Method for producing plateable hepatocytes and application thereof

Abstract

One purpose of the present invention is to obtain plateable hepatocytes from suspension hepatocytes without using feeder cells. The present invention provides a method for producing plateable hepatocytes from suspension hepatocytes, the method comprising culturing the suspension hepatocytes in the presence of at least five selected from (ii) ROCK inhibitors, TGF-β receptor inhibitors, GSK-3β inhibitors, EGFs, HGFs, sphingophospholipids, and glycerophospholipids.

Description

Method for producing adherent hepatocytes and its applications

[0001] The present invention relates to a technique for improving the adhesive ability of hepatocytes.

[0002] The liver plays a central role in drug metabolism. Drug metabolism studies are performed using primary cultured cells (primary hepatocytes) obtained from living livers. When hepatocytes obtained from living livers are seeded and cultured, they are known to form either plateable cells, which firmly adhere to plates, or suspension cells, which either fail to adhere or only partially adhere. Adherent cells can generally be cultured for 4–5 days and are highly versatile, being useful for cell-cell or cell-matrix interactions, cytochrome P450 (CYP) enzyme induction experiments, cholestatic toxicity tests using microbile duct formation, and drug transport evaluation using Transwell. On the other hand, suspension cells cannot adhere to plates or only partially adhere to plates. Therefore, although enzyme activity such as CYP can be measured, they cannot be used for CYP induction experiments, cholestatic toxicity tests, or Transwell experiments. Furthermore, the use of suspension cells is limited because they die within a few hours and cannot be cultured for long periods.

[0003] Therefore, it is desirable to perform each test using adherent primary hepatocytes. However, it is not known whether primary hepatocytes obtained from living livers will become adherent or suspension cells until they are seeded and cultured, and the reason for this is not clear. Furthermore, due to their versatility, adherent cells are more expensive than suspension cells. For these reasons, a technology to induce the conversion of suspension cells to adherent cells is needed.

[0004] Patent Document 1 describes a method for preparing seeded human hepatocytes, including: (a) applying human hepatocytes to a surface in the presence of feeder cells, the feeder cells being endothelial cells and fibroblasts; (b) co-culturing the applied hepatocytes with the feeder cells after step (a); and (c) forming one or more hepatocyte clusters on the feeder cells by the co-cultured hepatocytes, the feeder cells attaching to the surface, and at least 85% of the co-cultured hepatocytes being in one or more hepatocyte clusters, thereby preparing seeded human hepatocytes. However, this method requires the use of feeder cells, which raises concerns about cumbersome procedures, reduced experimental reproducibility, and the risk of cell infection. Furthermore, when various tests are performed using cells obtained by co-culture with feeder cells, the function of the feeder cells may directly affect the test evaluation. Furthermore, when fibroblasts are used, the number of fibroblasts may increase or detach, which may lead to problems with the accuracy of quantitative tests performed using cells obtained by co-culture with fibroblasts. For these reasons, methods using feeder-free culture are required.

[0005] Furthermore, Patent Document 2 describes a method for culturing primary hepatocytes, which includes culturing one or more hepatocytes in contact with an extracellular matrix (ECM) in the presence of an expansion medium containing a basal medium supplemented with one or more Wnt signal inhibitors, one or more receptor tyrosine kinase ligands, and one or more epithelial phenotype stabilizers. Non-Patent Document 1 demonstrates that cell proliferation and reprogramming can be induced by culturing primary hepatocytes (adherent type) in a medium containing YACs (Y27632, A83-01, and CHIR99021) using a collagen-coated culture dish. However, these methods do not demonstrate the induction of suspension cells into adherent cells. Furthermore, the present inventors cultured suspension human primary hepatocytes on type 1 collagen plates in the presence of YACs using a method similar to Non-Patent Document 1, but found that this did not result in the induction of adherent cells.

[0006] U.S. Patent No. 11,535,827 International Publication No. WO2023 / 076292

[0007] Katsuda T, Matsuzaki J, Yamaguchi T, Yamada Y, Prieto-Vila M, Hosaka K, Takeuchi A, Saito Y, Ochiya T. Generation of human hepatic progenitor cells with regenerative and metabolic capacities from primary hepatocytes. Elife. 2019 Aug 8;8:e47313. doi: 10.7554 / eLife.47313.

[0008] An object of the present invention is to obtain adherent hepatocytes from floating hepatocytes without using feeder cells.

[0009] Another object of the present invention is to use the obtained adherent hepatocytes in various tests relating to drug metabolism and the like.

[0010] The present inventors conducted extensive research to solve the above problems and discovered that adhesive hepatocytes can be produced by culturing floating hepatocytes under specific conditions, thereby completing the present invention.

[0011] That is, the present invention relates to the following: [1] A method for producing adherent hepatocytes from suspension hepatocytes, comprising: (ii) culturing suspension hepatocytes in the presence of at least five substances selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, epidermal growth factor (EGF), hepatocyte growth factor (HGF), sphingophospholipids, and glosserophospholipids. [2] The method according to [1], comprising: (i) culturing suspension hepatocytes in the presence of at least four substances selected from a ROCK inhibitor, EGF, HGF, sphingophospholipids, and glosserophospholipids prior to the culturing in (ii). [3] The method according to [1] or [2], wherein the culturing in (ii) comprises forming spheroids from suspension hepatocytes. [4] The method according to any one of [1] to [3], wherein the culturing in (i) and (ii) is performed on a culture surface coated with a basement membrane component. [5] The method according to any one of [1] to [4], comprising, following the culture of (ii), (iii) culturing in the presence of at least nine species selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, a cAMP signal activator, EGF, HGF, sphingophospholipids, glycerophospholipids, a γ-secretase inhibitor, oncostatin M, and dexamethasone. [6] The method according to any one of [1] to [5], comprising performing a passaging operation after the culture of (ii). [7] The method according to any one of [1] to [6], comprising cryopreserving and thawing the hepatocytes after the culture of (ii). [8] The method according to any one of [1] to [7], wherein the sphingophospholipid is sphingosine-1-phosphate or a salt thereof, and the glycerophospholipid is lysophosphatidic acid or a salt thereof. [9] The method according to any one of [1] to [8], wherein the ROCK inhibitor is Y27632, the TGFβ receptor inhibitor is A83-01, and the GSK-3β inhibitor is CHIR99021.

[10] The method according to any one of [1] to [9], wherein the floating hepatocytes are floating primary hepatocytes.

[11] The method according to any one of [1] to

[10] , wherein the floating hepatocytes are human floating primary hepatocytes.

[12] A method for measuring the metabolism of a test substance, comprising the following steps (1) to (3): (1) obtaining adherent hepatocytes by the preparation method described in any one of [1] to

[11] ; (2) contacting the adherent hepatocytes obtained in step (1) with a test substance; (3) measuring the metabolism of the test substance.

[13] A method for inducing metabolic enzymes in hepatocytes, comprising the following steps (1) and (2): (1) obtaining adherent hepatocytes by the preparation method described in any one of [1] to

[11] ; (2) contacting the adherent hepatocytes obtained in step (1) with a metabolic enzyme inducer.

[14] The method described in

[13] , which comprises sandwich culturing the adherent hepatocytes obtained in step (1).

[15] A method for producing hepatocyte spheroids, comprising the following steps (1) and (2): (1) obtaining adherent hepatocytes by the production method described in any one of [1] to

[11] ; (2) culturing the adherent hepatocytes obtained in step (1) to form hepatocyte spheroids.

[16] A method for two-dimensional culture on a cell culture insert, comprising the following steps (1) to (3): (1) obtaining adherent hepatocytes by the production method described in any one of [1] to

[11] ; (2) converting the adherent hepatocytes obtained in step (1) into single cells; (3) two-dimensionally culturing the hepatocytes converted into single cells in step (2) on a cell culture insert such as Transwell.

[17] The method described in

[16] , wherein the culture in step (3) is performed on a culture surface coated with a basement membrane component.

[18] A medium for use in the method according to any one of [1] to

[11] , comprising at least four selected from a ROCK inhibitor, EGF, HGF, sphingophospholipids, and glosserophospholipids.

[19] A medium for use in the method according to any one of [1] to

[11] , comprising at least five selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipids, and glosserophospholipids.

[20] A kit comprising the medium according to

[18] , the medium according to

[19] , and suspension hepatocytes.

[0012] According to the present invention, adherent hepatocytes can be prepared from floating hepatocytes.

