Methods for assessing hepatotoxicity

Abstract

To provide methods for highly sensitive toxicity assessment using cultured hepatocytes.SOLUTION: The present invention comprises a method of assessing hepatotoxicity, the method comprising: step 1 of culturing a hepatocyte α to obtain a hepatocyte β; step 2 of culturing the hepatocyte β obtained in step 1 in the presence and absence of a test substance; step 3 of analyzing the cytotoxicity index in the medium or hepatocyte β after the culture in step 2; and step 4 of comparing the cytotoxicity indexes, the steps 1 and 2 being carried out in a culture vessel in which part or all of the culture surface is formed from a substrate containing a 4-methyl-1-pentene polymer (A).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating hepatotoxicity. [Background technology]

[0002] Chemical substances contained in foods and pharmaceuticals taken into the body are converted into water-soluble substances and excreted from the body through a series of metabolic reactions mediated by various drug-metabolizing enzymes, including phase 1 drug-metabolizing enzymes such as cytochrome P450, and phase 2 drug-metabolizing enzymes such as conjugating enzymes that conjugate sulfate, glucuronic acid, and glutathione. Drug metabolism takes place in the liver, kidneys, and intestines, and because many drug-metabolizing enzymes are present in the liver, the liver plays an important role in the metabolism of chemicals, especially toxic substances.

[0003] In pharmaceutical development, drug metabolism tests using cultured hepatocytes are conducted to evaluate the toxicity and drug sensitivity of drug candidates or pharmaceuticals. For more accurate evaluation, it is desirable that the cultured hepatocytes reproduce the in vivo environment as closely as possible.

[0004] In vivo, hepatocytes form a three-dimensional network to function as a liver. For example, it has been reported that when hepatocytes are cultured on conventional plates, the activity of cytochrome P450 decreases over time (Non-Patent Document 1).

[0005] Under these circumstances, three-dimensional culture methods are being developed in which cultured hepatocytes form a three-dimensional network. For example, Patent Document 1 discloses a culture material having predetermined properties containing a 4-methyl-1-pentene polymer (X), a culture vessel formed from the culture material, and a culture method for cells using the culture vessel, and reports that the culture method enables three-dimensional culture of hepatocytes. However, the document does not propose any toxicity assessment of chemical substances using hepatocytes cultured by the culture method. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2020 / 256079 [Non-patent literature]

[0007] [Non-Patent Document 1] Ulvestad et al.,Biochem Pharmacol. 2013;86:691-702. Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to provide a method for highly sensitive toxicity evaluation using cultured hepatocytes. As a result of extensive research to solve the above problems, the present inventors discovered that by using cultured hepatocytes cultured in a specified culture vessel, the drug metabolism function of the hepatocytes is enhanced and their sensitivity to toxicity is increased compared to when hepatocytes cultured in conventional culture vessels are used, thereby enabling highly sensitive toxicity evaluation of chemical substances, and thus completed the present invention. [Means for solving the problem]

[0009] That is, the present invention includes the following [1] to

[11] . [1] A method for evaluating hepatotoxicity, comprising step 1 of culturing hepatocytes α to obtain hepatocytes β, step 2 of culturing the hepatocytes β obtained in step 1 in the presence and absence of a test substance, step 3 of analyzing the cytotoxicity index in the medium or in the hepatocytes β after the culture in step 2, and step 4 of comparing the cytotoxicity index, wherein steps 1 and 2 are carried out in a culture vessel formed from a substrate containing a 4-methyl-1-pentene polymer (A) in part or in its entirety on the culture surface. [2] The method for evaluating hepatotoxicity described in [1], wherein in step 2, the hepatocyte beta cells are cultured in the presence of an inhibitor that inhibits at least one selected from phase 1 drug-metabolizing enzymes, phase 2 drug-metabolizing enzymes, and transporters.

[0010] [3] The method for evaluating hepatotoxicity according to [1] or [2], wherein the 4-methyl-1-pentene polymer (A) is a homopolymer of 4-methyl-1-pentene or a copolymer of 4-methyl-1-pentene and at least one selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).

[0011] [4] A method for evaluating hepatotoxicity according to any one of [1] to [3], wherein the expression of at least one gene selected from a phase 1 drug-metabolizing enzyme, a phase 2 drug-metabolizing enzyme, and a transporter is increased in the hepatocyte beta cells obtained in step 1. [5] The method for evaluating hepatotoxicity according to any one of [2] to [4], wherein the phase 1 drug-metabolizing enzyme is at least one enzyme belonging to the cytochrome P450 subfamily selected from CYP1A, CYP3A, CYP2C, and CYP2D. [6] The method for evaluating hepatotoxicity according to any one of [2] to [4], wherein the phase 2 drug-metabolizing enzyme is an enzyme belonging to at least one species selected from the UDP-glucuronyltransferase superfamily, the sulfotransferase superfamily, the glutathione transferase superfamily, and the acetyltransferase superfamily. [7] A method for evaluating hepatotoxicity described in any of [2] to [4], wherein the transporter is at least one selected from transporters belonging to the NTCP, BSEP, and MRP subfamilies, transporters belonging to the MDR subfamily, and transporters belonging to the OATP family.

[0012] [8] The method for evaluating hepatotoxicity according to any one of [1] to [7], wherein the hepatocyte β forms aggregates. [9] The method for evaluating hepatotoxicity described in [8], wherein the aggregates include bile canaliculi.

[0013]

[10] A method for culturing hepatocytes used in the method for evaluating hepatotoxicity described in any one of [1] to [9], comprising a step of culturing hepatocytes in a culture vessel in which part or all of the culture surface is formed from a substrate containing 4-methyl-1-pentene polymer (A).

[0014]

[11] A culture vessel having a culture surface partially or entirely formed from a substrate containing a 4-methyl-1-pentene polymer (A), and hepatocytes β; A kit for evaluating hepatotoxicity, comprising: an inhibitor that inhibits at least one selected from a phase 1 drug-metabolizing enzyme, a phase 2 drug-metabolizing enzyme, and a transporter. [Effects of the Invention]

[0015] According to the present invention, it is possible to perform toxicity evaluation with high sensitivity for chemical substances. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 is a graph comparing LDH activity when hepatocytes were cultured on T plates or PS plates and exposed to rotenone in the presence or absence of the CYP inhibitor 1-ABT. [Figure 2] FIG. 2 is a graph comparing LDH activity when hepatocytes were cultured on T plates or PS plates and exposed to phenformin. [Figure 3] FIG. 3 is a graph comparing LDH activity when hepatocytes were cultured on T plates or PS plates and exposed to bile acids in the presence or absence of cyclosporin A, a BSEP inhibitor. [Figure 4] Figure 4 is a graph comparing LDH activity when hepatocytes were cultured on T plates or PS plates in the presence or absence of the CYP inhibitor 1-ABT and exposed to flutamide or clopidogrel (DMSO as a control). [Figure 5]FIG. 5 is a graph comparing the gene expression levels and activities of phase 1 drug-metabolizing enzymes in hepatocytes cultured on T plates and PS plates. [Figure 6] FIG. 6 is a graph comparing the gene expression levels and activities of phase II drug-metabolizing enzymes in hepatocytes cultured on T plates and PS plates. [Figure 7] FIG. 7 is a graph comparing the levels of transporter gene expression in hepatocytes cultured on T plates and PS plates. [Figure 8] FIG. 8 shows fluorescence micrographs of hepatocytes cultured on T plates or PS plates, immunostained for MRP2 and F-actin, which are indicators of bile canalicular structure. [Figure 9] 9 is a graph comparing the amount of lactate released into the medium from hepatocytes cultured on T plates and PS plates. The horizontal axis indicates the number of days of culture. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention will be described in detail below. A first aspect of the present invention is a method for evaluating hepatotoxicity, comprising step 1 of culturing hepatocytes α to obtain hepatocytes β, step 2 of culturing the hepatocytes β obtained in step 1 in the presence and absence of a test substance, step 3 of analyzing a cytotoxicity index in the medium or in the hepatocytes β after the culture in step 2, and step 4 of comparing the cytotoxicity indexes, wherein steps 1 and 2 are carried out in a culture vessel whose culture surface is formed, in part or in whole, from a substrate containing 4-methyl-1-pentene polymer (A).

[0018] [Process 1] Step 1 of the present invention is a step of culturing hepatocytes α in a culture vessel (hereinafter also referred to as "culture vessel (T)") formed from a substrate containing 4-methyl-1-pentene polymer (A) in part or in its entirety on the culture surface to obtain hepatocytes β.

[0019] (hepatocyte α) Hepatocyte α in the present invention may be any cell present in the liver, including hepatocytes, and specifically includes vascular endothelial cells, vascular smooth muscle cells, adipocytes, blood cells, hepatic mononuclear cells, hepatic macrophages (including Kupffer cells), hepatic stellate cells, intrahepatic bile duct epithelial cells, embryonic sac fibroblasts, etc. The hepatocytes are a cell population containing, for example, 20% or more, 30% or more, 40% or more, or 50% or more hepatic parenchymal cells.

[0020] Hepatocyte α may be a cell population containing cell types other than hepatocytes, for example, a cell population containing 20% ​​or more, 30% or more, 40% or more, or 50% or more hepatocytes.

