Bile canaliculi maintenance medium and method for maintaining bile canaliculi using same

Abstract

The present invention addresses the problem of providing a culture medium for long-term culture of hepatocytes while maintaining the bile canaliculi thereof. The present invention relates to a medium for culturing hepatocytes while maintaining the bile canaliculi that contains, as an active ingredient, one or more selected from the group consisting of AMPK activators, Epac activators, apoptosis inhibitors, and antioxidants, and to a method for maintaining the bile canaliculi that includes culturing hepatocytes using the medium.

Description

Medium for maintaining bile canaliculi and method for maintaining bile canaliculi using the same

[0001] The present invention relates to a medium for maintaining bile canaliculi and a method for maintaining bile canaliculi using the same.

[0002] Bile canaliculi, which function as an excretion pathway for bile produced in the liver, are also one of the major elimination pathways for pharmaceuticals. In addition, drug-induced liver injury caused by inhibition of bile excretion by pharmaceuticals and retention (cholestasis) in hepatocytes is one of the major causes of discontinuation of pharmaceutical development. Therefore, in pharmaceutical development, evaluation of bile excretion of candidate compounds and risk assessment of cholestasis are performed.

[0003] In the above evaluations, in vivo tests using experimental animals are performed, and bile collected by inserting a cannula into the common bile duct of rats is analyzed and evaluated. In particular, in the cholestasis evaluation, a test in which a candidate compound is administered multiple times is required. In order to evaluate this by an in vitro test, it is necessary to maintain the cells forming bile canaliculi in long-term culture. Although research using human primary cultured hepatocytes, iPS-derived hepatocytes, etc. is active for cells, there is still no medium having sufficient quality that can be cultured for a long time while maintaining bile canaliculi.

[0004] Non-patent document 1 describes a study using rat hepatocytes in which the activation of AMPK and LKB1 controls bile canal formation and maintenance. It states that the tubular structure, which was fully formed on day 6 of culture, remained unchanged until day 14 of culture, after which the cells deteriorated. Non-patent document 2 describes a study using primary human cultured hepatocytes in which the addition of the caspase inhibitor Z-VAD-FMK inhibited apoptosis and promoted transporter function and bile canal formation. It states that the cells were cultured for about one week. Non-patent document 3 reports an investigation into the mechanism by which the bile canal network is constructed by the addition of bile acids. It states that the cells were cultured for six days. Non-patent document 4 describes culturing human iPS cell-derived hepatocytes (commercially available iCell® Hepatocytes (FUJIFILM Cellular Dynamics)) using commercially available Cellartis® Enhanced hiPS-HEP Long-Term Maintenance Medium (LTM medium) (Takara Bio Inc.), and confirming the structure and function of bile canaliculi even on day 30 of culture. However, the time elapsed since bile canaliculi formation and their active ingredients are not clarified. Non-patent documents 5 and 6 describe culturing primary human hepatocytes using commercially available LTM medium, and confirming the structure and function of bile canaliculi even on day 21 or day 25 of culture. However, the time elapsed since bile canaliculi formation and their active ingredients are not clarified.

[0005] Dong Fu et al., “Regulation of bile canalicular network formation and maintenance by AMP-activated protein kinase and LKB1”, Journal of Cell Science 123, 3294-3302.Yoko Sakai et al., “Culture methods focusing on bile canalicular formation using primary human hepatocytes in short time”, research square.Dong Fu et al., “Bile acid stimulates hepatocyte polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway”, PNAS. 2011, vol. 108, no. 4, p.1403-1408Shinichiro Horiuchi et al., “Construction of a culture protocol for functional bile canaliculi formation to apply human iPS cell-derived hepatocytes for cholestasis evaluation”, Scientific Reports, 2022, 12:15192Takashi Kitaguchi et al., “Construction of extended and functional bile canaliculi using long-term sandwich-cultured cryopreserved human hepatocytes and the application of hepatocytes for predicting the biliary excretion of pharmaceutical and food-related compounds”, J. Toxicol. Sci., 2023, Vol.48, No.5, 251-261Shinichiro Horiuchi et al., “Formation of functional, extended bile canaliculi, and increased bile acid production in sandwich-cultured human cryopreserved hepatocytes using commercially available culture medium”, Archives of Toxicology, 2024, 98:2605-2617.

[0006] The object of the present invention is to provide a culture medium of sufficient quality to allow hepatocytes to be cultured for a long period of time while maintaining the bile canals.