[0013] Figure 1 shows an overview of the experimental protocol for Example 1. Day 0 indicates the day the cells were seeded. Figure 2 shows morphological images of Lot 1 HPHs (human primary hepatocytes) before and after medium replacement 3 hours after seeding. Figure 3 shows morphological images of each of the three lots of HPHs 10 days after seeding. Figure 4 shows morphological images of Lot 1 HPHs after a metabolic enzyme induction test. Figure 5 shows the results of an induction test of mRNA expression of each metabolic enzyme in HPHs. Each value represents the mRNA expression level corrected for the expression level of a reference gene (HPRT: hypoxanthine-guanine phosphoribosyltransferase). Omep: omeprazole; Rif: rifampicin; PB: phenobarbital; 0 hrs sHPHs: suspension-type human primary hepatocytes immediately after thawing; 0 hrs plated: adherent-type human primary hepatocytes immediately after thawing. Figure 6 shows an overview of the experimental protocol for Example 2. Day 0 in the lower panel indicates the time point at which the passaging procedure was performed on Day 8 in the upper panel. Figure 7 shows morphological images of HPHs from Lot 1 after a metabolic enzyme induction test on Day 7 after passaging. Figure 8 shows the results of measuring the metabolic activity of CYP3A4 using HPHs from Lot 1 after a metabolic enzyme induction test on Day 7 after passaging. Each value represents the amount of 1-OH MDZ (1-hydroxymidazolam), a metabolite of midazolam (a CYP3A4 substrate), quantified by LC-MS / MS in each culture supernatant. Figure 9 shows morphological images of HPHs after re-culture after cryopreservation. Figure 10 shows an outline of the experimental protocol for Example 3. Day 0 in the lower panel indicates the time point at which the passaging procedure was performed on Day 8 in the upper panel. Figure 11 shows morphological images of sandwich-cultured HPHs from Lot 1 on Day 7 after passaging. Figure 12 shows the results of an induction test of mRNA expression of each metabolic enzyme in sandwich-cultured HPHs from Lot 1 on day 7 after passaging. The graph notation is the same as in Figure 5. Figure 13 shows the results of measuring the metabolic activity of CYP3A4 using sandwich-cultured HPHs from Lot 1 after a metabolic enzyme induction test on day 7 after passaging. The graph notation is the same as in Figure 8. Figure 14 shows morphological images of sandwich-cultured HPHs from Lot 2 on day 7 after passaging.Figure 15 shows the results of an induction test of mRNA expression of metabolic enzymes in sandwich-cultured HPHs from Lot 2 on day 7 after passaging. The graph notation is the same as in Figures 5 and 12. Figure 16 shows the results of measuring the metabolic activity of CYP3A4 using sandwich-cultured HPHs from Lot 2 after a metabolic enzyme induction test on day 7 after passaging. The graph notation is the same as in Figures 8 and 13. Figure 17 shows an outline of the experimental protocol for Example 3. Day 0 in the lower panel corresponds to the time point of passaging on Day 8 in the upper panel. Figure 18 shows morphological images of 3D spheroids induced from HPHs on day 20 after passaging. Figure 19 shows morphological images of hepatocytes induced from 3D spheroids by 2D expansion culture on cell culture inserts on day 18 after passaging, after a metabolic enzyme induction test. Figure 19A shows the results for media, DMSO, and omeprazole. Figure 19B shows the results for phenobarbital (PB), CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime), and rifampicin. Figure 20 shows the results of a metabolic enzyme induction test using cells induced from 3D spheroids by two-dimensional expansion culture on cell culture inserts, 18 days after subculturing. The notation used in the graphs is the same as in Figures 5, 12, and 15. Figure 21 shows the results of fluorescent immunostaining using cells induced from 3D spheroids by two-dimensional expansion culture on cell culture inserts, 18 days after subculturing. A. DAPI: nucleus (blue), BSEP (Bile Salt Export Pump; ABCB11) efflux transporter: bile acid efflux transporter (green), E-CAD: E-cadherin (red), merged image. B. DAPI: nucleus (blue), NTCP influx transporter: sodium taurocholate cotransporter (green), ZO-1 tight junction: tight junction molecule ZO-1 (red), merged image. Figure 22 shows an outline of the experimental protocol in Example 5.Day 0 in the lower panel represents the time point at which cryopreservation and subculture were performed on Day 5 in the upper panel. Figure 23 shows the results of cell morphology (3D culture, cryopreservation, and subculture) and CDFDA assay in Example 5. 1. Spheroid generation using EZSPHERE, 2. Subsequent suspension culture of spheroids on ultra-low attachment plates, 3. Microbial excretory activity using MRP2, 4. Spheroids dissociated into single cells and then subcultured (2D culture), and 5. Spheroids dissociated into single cells, then frozen and subcultured (2D culture). Figure 24 shows the analysis of mRNA expression of metabolic enzymes and drug transporters in sandwich-cultured HPHs cultured in 2D from 3D spheroids on day 3 after subculture. Each value represents the mRNA expression level corrected for the expression level of the reference gene (HPRT: hypoxanthine-guanine phosphoribosyltransferase). pHPHs were obtained after 48 hours of culture of adherent human primary hepatocytes. Figure 25A shows an outline of the experimental protocol in Example 6. Figure 25B shows the results of evaluating caspase 3 mRNA expression. M2: Medium 2, Y: Y27632, L: lysophosphatidic acid, A: A83-01, CH: CHIR99021. Figure 26 shows an outline of the experimental protocol in Example 7. Figure 27 shows morphological images of HPHs. L1: Lot 1, L2: Lot 2, Normal protocol: normal protocol, -A, CH: without A83-01 and CHIR99021, -Y: without Y27632, -A: without A83-01, -CH: without CHIR99021. Figure 28 shows morphological images of HPHs. L1: Lot 1, L2: Lot 2, -HGF: No HGF, -S: No D-erythro-sphingosine-1-phosphate, -LPA: No lysophosphatidic acid, -vitamin C: No vitamin C, -EGF: No EGF, -FBS: No FBS. Figure 29 shows morphological images of HPHs. L1: Lot 1, L2: Lot 2, -FBS (+B27-Vitamin A): No FBS and B27 without vitamin A. iMatrix 511 (laminin) was used to precoat the porous membrane of the cell culture insert instead of Matrigel.Collagen was used to precoat the porous membrane of cell culture inserts with collagen Type I instead of Matrigel. Figure 30 shows the results of evaluating the mRNA expression of albumin, CYP3A4, and CYP1A2 in HPHs. Figure 31 shows an outline of the experimental protocol in Example 8. Figure 32 shows morphological images of HPHs on day 10. L1: Lot 1. Figure 33 shows the results of a test to induce CYP3A4 mRNA expression in HPHs. Figure 34 shows the results of a test to induce CYP1A2 mRNA expression in HPHs.

[0014] <Method for Producing Adherent Hepatocytes> In one aspect, the present invention relates to a method for producing adherent hepatocytes from floating hepatocytes. This method allows for the production of adherent hepatocytes from floating hepatocytes. Adherent hepatocytes can be cultured for a long period of time and are useful for various evaluation experiments such as drug metabolism and drug transport.

[0015] In one embodiment, the method comprises culturing the suspension hepatocytes in the presence of (ii) at least five selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, epidermal growth factor, hepatocyte growth factor, sphingophospholipids, and glycerophospholipids.

[0016] [Floating hepatocytes] In the present invention, floating hepatocytes refer to hepatocytes that cannot adhere to a culture surface such as a plate, or that have a morphology different from that of adhesive hepatocytes even if they have partial adhesive ability. In one embodiment, in the present invention, floating hepatocytes refer to hepatocytes that cannot adhere to other cells or extracellular matrix, or that have partial adhesive ability but have a morphology different from that of adhesive hepatocytes.

[0017] In one embodiment, the hepatocytes of the present invention refer to cells that constitute the liver, such as hepatic parenchymal cells (cells that perform major liver functions such as bile production and drug metabolism), bile duct epithelial cells, hepatic sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells, etc. In one embodiment, the hepatocytes of the present invention are hepatic parenchymal cells.

[0018] The hepatocytes are preferably primary hepatocytes, more preferably human primary hepatocytes (HPHs). Primary hepatocytes are hepatocytes directly collected from a living mammal or differentiated from stem cells collected from a living mammal, and human primary hepatocytes are hepatocytes directly collected from a human or differentiated from stem cells collected from a human.

[0019] Suspension hepatocytes generally can only remain viable for about five hours, making them difficult to use in experiments involving the induction of drug-metabolizing enzymes such as cytochrome P450 (CYP) enzymes, cholestatic toxicity tests using microbile duct formation, and drug transport evaluation tests using Transwell.