[0021] Hepatocyte α may be normal cells, cancer cells, primary cultured hepatocytes, established and continuous hepatocyte lines, or hepatocytes differentiated from iPS cells or ES cells, but is preferably primary cultured hepatocytes. The type of established and continuous hepatocyte line is not particularly limited, but examples include SSP-25, RBE, HepG2, TGBC50TKB, HuH-6, HuH-7, ETK-1, Het-1A, PLC / PRF / 5, Hep3B, SK-HEP-1, C3A, THLE-2, THLE-3, HepG2 / 2.2.1, SNU-398, SNU-449, SNU-182, SNU-475, SNU-387, SNU-423, FL62891, and DMS153.

[0022] Hepatocytes α are preferably mammalian-derived hepatocytes, more preferably human, bovine, canine, feline, porcine, miniature pig, rabbit, hamster, rat, or mouse-derived hepatocytes, and even more preferably human, rat, mouse, or bovine-derived hepatocytes.

[0023] (culture) In the present invention, the term "culture" is used in a broad sense to include not only the proliferation and maintenance of hepatocytes α and hepatocytes β (hereinafter referred to as "hepatocytes, etc.") but also processes such as seeding, passaging, differentiation induction, and self-organization induction of hepatocytes, etc. The method for culturing hepatocytes, etc. in a medium is not particularly limited, and may be performed according to known methods such as sandwich culture, or commercially available media or culture kits may be used.

[0024] Hepatocytes and the like may be cultured by either adherent culture or suspension culture. Adherent culture is preferred because oxygen can be efficiently supplied to the medium in the culture vessel (T) via the substrate containing the 4-methyl-1-pentene polymer (A).

[0025] There are no particular limitations on the medium used for culturing hepatocytes, and a medium may be selected depending on the characteristics of the hepatocytes. Examples of suitable media include basal cell culture media, differentiation media, and media specifically designed for primary culture, such as Williams' Medium E (WME), Eagle's Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle's Medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI 1640, Ham's F-12, and MCDB medium, as well as mixtures thereof. Furthermore, media supplemented with serum, various growth factors, differentiation inducers, antibiotics, hormones, amino acids, sugars, salts, minerals, metals, vitamins, and the like may also be used, with the addition of sugars being preferred.

[0026] The sugar source in the medium can be any sugar source, but glucose, galactose, fructose, and mannose are preferred, with glucose being more preferred in terms of easy medium availability. The type of medium may be changed during the culture period; for example, a medium containing glucose as a sugar source may be changed to a medium containing galactose as a sugar source, or a medium containing galactose as a sugar source may be changed to a medium containing glucose as a sugar source. The sugar source may be changed as desired during the culture period. Furthermore, two or more of these sugar sources may be used in combination.

[0027] The amount of medium used for culturing hepatocytes and the like is not particularly limited, but when added to the culture vessel (T), the longest vertical distance from the culture surface of the culture vessel to the top surface of the medium is preferably 1 mm or more and 30 mm or less, more preferably 2 mm or more and 20 mm or less, and even more preferably 2 mm or more and 10 mm or less.

[0028] The culture period for hepatocytes α may be appropriately determined depending on the type of medium and culture conditions, as long as the hepatocytes α are able to form a two-dimensional or three-dimensional structure. The culture period is preferably 1 to 21 days, more preferably 3 to 14 days, and even more preferably 5 to 10 days. The frequency of medium change during the culture period is not particularly limited, but it is preferable to change the medium every day. The culture temperature is not particularly limited, but is usually about 25 to 40°C.

[0029] (hepatocyte β) Hepatocyte β is hepatocyte obtained by culturing the above-mentioned hepatocyte α. There are no particular limitations on size, morphology, or cell number, but it is preferable that the hepatocyte β form aggregates. An aggregate is a collection of cells with a three-dimensional shape in which two or more hepatocyte α cells are stacked in two or more layers, and with well-developed cell-cell interactions and extracellular matrix, it more closely resembles the in vivo state. It is preferable that bile canaliculi are formed in the aggregate. Bile canaliculi formation can be confirmed by microscopic observation using immunostaining with antibodies specific to transporters present in hepatocytes forming the bile canaliculi, such as MRP2 (Multidrug-resistant protein 2), BSEP (Bile salt export pump), NTCP (Na+ taurocholate cotransporting polypeptide), MDR1 (Multidrug-resistant 1), and OATP (Organic anion transporting polypeptide).

[0030] Preferably, increased expression of at least one gene selected from phase 1 drug-metabolizing enzymes, phase 2 drug-metabolizing enzymes, and transporters is observed in hepatocyte β. The increased gene expression can be determined by, for example, using a polystyrene cell culture vessel instead of the culture vessel (T) used in the present invention, culturing hepatocytes under the same experimental conditions as in the culture methods of steps 1 and 2, and observing a higher expression level than that of a control, preferably a significantly higher expression level. Gene expression levels can be measured by known methods, such as quantitative RT-PCR.

[0031] Preferably, hepatocyte β exhibits increased activity of at least one enzyme selected from phase 1 and phase 2 drug-metabolizing enzymes. The increased enzyme activity can be determined by, for example, using a polystyrene cell culture vessel instead of the culture vessel (T) used in the present invention and culturing hepatocytes under the same experimental conditions as in the culture method of steps 1 and 2, and measuring the activity of the same enzyme as a control; the increased enzyme activity is preferably significantly higher than that of the control. Enzyme activity can be assessed by known methods; for example, a compound to be metabolized (parent compound) can be used as a probe for a specific metabolic enzyme, and the amount of metabolite produced can be measured, for example, by LC-MS / MS, and the resulting amount of metabolite produced can be used as an index for activity assessment.

[0032] (Phase 1 drug-metabolizing enzymes) Phase 1 drug-metabolizing enzymes are a group of enzymes involved in phase 1 reactions that do not significantly change the molecular weight of a target substance or reduce it through degradation, such as hydrolysis of esters, oxidation, and reduction. Examples of phase 1 drug-metabolizing enzymes include enzymes belonging to the CYP (cytochrome P450) family, such as CYP1, CYP2, CYP3, and CYP4, as well as enzymes and molecular species belonging to these subfamilies. Examples of CYP subfamilies include CYP1A, CYP1B, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E, CYP2J, CYP3A, CYP4A, and CYP4B. Examples of CYP molecular species include CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D4, CYP2D6, CYP2E1, CYP3A1, CYP3A4, CYP3A5, and CYP3A7. Among these, the phase 1 drug-metabolizing enzyme is preferably an enzyme belonging to at least one subfamily selected from CYP1A, CYP1B, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E, CYP2J, CYP3A, CYP4A, and CYP4B, more preferably an enzyme belonging to at least one subfamily selected from CYP1A, CYP3A, CYP2C, and CYP2D, and even more preferably CYP1A2, CYP2D4, and CYP3A1.

[0033] The reason why hepatotoxicity, for example, mitochondrial toxicity, can be evaluated with high sensitivity by culturing hepatocyte β using the culture vessel (T) described below is unclear, but it is speculated as follows. Normally, under culture conditions where oxygen supply to hepatocytes is insufficient, energy production in mitochondria is reduced, resulting in a reduced sensitivity to mitochondrial toxicity. On the other hand, it is speculated that when hepatocytes are cultured using the vessel (T), sufficient oxygen is supplied to the hepatocytes, energy production in mitochondria increases, and sensitivity to mitochondrial toxicity increases. Note that, for example, the amount of lactic acid released by hepatocytes into the culture medium under anaerobic conditions can be used as an indicator of oxygen supply to hepatocytes.

[0034] (Phase 2 drug-metabolizing enzymes) Phase 2 drug-metabolizing enzymes are a group of enzymes involved in phase 2 reactions (conjugation reactions) that add endogenous substances such as sulfate, acetate, glutathione, and glucuronic acid to increase molecular weight. Phase 2 drug-metabolizing enzymes are not particularly limited, but are preferably at least one enzyme selected from the UDP-glucuronyltransferase superfamily (UGT superfamily), sulfotransferase superfamily (SULT superfamily), glutathione transferase superfamily (GST superfamily), and acetyltransferase superfamily (NAT superfamily).

[0035] As the enzyme belonging to the UGT superfamily, an enzyme belonging to at least one family selected from UGT1, UGT2, and UGT3 is preferred, an enzyme belonging to at least one subfamily selected from UGT1A and UGT2B is more preferred, and at least one enzyme selected from UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 is more preferred.

[0036] As enzymes belonging to the sulfotransferase superfamily, enzymes belonging to at least one family selected from SULT1 and SULT2 are preferred, enzymes belonging to at least one subfamily selected from SULT1A, SULT1B, SULT1C, SULT1E, and SULT2A are more preferred, and at least one enzyme selected from SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1E1, and SULT2A1 are even more preferred.

[0037] As the enzyme belonging to the glutathione transferase superfamily, an enzyme belonging to at least one family selected from GSTA1, GSTA2, GSTM1 and GSTT1 is preferred.

[0038] As the enzyme belonging to the acetyltransferase superfamily, an enzyme belonging to at least one family selected from NAT1 and NAT2 is preferred.