[0007] In order to solve the above problems, the inventors diligently conducted research and discovered that when hepatocytes are cultured in a culture medium containing one or more selected from the group consisting of AMPK activators, Epac activators, apoptosis inhibitors, and antioxidants as active ingredients, they can be cultured for a long period of time while maintaining bile canal function. Further research led to the completion of the present invention.

[0008] The present invention relates to the following inventions: [1] A culture medium for culturing hepatocytes while maintaining bile canaliculi, comprising one or more selected from the group consisting of AMPK activators, Epac activators, apoptosis inhibitors, and antioxidants as active ingredients. [2] The culture medium according to [1], wherein the AMPK activator is resveratrol. [3] The culture medium according to [1] or [2], wherein the Epac activator is Sp-8-BnT-cAMPS. [4] The culture medium according to any one of [1] to [3], wherein the apoptosis inhibitor is 2,2'-methylenebis(1,3-cyclohexanedione). [5] The culture medium according to any one of [1] to [4], wherein the antioxidant is N-acetylcysteine. [6] A culture medium for culturing hepatocytes while maintaining bile canaliculi, comprising resveratrol as an active ingredient. [7] A method for maintaining bile canaliculi, comprising culturing hepatocytes using a culture medium described in any one of the above items [1] to [6].

[0009] When hepatocytes are cultured using the culture medium of the present invention, bile canals can be maintained at a high structural and functional level for a long period of time (at least 32 days after the formation of bile canals). Therefore, they can be used as model cells for evaluating the bile excretion of candidate compounds and assessing the risk of cholestasis in drug development.

[0010] Figure 1 shows phase-contrast microscope images at day 14 of culture in the culture medium of the present invention, the base medium, and the medium for bile canal formation. The scale bar represents 100 μm. Figure 2A shows the accumulation of phase-contrast microscope images (1st row), CDFDA (fluorescence microscope image; 2nd row), and Tauro-nor-THCA-24-DBD (fluorescence microscope image; 3rd row) in bile canals at days 7, 14, and 21 of culture in the base medium. The scale bar represents 100 μm. Figure 2B shows the accumulation of phase-contrast microscope images (1st row), CDFDA (fluorescence microscope image; 2nd row), and Tauro-nor-THCA-24-DBD (fluorescence microscope image; 3rd row) in bile canals at days 7, 14, and 21 of culture in the culture medium of the present invention. The scale bar represents 100 μm. Figure 3A shows phalloidin stained images at days 3, 7, 14, 21, 28, and 35 of culture in the culture medium of the present invention. Figure 3B shows the accumulation of CDFDA (fluorescence micrograph) in bile canaliculi on days 3, 7, 14, 21, 28, and 35 of culture in the culture medium of the present invention. The scale bar represents 100 μm. Figure 4 shows the accumulation of CDFDA (fluorescence micrograph; 2nd row) in bile canaliculi on days 7, 14, 21, and 28 of culture when human hepatocytes derived from chimeric mice were cultured in the culture medium of the present invention. The scale bar represents 100 μm. Figure 5 shows the accumulation of CDFDA (fluorescence micrograph; 2nd row) and Tauro-nor-THCA-24-DBD (fluorescence micrograph; 3rd row) in bile canaliculi on day 14 of culture in culture media to which each component was added to the base medium. The scale bar represents 100 μm. Figure 6 shows the accumulation of CDFDA (fluorescence microscopy image) in bile canaliculi on day 10 of culture (Lot. CAK) in media to which each component was added to the base medium. The scale bar represents 100 μm. Figure 7 shows the accumulation of CDFDA (fluorescence microscopy image; second row) and Tauro-nor-THCA-24-DBD (fluorescence microscopy image; third row) in bile canaliculi on day 14 of culture in media to which each inhibitor was added to the base medium. The scale bar represents 100 μm.

[0011] The culture medium of the present invention is a culture medium for culturing hepatocytes while maintaining bile canaliculi, and contains one or more active ingredients selected from the group consisting of AMPK activators, Epac activators, apoptosis inhibitors, and antioxidants. In particular, the culture medium of the present invention can be used for long-term culture of hepatocytes while structurally and functionally maintaining bile canaliculi. Human-derived hepatocytes are preferred as hepatocytes that form bile canaliculi, but hepatocytes derived from animals other than humans may also be used. Examples of animals other than humans include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, cattle, horses, goats, and monkeys.