[0020] [Adherent hepatocytes] In the present invention, "adherent hepatocytes" refers to hepatocytes that exhibit high cell adhesion to a culture surface such as a plate and exhibit a paving-stone-like morphology in two-dimensional culture. In one embodiment, in the present invention, "adherent hepatocytes" refers to hepatocytes that exhibit high cell adhesion to other cells or extracellular matrix and exhibit a paving-stone-like morphology in two-dimensional culture.

[0021] It is known that adherent hepatocytes can generally be cultured for about 4 to 5 days, but the adherent hepatocytes obtained by the present invention can be cultured for a longer period of time.

[0022] The preparation of adherent hepatocytes from suspension hepatocytes can be evaluated by one or more of the following: 1) the production of hepatocytes capable of adhering to a culture surface such as a plate; 2) the production of hepatocytes capable of adhering to other cells or extracellular matrix; and 3) a change in cell morphology. The change in cell morphology is based on the difference in morphology between adherent hepatocytes and suspension hepatocytes. When suspension hepatocytes are cultured in a conventional culture dish, they generally show no or little morphological change even after culture and maintain a spherical shape similar to that immediately after seeding, whereas when cultured in a conventional culture dish, adherent hepatocytes generally have a flatter (cobblestone-like) shape than suspension hepatocytes. Therefore, 3) the change in cell morphology may be that they become flatter (cobblestone-like) than suspension hepatocytes. The change in cell morphology can be confirmed using any microscope suitable for observing cells.

[0023] In the present invention, when "adherent hepatocytes are produced," it is not necessary that all cells in the cell population used for culture be obtained as adherent hepatocytes; it is sufficient that at least a portion of the cells be obtained as adherent hepatocytes.

[0024] Furthermore, a cell population of adherent hepatocytes may contain cells that do not have adhesive ability (for example, about several percent to 20%), and the production method of the present invention can also be used for the purpose of obtaining adherent hepatocytes from such cells that do not have adhesive ability.

[0025] (ii) Culturing in the presence of at least five selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, epidermal growth factor, hepatocyte growth factor, sphingophospholipids, and glosserophospholipids. The method of the present invention for producing adherent hepatocytes comprises culturing suspension hepatocytes in the presence of at least five selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, epidermal growth factor (EGF), hepatocyte growth factor (HGF), sphingophospholipids, and glosserophospholipids. (ii) "In the presence of at least five selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipids, and glosserophospholipids" is synonymous with a culture system containing at least five selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipids, and glosserophospholipids (hereinafter, in the present invention, "in the presence of (predetermined components)" similarly refers to a culture system containing (predetermined components). In the present invention, a culture system may include a medium, a culture substrate, a culture vessel, etc. In one embodiment, "culturing in the presence of at least five selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipids, and glosserophospholipids" refers to culturing in a medium containing at least five selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipids, and glosserophospholipids. The medium may be any medium that can generally be used for culturing hepatocytes, such as Dulbecco's modified Eagle's medium (DMEM).

[0026] In the present invention, when the concentration of a particular component in a culture system or medium is specified, it is not necessary to maintain that concentration throughout the culture period, but it means that the concentration is maintained at the start of the culture.

[0027] (ROCK Inhibitors) Any ROCK (Rho-associated protein kinase: Rho kinase) inhibitor can be used as long as it has the effect of inhibiting Rho kinase, with Y27632 being preferred. The concentration of the ROCK inhibitor (in the case of Y27632) added to the culture system is, for example, 0.1 to 50 μM, preferably 0.5 to 30 μM, and more preferably 1 to 20 μM. When using a ROCK inhibitor other than Y27632, the concentration of the inhibitor added to the culture system can be determined by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the ROCK inhibitor used and Y27632. Furthermore, whether the selected concentration range is appropriate can be confirmed by preliminary experiments.

[0028] (TGFβ Receptor Inhibitor) The TGFβ receptor inhibitor may preferably be one that exhibits inhibitory activity against one or more of the TGFβ receptors ALK4, ALK5, and ALK7. Examples of such inhibitors include A83-01, SB431542, SB-505124, SB525334, D4476, ALK5 inhibitor, LY2157299, LY364947, GW788388, and RepSox, with A83-01 being particularly preferred. Alternatively, a peptide compound (alternative peptide compound) having TGFβ inhibitory activity may be used instead of the TGFβ receptor inhibitor. The peptide compound having TGFβ inhibitory activity may be a TGFβ inhibitory peptide, i.e., one that binds to TGFβ and inhibits the binding of TGFβ to the TGFβ receptor, or a TGFβ receptor inhibitory peptide, i.e., one that binds to the TGFβ receptor and inhibits the binding of TGFβ to the TGFβ receptor. Examples of TGFβ inhibitory peptides include PG-002 (PeptiGrowth), a TGFβ1 inhibitor peptide. The concentration of the TGFβ receptor inhibitor added to the culture system (in the case of A83-01) may be, for example, 0.05 to 10 μM, preferably 0.1 to 5 μM, and more preferably 0.5 to 3 μM. When using a compound different from A83-01, the concentration can be determined by one skilled in the art based on the above concentration range, taking into account the differences (particularly activity) between the properties of the compound used and those of A83-01. Furthermore, whether the set concentration range is appropriate can be confirmed by a preliminary experiment based on the examples described below.

[0029] (GSK-3β Inhibitor) Any GSK-3β inhibitor can be used as long as it has the effect of inhibiting GSK (glycogen synthase kinase)-3, and preferably CHIR99021. The concentration of the GSK-3β inhibitor (in the case of CHIR99021) added to the culture system is, for example, 0.1 to 20 μM, preferably 0.5 to 10 μM, and more preferably 1 to 5 μM. Note that when using a GSK-3β inhibitor other than CHIR99021, the concentration added to the culture system can be set by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the GSK-3β inhibitor used and CHIR99021. Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0030] (Epidermal Growth Factor) Epidermal growth factor (EGF) is a protein characterized by binding to epidermal growth factor receptors (EGFR) present on the cell surface of various cells, thereby inducing cell growth and proliferation. Epidermal growth factor is also referred to as epidermal growth factor, epidermal cell growth factor, epidermal cell growth factor, etc. In the present invention, EGF preferably has a human amino acid sequence, but is not particularly limited thereto. EGF may be produced from human or animal cells using genetic recombination technology, or may be a peptide compound. Peptide compounds are chemically synthesized peptide compounds that may have activity equivalent to or greater than that of biologically derived peptides. Peptide compounds are also sometimes referred to as alternative peptide compounds. Peptide compounds will continue to be developed in the future, and it is naturally expected that they will be used by those skilled in the art.

[0031] The concentration of EGF added to the culture system may be, for example, 3 to 100 ng / mL, preferably 5 to 50 ng / mL, and more preferably 10 to 30 ng / mL. In the present invention, it is preferable that the EGF has a human amino acid sequence, but is not particularly limited.

[0032] (Hepatocyte Growth Factor) Hepatocyte growth factor (HGF) is a protein characterized by binding to the receptor tyrosine kinase Met and inducing biological activities such as cell proliferation, survival promotion, and migration. In the present invention, HGF preferably has a human amino acid sequence, but is not particularly limited thereto. HGF may be produced from human or animal cells by genetic recombination technology, or may be an alternative peptide compound. An example of an alternative peptide compound to HGF is PG-001 available from PeptiGrowth, Inc.

[0033] The concentration of HGF added to the culture system may be, for example, 1 to 200 ng / mL, preferably 3 to 100 ng / mL, more preferably 10 to 50 ng / mL, and even more preferably 15 to 30 ng / mL.

[0034] (Sphingophospholipids) Sphingophospholipids are compounds in which a phosphate group is bound to a sphingoid base (a long-chain aliphatic amino acid having a hydroxyl group). Examples of sphingophospholipids include sphingosine-1-phosphate (S1P), sphingomyelin, and salts thereof. In a preferred embodiment, the sphingophospholipid is S1P having the structure shown below or a salt thereof.

[0035]

[0036] The concentration of sphingophospholipid (in the case of S1P) added to the culture system is, for example, 0.01 to 50 μM, preferably 0.05 to 30 μM, more preferably 0.1 to 10 μM, and even more preferably 0.5 to 5 μM. When using a sphingophospholipid other than S1P, the concentration added to the culture system can be determined by one skilled in the art in accordance with the above concentration range, taking into account the differences in properties (particularly activity) between the sphingophospholipid used and S1P. Furthermore, whether the determined concentration range is appropriate can be confirmed by preliminary experiments.