[0039] (Transporter) Transporters are proteins involved in the uptake of drugs from the blood into the liver and the excretion of drugs and metabolites from the liver into bile. Examples of transporters include transporters that take up drugs and transporters that excrete drugs through bile. Examples of the former include transporters belonging to the NTCP (Sodium taurocholate co-transporting polypeptide), OATP (Organic anion transporting polypeptide) family, and OCT (Organic anion transporter) subfamily. Examples of the latter include transporters belonging to the MRP (Multidrug resistance protein) subfamily, BCRP (Breast cancer resistant protein), BSEP (Bile salt export pump), and MDR (Multidrug resistance protein) subfamily. The transporter is not particularly limited, but is preferably at least one selected from the NTCP, BSEP, MRP subfamily, MDR subfamily, and OATP family.

[0040] Preferred transporters belonging to the MRP subfamily are MRP2, MRP3, and MRP4. Preferred transporters belonging to the MDR subfamily are MDR1 and MDR3. Preferred transporters belonging to the OATP family are, for example, OATP1, OATP2, and OATP4, with OATP1B1 and OATP1B3 being more preferred. Preferred transporters belonging to the OCT subfamily are OCT1.

[0041] Culturing hepatocyte β cells using the culture vessel (T) described below can increase the expression of genes related to the above transporters, making it possible to evaluate cholestatic toxicity, among other types of hepatotoxicity, with greater sensitivity.

[0042] [Process 2] Step 2 of the present invention is a step of culturing the hepatocytes β obtained in step 1 above in the presence and absence of a test substance. The test substance to be evaluated for hepatotoxicity may be one type or two or more types. When evaluating two or more types of test substances, the hepatocytes β may be cultured with each test substance separately, or multiple test substances may be cultured with the hepatocytes β simultaneously.

[0043] Hepatocytes β may be cultured in the presence of an inhibitor that inhibits at least one selected from phase 1 drug-metabolizing enzymes, phase 2 drug-metabolizing enzymes, and transporters. Culturing hepatocytes β in the presence of the inhibitor using a culture vessel (T) described below increases the toxicity sensitivity of hepatocytes β compared to when conventional culture vessels are used, making it possible to evaluate hepatotoxicity at low concentrations of the test substance. The above inhibitor may be used as the test substance, and in this case, hepatocytes β may be cultured in the presence of an inhibitor that inhibits at least one selected from phase 1 drug-metabolizing enzymes, phase 2 drug-metabolizing enzymes, and transporters, other than the inhibitor used as the test substance.

[0044] Phase 1 drug-metabolizing enzyme inhibitors are not particularly limited and may be selected appropriately depending on the type of CYP to be inhibited. Examples include furaferin for CYP1A2, sulfaphenazole for CYP2C9, quinidine for CYP2D6, and ketoconazole for CYP3A4. 1-Aminobenzotriazole (1-ABT), which irreversibly binds to the heme iron contained in the CYP family and inhibits enzyme activity, may also be used.

[0045] The inhibitor of a phase 2 drug-metabolizing enzyme is not particularly limited and may be appropriately selected depending on the type of enzyme to be inhibited. Examples include borneol for UGT, pentachlorophenol for SULT, and ethacrynic acid for GST.

[0046] The transporter inhibitor is not particularly limited and may be appropriately selected depending on the type of transporter to be inhibited. Examples include cyclosporin A, flutamide, and clopidogrel for BSEP, and verapamil for MDR1.

[0047] The culture method is not particularly limited as long as it allows hepatocytes β to be exposed to a test substance or a control substance and cultured, but for example, the test substance or control substance adjusted to a predetermined concentration may be added to a medium and then hepatocytes β are seeded and cultured, or the test substance or control substance may be added to the medium in which hepatocytes β are cultured to a predetermined concentration and then cultured. The medium may be selected depending on the characteristics of the test substance and hepatocytes, and the medium exemplified in step 1 may be used.

[0048] The culture time is not particularly limited, but is, for example, 6 to 168 hours, preferably 24 to 120 hours, and more preferably 24 to 72 hours. The culture temperature is not particularly limited, but is usually about 25 to 40°C.

[0049] [Step 3] Step 3 is a step of analyzing a cytotoxicity index in the medium or hepatocyte β after the culture in step 2. The cytotoxicity index is not particularly limited, but examples thereof include LDH (lactate dehydrogenase) activity, succinate tetrazolium reductase activity, and esterase activity. Alternatively, cell viability calculated from these activity values ​​by a known method may be used as an index.

[0050] LDH activity can be measured by known methods, but may also be measured using commercially available kits such as the LDH Cytotoxicity Detection Kit (manufactured by Takara Bio Inc.) and the Cytotoxicity LDH Assay Kit-WST (manufactured by Dojindo Laboratories, Inc.). LDH activity is an enzyme present in the cytoplasm that normally remains in the cytoplasm but is released into the medium when the cell membrane is damaged. The released LDH is stable and is measured as an indicator of the number of dead cells or cells with damaged cell membranes. In other words, high LDH activity indicates a large number of dead or damaged cells.

[0051] Succinate tetrazolium reductase activity can be measured by known methods, but may also be measured using commercially available kits such as CytoSelec™ MTT Cell Proliferation Assay (Cosmo Bio Co., Ltd.) and MTT Cell Proliferation Kit (Funakoshi Co., Ltd.).

[0052] Esterase activity can be measured by known methods, but may also be measured using a commercially available kit such as Cell Counting Kit-F (manufactured by Dojindo Laboratories, Ltd.).

[0053] [Step 4] Step 4 is a step of comparing the cytotoxicity indexes, in other words, a step of comparing and considering the cytotoxicity indexes obtained by the analysis in step 3 in the presence and absence of the test substance, and determining the level of hepatotoxicity of the test substance. The criterion for judgment is whether the cytotoxicity index in the presence of the test substance is higher or lower than the cytotoxicity index in the absence of the test substance. In this case, for example, when a t-test is performed on the results of measuring the cytotoxicity index values ​​of three or more samples per group, P<0.05 is preferred, P<0.001 is more preferred, and P<0.0001 is even more preferred.

[0054] [Culture container] In the present invention, a culture vessel (culture vessel (T)) used for culturing hepatocytes or the like is formed from a base material whose culture surface is partly or entirely made of a 4-methyl-1-pentene polymer (A).

[0055] In the present invention, the term "culture vessel" refers to any vessel used for culturing cells. Various known culture vessels can be used as the culture vessel, and there are no particular limitations on the shape or size. Examples of the culture vessel include dishes, flasks, plates, bottles, bags, and tubes. Culture vessels are usually used in devices such as incubators, mass culture devices, or perfusion culture devices.

[0056] The culture surface means a surface that is in contact with a medium and / or cells when culturing cells, or a surface that will be in contact with a medium and / or cells.

[0057] The culture vessel (T) is preferably a culture vessel whose bottom surface comprises a culture surface in order to retain or store the culture medium. When the culture vessel (T) is, for example, a dish, flask, or plate, the bottom surface comprises the culture surface, and therefore at least a portion or all of the bottom surface among the bottom, side, and top surfaces thereof is formed from a substrate containing a 4-methyl-1-pentene polymer (A). When at least a portion or all of the bottom surface is formed from a substrate containing a 4-methyl-1-pentene polymer (A), oxygen can be efficiently supplied into the culture medium via the substrate containing the 4-methyl-1-pentene polymer (A), making it easier to maintain or improve the drug-metabolizing function of hepatocytes and the like in the culture medium. Furthermore, it becomes easier to culture hepatocytes and the like at high density while maintaining this function.

[0058] The entire culture surface of the culture vessel (T) is preferably formed from a substrate containing a 4-methyl-1-pentene polymer (A). That is, when the culture vessel (T) is, for example, a dish, a flask, or a plate, the bottom surface comprises the culture surface, and therefore, of the bottom, side, and top surfaces, the entire bottom surface is preferably formed from a substrate containing a 4-methyl-1-pentene polymer (A).

[0059] The thickness of the substrate containing the 4-methyl-1-pentene polymer (A) is not particularly limited, but is preferably 20 μm to 400 μm, more preferably 20 μm to 300 μm, and even more preferably 20 μm to 200 μm. The thickness of the substrate containing the 4-methyl-1-pentene polymer (A) is appropriately selected depending on the shape of the culture vessel (T), and by adjusting the thickness within the above range, it is easy to obtain an appropriate oxygen concentration in the culture medium necessary for hepatocytes and the like to grow and express drug-metabolizing functions, and it is also easy to obtain sufficient strength as a culture vessel (T).

[0060] The culture vessel (T) is preferably a culture vessel having at least one well, more preferably a plate having at least one well, and even more preferably a plate having 6, 12, 24, 48, 96, 384, or 1536 wells. Generally, culture vessels having a well-like depression on the bottom surface require a thick bottom to stabilize the complex shape of the bottom, making it difficult to adequately supply oxygen to hepatocytes and the like. When the bottom surface is formed from a substrate containing 4-methyl-1-pentene polymer (A), the shape is stable and sufficient oxygen is supplied to hepatocytes and the like, even in plates having 1, 6, 12, 24, 48, 96, 384, or 1536 wells.

[0061] The shape of the bottom of the culture vessel (T) is not particularly limited, and examples include a flat bottom (F bottom), a round bottom (U bottom), a conical bottom (V bottom), and a flat bottom with a curved edge. When processing a round bottom (U bottom), a flat bottom (F bottom), a conical bottom (V bottom), or a flat bottom with a curved edge, it can be processed in one step using general injection molding or press molding, or it can be produced by first preparing a film or sheet and then performing secondary processing such as vacuum forming or pressure forming. The shape of the bottom is selected depending on the purpose of the culture, but a flat bottom (F bottom) is usually desirable for two-dimensional culture of hepatocytes, etc., and a round bottom (U bottom) or conical bottom (V bottom) is usually desirable for three-dimensional culture.

