 cells (Oshikata-Miyazaki and Takezawa, 2016). The vitrigel membrane provides a scaffold composed of high-density collagen fibrils, resulting in a culture substratum that is highly adhesive for various anchorage-dependent cells (Takezawa et al., 2007, 2004). However, a culture method using a CVM chamber had not previously been used with freshly isolated chimeric human hepatocytes from liver-humanized mice, i.e. PXB-mice®.

In this study, we established a culture method in which we used a CVM chamber and PXB-cells to maintain the liver-specific functions including CYP activities at high levels for a prolonged period. Our data suggest that our vitrigel culture method using PXB-cells could be a powerful in vitro tool to predict the pharmacokinetic and toxicological properties of drug candidates in human.

MATERIALS AND METHODS

Reagents

Dulbecco’s modified Eagle medium (DMEM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), antibiotics (penicillin and streptomycin), and LIVE/DEAD Viability/Cytotoxicity kit were purchased from Life Technologies Corp. (Grand Island, NY, USA). Fetal bovine serum (FBS), Hanks’ balanced salt solution (HBSS), Williams’ medium E, and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Human Albumin ELISA Quantitation Set was purchased from Bethyl Laboratories (Montgomery, TX, USA). QuantiChrom^TM Urea Assay Kit was purchased from BioAssay Systems (Hayward, CA, USA). All other materials and chemicals not specified above were of the highest grade.

Preparation of collagen vitrigel membrane chambers

A CVM chamber is currently a commercially available product with the name of “ad-MED Vitrigel®”. The ad-MED Vitrigel® was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). The CVM chamber was set in the well of a 12-well plate. Immediately before seeding the cells, the CVM of each chamber was rehydrated for 10 min by pouring 1.5 mL and 0.2 mL of a medium (Medium A: DMEM containing 10% FBS, 20 mmol/L HEPES, 100 IU/mL penicillin G, and 100 µg/mL streptomycin) outside and inside the chamber, respectively.

Ethics statements

All in vivo experiments and protocols for animal studies were approved by the Laboratory Animal Ethics Committee at PhoenixBio Co., Ltd.

Preparation of PXB-cells

Cryopreserved human hepatocytes (Hispanic, 2-year old, female) were purchased from BD Bioscience (San Jose, CA, USA). The hepatocytes were thawed and transplanted into cDNA-urokinase-type plasminogen activator-transgenic/severely combined immunodeficient mice to prepare PXB-mice® as described previously (Tateno et al., 2015). The PXB-cells were isolated from PXB-mice® with blood human albumin level of higher than 12 mg/mL by a two-step collagenase perfusion method (Yamasaki et al., 2010). PXB-cells were a mixture of human and mouse hepatocytes, and PXB-cells constituted approximately 90% of human hepatocytes and the rest were mice hepatocytes and non-parenchymal cells. The number of alive and dead cells were counted by trypan-blue exclusion test, and viability of cells were calculated. Then the cells were suspended in the Medium A.

Culture of PXB-cells

After Medium A inside the chamber was removed, 0.3 mL of a cell suspension of PXB-cells in medium A was poured in to the CVM chamber at a density of 2.1 × 10⁵ viable cells/well on the liquid-liquid interface in accordance with the vitrigel culture procedure (Oshikata-Miyazaki and Takezawa, 2016), and the PXB-cells in CVM chamber were cultured for 29 days at 37°C in a humidified atmosphere of 5% CO₂ and 95% air (vitrigel culture). The both media inside and outside the chamber were changed on Days 1, 4, 7, 10, 14, 17, 21, 24, and 27 with fresh culture medium (dHCGM medium: DMEM supplemented with 10% FBS, 20 mmol/L HEPES, 15 µg/mL L-proline, 0.25 µg/mL insulin, 50 nmol/L Dexamethasone, 44 mmol/L NaHCO₃, 5 ng/mL EGF, 0.1 mmol/L ascorbic acid 2-phosphate, 100 IU/mL penicillin G, 100 µg/mL streptomycin, and 2% DMSO). Simultaneously, 0.2 mL of a cell suspension of PXB-cells in medium A was seeded at a density of 1.6 × 10⁵ cells/well in the well of a collagen coating 48-well plate (BD Biosciences, Bedford, MA, USA), and the PXB-cells on the collagen coating 48-well plate were cultured for 21 days at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. The medium in the well was replaced with fresh dHCGM medium on Days 1, 4, 7, 10, 14, and 17 (conventional monolayer culture).