[0037] (Glycerophospholipid) Glycerophospholipid is a substance in which phosphoric acid and fatty acid are bound to glycerol via hydroxyl groups. In the glycerophospholipid of the present invention, the hydroxyl groups to which the phosphoric acid and fatty acid are bound may be located at any position on the glycerol. Examples of glycerophospholipids include lysophosphatidic acid (LPA), phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and salts thereof. In a preferred embodiment, the glycerophospholipid is LPA having the structure shown below or a salt thereof.

[0038]

[0039] The concentration of glycerophospholipid (in the case of LPA) added to the culture system is, for example, 0.05 to 100 μM, preferably 0.1 to 50 μM, more preferably 0.5 to 30 μM, and even more preferably 1 to 10 μM. When using a glycerophospholipid other than LPA, the concentration to be added to the culture system can be determined by a person skilled in the art in accordance with the above concentration range, taking into account the differences in properties (particularly activity) between the glycerophospholipid used and LPA. Furthermore, whether the determined concentration range is appropriate can be confirmed by preliminary experiments.

[0040] When either or both of sphingophospholipid and glycerophospholipid are salt, the salt can be acidic or basic salt.Basic salts include, but are not limited to, alkali metal salts such as sodium, potassium, etc.; alkaline earth metal salts such as calcium, magnesium, etc.; ammonium salts; and salts of nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, N,N'-dibenzylethylenediamine, etc. Examples of acid salts include, but are not limited to, mineral acid salts such as hydrochloride, hydrobromide, nitric acid, and sulfuric acid; organic carboxylic acid salts such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid, and trifluoroacetic acid; and sulfonic acid salts such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid.

[0041] (Other Components) The culture in (ii) may be performed in the presence of any other appropriate components in addition to the factors described above. Examples of suitable components include, but are not limited to, serum, antibiotics, amino acids, vitamins, growth factors, and ROCK inhibitors.

[0042] The serum may be, for example, serum derived from a mammal, preferably fetal bovine serum (FBS). The concentration of FBS added to the culture system may be, for example, 0.05 to 20%, preferably 0.1 to 10%, and more preferably 0.5 to 5%. Alternatively, KnockOut serum (KSR) may be used as a serum substitute. TM It is also possible to use B27 supplements with vitamin A removed, such as commercially available B-27 (Serum Replacement, Gibco). TMSupplement (50X), minus vitamin A (Gibco) can be used.

[0043] The antibiotic may be, for example, penicillin, streptomycin, ampicillin, puromycin, gentamicin, etc., with penicillin and streptomycin being preferred. The concentration of penicillin and streptomycin added to the culture system is, for example, 1 / 100 of a commercially available penicillin-streptomycin solution for culture (×100) (final concentration ×1). The antibiotic may also have antifungal activity. The concentration of the antibiotic added to the culture system may be a concentration commonly used in cell culture.

[0044] The amino acids are preferably essential amino acids such as L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-glycine, L-proline, and L-serine, as well as L-alanyl-L-glutamine. The concentration of amino acids added to the culture system is, for example, 1 / 50 or 1 / 100 of the amount of a commercially available essential amino acid solution (x50) or L-glutamine solution (x100) (final concentration x1). The concentration of amino acids added to the culture system may be a concentration commonly used in cell culture.

[0045] Examples of vitamins include vitamin C (ascorbic acid), vitamin B1 (thiamine), vitamin B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), B6 ​​(pyridoxine), B7 (biotin), B12 (cobalamin), folic acid, vitamin A, and vitamin E. In a preferred embodiment, the culture system contains ascorbic acid or a derivative thereof. Examples of ascorbic acid derivatives include phosphorylated ascorbic acid and metal salts thereof. The concentration of ascorbic acid or a derivative thereof added to the culture system is, for example, 1 to 100 ng / mL, preferably 3 to 50 ng / mL, and more preferably 5 to 30 ng / mL. Vitamins other than ascorbic acid or a derivative thereof may be added to the culture system at concentrations commonly used in cell culture.

[0046] In a preferred embodiment, in the culture of (ii), the at least five selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipids, and glosserophospholipids include a ROCK inhibitor, EGF, HGF, sphingophospholipids, and glosserophospholipids, or include at least six selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipids, and glosserophospholipids. From the viewpoint of quickly obtaining intercellular adhesion, the culture of (ii) is preferably cultured in the presence of a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipids, and glosserophospholipids.

[0047] In one embodiment, the culture system in the culture (ii) does not have a composition containing any combination of one or more selected from the group consisting of nicotinamide, ascorbic acid-2-phosphate, dexamethasone, and 10% or more FBS.

[0048] (Culture Period) The culture period (ii) is not particularly limited, but the lower limit may be, for example, 3 days or more, 4 days or more, 5 days or more, preferably 6 days or more, more preferably 7 days or more, or 8 days or more, 9 days or more, 10 days or more, or even longer. On the other hand, the upper limit may be, for example, 35 days or less, 30 days or less, 25 days or less, 20 days or less, 15 days or less, or 10 days or less, and is not particularly limited as long as the adherent hepatocytes can be maintained in a viable state. Any of the upper and lower limits of the above-mentioned periods may be combined to express the period (range).

[0049] (i) Culturing in the Presence of At Least Four Selected from a ROCK Inhibitor, EGF, HGF, Sphingophospholipids, and Glucerophospholipids In one embodiment, the method of the present invention for producing adherent hepatocytes comprises culturing suspension hepatocytes in the presence of at least four selected from a ROCK inhibitor, EGF, HGF, sphingophospholipids, and glucerophospholipids prior to the culturing step (ii). (i) "In the presence of at least four selected from a ROCK inhibitor, EGF, HGF, sphingophospholipids, and glucerophospholipids" is synonymous with a culture system containing at least four selected from a ROCK inhibitor, EGF, HGF, sphingophospholipids, and glucerophospholipids (hereinafter, in the present invention, "in the presence of (predetermined components)" similarly refers to a culture system containing (predetermined components). In the present invention, the culture system may include a medium, a culture substrate, a culture vessel, etc. In one embodiment, "culturing in the presence of at least four selected from a ROCK inhibitor, EGF, HGF, sphingophospholipids, and glycerophospholipids" refers to culturing in a medium containing at least four selected from a ROCK inhibitor, EGF, HGF, sphingophospholipids, and glycerophospholipids. The medium may be any medium generally used for culturing hepatocytes, such as Dulbecco's modified Eagle's medium (DMEM).

[0050] The ROCK inhibitor, EGF, HGF, sphingophospholipids, and glycerophospholipids or other components in the culture of (i) can be selected as described for the culture of (ii), but the conditions do not need to be the same as those for the culture of (ii).

[0051] From the viewpoint of quickly obtaining intercellular adhesion, the culture in (i) is preferably cultured in the presence of a ROCK inhibitor, EGF, HGF, sphingophospholipid, and glycerophospholipid.

[0052] In one embodiment, the culture system in the culture of (i) does not have a composition containing any combination of one or more selected from the group consisting of Y27632, A83-01, CHIR99021, nicotinamide, ascorbic acid-2 phosphate, dexamethasone, and 10% or more FBS.

[0053] (Culture Period) The culture period of (i) is not particularly limited, but the lower limit may be, for example, 30 minutes or more, 1 hour or more, preferably 2 hours or more, more preferably 3 hours or more, or 5 hours or more, 10 hours or more, 15 hours or more, 20 hours or more, and even more preferably 24 hours or more. On the other hand, the upper limit may be, for example, 5 days or less, 4 days or less, 3 days or less, preferably 2 days or less, more preferably 1 day (24 hours) or less, or 20 hours or less, 15 hours or less, or 10 hours or less. Any of the upper and lower limits of the above-mentioned periods may be combined to represent a period (range).

[0054] In one embodiment, the culture in (ii) comprises forming spheroids from suspended hepatocytes. By including the formation of spheroids, the culture period can be shortened and the culture in (i) can be eliminated.

[0055] Spheroids can be formed by any suitable method known to those skilled in the art, and are generally formed by three-dimensional culture using a culture vessel equipped with a low- or non-cell-adhesive culture surface (e.g., a culture surface that has been imparted with low- or non-cell-adhesive properties by treatment / bonding with a polymer material, hydrogel, etc.). Examples of such culture vessels include culture vessels with multiple wells of uniform shape and size formed on the culture surface (commonly referred to as pattern plates; specific examples include EZSPHERE (registered trademark) available from AGC Technoglass Co., Ltd. and Elplasia available from Kuraray Co., Ltd.).

[0056] The period of culture in (ii), when including the formation of spheroids, can be 6 days or less, 5 days or less, 4 days or less, or 3 days or less, or may be even shorter if spheroid formation is possible.