[0062] The portions of the culture vessel (T) other than the culture surface may be made of a material other than the base material containing the 4-methyl-1-pentene polymer (A). The material is not particularly limited, and known materials can be used. Examples of such materials include polystyrene (PS), polydimethylsiloxane (PDMS), thermosetting resins, cyclic olefin polymers, cyclic olefin copolymers, and glass.

[0063] At least the culture surface of the culture vessel (T) may be coated with a natural polymer material, a synthetic polymer material, or an inorganic material. The coating can be carried out by a known method.

[0064] The coated culture vessel (T) exhibits superior adhesion and proliferation properties for hepatocytes and the like. This is thought to be because the natural polymeric material, synthetic polymeric material, or inorganic material coated on the culture surface serves as a scaffold for hepatocytes and the like. Therefore, when attaching and culturing hepatocytes and the like, it is a preferred embodiment to use the culture vessel (T) after coating it with a natural polymeric material, synthetic polymeric material, or inorganic material.

[0065] The natural polymer material, synthetic polymer material, or inorganic material is not particularly limited, and examples of natural polymer materials include collagen, gelatin, alginic acid, glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, fibronectin, laminin, fibrinogen, osteopontin, tenascin, vitronectin, thrombospondin, agarose, elastin, keratin, chitosan, fibrin, fibroin, and sugars; synthetic polymer materials include polyglycolic acid, polylactic acid, polyethylene glycol, polycaprolactone, synthetic peptides, synthetic proteins, polyhydroxyethyl methacrylate, and polyethyleneimine; and inorganic materials include β-tricalcium phosphate and calcium carbonate.

[0066] Examples of the natural polymeric material, synthetic polymeric material, or inorganic material include vitrigel, which is obtained by vitrifying a hydrogel such as a conventional extracellular matrix component and then rehydrating it, such as collagen vitrigel, which is composed of a high-density collagen fiber network made from collagen, one of the extracellular matrix components.

[0067] From the viewpoints of improving the adhesiveness and proliferation of hepatocytes and maintaining the drug metabolism function of hepatocytes for a longer period of time, coating with proteins or peptides such as collagen, gelatin, laminin, polylysine is preferred, coating with laminin, collagen, or polylysine is more preferred, and coating with collagen is even more preferred. These coatings may be used alone or in combination of two or more.

[0068] At least the culture surface of the culture vessel (T) may be processed. Examples of surface processing include surface modification treatments such as forming a concave-convex structure, hydrophilization treatment, and hydrophobic treatment.

[0069] The method used for surface modification is not particularly limited, but examples include hydrophilization treatments such as corona treatment, plasma treatment, ozone treatment, and ultraviolet treatment; hydrophobic treatments such as esterification, silylation, and fluorination; surface graft polymerization, chemical vapor deposition, etching; addition of specific functional groups such as hydroxyl groups, amino groups, sulfonic groups, thiol groups, and carboxyl groups; treatments using specific functional groups such as silane coupling, titanium coupling, and zirconium coupling; surface roughening using oxidizing agents; and physical treatments such as rubbing and sandblasting. These surface modification treatments may be performed alone or in combination of two or more. When performing surface modification treatments, it is preferable to perform them on at least the culture surface.

[0070] It is preferable to hydrophilize at least the culture surface of the culture vessel (T), and more preferably to perform corona treatment or plasma treatment. Hydrophilizing the surface of the culture vessel (T) increases the wettability of the surface of the culture vessel (T), improving adhesion between the culture vessel (T) and hepatocytes and the like, allowing hepatocytes and the like to grow uniformly on the surface of the culture vessel (T). Furthermore, hydrophilizing the surface of the culture vessel (T) makes it easier to coat natural polymeric materials, synthetic polymeric materials, or inorganic materials on the culture surface of the culture vessel (T). In particular, it makes it easier to uniformly load and adhere natural polymeric materials, synthetic polymeric materials, or inorganic materials onto the culture surface of the culture vessel (T). Even after loading, the natural polymeric materials, synthetic polymeric materials, or inorganic materials do not peel off when washed with saline or in a cell culture environment, allowing them to maintain a stable initial state and be used for cell culture.

[0071] When plasma treatment is performed, nitrogen, hydrogen, helium, oxygen, argon, etc. are used as the entraining gas, and preferably at least one gas selected from nitrogen, helium, and argon is selected.

[0072] The culture vessel (T) may be disinfected or sterilized to prevent contamination. The disinfection or sterilization method is not particularly limited, and examples thereof include physical disinfection methods such as steam circulation, boiling, intermittent irradiation, and ultraviolet light; chemical disinfection methods using gases such as ozone or disinfectants such as ethanol; heat sterilization methods such as high-pressure steam and dry heat; irradiation sterilization methods such as gamma ray sterilization and high-frequency sterilization; and gas sterilization methods such as ethylene oxide gas and hydrogen peroxide gas plasma. Among these, ethanol disinfection, high-pressure steam sterilization, gamma ray sterilization, and ethylene oxide gas sterilization are preferred because of their simple operation and ability to achieve sufficient sterilization. These disinfection or sterilization methods may be performed alone or in combination of two or more.

[0073] The culture surface of the culture vessel (T) preferably has a water contact angle of 50° to 100°, more preferably 55° to 100°, and even more preferably 60° to 100°. Another preferred embodiment of the water contact angle is 84° or less, more preferably 50° to 84°.

[0074] By adjusting the water contact angle of the culture surface of the culture vessel (T) within the above range, for example, hepatocytes and the like can be more easily attached to the culture surface and can grow uniformly on the culture surface. Furthermore, it is easier to coat a natural polymer material, synthetic polymer material, or inorganic material uniformly on the culture surface of the culture vessel (T) and ensure adhesion. Even after coating, the natural polymer material, synthetic polymer material, or inorganic material does not peel off when washed with physiological saline or in the cell culture environment, and can be used for cell culture while maintaining a stable initial state.

[0075] The method for measuring the water contact angle is not particularly limited, and known methods can be used, but the sessile drop method is preferred. The water contact angle can be measured, for example, in accordance with Japanese Industrial Standard JIS-R3257 (testing method for wettability of substrate glass surfaces), by dropping a water droplet of 4 μL or less, which can be considered spherical, under constant temperature and humidity conditions of 25±5° C. and 50±10% onto the surface of a culture vessel or a measurement sample made of the same material as the culture vessel, and measuring the angle of the contact interface between the measurement sample and the water droplet within 1 minute immediately after the water droplet contacts the surface of the measurement sample using the sessile drop method.

[0076] The method for producing the culture vessel (T) is not particularly limited, and neither is the equipment used for production. When the entire culture vessel is formed from a substrate containing a 4-methyl-1-pentene polymer (A), for example, a film or sheet containing the 4-methyl-1-pentene polymer (A) is formed, and the film or sheet is molded as needed to produce a culture vessel in the desired shape. The culture vessel can also be obtained by direct molding using methods such as extrusion molding, solution casting, injection molding, and blow molding. When only a portion of the culture vessel is formed from a substrate containing a 4-methyl-1-pentene polymer (A), for example, a film or sheet containing the 4-methyl-1-pentene polymer (A) is formed, and the film or sheet is appropriately bonded to another substrate to obtain a culture vessel. The bonding method is not particularly limited, and the substrate containing the 4-methyl-1-pentene polymer (A) and the other substrate may be integrally formed, or may be bonded to each other via an adhesive or pressure-sensitive adhesive.

[0077] Specific examples of methods for forming the film or sheet include the usual inflation method and T-die extrusion method. The production is usually carried out under heating. When the T-die extrusion method is used, the extrusion temperature is preferably 100°C to 400°C, and more preferably 200°C to 300°C. The roll temperature is preferably 45°C to 75°C, and more preferably 55°C to 65°C.

[0078] The film or sheet may be produced by a solution casting method in which the 4-methyl-1-pentene polymer (A) is dissolved in a solvent, poured onto a resin or metal, and slowly dried while leveling to form a film (sheet). There are no particular limitations on the solvent used, and hydrocarbon solvents such as cyclohexane, hexane, decane, and toluene may be used. Furthermore, two or more solvents may be mixed, taking into consideration the solubility of the 4-methyl-1-pentene polymer (A) and drying efficiency. The polymer solution can be applied by a method such as table coating, spin coating, dip coating, die coating, spray coating, bar coating, roll coating, or curtain flow coating, followed by drying and peeling to form a film or sheet.

[0079] (4-methyl-1-pentene polymer (A)) In the present invention, the 4-methyl-1-pentene polymer (A) is preferably a homopolymer of 4-methyl-1-pentene or a copolymer of 4-methyl-1-pentene and at least one selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).

[0080] The copolymer may be any of a random copolymer, an alternating copolymer, a block copolymer, and a graft copolymer. As a copolymer of 4-methyl-1-pentene and another monomer, a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) is preferred because it has high strength and is resistant to tearing, cracking, and bending even when used as a substrate.