[0012] Hepatocytes may be human liver cancer-derived cell lines, human hepatocytes derived from chimeric mice, hepatocytes differentiated and induced from pluripotent stem cells, or hepatocytes further matured from such hepatocytes. However, preferably, hepatocytes collected from living organisms, such as primary cultured hepatocytes, are used. Here, the meaning of "primary cultured hepatocytes" can be interpreted in the sense commonly used by those skilled in the art, but it can also be defined as hepatocytes cultured by first seeding tissue or cells collected from living organisms. Primary cultured hepatocytes include plateable and suspension types, but either type of primary cultured hepatocyte may be used in this invention.

[0013] The culture medium of the present invention comprises a general basal culture medium and one or more selected from the group consisting of AMPK activators, Epac activators, apoptosis inhibitors, and antioxidants.

[0014] Examples of basal culture media include BME medium, BGjB medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM medium, IMDM medium, Medium 199 medium, Eagles MEM medium, αMEM medium, DMEM medium, Ham medium, RPMI 1640 medium, Fischer's medium, William's E medium, and mixtures thereof, but are not particularly limited as long as they can be used for culturing animal cells. These media are commercially available and readily available.

[0015] In one embodiment, the culture medium of the present invention may contain an AMPK activator. The AMPK activator is not particularly limited, but examples include resveratrol, akadesin, metformin, nilotinib, phenformin hydrochloride, bempedoic acid, A-769662, and GSK621. The AMPK activator is preferably resveratrol. The AMPK activator is added in a concentration of preferably 0.1 to 100 μmol / L, and more preferably 1 to 50 μmol / L.

[0016] In one embodiment, the culture medium of the present invention may contain an Epac activator. The Epac activator is not particularly limited, but examples include Sp-8-BnT-cAMPS (S-220), Sp-8-BnT-2'-O-Me-cAMPS, 8-CPT-2Me-cAMP, 8-pCPT-2'-O-Me-cAMP-AM, and glucagon. The Epac activator is preferably Sp-8-BnT-cAMPS (S-220). The Epac activator is added in a concentration of preferably 0.01 to 100 μmol / L, and more preferably 0.1 to 10 μmol / L.

[0017] In one embodiment, the culture medium of the present invention may contain an apoptosis inhibitor. The apoptosis inhibitor is not particularly limited, but examples include 2,2'-methylenebis(1,3-cyclohexanedione), Z-VAD-FMK, Z-VAD(OMe)-FMK, PMA, and sodium orthovanadate. The apoptosis inhibitor is preferably 2,2'-methylenebis(1,3-cyclohexanedione). The apoptosis inhibitor is added in a concentration of preferably 0.1 to 100 μmol / L, and more preferably 5 to 20 μmol / L.

[0018] In one embodiment, the culture medium of the present invention may contain an antioxidant. The antioxidant is not particularly limited, but examples include N-acetylcysteine, L-ascorbic acid, bilirubin, butylhydroxytoluene (BHT), coenzyme Q10, curcumin, epigallocatechin, reduced glutathione, and hesperidin. The antioxidant is preferably N-acetylcysteine. The antioxidant is added in a concentration of preferably 0.1 to 10 mmol / L, and more preferably 0.5 to 2 mmol / L.

[0019] The culture medium of the present invention may also contain other additives, such as serum albumin (e.g., bovine serum albumin or human serum albumin), lipids, amino acids (e.g., non-essential amino acids), vitamins, sugars, hormones, growth factors, cytokines, insulin, transferrin, serum substitutes, 2-mercaptoethanol, pyruvate, dimethyl sulfoxide (DMSO), adenylyl cyclase activators (e.g., forskolin), buffers (e.g., HEPES), inorganic salts, antibiotics (e.g., penicillin or streptomycin), or antibacterial agents (e.g., amphotericin B).

[0020] In one embodiment, the culture medium of the present invention does not contain one or more components selected from oncostatin M (OsM), hepatocyte growth factor (HGF), and dexamethasone (DEX). Preferably, the culture medium of the present invention does not contain any of OsM, HGF, and DEX.

[0021] In one embodiment, the method for maintaining bile canaliculi of the present invention includes seeding hepatocytes and then forming bile canaliculi. Herein, in one embodiment, the hepatocytes are primary cultured hepatocytes. In one embodiment, the method for maintaining bile canaliculi of the present invention is a method for maintaining culture of hepatocytes that have already formed bile canaliculi.