Cytochrome P450 activity

CYP activities on Days 0 (suspension assay just after preparation of PXB-cells (the mixture of human and mouse hepatocytes)), 7, 10, 14, 17, and 21 were compared in conventional monolayer culture and vitrigel culture methods. CYP isoforms tested are CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A. To measure the specific activity of different CYP isoforms, a cocktail mixture of probe substrates (phenacetin (50 μmol/L) for CYP1A2, tolbutamide (250 μmol/L) for CYP2C9, S-mephenytoin (100 μmol/L) for CYP2C19, bufuralol (25 μmol/L) for CYP2D6, and midazolam (50 μmol/L) for CYP3A) was added for each metabolic assay, and formed metabolites (acetaminophen, hydroxytolbutamide, 4’-hydroxymephenytoin, 1’-hydroxybufuralol, and 1’-hydroxymidazolam, respectively) were quantified by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The cocktail mixture of probe substrates was diluted in Williams’ medium E to prepare assay medium, and assay medium was preheated at 37°C before start of incubation.

In the vitrigel culture, prior to assay, each vitrigel membrane was inserted in option ring (Kanto Chemical, Tokyo, Japan), and dHCGM medium was removed from the PXB-cells, followed by washing with 400 μL of preheated PBS. Then, PBS was removed from the PXB-cells, and the second washing was conducted with 100 μL of Williams’ medium E preheated at 37°C. After removal of Williams’ medium E from the PXB-cells, 100 μL of Williams’ medium E preheated at 37°C was added to the PXB-cells, and CYP reaction was initiated by adding 100 μL of assay medium in each vitrigel chamber (n = 3).

In the conventional monolayer culture, the PXB-cells were washed with 400 μL of PBS preheated at 37°C, followed by washing with 100 μL of Williams’ medium E preheated at 37°C. After removal of Williams’ medium E from the PXB-cells, 75 μL of Williams’ medium E preheated at 37°C was added to the PXB-cells, and the CYP reaction was initiated by the addition of 75 μL of assay medium in each well (n = 3) of collagen-coating plates.

For the suspension assay on Day 0, the PXB-cells were suspended in 125 μL of Williams’ medium E preheated at 37 °C, and the reaction was initiated by the addition of 125 μL of assay medium in each well (n = 3) of non-collagen-coating plates.

All cultures and CYP assays were conducted at 37°C in humidified atmosphere containing 95% air and 5% CO₂. Samples (20 μL) were taken from each well or vitrigel chamber at 10 and 60 minutes sequentially (n = 3/each day /suspension assay or each culture method) and kept in the freezer (−20°C) until analysis by LC-MS/MS. The number of cells seeded at the beginning was applied for each metabolic assay. The values of each CYP activity in the conventional culture and the vitrigel culture were corrected by the cell numbers of at 1.6 × 10⁵ cells / well and 2.1 × 10⁵ cells / well, respectively.

LC-MS/MS analysis

For analyses, the frozen samples were thawed at room temperature and added to 100 μL of acetonitrile (acetonitrile/methanol, 7/3 [v/v]) containing internal standard (IS). The samples were mixed with vortex and centrifuged at 3,000 rpm for 10 min, and the supernatants were analyzed by LC-MS/MS. Niflumic acid was used as an IS in LC-MS/MS analyses for all CYP activities. For all metabolites from each probe substrate, a mobile phase was a gradient with 0.02% formic acid in water (A) and 0.02% formic acid in acetonitrile (B) with a flow rate of 0.5 mL/min. The initial composition of the mobile phase was 2% of B for 0.5 min, followed by a linear gradient to 99% of B over 2 min, held at 99% of B for 2.5 min, and back to 2% of B in 0.01 min and then allowed to re-equilibrate to the initial conditions. The total run time was 6.0 min. Separation was carried out using an L-Column ODS (2.1 mm × 150 mm) (Chemicals Evaluation and Research Institute, Tokyo, Japan) and eluted fractions were directly passed through an API4000 or API3200 MS/MS (AB Sciex, Waltham, MA, USA) equipped with an electrospray ionization source operating in the positive ion mode. The ionization mode, parent ion, and product ion were as follows: acetaminophen m/z = 152.1 [M+H]+ to 109.9, hydroxytolbutamide, m/z = 287.0 [M+H]+ to 171.1, 4’-hydroxymephenytoin m/z = 235.3 [M+H]+ to 150.1, 1’-hydroxybufuralol m/z = 278.1 [M+H]+ to 159.1, 1’-hydroxymidazolam m/z = 342.2 [M+H]+ to 203.0, and niflumic acid (internal standard) m/z = 283.0 [M+H]+ to 245.0 for API4000 or 282.9 [M+H]+ to 145.3 for API3200.