[0081] Examples of the olefin include ethylene, propylene, 1-butene, 1-hexene, 1-heptene, 1-octene, 1-decene, 1-tetradecene, 1-hexadecene, 1-heptadecene, 1-octadecene, and 1-eicosene. The olefin can be appropriately selected depending on the physical properties required for the substrate. For example, from the viewpoints of appropriate oxygen permeability and excellent rigidity, the olefin is preferably an α-olefin having 8 to 18 carbon atoms, and more preferably at least one selected from 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-heptadecene, and 1-octadecene. When the carbon number of the olefin is within the above range, the polymer has better processability and tends to be less susceptible to poor appearance of the substrate due to cracks or edge breakage. Furthermore, the rate of defective substrates is reduced.

[0082] The olefin may be one or more of these. From the viewpoint of material strength, the carbon number is preferably 2 or more, and more preferably 10 or more. When combining two or more different α-olefins, it is particularly preferable to combine at least one selected from 1-tetradecene and 1-hexadecene with at least one selected from 1-heptadecene and 1-octadecene.

[0083] The content of structural units derived from 4-methyl-1-pentene in the 4-methyl-1-pentene polymer (A) is preferably 60 to 100 mol %, more preferably 80 to 99.5 mol %, and even more preferably 85 to 98 mol %.

[0084] When the 4-methyl-1-pentene polymer (A) is a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene), the content of structural units derived from at least one olefin selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) in the copolymer is preferably 0 to 40 mol%, more preferably 0.5 to 20 mol%, and even more preferably 2 to 15 mol%. The content of these structural units is calculated based on the total amount of repeating structural units in the 4-methyl-1-pentene polymer (A) being 100 mol%. A structural unit content within the above range provides a uniform culture surface with excellent processability, and the film has a good balance between toughness and strength, resulting in less deflection.

[0085] The 4-methyl-1-pentene polymer (A) may contain structural units (hereinafter also referred to as "other structural units") other than the structural units derived from 4-methyl-1-pentene and the structural units derived from ethylene and the α-olefin having 3 to 20 carbon atoms, as long as the effects of the present invention are not impaired. The content of the other structural units is, for example, 0 to 10.0 mol%. When the 4-methyl-1-pentene polymer (A) contains other structural units, the other structural units may be one type or two or more types.

[0086] Examples of monomers that derive other structural units include cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl-containing olefins, and halogenated olefins. Examples of the cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl-containing olefins, and halogenated olefins that can be used include the compounds described in paragraphs

[0035] to

[0041] of JP 2013-169685 A.

[0087] 4-Methyl-1-pentene polymer (A) typically has a melting point of 200°C to 240°C and is highly heat-resistant. Furthermore, because it does not undergo hydrolysis and has excellent water resistance, boiling water resistance, and steam resistance, substrates containing 4-methyl-1-pentene polymer (A) can be sterilized by high-pressure steam. 4-Methyl-1-pentene polymer (A) also has high visible light transmittance (usually 90% or more) and does not emit autofluorescence, making it easy to observe cultured cells when used with substrates containing 4-methyl-1-pentene polymer (A). 4-Methyl-1-pentene polymer (A) is heat-sealable, allowing for easy thermal fusion not only between itself but also with other materials. Furthermore, because it is thermoformable, it can be easily molded into culture vessels of any shape, including by imprinting or insert molding.

[0088] The weight-average molecular weight (Mw) of the 4-methyl-1-pentene polymer (A), measured by gel permeation chromatography (GPC) using standard polystyrene as a reference material, is preferably 10,000 to 2,000,000, more preferably 20,000 to 1,000,000, and even more preferably 30,000 to 500,000. The sample concentration during GPC measurement can be, for example, 1.0 to 5.0 mg / ml. The molecular weight distribution (Mw / Mn) of the 4-methyl-1-pentene polymer (A) is preferably 1.0 to 30, more preferably 1.1 to 25, and even more preferably 1.1 to 20. The solvent used in GPC is preferably orthodichlorobenzene.

[0089] By setting the weight-average molecular weight (Mw) to the above upper limit or less, in the molding method of the 4-methyl-1-pentene polymer (A) described below, the occurrence of defects such as gelling in the film produced by melt molding is easily suppressed, and a film with a uniform surface can be easily produced. Furthermore, when produced by a solution casting method, the solubility in a solvent is improved, and defects such as gelling in the film are easily suppressed, and a film with a uniform surface can be easily produced.

[0090] Furthermore, by setting the weight-average molecular weight (Mw) to the above lower limit or more, the culture vessel tends to have sufficient strength. Furthermore, by setting the molecular weight distribution within the above range, stickiness on the surface of the culture vessel produced tends to be suppressed, and the toughness of the culture vessel tends to be sufficient, making it easier to suppress bending during molding and cracking during cutting.

[0091] When two or more types of 4-methyl-1-pentene polymers (A) are used, the weight average molecular weight (Mw) and molecular weight distribution (Mw / Mn) of the 4-methyl-1-pentene polymer (A) may be within the above-mentioned ranges, respectively.

[0092] The 4-methyl-1-pentene polymer (A) has an oxygen permeability coefficient of 100 to 2500 cm 3 ×mm / (m 2 × 24h × atm)], and 1000 to 2500 [cm 3 ×mm / (m 2 When the oxygen permeability coefficient is in the above range, the oxygen permeability is excellent, so that the hepatocytes etc. can maintain a good morphology and can easily proliferate efficiently depending on the culture period.

[0093] Specifically, the oxygen permeability coefficient can be measured by the following method. A measurement sample is prepared from a film composed of the 4-methyl-1-pentene polymer (A), and the oxygen permeability coefficient [cm ] at a temperature of 23°C and humidity of 0% is measured by a differential pressure gas permeability measurement method. 3 ×mm / (m 2× 24h × atm)] is measured. The equipment used for the measurement is not particularly limited as long as it uses a differential pressure gas permeability measurement method, and an example is the differential pressure gas permeability measurement device MT-C3 manufactured by Toyo Seiki Seisakusho. The measurement sample is prepared by cutting a 90 × 90 mm test piece from a 50 μm thick film made of 4-methyl-1-pentene polymer (A), and the measurement section diameter is 70 mm (permeation area is 38.46 cm 2 Because the oxygen permeability is high, an aluminum mask is placed on the sample in advance to reduce the actual permeation area to 5.0 cm. 2 The measurement sample may or may not have undergone microfabrication or surface modification treatment, but it is preferable that no treatment has been performed. The oxygen permeability [cm 3 / (m 2 ×24h×atm)].

[0094] The 4-methyl-1-pentene polymer (A) may be used alone or in combination of two or more.

[0095] Commercially available 4-methyl-1-pentene polymers (A) can also be used. Specific examples include TPX MX001, MX002, MX004, MX0020, MX021, MX321, RT18, RT31, and DX845 (all trademarks) manufactured by Mitsui Chemicals, Inc. Furthermore, 4-methyl-1-pentene polymers (A) manufactured by other manufacturers that satisfy the above requirements can also be preferably used. These commercially available products can be used alone or in combination of two or more.

[0096] Because 4-methyl-1-pentene polymer (A) has the excellent properties described above, culture vessels formed of a substrate in which part or all of the culture surface is made of 4-methyl-1-pentene polymer (A) do not adversely affect culture, and have good shape stability, light transparency, moldability, and oxygen permeability, and can be sterilized, making them extremely excellent culture vessels for culturing hepatocytes, etc.

[0097] The method for producing the 4-methyl-1-pentene polymer (A) may be any method capable of polymerizing 4-methyl-1-pentene, an olefin, or other monomers. Furthermore, a chain transfer agent, such as hydrogen, may be present in the presence of the polymer to control the molecular weight and molecular weight distribution. The equipment used for the production is not limited. The polymerization method may be a known method, such as a gas-phase method, a slurry method, a solution method, or a bulk method. The slurry method or the solution method is preferred. Furthermore, the polymerization method may be a single-stage polymerization method or a multi-stage polymerization method, such as a two-stage polymerization method, in which multiple polymers with different molecular weights are blended into the polymerization system. Whether single-stage or multi-stage polymerization is used, when hydrogen is used as a chain transfer agent, it may be added all at once or in portions, for example, at the initial, middle, or final stages of polymerization. The polymerization may be carried out at room temperature or, if necessary, heated. From the viewpoint of polymerization efficiency, the polymerization is preferably carried out at 20°C to 80°C, and particularly preferably at 40°C to 60°C. There are no limitations on the catalyst used in the production, but from the viewpoint of polymerization efficiency, it is preferable to use, for example, the solid titanium catalyst component (I) described in WO 2006 / 054613.

[0098] When the substrate containing a 4-methyl-1-pentene polymer (A) is a composition containing a 4-methyl-1-pentene polymer (A), the 4-methyl-1-pentene polymer (A) is preferably present in an amount of 90% by mass or more but less than 100% by mass, more preferably 95% by mass or more but less than 100% by mass, and particularly preferably 99% by mass or more but less than 100% by mass, based on 100% by mass of the composition. The inclusion of a large amount of components other than the 4-methyl-1-pentene polymer (A) not only reduces oxygen permeability but also leads to reduced transparency and strength. Examples of components other than the 4-methyl-1-pentene polymer (A) include additives such as heat stabilizers, light stabilizers, processing aids, plasticizers, antioxidants, lubricants, antifoaming agents, antiblocking agents, colorants, modifiers, antibacterial agents, antifungal agents, and antifogging agents.