[0022] The hepatocyte culture method is typically carried out in a CO2 incubator at a culture temperature suitable for hepatocyte culture (usually 30-40°C, preferably around 37°C). From the viewpoint of the structure and function of bile canaliculi, the specific culture period is usually 1 to 60 days, preferably 2 to 32 days, and more preferably 4 to 18 days, after bile canaliculi formation and switching to the culture medium of the present invention.

[0023] The method for maintaining bile canaliculi of the present invention may, in one embodiment, include, but is not limited to, the step of layering a solubilized basement membrane extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma onto hepatocytes. Examples of solubilized basement membranes extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma include Matrigel® (Corning).

[0024] In one embodiment, the method for maintaining bile canaliculi of the present invention may include, but is not limited to, a step of layering an extracellular matrix on hepatocytes. Examples of the extracellular matrix include collagen, in addition to the solubilized basement membrane extracted from the Engelblesse-Holm swarm (EHS) mouse sarcoma described above.

[0025] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0026] 1. Materials and Methods [Preparation of Base Medium] This medium was prepared using William's E as the base medium, according to the medium composition shown in Table 1.

[0027] [Preparation of culture medium for bile canal formation] This culture medium was prepared using William's E as the base medium, according to the culture medium composition shown in Table 2.

[0028] [Preparation of the Culture Medium of the Invention] The culture medium of the invention was prepared using William's E as the base medium, according to the culture medium composition shown in Table 3.

[0029] [Hepatocyte culture for bile canal formation] Frozen stocks of primary human hepatocytes (PHH) (BioIVT, Lot.SBF, Lot.WYZ, and Lot.CAK) were thawed using commercially available thawing medium, and the cell count was measured using trypan blue. PHH cells were placed in 1.0 × 10⁶ well plates coated with collagen I. 5 Cells were seeded in a capillary bile duct formation medium at a seeding density of cells / well, resulting in a medium volume of 75 μL / well. The cells were cultured at 37°C under 5% CO2 conditions. Four hours, one day, and two days after seeding, the entire medium was changed to 75 μL / well using capillary bile duct formation medium, and the cells were cultured until day 3. Chimeric mouse-derived human hepatocytes (Fresh Suspended Ready-to-plate HepaSH®, model number FSSH001, K.A.C. Co., Ltd.) were processed using OneStep PHep Thawing Medium for hepatocytes (model number MIL130C), sold by the same company, to remove dead cells according to the recommended protocol. After suspension in the company's Seeding Medium (model number MIL221), the cell count was measured using trypan blue. After removing the supernatant by centrifugation, capillary bile duct formation medium was added to the remaining cell pellet to prepare a cell suspension. The cells were then placed in a collagen I-coated 96-well plate with a PHH of 1.0 × 10⁻⁶. 5 Cells were seeded using a medium for bile canaliculi formation at a seeding density of cells / well, resulting in a medium volume of 75 μL / well. The cells were then cultured at 37°C under 5% CO2 conditions. Four hours, one day, two days, and three days after seeding, the entire medium was changed using the same medium for bile canaliculi formation to maintain a volume of 75 μL / well. The cells were cultured until day 4.

[0030] [Maintenance Culture of Capillary Bileduct-Forming Hepatocytes] Lot. PHH was cultured using capillary bile duct formation medium until day 3. From day 3 onwards, each medium (base medium listed in Table 1, base medium with each component or inhibitor added, capillary bile duct formation medium listed in Table 2, and the medium of the present invention listed in Table 3) was used for culture under conditions of 37°C and 5% CO2. Lot. CAK underwent a complete medium change to 75 μL / well using each medium on days 3 and 6-9, and was cultured until day 10. Lot. SBF underwent a complete medium change to 75 μL / well using each medium on days 3, 6-10, 13-17, and 20, and was cultured until day 21. Lot. WYZ underwent the above medium change schedule using the medium of the present invention, as well as a complete medium change to 75 μL / well using the medium of the present invention on days 20-24, 27-31, and 34, and was cultured until day 35. Human hepatocytes derived from chimeric mice were cultured using a medium for bile canal formation until day 4. From day 4 onwards, they were cultured at 37°C under 5% CO2 conditions using the culture medium of the present invention as described in Table 3. On days 4-8, 11-15, 18-21, and 24-27, the entire culture medium was changed to 75 μL / well using each medium, and the cells were cultured until day 28. Of these, on days 1, 8, 15, and 22, the culture medium was changed with the culture medium of the present invention supplemented with Matrigel® (Corning Corporation) at a final concentration of 0.25 mg / mL.