[0057] The culture step (ii) may further include dissociating the spheroids into single cells. Dissociation into single cells can be performed, for example, using a cell dissociation solution. The cell dissociation solution may contain proteolytic enzymes such as trypsin-EDTA, collagenase IV, and metalloproteases, either alone or in combination, and is preferably one with minimal cytotoxicity. Examples of such cell dissociation solutions include TrypLE (Invitrogen) and Accutase (MILLIPORE).

[0058] [Passaging] After culturing in (ii), a passaging procedure may be performed. Conventionally, passaging has generally been difficult in culturing adherent hepatocytes derived from frozen human hepatocytes. Therefore, the ability to passage the adherent hepatocytes obtained by the present invention is advantageous. The number of passaging procedures may be, for example, 1 to 5 times, preferably 1 to 3 times, and more preferably 1 to 2 times during the culture period. Passaging may be performed according to methods known to those skilled in the art. For example, upon reaching confluence or subconfluence, a portion of the cells may be harvested and transferred to another culture vessel, and the culture may continue. Cell recovery may be performed using a cell dissociation solution. Examples of cell dissociation solutions include proteolytic enzymes such as trypsin-EDTA, collagenase IV, and metalloproteases, either alone or in combination. Less cytotoxicity is preferred. Commercially available cell dissociation solutions include Dispase (Eidea), TrypLE (Invitrogen), and Accutase (MILLIPORE).

[0059] [Cryopreservation] In one embodiment, the method of the present invention for producing adherent hepatocytes may include cryopreserving the hepatocytes after the culture step (ii) and then thawing them. Conventionally, cryopreserving adherent hepatocytes and then thawing them for use has generally been difficult, so the ability to cryopreserve and then culture the adherent hepatocytes obtained by the present invention after thawing is an advantage. Cryopreservation can be performed according to methods known to those skilled in the art. For example, cells can be suspended to an appropriate cell density in a medium suitable for cryopreservation, such as complete medium containing a cryoprotectant such as dimethyl sulfoxide (DMSO), and then placed in a cryovial or other cryopreservation container and stored at -80°C or below. The cryopreservation period is not particularly limited as long as the function of the adherent hepatocytes is not impaired. Thawing ... thawed by transferring the cryopreservation container containing the cells to a 37°C water bath and gently swirling the container.

[0060] (iii) Culturing in the presence of at least nine selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, a cAMP signal activator, EGF, HGF, sphingophospholipids, glosserophospholipids, gamma-secretase inhibitors, oncostatin M, and dexamethasone. In one embodiment, the method of the present invention for producing adherent hepatocytes comprises (ii) culturing suspension hepatocytes in the presence of at least five selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipids, and glosserophospholipids; and subsequently (iii) culturing in the presence of at least nine selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, a cAMP signal activator, EGF, HGF, sphingophospholipids, glosserophospholipids, gamma-secretase inhibitors, oncostatin M, and dexamethasone. The culture in (iii) is not essential for obtaining adherent hepatocytes from non-adherent hepatocytes, but it is thought to be able to mature the adherent hepatocytes obtained by the culture in (ii).

[0061] In the culture of (iii), the factors or other components such as ROCK inhibitors, TGFβ receptor inhibitors, GSK-3β inhibitors, EGF, HGF, sphingophospholipids, and glycerophospholipids can be selected or determined in the same manner as in the cultures of (i) and (ii) above. Note that these selections or determinations may be made independently of the conditions for the cultures of (i) and (ii).

[0062] The TGFβ receptor inhibitor can be selected or determined independently of the cultures (i) and (ii) described above, as in the cultures (i) and (ii). However, one or more selected from A83-01 and SB431542 are particularly preferred. Alternatively, an alternative peptide compound with TGFβ inhibitory activity may be used instead of the TGFβ receptor inhibitor. The concentration of the TGFβ receptor inhibitor added to the culture system may be, for example, 0.05 to 10 μM in the case of A83-01, preferably 0.1 to 5 μM, and more preferably 0.5 to 3 μM. The concentration of SB431542 added to the culture system may be, for example, 0.5 to 50 μM, preferably 1 to 30 μM, and more preferably 5 to 20 μM. When using a compound other than A83-01 or SB431542, the concentration can be determined within the above concentration ranges by one skilled in the art, taking into account the characteristics of the compound used and the differences in the properties (particularly differences in activity) of the exemplified compounds. Furthermore, whether the set concentration range is appropriate or not can be confirmed by a preliminary experiment based on the examples described below.

[0063] Any gamma-secretase inhibitor can be used as long as it has the effect of inhibiting gamma-secretase, such as DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester), dibenzazepine, etc., with DAPT being preferred. The concentration of the gamma-secretase inhibitor (in the case of DAPT) added to the culture system may be, for example, 0.1 to 50 μM, preferably 0.5 to 30 μM, and more preferably 0.1 to 20 μM. When using a gamma-secretase inhibitor other than DAPT, the concentration of the inhibitor added to the culture system can be determined by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the gamma-secretase inhibitor used and DAPT. Whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0064] The concentration of oncostatin M added to the culture system may be, for example, 1 to 50 ng / mL, preferably 5 to 40 ng / mL, and more preferably 10 to 30 ng / mL.The concentration of dexamethasone added to the culture system may be, for example, 0.1 to 50 μM, preferably 0.5 to 30 μM, and more preferably 0.1 to 20 μM.

[0065] The cAMP signal activator may be one or more selected from the group consisting of cAMP derivatives, cAMP degrading enzyme inhibitors and cAMP activators, and is preferably a cAMP activator.

[0066] As cAMP derivatives, for example, PKA activators (e.g., 8-Br-cAMP (8-Bromoadenosine-3′,5′-cyclic monophosphate sodium salt, CAS Number : 76939-46-3), 6-Bnz-cAMP (N6-Benzoyladenosine-3',5'-cyclic monophosphate sodium salt salt, CAS Number : 1135306-29-4), cAMPS-Rp ((R)-Adenosine, cyclic 3',5'-(hydrogenphosphorothioate)triethylammonium salt, CAS Number : 151837-09-1), cAMPS-Sp ((S)-Adenosine, cyclic 3',5'-(hydrogenphosphorothioate)triethylammonium salt, CAS Number : 93602-66-5), Dibutyryl-cAMP (N6,O2'-Dibutyryl adenosine 3',5'-cyclic monophosphate sodium salt salt, CAS Number : 16980-89-5), 8-Cl-cAMP (8-Chloroadenosine- 3', 5'- cyclic monophosphate salt, CAS Number : 124705-03-9)), Epac activators (Rp-8-Br-cAMPS (8-Bromoadenosine 3',5'-cyclic Monophosphothioate, Rp-Isomer...Examples of cAMP derivatives that can be used include 8-CPT-cAMP (8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate sodium salt, CAS Number: 129735-00-8), 8-CPT-cAMP (8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, CAS Number: 93882-12-3), and 8-pCPT-2'-O-Me-cAMP (8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium, CAS Number: 634207-53-7). The concentration of the cAMP derivative added (in the case of 8-Br-cAMP) may be, for example, 0.1 mM to 10 mM, preferably 0.2 mM to 5 mM, and more preferably 0.5 mM to 2 mM. When using an example compound, i.e., a compound other than 8-Br-cAMP, the concentration of the compound can be determined by a person skilled in the art based on the above concentration range, taking into account the differences (particularly activity) between the properties of the compound used and the example compound (8-Br-cAMP). Furthermore, whether the selected concentration range is appropriate can be confirmed by preliminary experiments.

[0067] cAMP degrading enzyme inhibitors include IBMX (3-isobutyl-1-methylxanthine) (MIX), Theophylline, Papaverine, Pentoxifylline (Trental), KS-505 , 8-Methoxymethyl-IBMX, Vinpocetine (TCV-3B), EHNA, Trequinsin (HL-725), Lixazinone (RS-82856), (LY-186126), Cilostamide (OPC3689), Bemoradan (RWJ-22867), Anergrelide (BL4162A), Indolidan (LY195115), Cilostazol (OPC-13013), Milrinone (WIN47203), Siguazodan (SKF-94836), 5-Methyl-imazodan (CI 930), SKF-95654, Pirilobendan (UD-CG Examples of cAMP inhibitors include AR-L 115 BS), Enoximone (MDL 17043), Imazodan (CL 914), SKF-94120, Vesnarinone (OPC 8212), Rolipram (Ro-20-1724), (ZK-62711), Denbufylline, Zaprinast (M&B-22,948), Dipyridamole, Zaprinast (M&B-22,948), Dipyridamole, Zardaverine, AH-21-132, and Sulmazol (AR-L 115 BS). The concentration of the cAMP inhibitor (in the case of IBMX) may be, for example, 0.05 mM to 5 mM, preferably 0.1 mM to 3 mM, and more preferably 0.2 mM to 1 mM. When using an exemplified compound, i.e., a compound other than IBMX, the concentration of the compound to be added can be determined by a person skilled in the art in accordance with the above concentration range, taking into consideration the differences (particularly the differences in activity) between the properties of the compound used and the exemplified compound (IBMX). Whether the set concentration range is appropriate can be confirmed by a preliminary experiment.