[0099] One embodiment of the present invention is a method for culturing hepatocytes used in the above-mentioned method for evaluating hepatotoxicity, comprising the step of culturing hepatocytes in a culture vessel formed of a substrate whose culture surface is partially or entirely composed of a 4-methyl-1-pentene polymer (A). The step of culturing hepatocytes in the culture vessel in the culture method comprises the above-mentioned steps 1 and 2. The definitions of each term are the same as those defined above.

[0100] One embodiment of the present invention is a kit for evaluating hepatotoxicity, comprising a culture vessel whose culture surface is formed entirely or partially from a substrate containing a 4-methyl-1-pentene polymer (A), hepatocyte β cells, and an inhibitor that inhibits at least one selected from a phase 1 drug-metabolizing enzyme, a phase 2 drug-metabolizing enzyme, and a transporter. The definitions of each term are the same as those defined above.

[0101] The kit for evaluating hepatotoxicity may optionally contain a medium for culturing hepatocyte β, a solvent for adjusting the concentration of the inhibitor, and the like. [Example]

[0102] The present invention will be explained in more detail below based on examples, but the present invention is not limited to these examples and can be practiced with appropriate modifications within the scope of the present invention.

[0103] [Test conditions] The test conditions are described below. [LC-MS / MS] One hundred microliters of culture supernatant was mixed with 1 mL of MilliQ water containing 2–20 ng / mL of internal standard (phenytoin or chlorpropamide). The phosphate buffer solution was prepared by mixing 200 mM NaH2PO4·2H2O (in MilliQ water) and 200 mM Na2HPO4 (in MilliQ water) to pH 7.0. After centrifugation at 12,000 xg for 10 minutes at 4°C, 1 mL of the supernatant was collected and subjected to solid-phase extraction using an Oasis HLB cartridge (Waters). The sample was analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system (QTRAP 4500, Sciex) connected to a Prominence high-performance liquid chromatography (HPLC) instrument (Shimadzu Corporation). Separation was performed at 40°C using a YMC-Triart C18 column (50 × 2.0 mm internal diameter, 3 μm particle size; YMC Corporation). The flow rate was set to 0.2 mL / min, and the HPLC and MS analysis conditions were as follows. The data were analyzed using MultiQuant software (Sciex).

[0104] (HPLC conditions) Mobile phase A: 0.1% formic acid (in MilliQ water) Mobile phase B: 0.1% formic acid (in acetonitrile)

[0105] [Table 1]

[0106] (MS analysis conditions) [Table 2]

[0107] [Manufacturing Example 1] (Base material manufacturing) TPX (registered trademark) (manufactured by Mitsui Chemicals, Inc.: molecular weight (Mw) = 428,000, molecular weight distribution (Mw / Mn) = 4.1), which is a 4-methyl-1-pentene polymer (A), was used and fed into a T-die extruder equipped with a full-flight screw that extrudes the substrate layer. The extrusion temperature was set to 270°C, the roll temperature to 60°C, and the roll rotation speed was varied to extrude and mold the film 1 having a thickness of 50 μm.

[0108] (Preparation of culture plates) The above film 1 was cut into an 8 cm x 12 cm size and plasma-treated using an atmospheric pressure plasma surface treatment device (manufactured by Sekisui Chemical Co., Ltd.) with the chamber filled with a nitrogen gas flow (treatment speed: 2 m / min, output: 4.5 kW, 2 round trips). The plasma-treated film 1 was used as the measurement sample and the water contact angle was measured. The water contact angle was measured in accordance with Japanese Industrial Standard JIS-R3257 (Test Method for Wettability of Substrate Glass Surfaces). A water droplet of 4 μL or less, which could be considered spherical, was placed on the surface of the measurement sample under constant temperature and humidity conditions of 25 ± 5°C and 50 ± 10%. The angle of the contact interface between the measurement sample and the water droplet was measured using the sessile drop method within 1 minute of the water droplet's contact with the sample surface. The water contact angle of the plasma-treated film 1 was 60.3°.

[0109] [Manufacturing Example 2] (Preparation of culture vessels) Plasma-treated film 1 was attached to the bottom of a 24-well polystyrene (PS) container frame and a 96-well polystyrene container frame via a medical adhesive (3M) to prepare 24-well culture plate 1 and 96-well culture plate 2, respectively. The plates were then packaged in gamma-resistant bags and sterilized by 10 kGy gamma irradiation. A 0.1 M hydrochloric acid solution (volumetric analysis grade, Fujifilm Wako Pure Chemical Industries) was diluted 100-fold with water for injection (Japanese Pharmacopoeia, Otsuka Pharmaceutical) to prepare a 0.001 M hydrochloric acid solution, which was then sterilized by filtration. A 0.05 mg / mL collagen solution (Cell Matrix Type I-P, derived from porcine tendon, Nitta Gelatin) was prepared using a 0.02 N acetic acid solution. 500 μL and 100 μL of the 0.05 mg / mL collagen coating solution were applied to each well of culture plate 1 and culture plate 2, which were made from 50 μm-thick film 1, respectively, and then excess collagen coating solution was removed. After leaving the plates to stand at room temperature for 60 minutes, they were washed with Dulbecco's PBS (-) and dried overnight at room temperature. These obtained culture plates were used as T plates (culture vessels (T) used in the present invention) in the following examples. The culture area of ​​one well in culture plate 1 and culture plate 2 was approximately 2 cm. 2 and approximately 0.4 cm 2 was

[0110] [Example 1] (Isolation and purification of rat primary hepatocytes) Male Sprague-Dawley rats (7-9 weeks old) were anesthetized with intraperitoneal administration of medecine (0.05 mg / body weight), midazolam (0.2 mg / body weight), and butorphanol (0.25 mg / body weight). Primary rat hepatocytes were isolated using a two-stage perfusion method as follows: First, perfusion with perfusion buffer (PB-1) (see Tables 3A1 and 3A2) was performed via the portal vein, followed by collagenase buffer (see Table 3B). The released cells were then filtered through 120-mesh and 200-mesh filters, and the filtrate was centrifuged at 50 g for 3 minutes at 4°C. The supernatant was discarded, and the cells were resuspended in 24 mL of WME (Williams' medium E, Life Technologies), 21.6 mL of Percoll® (Sigma-Aldrich), and 2.4 mL of 10x HBSS (Ca). 2+ The cells were resuspended in WME (see Table 3C) and mixed by inversion. The suspension was centrifuged at 50 g for 15 minutes at 4°C. The resulting pellet was resuspended in WME for plating (see Table 4A) and filtered through a 200-mesh filter. The filtrate was centrifuged at 50 g for 3 minutes at 4°C. The viability of the hepatocytes was determined by the tripan blue exclusion method. All hepatocyte purification experiments were performed under sterile conditions.

[0111] [Table 3]

[0112] (Rat primary hepatocyte culture) The T-plates used in the present invention, produced in Production Example 2, were washed with PBS before use. The hepatocytes isolated and purified above were suspended in WME for plating, and 1.25 × 10 cells were plated onto a 24-well T-plate and a 96-well T-plate, respectively. 5 cells / cm 2 The seeds were sown at a density of .

[0113] Approximately 2 hours later, the medium was replaced with WME for plating to remove non-adherent hepatocytes. 24 hours after seeding, the WME for plating was removed, and Matrigel diluted to 0.25 mg / mL with WME for glucose-based culture (see Table 4B) was added to each well of a 96-well T-plate on ice, and 500 μL was added to each well of a 24-well T-plate to initiate sandwich culture.

[0114] When culturing under glucose (Glu) conditions, the medium was replaced with WME for glucose-based culture every day until the fourth day of culture, when the experiment was conducted. The hepatocytes were cultured at 37°C in a CO2 incubator until the fifth day of culture, and primary cultured rat hepatocytes were obtained.

[0115] When culturing under galactose (Gal) conditions, the WME for glucose-based culture was replaced with WME for galactose-based culture (see Table 4C) 24 hours after seeding, and the medium was replaced with WME for galactose-based culture every day until the fourth day of culture, when the experiment was conducted. Hepatocytes were cultured at 37°C in a CO2 incubator until the fifth day of culture.

[0116] [Table 4]

[0117] In Table 4, fetal bovine serum was manufactured by Bio sera, antibiotic-antimycotic solution was manufactured by Nacalai Tesque, Inc., ITS Premix was manufactured by Corning, dexamethaone was manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., and Gluta MAX was manufactured by Thermo Fisher Scientific.

[0118] [Comparative Example 1] Primary cultured rat hepatocytes were obtained in the same manner as in Example 1, except that a comparative plate (hereinafter referred to as "PS plate") was used, which was prepared by sealing the bottom of a T plate with polystyrene Plateseal sealing film PS-PPO-100 (manufactured by Ina Optica Co., Ltd.) instead of the T plate.

[0119] [Example 2] [Toxicity Assessment 1] The toxicity of rotenone was evaluated by treating the primary cultured rat hepatocytes cultured in Example 1 with 1-aminobenzotriazole (1-ABT), a CYP inhibitor, using LDH activity (LDH release) as an indicator of cytotoxicity as follows. Note that rotenone is known to be metabolized and decomposed by CYP in vivo.

[0120] Primary rat hepatocytes were cultured on T-plates using a glucose-based culture medium containing no BSA (bovine serum albumin) and exposed to rotenone at concentrations of 0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, or 2 μM for 24 hours. DMSO was maintained at a concentration of 1% or less. 1-ABT (final concentration 250 μM) was added 30 minutes before rotenone exposure and then co-exposed to rotenone.