[0031] [Evaluation of excretion function into bile canaliculi by bile transporters] Lot. SBF of PHH was prepared by removing the culture medium on days 7, 14, and 21 of culture, adding 100 μL / well of 4 μmol / L CDFDA solution or 40 μmol / L Tauro-nor-THCA-24-DBD, and incubating in a CO2 incubator (5% CO2, 37°C) for 10 minutes (CDFDA) or 30 minutes (Tauro-nor-THCA-24-DBD). Subsequently, the samples were washed three times with HBSS(+), and 75 μL / well of the medium under each condition was added, followed by incubation in a CO2 incubator (5% CO2, 37°C) for 10 minutes (CDFDA) or 30 minutes (Tauro-nor-THCA-24-DBD), and then observed under a fluorescence microscope. Lot.CAK cultured in the culture medium of the present invention had its culture medium removed on day 10, and Lot.WYZ had its culture medium removed on days 3, 7, 14, 21, 28, and 35. A 4 μmol / L CDFDA solution was added, and the cells were incubated for 10 minutes. Subsequently, the cells were washed three times with HBSS(+), and 75 μL / well of the culture medium under each condition was added. The cells were then incubated in a CO2 incubator (5% CO2, 37°C) for 10 minutes, and observed under a fluorescence microscope. Human hepatocytes derived from chimeric mice cultured in the culture medium of the present invention also had their culture medium removed on days 7, 14, 21, and 28. A 4 μmol / L CDFDA solution was added, and the cells were incubated for 10 minutes. Subsequently, the cells were washed three times with HBSS(+), and 75 μL / well of the culture medium under each condition was added. The cells were then incubated in a CO2 incubator (5% CO2, 37°C) for 10 minutes, and observed under a fluorescence microscope.

[0032] [Verification of the effect of the culture medium of the present invention on maintaining bile canaliculi] After culturing for 3 days in a medium for bile canaliculi formation, maintenance culture was performed by switching to base medium, bile canaliculi formation medium, and the culture medium of the present invention. Phase-contrast microscopy observation and evaluation of excretion function by bile duct transporters were performed on culture days 7, 14, and 21. For Lot. WYZ of PHH cultured in the culture medium of the present invention, phalloidin staining (visualization of bile canaliculi structure) and evaluation of excretion function by bile duct transporters were performed on culture days 3, 7, 14, 21, 28, and 35.

[0033] [Effect Verification of Each Component of the Culture Medium of the Invention on Maintaining Bile Canaliculi] After culturing for 3 days in the culture medium for bile canaliculi formation, maintenance culture was performed by switching to the base medium, or the base medium supplemented with resveratrol, S-220, 2,2'-methylenebis(1,3-cyclohexanedione), or N-acetylcysteine, respectively. On day 14 of culture, phase-contrast microscopy observation and evaluation of excretory function by bile duct transporters were performed. Furthermore, after culturing Lot.CAK in a medium for bile canal formation for 3 days, maintenance culture was performed by switching to base medium, and then to base medium supplemented with AMPK activators (resveratrol, metformin, A-769662), Epac activators (S-220, glucagon, 8-pCPT-2'-O-Me-cAMP-AM), apoptosis inhibitors (2,2'-methylenebis(1,3-cyclohexanedione), Z-VAD-FMK, sodium orthovanadate), and antioxidants (N-acetylcysteine, curcumin, hesperidin), respectively. On day 10 of culture, the excretory function was evaluated by bile duct transporters.

[0034] [Evaluation of the effects of inhibitory components in the maintenance culture of hepatocytes forming bile canaliculi] After culturing for 3 days in a medium for bile canaliculi formation, maintenance culture was performed by switching to base medium, or base medium supplemented with HGF, OsM, or DEX, respectively. On day 14 of culture, phase-contrast microscopy observation and evaluation of excretory function by bile duct transporters were performed.

[0035] 2. Results [Maintenance Culture of Capillary Bile Duct-Forming Hepatocytes] On day 14 of culture, cell detachment was frequently observed under the maintenance culture conditions using the capillary bile duct-forming medium (Figure 1). On the other hand, no cell detachment was observed under other conditions, and intercellular interfaces were observed. In particular, cell morphology was good under the conditions of the culture medium of the present invention.