[0068] Examples of cAMP activators include forskolin, indomethacin, NKH477 (colforsin daropate), cell-derived toxic proteins (pertussis toxin, cholera toxin), PACAP-27, PACAP-38, and SKF83822, with forskolin being preferred. Examples of the concentration of a cAMP activator added to a culture system (forskolin) include 1-50 μM, preferably 3-30 μM, and more preferably 5-15 μM. When using an exemplified compound, i.e., a compound other than forskolin, the concentration to be added can be determined by one skilled in the art based on the above concentration range, taking into account the differences in the properties of the compound used and the properties of the exemplified compound (forskolin) (particularly differences in activity). The appropriateness of the selected concentration range can be confirmed by preliminary experiments similar to those described in the Examples below.

[0069] In a preferred embodiment, in the culture of (iii), the at least nine selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, a cAMP signal activator, EGF, HGF, sphingophospholipids, glycerophospholipids, γ-secretase inhibitors, oncostatin M, and dexamethasone include a ROCK inhibitor, a cAMP signal activator, EGF, HGF, sphingophospholipids, glycerophospholipids, γ-secretase inhibitors, oncostatin M, and dexamethasone, or include at least ten selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, a cAMP signal activator, EGF, HGF, sphingophospholipids, glycerophospholipids, γ-secretase inhibitors, oncostatin M, and dexamethasone. From the viewpoint of quickly obtaining intercellular adhesion, it is preferable that the culture in (iii) be carried out in the presence of a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, a cAMP signal activator, EGF, HGF, sphingophospholipids, glycerophospholipids, a γ-secretase inhibitor, oncostatin M, and dexamethasone.

[0070] In one embodiment, the culture system in the culture (iii) does not have a composition containing any combination of one or more selected from the group consisting of nicotinamide and ascorbic acid-2-phosphate.

[0071] (Culture Period) The culture period of (iii) is not particularly limited, but the lower limit may be, for example, 12 hours or more, 1 day or more, and preferably 2 days or more. On the other hand, the upper limit may be, for example, 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, or 3 days or less. Any of the upper and lower limits of the above-mentioned periods may be combined to represent a period (range).

[0072] (Culture Surface) Each of the cultures (i) to (iii) can be performed on a culture surface suitable for the present invention. The culture surface is not particularly limited as long as it is generally usable for cell culture, and may be, for example, a cell culture plate, flask, petri dish, dish, cell culture insert, etc. The culture surface may also be coated with any component suitable for cell differentiation, proliferation, adhesion, etc. In a preferred embodiment, the culture is performed on a culture surface coated with a basement membrane component. Examples of basement membrane components include laminin, type IV collagen, entactin, heparan sulfate proteoglycan, vitronectin, fibronectin, or fragments thereof. Examples of materials containing these basement membrane components include Matrigel (Corning) and iMatrix-511 (Matrixome), which contains the human laminin 511E8 fragment. Furthermore, the culture including the culture of (i) and the culture of (ii) can also be performed on a culture device generally referred to as an organ-on-a-chip (microphysiological system, MPS: biomimetic system) or the like.

[0073] (Other) Other culture conditions may be those generally employed in the culture of animal cells, such as 37°C and 5% CO2. The basal medium may be any medium suitable for the culture of animal cells, such as Iscove's Modified Dulbecco's Medium (IMDM) (Gibco, etc.), Ham's F12 medium (Sigma, Gibco, etc.), Dulbecco's Modified Eagle's Medium (DMEM) (Nacalai Tesque, Inc., Sigma, Gibco, etc.), Glasgow Essential Medium (Gibco, etc.), RPMI 1640 medium, etc. A preferred example of the basal medium is DMEM / F12 (a mixture of DMEM and Ham's F12 medium).

[0074] <Method for evaluating metabolism of a test substance> In one aspect, the present invention is a method for evaluating metabolism of a test substance, comprising: (1) obtaining adherent hepatocytes by the method of the present invention for producing adherent hepatocytes; (2) contacting the adherent hepatocytes obtained in step (1) with a test substance; and (3) measuring the metabolism of the test substance.

[0075] Test substances may be organic or inorganic compounds of various molecular sizes. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.)), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, and vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.). Existing or potential components of pharmaceuticals and nutritional foods are also suitable test substances. Plant extracts, cell extracts, culture supernatants, etc. may also be used as test substances. Two or more test substances may be added simultaneously to examine interactions, synergies, etc. between the test substances. Test substances may be naturally occurring or synthetic. In the latter case, efficient assay systems can be constructed using, for example, combinatorial synthesis techniques.

[0076] The period for contacting the adherent hepatocytes with the test substance in step (2) can be arbitrarily determined by those skilled in the art depending on the type and amount of the test substance, measurement conditions, etc. The contact period may be, for example, 10 minutes to 3 days, or 1 hour to 1 day, etc. The contact may be performed multiple times.

[0077] The measurement of the metabolism of the test substance in step (3) can be performed by any method known to those skilled in the art, including, for example, a method of measuring the amount of metabolites of the test substance after step (2). Metabolites of a test substance are substances produced by the metabolism of the test substance in the liver and are well known to those skilled in the art. For example, when midazolam is used as the test substance as shown in the Examples, its hepatic metabolite may be 1-hydroxymidazolam. The method for measuring the amount of metabolites may be selected by those skilled in the art, including, for example, mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry (LC-MS, LC-MS / MS), immunological techniques (e.g., fluorescent immunoassay (FIA)), enzyme immunoassay (EIA)), etc. The greater the amount of metabolite produced, the greater the extent to which the test substance has been metabolized.

[0078] <Method for inducing metabolic enzymes in hepatocytes> In one aspect, the present invention provides a method for inducing metabolic enzymes in hepatocytes, comprising: (1) obtaining adherent hepatocytes by the method of the present invention for producing adherent hepatocytes; and (2) contacting the adherent hepatocytes obtained in step (1) with a metabolic enzyme inducer. Because it is difficult to use suspension hepatocytes in metabolic enzyme induction tests, obtaining adherent hepatocytes according to the present invention allows for efficient metabolic enzyme induction tests.

[0079] Metabolic enzymes in hepatocytes include, for example, drug-metabolizing enzymes, such as the cytochrome P450 (CYP) family, uridine diphosphate-glucuronosyltransferase (UGT), and sulfotransferase (SULT). In particular, the CYP family is the main drug-metabolizing enzyme in hepatocytes. The CYP family is classified based on amino acid sequence homology, and many drugs are known to be substrates for CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4, among others. Furthermore, those skilled in the art are familiar with drugs (metabolic enzyme inducers) that induce the expression of metabolic enzymes in hepatocytes. For example, omeprazole, phenobarbital, and rifampicin are known as metabolic enzyme inducers for CYP1A2, CYP2B6, and CYP3A4, respectively.

[0080] The time for contacting the adherent hepatocytes with the metabolic enzyme inducer in step (2) can be appropriately determined by those skilled in the art depending on the types and amounts of the metabolic enzyme and metabolic enzyme inducer, measurement conditions, etc. The contact may be carried out in multiple batches.

[0081] The induction of metabolic enzymes in hepatocytes can be confirmed by methods known to those skilled in the art. For example, the gene expression of metabolic enzymes can be measured and compared between cases where adherent hepatocytes are contacted with a metabolic enzyme inducer and cases where they are not. Alternatively, the activity of metabolic enzymes can be measured and compared in both cases. Gene expression can be measured by methods known to those skilled in the art, such as quantitative PCR. The activity of metabolic enzymes can be measured, for example, by using a substrate specific to the metabolic enzyme of interest, reacting it with the metabolic enzyme under specified conditions, and measuring the metabolites.

[0082] In one embodiment, the method of the present invention for inducing metabolic enzymes in hepatocytes comprises sandwich culturing the adherent hepatocytes obtained in step (1). Sandwich culturing is a method in which cells are sandwiched between extracellular matrix components and cultured. Sandwich culturing is known to improve hepatocyte-specific functions such as drug metabolic activity. Sandwich culturing is also known to form a three-dimensional structure, enabling long-term maintenance of hepatocytes. Furthermore, sandwich culturing is known to localize efflux transporters on the bile canalicular membrane, enabling evaluation of biliary excretion of drugs. Non-limiting examples of extracellular matrix components that can be used for sandwich culturing include collagen gel and Matrigel.