[0121] The plate was centrifuged at 100g for 3 minutes, and 5 μL of the supernatant was transferred to a new 96-well plate (96-well flat-bottom PS test plate, Nerbe Plus) and diluted with 55 μL of MilliQ water. LDH activity was measured using a TaKaRa LDH cytotoxicity detection kit (TaKaRa Bio Inc.). 60 μL of the reaction mixture was added, and the 96-well plate containing the culture supernatant was shaken on a shaker while the reaction was carried out in the dark for 10 minutes. After that, 30 μL of 1N HCl was added as a reaction stop solution, and the absorbance was measured at 490 nm. A Labsystems Multiskan JX multiwell plate reader was used. LDH activity (LDH release) was calculated according to the following formula, using the absorbance of the culture supernatant of hepatocytes cultured in 0.25% Triton X-100 as the high control and the absorbance of the culture supernatant of hepatocytes cultured in up to 1% DMSO as the low control. The results are shown in Figure 1.

[0122] LDH release (% control) = (experimental value - low control) / (high control - low control) × 100

[0123] Comparative Example 2 Toxicity evaluation was carried out in the same manner as in Example 2, except that the rat primary cultured hepatocytes cultured in Comparative Example 1 were used instead of the rat primary cultured hepatocytes cultured in Example 1, and PS plates were used instead of T plates. The results are shown in Figure 1.

[0124] The results of Example 2 and Comparative Example 2 showed that when hepatocytes were not treated with 1-ABT, hepatocytes cultured on T plates had lower LDH activity (higher cell viability) than those cultured on PS plates, indicating that hepatocytes cultured on T plates had a higher ability to decompose rotenone and were closer to the in vivo environment. Furthermore, even when CYP activity was inhibited by treatment with 1-ABT, it was shown that cell survival rates were higher when hepatocytes were cultured on T plates than on PS plates for low concentrations of rotenone. This indicates that by using hepatocytes cultured on T plates, toxicity evaluation can be performed for low concentrations of test substances, taking into account drug metabolism in an environment closer to that in vivo.

[0125] [Example 3] [Toxicity evaluation 2] The toxicity of phenformin was evaluated in the same manner as in Example 2, except that the primary cultured rat hepatocytes were not exposed to 1-ABT and were exposed to 100 μM, 125 μM, 150 μM, 200 μM, 250 μM, or 300 μM of phenformin instead of rotenone. The results are shown in Figure 2.

[0126] Comparative Example 3 The toxicity of phenformin was evaluated in the same manner as in Comparative Example 2, except that primary cultured rat hepatocytes were not exposed to 1-ABT and were exposed to 100 μM, 125 μM, 150 μM, 200 μM, 250 μM, or 300 μM phenformin instead of rotenone. The results are shown in Figure 2. Note that phenformin is generally not easily metabolized in the liver in vivo, and it is thought that when exposed to hepatocytes, much of it is not broken down and exhibits cytotoxicity.

[0127] The results of Example 3 and Comparative Example 3 showed that the use of T plates enabled more accurate toxicity evaluation in an environment closer to that in vivo, as LDH activity increased due to the cytotoxicity of phenformin compared to the use of PS plates.

[0128] [Example 4] [Toxicity Assessment 3] Primary rat hepatocytes were cultured and LDH activity was calculated in the same manner as in Example 2, except that instead of adding 1-ABT and rotenone, cyclosporine A (CysA), an inhibitor of the transporter BSEP, was added to a final concentration of 10 μM, and bile acids (BA) were added to a concentration 50-200 times that of the bile acid mixture (in DMSO) (bile acids in human serum) with the composition shown in Table 5 below. The results are shown in Figure 3.

[0129] [Table 5]

[0130] Comparative Example 4 Toxicity evaluation was performed in the same manner as in Comparative Example 2, except that instead of adding 1-ABT and rotenone, cyclosporine A (CysA), an inhibitor of the transporter BSEP, was added to a final concentration of 10 μM, and bile acids (BA) were added to a bile acid mixture (in DMSO) with the composition shown in Table 5 below (bile acids in human serum) at a concentration 50-200 times higher. The results are shown in Figure 3. Among bile acids, there are highly toxic ones, such as glycochenodeoxycholic acid, deoxycholic acid, and lithocholic acid. However, in vivo, these are usually excreted from cells into bile by transporters such as BSEP, and bile acid levels in the liver are regulated.

[0131] The results of Example 4 and Comparative Example 4 showed that when hepatocytes were treated with cyclosporine A to inhibit BSEP, the use of T plates resulted in increased sensitivity to low concentrations of bile acids and increased LDH activity compared to the use of PS plates, allowing for more accurate toxicity evaluation.

[0132] [Example 5] [Toxicity Assessment 4] Primary rat hepatocytes cultured as described in Example 1 above, 4 days after seeding, were exposed to Flutamide (Tokyo Chemical Industry Co., Ltd.) or Clopidogrel (LKT Laboratories), and bile acids for 24 hours using a BSA-free WME for glucose-based culture. The DMSO concentration in the control group was kept below 1%. Cells were pretreated with 1-ABT (final concentration 250 μM) for 30 minutes prior to the exposure, followed by co-exposure with Clopidogrel and bile acids, Flutamide and bile acids, or DMSO. The bile acid exposure concentration was set at 150-fold the bile acid mixture listed in Table 5 above. After incubation, LDH activity was measured as described in Example 2. The results are shown in Figure 4.

[0133] Comparative Example 5 Primary rat hepatocytes cultured in Comparative Example 1 on day 4 after seeding were exposed to Flutamide (Tokyo Chemical Industry Co., Ltd.) or Clopidogrel (LKT Laboratories) and bile acids for 24 hours using a BSA-free WME for glucose-based culture. The DMSO concentration in the control group was kept below 1%. Cells were pretreated with 1-ABT (final concentration 250 μM) for 30 minutes prior to the exposure, followed by co-exposure with Clopidogrel and bile acids, Flutamide and bile acids, or DMSO. The bile acid exposure concentration was set at 150-fold the bile acid mixture listed in Table 5 above. After incubation, LDH activity was measured as in Comparative Example 2. The results are shown in Figure 4.

[0134] The results of Example 5 and Comparative Example 5 show that in the Flutamide-exposed system, the tendency for increased LDH activity in the exposed group compared to the non-exposed group was more pronounced when T plates were used than when PS plates were used. Flutamide is known to enhance bile acid-dependent toxicity by metabolic inhibition. When T-plates were used, 1-ABT enhanced bile acid-dependent hepatotoxicity, demonstrating that toxicity evaluation taking into account drug metabolism of the test substance can be performed more accurately.

[0135] Furthermore, in the Clopidogrel-exposed system, a tendency for LDH activity to be reduced in the 1-ABT-exposed group compared to the 1-ABT-unexposed group was observed when T plates were used. It is known that clopidogrel inhibits the metabolism of clopidogrel, which reduces bile acid-dependent toxicity. The use of T-plates demonstrated that 1-ABT reduced bile acid-dependent hepatotoxicity, demonstrating that the use of T-plates allows for more accurate evaluation of toxicity due to metabolites of test substances.

[0136] [Example 6] [Phase 1: Measurement of gene expression levels and activity evaluation of drug-metabolizing enzymes] (1) Gene expression measurement The gene expression levels of CYP, a phase 1 drug-metabolizing enzyme, were measured as follows. Sepasol was added to the cultured hepatocytes obtained in Example 1 at 0.5 mL / well to detach the cells. The cells were then transferred to Eppendorf tubes (registered trademark) and allowed to stand at room temperature for 5 minutes. 100 μL of chloroform was added and mixed. The cells were then allowed to stand at room temperature for 5 minutes, and centrifuged at 12,000 g and 4°C for 15 minutes. 200 μL of the upper aqueous layer was transferred to a new centrifuge tube, and 200 μL of isopropanol was added and mixed. The cells were allowed to stand at room temperature for 10 minutes, and then centrifuged at 12,000 g and 4°C for 10 minutes. The supernatant was removed, and 500 μL of 75% ethanol was added to wash the precipitate. After centrifugation at 12,000 g and 4°C for 5 minutes, the supernatant was discarded, and the precipitate was completely dried. An appropriate amount of DEPC water was added to adjust the total RNA concentration to 300-500 ng / μL.

[0137] Reverse transcription of total RNA was performed using ReverTra Ace qPCR RT Master Mix (Toyobo Co., Ltd.) by mixing 2 μL of 5x RT Master Mix with 1 μg of RNA template and adjusting the reaction mixture to 10 μL with nuclease-free water. The reaction mixture was then incubated at 37°C for 15 minutes, 50°C for 5 minutes, 98°C for 5 minutes, and then maintained at 14°C using LifeECO (Bioer Technology).

[0138] Real-time PCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo Co., Ltd.) by mixing 5 μL of THUNDERBIRD SYBR qPCR Mix, 1 μL of cDNA (reverse transcription product diluted 10-fold with MilliQ water), 2 μL Primer Mix (a mixture of the forward and reverse primers in Table 6 with a final concentration of 1.5 μM each), and 2 μL of MilliQ water. The reaction mixture was added to an 8-tube tube or a 96-well plate (LightCycler480 Multiwell Plate 96-well, Roche), gently mixed to homogenize, and then performed using a LightCycler Nano Real-Time PCR System (Roche Diagnostics) under the conditions listed in Table 7. The results are shown in the upper panel of Figure 5.