Biochemical assay

The levels of albumin secretion and urea synthesis in the conditioned medium accumulated for 3 weeks in conventional monolayer culture and vitrigel culture methods were measured in accordance with each manufacturer’s protocol for Human Albumin ELISA Quantitation Set and QuantiChrom^TM Urea Assay Kit, respectively. Then, the cumulative values for the levels of albumin secretion and urea synthesis per 1.0 × 10⁶ cells were calculated.

Cell morphology and viability

The morphology of PXB-cells was periodically observed with a phase-contrast microscope. Also, the cell viability in the vitrigel culture method was confirmed with a fluorescent microscope after staining the cells by using LIVE/DEAD Viability/Cytotoxicity Kit composed of calcein-AM and ethidium homodimer-1 as previously reported (Oshikata-Miyazaki and Takezawa, 2016).

Statistical analysis

Quantifiable data are presented as the mean ± the standard deviation (S.D.). Data obtained from single experiment were analyzed by the unpaired t-test, where the values of P < 0.05 were considered statistically significant.

RESULTS

CYP activity

We treated PXB-cells in both conventional monolayer and our vitrigel cultures with a cocktail mixture of probe substrates for 5 major CYP isoforms and monitored the formation of the various metabolites by using LC-MS/MS (Fig. 1). The CYP activities on Day 4 in both conventional monolayer culture and vitrigel culture decreased compared to those on Day 7 due to an inappropriate culture environment just after seeding PXB-cells (data not shown). In conventional monolayer culture, the activities of all 5 CYP isoforms evaluated peaked on Day 10 and gradually decreased thereafter. In contrast, all 5 CYP activities during Days 14 through 21 in vitrigel culture were equal to or higher than those of conventional monolayer culture throughout this period (Figs. 1A-E). In addition, we compared the CYP activities in PXB-cell suspension freshly prepared on Day 0 with those in both conventional monolayer and vitrigel cultures. Overall, all CYP activities in conventional monolayer culture were lower than those in the PXB-cell suspension, although CYP3A and CYP2C9 activities during Days 10 through 14 were nearly equivalent to or higher than those in the PXB-cell suspension (Fig. 1F). However, the activities of CYP3A, CYP2C9, and CYP2C19 between Days 14 and 17 in the vitrigel culture were approximately 2- to 3-fold as high as those in the PXB-cell suspension (Fig. 1G). In contrast, the activities of both CYP1A2 and CYP2D6 between Days 7 and 21 were decreased to less than half of those on Day 0. These results indicate that the vitrigel culture method can maintain CYP metabolic activities for at least 3 weeks and that the activity levels except for CYP1A2 and CYP2D6 were comparable to those of the PXB-cell suspension freshly prepared on Day 0.

Fig. 1

CYP-dependent metabolic activity of PXB-cells in each culture method for 3 weeks. CYP activities on Days 7, 10, 14, 17, and 21 were compared between conventional monolayer culture (blue bar) and vitrigel culture methods (red bar). PXB-cells were incubated with a mixture of the probe substrates ((A) phenacetin (CYP1A2, 50 μmol/L), (B) tolbutamide (CYP2C9, 250 μmol/L), (C) S-mephenytoin (CYP2C19, 100 μmol/L), (D) bufuralol (CYP2D6, 25 μmol/L), and (E) midazolam (CYP3A, 50 μmol/L)). Formed metabolites from each probe substrate (acetaminophen, hydroxytolbutamide, 4’-hydroxymephenytoin, 1’-hydroxybufuralol, and 1’-hydroxymidazolam) were quantified by LC-MS/MS. Data represent the mean ± S.D. (n = 3 from single experiment, * indicates P < 0.05, ** indicates P < 0.01 vs. conventional monolayer culture, unpaired t-test). CYP activities in suspended PXB-cells prepared on Day 0 (suspension assay) were compared with those on Days 7, 10, 14, 17, and 21 in conventional monolayer culture method (F) or vitrigel culture method (G). The ratio activity was calculated by dividing the mean value of metabolic activity in the conventional monolayer culture method or vitrigel culture method by those in the suspension assay on Day 0 (n = 3 from single experiment; *P < 0.05, **P < 0.01 vs. suspension assay on Day 0, unpaired t-test).