[0036] [Verification of the effect of the culture medium of the present invention on maintaining bile canaliculi] Furthermore, on culture days 7, 14, and 21, the maintenance culture conditions using the culture medium of the present invention showed significantly greater accumulation of CDFDA and Tauro-nor-THCA-24-DBD in the bile canaliculi compared to the conditions using the base medium (Figures 2A and 2B). In addition, visualization with phalloidin, which specifically stains F-actin that forms the bile canaliculi structure, showed that the maintenance of the bile canaliculi structure was observed from culture day 3 to day 35 using the culture medium of the present invention with a different cell lot, and significant accumulation of CDFDA in the bile canaliculi was also observed (Figures 3A and 3B). In human hepatocytes derived from chimeric mice that were maintained cultured using the culture medium of the present invention, good polygonal cell morphology characteristic of hepatocytes was observed in phase-contrast microscopy images on culture days 7, 14, 21, and 28. Accumulation of CDFDA in the bile canaliculi was also observed on culture days 7, 14, 21, and 28 (Figure 4).

[0037] [Verification of the effects of each component of the culture medium of the present invention on the maintenance of bile canales] On day 14 of culture, maintenance culture conditions using media supplemented with resveratrol, S-220, 2,2'-methylenebis(1,3-cyclohexanedione), or N-acetylcysteine ​​showed significantly greater accumulation of CDFDA and Tauro-nor-THCA-24-DBD in the bile canales compared to conditions using only the base medium (Figure 5). On day 10 of culturing Lot.CAK, maintenance culture conditions using media supplemented with AMPK activators (resveratrol, metformin, A-769662), Epac activators (S-220, glucagon, 8-pCPT-2'-O-Me-cAMP-AM), apoptosis inhibitors (2,2'-methylenebis(1,3-cyclohexanedione), Z-VAD-FMK, sodium orthovanadate), and antioxidants (N-acetylcysteine, curcumin, hesperidin) showed significantly greater accumulation of CDFDA in the bile canaliculi compared to culture conditions using the base medium (Figure 6).

[0038] [Evaluation of the effects of inhibitory components in the maintenance culture of hepatocytes forming bile canaliculi] On day 14 of culture, under maintenance culture conditions using a base medium supplemented with HGF, OsM, or DEX, almost no accumulation of CDFDA and Tauro-nor-THCA-24-DBD in the bile canaliculi was observed (Figure 7).

[0039] 3. Discussion The results of maintenance culture of hepatocytes forming bile canaliculi suggested that the culture medium for bile canaliculi formation is unsuitable for maintenance culture. Furthermore, the results obtained from the above under culture conditions with resveratrol, S-220, 2,2'-methylenebis(1,3-cyclohexanedione), or N-acetylcysteine ​​added to the base medium, and under the conditions of the present invention, suggested that resveratrol, S-220, 2,2'-methylenebis(1,3-cyclohexanedione), and N-acetylcysteine ​​contribute to the long-term maintenance of bile canaliculi structure. In addition, the results of the effect verification of each inhibitory component in the maintenance culture of hepatocytes forming bile canaliculi suggested that HGF, OsM, and DEX inhibit the long-term maintenance of bile canaliculi structure.

[0040] This invention enables the long-term maintenance culture of hepatocytes that form bile canals while maintaining their bile canal structure. Hepatocytes that form bile canals and are cultured according to this invention can be used in a wide range of fields, including pharmacokinetic studies such as evaluation of bile excretion of candidate drugs in non-clinical trials, safety studies such as evaluation of cholestasis, and research.

Claims

A culture medium for culturing hepatocytes while maintaining bile canaliculi, the medium comprising one or more active ingredients selected from the group consisting of AMPK activators, Epac activators, apoptosis inhibitors, and antioxidants. 　 The culture medium according to claim 1, wherein the AMPK activator is resveratrol. The culture medium according to claim 1, wherein the Epac activator is Sp-8-BnT-cAMPS. 　 The culture medium according to claim 1, wherein the apoptosis inhibitor is 2,2'-methylenebis(1,3-cyclohexanedione). The culture medium according to claim 1, wherein the antioxidant is N-acetylcysteine. 　 A culture medium for culturing hepatocytes while maintaining bile canaliculi, the culture medium comprising resveratrol as an active ingredient. 　 A method for maintaining bile canaliculi, comprising culturing hepatocytes using a culture medium according to any one of claims 1 to 6.

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