[0083] <Method for Producing Hepatocyte Spheroids> In one aspect, the present invention provides a method for producing hepatocyte spheroids, comprising: (1) obtaining adherent hepatocytes by the method of the present invention for producing adherent hepatocytes; and (2) culturing the adherent hepatocytes obtained in step (1) to form hepatocyte spheroids. Generally, adherent hepatocytes have difficulty adhering to each other once detached from the culture surface. On the other hand, the adherent hepatocytes obtained by the present invention are capable of adhering to each other even after detachment from the culture surface, thereby offering the advantage of enabling the production of hepatocyte spheroids by three-dimensional culture.

[0084] Suspension culture is suitable for step (2) of culturing adherent hepatocytes to form hepatocyte spheroids. Suspension culture typically involves the use of a culture vessel equipped with a low- or non-adhesive culture surface (e.g., a culture surface imparted with low- or non-adhesive properties by treatment / bonding with a polymer material or hydrogel, etc.), and the cells are cultured away from the culture surface (i.e., in a suspended state). The culture vessel used for suspension culture is not particularly limited, and examples include dishes, flasks, multiwell plates, tubes, trays, and culture bags. Multiple spheroids are formed simultaneously using a culture vessel with multiple wells of uniform shape and size formed on the culture surface (commonly referred to as a pattern plate; specific examples include EZSPHERE (registered trademark) from AGC Technoglass Co., Ltd. and Elplasia from Kuraray Co., Ltd.). This allows for efficient spheroid formation. The composition of the medium used in step (2) is not particularly limited as long as it is suitable for forming hepatocyte spheroids, but it may be the same medium as that used in step (1) for producing adherent hepatocytes.

[0085] In one embodiment, the culture in step (2) is a sandwich culture.

[0086] The period (culture period) of step (2) may be, for example, 10 to 35 days, preferably 12 to 30 days, and more preferably 15 to 25 days. If this period is too short, spheroids of sufficient size will not be formed. On the other hand, if this period is too long, the spheroids will grow larger than necessary, which may cause necrosis of the cells inside.

[0087] <Method for two-dimensionally culturing hepatocyte spheroids> In one embodiment, the present invention provides a method for two-dimensionally culturing hepatocyte spheroids, comprising: (1) obtaining adherent hepatocytes by the method of the present invention for producing adherent hepatocytes; (2) converting the adherent hepatocytes obtained in step (1) into single cells; and (3) culturing the hepatocytes converted into single cells in step (2) to produce hepatocyte spheroids. Generally, once adherent hepatocytes are converted into single cells (i.e., cells are no longer adherent to each other), it is difficult to re-adhere the cells. On the other hand, the adherent hepatocytes obtained by the present invention are capable of adhering to each other even after conversion into single cells, which offers the advantage of enabling the production of hepatocyte spheroids, converting the resulting hepatocyte spheroids into single cells, and then expanding and culturing them in two dimensions.

[0088] The dissociation of adherent hepatocytes into single cells in step (2) can be carried out using, for example, a cell dissociation solution. The cell dissociation solution may contain proteolytic enzymes such as trypsin-EDTA, collagenase IV, and metalloproteases, either singly or in combination, and is preferably one with minimal cytotoxicity. Examples of such cell dissociation solutions include TrypLE (Invitrogen) and Accutase (MILLIPORE).

[0089] In one embodiment, the step (2) of forming single cells may be performed using hepatocyte spheroids obtained by the present invention.

[0090] In one embodiment, the culture in step (2) is a sandwich culture.

[0091] Step (3), which involves culturing the hepatocytes isolated in step (2) to form hepatocyte spheroids, can be performed on any culture surface. The culture surface can be any surface commonly used for cell culture, including cell culture plates, flasks, petri dishes, dishes, and cell culture inserts (e.g., Transwell). Preferably, the culture surface is coated with a basement membrane component. Examples of basement membrane components include laminin, type IV collagen, entactin, heparan sulfate proteoglycan, vitronectin, fibronectin, and fragments thereof. Examples of materials containing these basement membrane components include Matrigel (Corning).

[0092] The composition of the medium used in step (3) is not particularly limited as long as it is suitable for disaggregating hepatocyte spheroids into single cells and culturing them in two dimensions, including the step of producing hepatocyte spheroids. However, it may be the same medium as that used in step (1) of producing adherent hepatocytes.

[0093] <Culture Medium> In one embodiment, the present invention relates to a culture medium for use in the method for producing adherent hepatocytes of the present invention. In one embodiment, the culture medium of the present invention contains at least four substances selected from a ROCK inhibitor, EGF, HGF, sphingophospholipids, and glycerophospholipids (hereinafter, this culture medium will be referred to as culture medium (i)). In another embodiment, the culture medium of the present invention contains at least five substances selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipids, and glycerophospholipids (hereinafter, this culture medium will be referred to as culture medium (ii)).

[0094] The concentration of HGF in the medium of the present invention (hereinafter, the term "medium of the present invention" refers to one or more of medium (i) and medium (ii) unless otherwise specified. Note that the term "medium of the present invention" does not specify that medium (i) and medium (ii) are under the same conditions, and the compositions of medium (i) and medium (ii) are determined independently) may be, for example, 1 to 200 ng / mL, preferably 3 to 100 ng / mL, more preferably 10 to 50 ng / mL, and even more preferably 15 to 30 ng / mL.

[0095] The EGF in the medium of the present invention may be selected as described in the description of the method for producing adherent hepatocytes herein. The concentration of EGF in the medium of the present invention may be, for example, 3 to 100 ng / mL, preferably 5 to 50 ng / mL, and more preferably 10 to 30 ng / mL.

[0096] The sphingophospholipid in the medium of the present invention may be selected as described herein in the description of the method for producing adherent hepatocytes. The concentration of the sphingophospholipid (in the case of S1P) in the medium of the present invention may be, for example, 0.01 to 50 μM, preferably 0.05 to 30 μM, more preferably 0.1 to 10 μM, and even more preferably 0.5 to 5 μM. When using a sphingophospholipid other than S1P, the concentration in the medium can be determined by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) of the sphingophospholipid used and S1P. Furthermore, whether the determined concentration range is appropriate can be confirmed by preliminary experiments.

[0097] The glycerophospholipid in the medium of the present invention may be selected as described herein in the description of the method for producing adherent hepatocytes. The concentration of glycerophospholipid (in the case of LPA) in the medium of the present invention may be, for example, 0.05 to 100 μM in the medium, preferably 0.1 to 50 μM, more preferably 0.5 to 30 μM, and even more preferably 1 to 10 μM. When using a glycerophospholipid other than LPA, the concentration in the medium can be set by one skilled in the art in accordance with the above concentration range, taking into account the differences in properties (particularly activity) between the glycerophospholipid used and LPA. Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0098] The ROCK inhibitor in media (i) and (ii) may be selected as described in the description of the method for producing adherent hepatocytes herein. The concentration of the ROCK inhibitor (in the case of Y27632) in the medium of the present invention may be, for example, 0.1 to 50 μM in the medium, preferably 0.5 to 30 μM, and more preferably 1 to 20 μM. When using a ROCK inhibitor other than Y27632, the concentration in the medium can be set by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the ROCK inhibitor used and Y27632. Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0099] The TGFβ receptor inhibitor in medium (ii) may be selected as described in the description of the method for producing adherent hepatocytes herein. The concentration of the TGFβ receptor inhibitor (in the case of A83-01) in the medium of the present invention may be, for example, 0.05 to 10 μM, preferably 0.1 to 5 μM, and more preferably 0.5 μM to 3 μM. When a compound other than A83-01 is used, the concentration in the medium can be set by one skilled in the art based on the above concentration range, taking into account the differences in the properties of the compound used and the properties of A83-01 (particularly differences in activity). Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments based on the examples described below.

[0100] The GSK-3β inhibitor in medium (ii) may be selected as described in the description of the method for producing adherent hepatocytes herein. The concentration of the GSK-3β inhibitor (in the case of CHIR99021) in the medium of the present invention may be, for example, 0.1 to 20 μM in the medium, preferably 0.5 to 10 μM, and more preferably 1 to 5 μM. When a GSK-3β inhibitor other than CHIR99021 is used, the concentration in the medium can be set by a person skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the GSK-3β inhibitor used and CHIR99021. Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0101] The medium of the present invention may further contain components suitable for the method of producing adherent hepatocytes of the present invention, such as, but not limited to, serum or serum substitutes, antibiotics, amino acids, vitamins, growth factors, and ROCK inhibitors.