Hepatic functions of PXB-cells

To quantitatively assess the liver-specific functions of PXB-cells in conventional monolayer and vitrigel cultures, we measured albumin secretion (a marker of protein synthesis) and urea synthesis (nitrogen metabolism). In conventional monolayer culture, albumin secretion increased gradually throughout the culture period; in contrast, albumin secretion rose sharply until Day 14 and then plateaued in vitrigel culture (Fig. 2A). Similarly, the amount of urea synthesized in the vitrigel culture was greater than that in the conventional monolayer culture method (Fig. 2B). In the vitrigel culture method, liver-specific functions such as albumin secretion and urea synthesis were maintained at increased levels for 3 weeks and peak level was observed on Day 14, which is different from those in conventional monolayer culture.

Fig. 2

Time course changes of cumulative albumin secretion and urea synthesis. The time course changes of albumin secretion (A) and urea synthesis (B) were measured. Data represent the mean ± S.D. (n = 3 from single experiment, **P < 0.01 on Day 21, unpaired t-test).

Morphology and viability of PXB-cells

Within the first 7 days after plating, PXB-cells in the vitrigel culture formed confluent monolayers with scattered clumps of aggregated cells (Fig. 3A). The cellular morphology in the monolayer areas was characteristic of parenchymal hepatocytes, with hepatic cord-like patterns demonstrating typical features of bile canaliculi; these morphologic features, including the cellular aggregates, were maintained for as long as 21 days (Figs. 3A-C). PXB-cells cultured for 21 days were almost alive in monolayer and were dead in piled-up aggregates (Fig. 3D). When the culture period was increased to 24 days, the cell monolayers gradually detached from the CVM and formed aggregates containing many dead cells; healthy fibroblast-like cells then appeared (Figs. 3E and 3F). PXB-cells in conventional monolayer culture likewise maintained appropriate hepatic architecture for at least 21 days (data not shown).

Fig. 3

Microscopic observation of PXB-cells cultured in the vitrigel culture method. The cells were cultured for 7 days (A), 14 days (B), 21 days (C and D) and 29 days (E and F) and observed with a phase-contrast microscope (A-C and E) and a fluorescent microscope (D and F) after staining the cells with calcein (green) and ethidium homodimer-1 (red). Fluorescent microphotographs are the same visual field of phase-contrast ones (C-F). Arrows and dotted frames indicate typical bile canaliculus-like aspects and piled-up cell aggregates, respectively (A-D). Asterisk symbols represent healthy fibroblast-like cells. Scale-bar represents 100 μm.

DISCUSSION

In this study, we evaluated the utility of our novel vitrigel culture method, which is based on CVM chambers (ad-MED Vitrigel®), as a new system for assessing drug metabolism and enhancing the liver-specific functions of hepatocytes. Unlike conventional monolayer culture, vitrigel culture stably maintained CYP activities for as long as 21 days (Figs. 1A-E). In particular, the CYP3A, CYP2C9, and CYP2C19 activities from Day 7 to 21 in vitrigel culture were comparable to or higher than those in Day-0 PXB-cell suspension (Fig. 1G). Suspensions of hepatocytes retain their highest metabolic activity immediately after isolation, and these activities gradually decrease thereafter (Gebhardt et al., 2003). Therefore, our current results are noteworthy and suggest that the vitrigel culture method establishes a biological environment that can highly enhance CYP metabolic activities. Although the activities of CYP1A2 and CYP2D6 were higher in PXB-cell suspension on Day 0 than in either type of culture, they tended to be higher in vitrigel culture than in conventional monolayer (Figs. 1A and 1D). The differences in CYP metabolic activities were recognized. Regardless, the current data suggest that our CVM chamber-based vitrigel culture method enhances the metabolic activities of all CYP isoforms examined. For the target values in this method, the most frequently observed value ranges (MFRs) of the activities of major drug metabolizing enzymes (Araki et al., 2016) were approximate references. Although the activities of CYP2C9 and CYP2D6 cannot be referenced because these substrates tested were different, the activity of CYP1A2 in vitrigel culture was approximately half of the value of MFR, and the activities of CYP2C19 and CYP3A were higher.

In addition, vitrigel culture showed increased liver function, represented by albumin secretion and urea synthesis, for as long as 3 weeks compared to conventional monolayer culture (Fig. 2). Interestingly, the daily secretion rate of albumin in vitrigel culture had the largest slope between Day 10 and Day 14, as CYP1A2, CYP2C9, and CYP2C19 activities showed their maxima on Day 14. Although CYP2D6 and CYP3A activities peaked on Day17 in vitrigel culture, high levels of all 5 CYP activities were maintained until Day 21 compared to the conventional monolayer culture. These results indicate that the prolonged exponential increase in albumin secretion in vitrigel culture was associated with dramatic increases in CYP activities. Furthermore, because the vitrigel culture method also sufficiently activated urea synthesis (a marker of liver-specific function), other bioenergetics pathways (e.g., tricarboxylic acid cycle, ornithine cycle) may be induced in this system as well, thus further promoting the CYP metabolic pathway. Additional study is needed to confirm this hypothesis.