[0139] [Table 6]

[0140] [Table 7]

[0141] (2) Enzyme activity measurement The drug-metabolizing activity of CYP was evaluated as follows. The compounds in Table 8 were used as probes for drug-metabolizing enzymes, and each compound was dissolved in all-free WME (WME without supplement). All compounds were dissolved in DMSO, with the DMSO concentration adjusted to 1% or less. The hepatocytes obtained in Example 1 were exposed to the lysate for 90 minutes at the concentrations shown in Table 8. The supernatant was then recovered, and the metabolites produced were measured by LC-MS / MS using the method described above to determine the amount produced, which was used as an index of activity. The results are shown in the lower panel of Figure 5.

[0142] [Table 8]

[0143] Comparative Example 6 In Example 6, gene expression levels and enzyme activities were measured in the same manner as in Example 6, except that the cultured hepatocytes obtained in Comparative Example 1 were used instead of the cultured hepatocytes obtained in Example 1. The results are shown in Figure 5.

[0144] The results of Example 6 and Comparative Example 6 showed that the expression level of CYP genes was higher in hepatocytes cultured on T plates than in those cultured on PS plates. Furthermore, no effect was observed when glucose in the medium was replaced with galactose as the sugar source. Furthermore, the activity of CYP was higher in hepatocytes cultured on T plates than in those cultured on PS plates. Furthermore, no effect was observed when glucose in the medium was replaced with galactose as the sugar source.

[0145] [Example 7] [Phase 2: Measurement of gene expression levels and activity evaluation of drug-metabolizing enzymes] (1) Gene expression measurement The gene expression levels of phase 2 drug-metabolizing enzymes UGTs and SULTs were measured in the same manner as in Example 6, except that the primers in Table 6 were replaced with the primers in Table 9. The results are shown in the upper panel of Figure 6.

[0146] [Table 9]

[0147] (2) Enzyme activity The enzyme activities of phase 2 drug-metabolizing enzymes, UGTs and SULTs, were measured in the same manner as in Example 6, except that the conditions in Table 8 were changed to those in Table 10. The results are shown in the lower panel of Figure 6.

[0148] [Table 10]

[0149] Comparative Example 7 In Example 7, gene expression levels and enzyme activities were measured in the same manner as in Example 7, except that the cultured hepatocytes obtained in Comparative Example 1 were used instead of the cultured hepatocytes obtained in Example 1. The results are shown in Figure 6.

[0150] The results of Example 7 and Comparative Example 7 showed that the enzyme activity of hepatocytes cultured on T plates was higher than that of hepatocytes cultured on PS plates. Furthermore, no effect was observed when glucose in the medium was replaced with galactose as the sugar source.

[0151] [Example 8] [Measurement of transporter gene expression levels] The expression levels of the transporter genes Ntcp, Bsep, and Oatp4 were measured in the same manner as in Example 6, except that the primers in Table 6 were replaced with the primers in Table 11. The results are shown in FIG.

[0152] [Table 11]

[0153] [Comparative Example 8] In Example 8, the expression levels of Ntcp, Bsep, and Oatp4 were measured in the same manner as in Example 8, except that the cultured hepatocytes obtained in Comparative Example 1 were used instead of the cultured hepatocytes obtained in Example 1. The results are shown in Figure 7.

[0154] The results of Example 8 and Comparative Example 8 showed that gene expression levels were higher in hepatocytes cultured on T plates than in those cultured on PS plates. Furthermore, no effect was observed when glucose in the medium was replaced with galactose as the sugar source.

[0155] [Example 9] [Evaluation of bile canaliculi structure] Using the cultured hepatocytes obtained in Example 1, immunostaining of MRP2, a transporter expressed in bile canaliculi, and F-actin, a cytoskeleton, was performed using the antibodies and staining reagents listed in Table 12 as follows.

[0156] [Table 12]

[0157] The hepatocytes were washed twice with PBS and fixed by immersion in 4% paraformaldehyde (PFA) for 15 minutes. After washing twice with PBS, they were permeabilized by immersion in 0.5% triton (in PBS) for 10 minutes. After washing twice with PBS, they were blocked by immersion in 1% BSA (in PBS) for 30 minutes. The primary antibody diluted in 0.1% BSA (in PBS) was incubated for 1 hour at room temperature. After washing twice with PBS, the secondary antibody diluted in 0.1% BSA (in PBS) was incubated for 1 hour at room temperature in the dark. After incubation, the cells were washed twice with PBS, and images were captured using a BZ-X710 (Keyence). The results are shown in Figure 8.

[0158] Comparative Example 9 The bile canaliculus structure was evaluated in the same manner as in Example 9, except that the cultured hepatocytes obtained in Comparative Example 1 were used instead of the cultured hepatocytes obtained in Example 1. The results are shown in Figure 8.

[0159] The results of Example 9 and Comparative Example 9 showed that co-staining areas (orange to yellow areas) of MRP2 (green) and F-actin (red), which indicate bile canalicular structures, were more prominent in hepatocytes cultured on T plates than in those cultured on PS plates, indicating that culturing on T plates resulted in the formation and elongation of bile canalicular structures. Furthermore, no effect was observed when glucose in the medium was replaced with galactose as a sugar source.

[0160] [Test Example 1] [Cell Evaluation] As in Example 1, the amount of lactate released into the medium from each type of hepatocyte cultured in a 24-well T-plate under glucose conditions or galactose conditions with galactose as the sugar source was measured using a lactate measurement kit (Dojindo Laboratories, Inc.) as follows: The culture supernatant was diluted 50-fold with MilliQ water, and 25 μL was transferred to a 96-well T-plate. 25 μL of working solution was added to each well, and the mixture was stirred on a shaker in the dark for 2 minutes. The mixture was then incubated at 37°C for 30 minutes, and the absorbance was measured at a wavelength of 450 nm. The results are shown in Figure 9.

[0161] [Comparative Test Example 1] Cell evaluation was carried out in the same manner as in Test Example 1, except that the primary cultured rat hepatocytes obtained in Comparative Example 1 were used instead of the primary cultured rat hepatocytes obtained in Example 1. The results are shown in Figure 9. In the figure, PS indicates PS plates, T indicates T plates, Glu indicates glucose conditions, and Gal indicates galactose conditions.

[0162] The results of Test Example 1 and Comparative Test Example 1 showed that hepatocytes cultured on T plates had lower lactate levels than hepatocytes cultured on PS plates, indicating that T plates provide a greater amount of oxygen to cells and activate mitochondrial function than PS plates, and that the cultured hepatocytes are in an environment closer to that in vivo. This was also true when glucose or galactose was used as the sugar source when culturing on T plates.

Claims

1. Step 1: culturing hepatocytes α to obtain hepatocytes β; Step 2: culturing the hepatocyte β obtained in step 1 in the presence and absence of a test substance; Step 3: analyzing a cytotoxicity index in the medium or in the hepatocyte β after the culture in Step 2; and Step 4 includes comparing the cytotoxicity indexes; The steps 1 and 2 are carried out in a culture vessel having a culture surface partly or entirely formed from a substrate containing a 4-methyl-1-pentene polymer (A), In the step 2, the hepatocyte β cells are cultured in the presence of an inhibitor that inhibits at least one selected from a phase 1 drug-metabolizing enzyme, a phase 2 drug-metabolizing enzyme, and a transporter; the 4-methyl-1-pentene polymer (A) is a homopolymer of 4-methyl-1-pentene or a copolymer of 4-methyl-1-pentene and at least one selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene); the phase 1 drug-metabolizing enzyme is at least one enzyme belonging to the cytochrome P450 subfamily selected from CYP1A, CYP3A, CYP2C, and CYP2D; the phase II drug-metabolizing enzyme is at least one enzyme selected from the UDP-glucuronyltransferase superfamily, the sulfotransferase superfamily, the glutathione transferase superfamily, and the acetyltransferase superfamily; The transporter is at least one selected from the group consisting of NTCP, BSEP, transporters belonging to the MRP subfamily, transporters belonging to the MDR subfamily, and transporters belonging to the OATP family. Methods for assessing hepatotoxicity.

2. The method for evaluating hepatotoxicity according to claim 1, wherein the expression of at least one gene selected from phase 1 drug-metabolizing enzymes, phase 2 drug-metabolizing enzymes, and transporters is increased in the hepatocyte beta obtained in step 1.

3. The method for evaluating hepatotoxicity according to claim 1 , wherein the hepatocyte β forms aggregates.

4. The method for evaluating hepatotoxicity according to claim 3 , wherein the aggregates comprise bile canaliculi.

5. The method includes culturing hepatocytes in a culture vessel in which a part or all of the culture surface is formed from a substrate containing a 4-methyl-1-pentene polymer (A). A method for culturing hepatocytes used in the method for evaluating hepatotoxicity according to any one of claims 1 to 4.

6. a culture vessel having a culture surface partly or entirely formed from a substrate containing a 4-methyl-1-pentene polymer (A); Hepatocyte β and an inhibitor that inhibits at least one selected from a phase 1 drug-metabolizing enzyme, a phase 2 drug-metabolizing enzyme, and a transporter; A kit for use in the method for evaluating hepatotoxicity according to any one of claims 1 to 4, comprising:

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