PXB-cells in vitrigel culture showed the characteristic morphology of parenchymal hepatocytes, with a hepatic cord-like pattern and bile canaliculus-like features; in addition, cells in monolayer regions of the culture remained alive for as long as 21 days (Fig. 3). In general, hepatocytes are differentiated and polarized cells that interact with each other, other types of cells, and extracellular matrices under physiological conditions. During the isolation process, the polarity of primary hepatocytes is disrupted, leading to rapid loss of liver-specific functions, including CYP activities, in conventional monolayer culture. Maintaining liver-specific functions is important in the establishment of in vitro tools used during drug discovery.

In our CVM chamber-based culture system, vitrigel culture method is simple static monolayer culture and CVM chambers are relatively easy to handle compared with spheroid culture or various co-culture systems. Since spheroid system is spontaneous self-aggregation of cells and often requires feeder cells, it is difficult to control spheroid formations and culture conditions. In the same way, a micropatterned hepatocyte-fibroblast co-culture system (Khetani and Bhatia, 2008) requires feeder cells, and because of the coexistence of two kinds of cells, it is necessary to evaluate which cell is attributed to the metabolic activities or toxicological properties in assay. Therefore, vitrigel culture method is superior to these culture systems in terms of simplicity, which can enhance liver specific functions only by monolayer culture. We surmise that improved nutrient supply through the CVM supports the liver-specific functions of PXB-cells during prolonged culture; the CVM can provide the pathway of chemicals as well as extracellular matrices in vivo. In addition, CYP activities in the vitrigel culture increased during the first 2 weeks of culture and remained at high levels for an additional week. Since suspended hepatocytes rapidly lose their liver-specific functions, including CYP activities (Rodríguez-Antona et al., 2002), it is important to maintain CYP activities for long term. Although the activities of CYP1A2 and CYP2D6 were lower than those of suspension assay on Day 0 (Fig. 1G), vitrigel culture method was superior to suspension assay in terms of the long maintenance of metabolic activities. These features suggest that our vitrigel culture method has significant merit in the prediction of the human pharmacokinetic profile of drug candidates, especially slowly metabolized compounds that require long-term exposure to reveal the metabolic profile. In this study, we focused on the fact that vitrigel culture method significantly improved the liver function compared with the conventional method, and we estimated the culture period when the CYP metabolic activities became maximum in the vitrigel culture. Thus, the study to investigate whether predicted clearance value calculated by the vitrigel culture correlates with the observed value of clinical data needs to be conducted in future. From a toxicological viewpoint, because the RMs produced by CYP isoforms reportedly reflect the hepatotoxicity of the compounds to which they are exposed (Thompson et al., 2016), the extended duration of high CYP activity in the vitrigel culture method provides the opportunity to predict or assess RM-induced hepatotoxicity. In particular, drug-induced liver injury (DILI) is one of the biggest clinical concerns in terms of serious adverse events (Murray et al., 2008; Navarro and Senior, 2006), and unpredictable severe DILI will lead to the withdrawal of a drug candidate from further development. Therefore, the risk of DILI due to candidate compounds should be assessed as early as possible in the drug discovery process. From this standpoint, an in vitro evaluation system with high prediction accuracy is strongly desired, and the results of our current study indicate that the unique and improved properties of our vitrigel culture method promote its application to toxicological assessment during drug discovery.

In summary, the results of our current study indicate that the novel culture method using CVM chambers enable PXB-cells to have higher CYP activities than those under simple static culture conditions. In addition, our vitrigel culture method maintains albumin secretion, urea synthesis, and appropriate hepatocyte morphology for as long as 3 weeks. These features suggest that the vitrigel culture method using PXB-cells could be a powerful tool for predicting the metabolism and hepatic toxicity of drugs in humans.

ACKNOWLEDGMENTS

This work was supported by the grant to Mr. T Matsumi and Ms. T Watadani from PhoenixBio for coordination of cell assay and Mr. H Yamaguchi from Kanto Chemical for preparation of a collagen vitrigel membrane.

Conflict of interest

The authors declare that this work was aided, in part, by the Japan Agency for Medical Research and Development (AMED) foundation.

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