[0102] The serum or serum substitute may be selected as described in the description of the method for producing adherent hepatocytes herein. The serum (in the case of FBS) concentration in the medium of the present invention may be, for example, 0.05 to 20% in the medium, preferably 0.1 to 10%, and more preferably 0.5 to 5%.

[0103] The antibiotic may be selected as described in the description of the method for producing adherent hepatocytes herein. The concentration of antibiotics (in the case of penicillin and streptomycin) in the medium of the present invention is, for example, 1 / 100 of the amount of a commercially available penicillin-streptomycin solution for culture (×100) (final concentration ×1). The concentration of antibiotics added to the culture system may be the concentration commonly used in cell culture.

[0104] The amino acids may be selected as described in the description of the method for producing adherent hepatocytes herein. The concentration of amino acids in the medium of the present invention is, for example, 1 / 50 or 1 / 100 of the amount of commercially available essential amino acid solution (x50) or L-glutamine solution (x100) (final concentration x1). The concentration of amino acids added to the culture system may be a concentration commonly used in cell culture.

[0105] Vitamins may be selected as described in the description of the method for producing adherent hepatocytes herein. The concentration of vitamins (in the case of ascorbic acid or its derivatives) in the medium of the present invention may be, for example, 1 to 100 ng / mL, preferably 3 to 50 ng / mL, and more preferably 5 to 30 ng / mL. The concentration of vitamins other than ascorbic acid or its derivatives in the medium may be a concentration commonly known to those skilled in the art of cell culture.

[0106] The ROCK inhibitor may be selected as described herein in the description of the method for producing adherent hepatocytes. The concentration of the ROCK inhibitor (in the case of Y27632) in the medium of the present invention may be, for example, 0.1 to 50 μM, preferably 0.5 to 30 μM, and more preferably 1 to 20 μM. When using a ROCK inhibitor other than Y27632, the concentration in the medium can be set by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the ROCK inhibitor used and Y27632. Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0107] From the viewpoint of quickly obtaining intercellular adhesion, the medium (i) preferably contains a ROCK inhibitor, EGF, HGF, sphingophospholipid, and glycerophospholipid.

[0108] In one embodiment, the medium (i) does not have a composition containing any combination of one or more selected from the group consisting of Y27632, A83-01, CHIR99021, nicotinamide, ascorbic acid-2 phosphate, dexamethasone, and 10% or more FBS.

[0109] In a preferred embodiment, medium (ii) contains a ROCK inhibitor, EGF, HGF, sphingophospholipid, and glosserophospholipid, or at least six selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipid, and glosserophospholipid. From the viewpoint of quickly obtaining intercellular adhesion, medium (ii) preferably contains a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipid, and glosserophospholipid.

[0110] In one embodiment, the medium (ii) does not have a composition containing any combination of one or more selected from the group consisting of nicotinamide, ascorbic acid-2 phosphate, dexamethasone, and 10% or more FBS.

[0111] In one embodiment, the medium of the present invention comprises at least nine substances selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, a cAMP signal activator, EGF, HGF, sphingophospholipids, glycerophospholipids, a gamma-secretase inhibitor, oncostatin M, and dexamethasone (hereinafter, this medium will be referred to as medium (iii)).

[0112] The ROCK inhibitor, TGFβ receptor inhibitor, GSK-3β inhibitor, cAMP signal activator, EGF, HGF, sphingophospholipid, and glycerophospholipid in medium (iii) can be selected or determined in the same manner as for medium (i) and medium (ii). Note that these selections or determinations may be made independently of medium (i) and medium (ii).

[0113] The TGFβ receptor inhibitor in medium (iii) may be selected as described in the description of the method for producing adherent hepatocytes herein. In the case of A83-01, the concentration of the TGFβ receptor inhibitor in medium (iii) may be, for example, 0.05 to 10 μM, preferably 0.1 to 5 μM, and more preferably 0.5 to 3 μM. In the case of SB431542, the concentration in medium (iii) may be, for example, 0.5 to 50 μM, preferably 1 to 30 μM, and more preferably 5 to 20 μM. When using a compound different from A83-01 or SB431542, the concentration in medium (iii) can be determined by one skilled in the art within the above concentration range, taking into account the differences (particularly differences in activity) between the properties of the compound used and those of A83-01 or SB431542. Furthermore, whether the set concentration range is appropriate or not can be confirmed by a preliminary experiment based on the examples described below.

[0114] The gamma-secretase inhibitor in medium (iii) may be selected as described in the description of the method for producing adherent hepatocytes herein. The concentration of the gamma-secretase inhibitor (in the case of DAPT) in medium (iii) may be, for example, 0.1 to 50 μM, preferably 0.5 to 30 μM, and more preferably 0.1 to 20 μM. When using a gamma-secretase inhibitor other than DAPT, the concentration in medium (iii) can be set by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the gamma-secretase inhibitor used and DAPT. Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0115] The oncostatin M concentration in the medium (iii) may be, for example, 1 to 50 ng / mL, preferably 5 to 40 ng / mL, and more preferably 10 to 30 ng / mL. The dexamethasone concentration in the medium (iii) may be, for example, 0.1 to 50 μM, preferably 0.5 to 30 μM, and more preferably 0.1 to 20 μM.

[0116] The cAMP signal activator in the medium (iii) may be one or more selected from the group consisting of a cAMP derivative, a cAMP degrading enzyme inhibitor, and a cAMP activator, and is preferably a cAMP activator. The cAMP signal activator in the medium (iii) may be selected as described in the description of the method for producing adherent hepatocytes herein.

[0117] The concentration of the cAMP derivative (in the case of 8-Br-cAMP) in medium (iii) may be, for example, 0.1 mM to 10 mM, preferably 0.2 mM to 5 mM, and more preferably 0.5 mM to 2 mM. When a cAMP derivative other than 8-Br-cAMP is used, the concentration in medium (iii) can be determined by one skilled in the art within the above concentration range, taking into account the differences in the properties of the cAMP derivative used and those of 8-Br-cAMP (particularly differences in activity). Whether the determined concentration range is appropriate can be confirmed by preliminary experiments.

[0118] The concentration of the cAMP inhibitor (in the case of IBMX) in the medium (iii) may be, for example, 0.05 mM to 5 mM, preferably 0.1 mM to 3 mM, and more preferably 0.2 mM to 1 mM. When a cAMP inhibitor other than IBMX is used, the concentration in the medium (iii) can be set by a person skilled in the art in accordance with the above concentration range, taking into consideration the differences in the properties of the cAMP inhibitor used and those of IBMX (particularly differences in activity). Whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0119] The concentration of the cAMP activator (forskolin) in medium (iii) may be, for example, 1 to 50 μM, preferably 3 to 30 μM, and more preferably 5 to 15 μM. When a cAMP activator other than forskolin is used, the concentration in medium (iii) can be determined by a person skilled in the art within the above concentration range, taking into account the differences in the properties of the cAMP activator used and those of forskolin (particularly differences in activity). Whether the determined concentration range is appropriate can be confirmed by a preliminary experiment similar to the examples described below.

[0120] In a preferred embodiment, medium (iii) contains a ROCK inhibitor, a cAMP signal activator, EGF, HGF, sphingophospholipids, glosserophospholipids, a gamma-secretase inhibitor, oncostatin M, and dexamethasone, or at least 10 selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, a cAMP signal activator, EGF, HGF, sphingophospholipids, glosserophospholipids, a gamma-secretase inhibitor, oncostatin M, and dexamethasone. From the viewpoint of quickly obtaining intercellular adhesion, medium (iii) preferably contains a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, a cAMP signal activator, EGF, HGF, sphingophospholipids, glosserophospholipids, a gamma-secretase inhibitor, oncostatin M, and dexamethasone.

[0121] In one embodiment, the medium (iii) does not have a composition containing any combination of one or more selected from the group consisting of nicotinamide and ascorbic acid-2-phosphate.

[0122] <Kit> In one embodiment, the present invention relates to a kit comprising the medium of the present invention and suspension hepatocytes. The number of suspension hepatocytes contained in the kit of the present invention is not particularly limited, and may be, for example, 1 × 10 4 ~1×10 8 cells, 1 × 10 5 ~1×10 7 The floating hepatocytes contained in the kit of the present invention may be frozen.

[0123] The kit of the present invention may include a container containing the medium of the present invention and a container containing suspended hepatocytes. These containers are not particularly limited as long as they are suitable for storing the medium or cells. The kit of the present invention may also include an instruction manual describing operating instructions such as the culture method.

[0124] The present invention will be explained in more detail below by way of examples, but the present invention should not be construed as being limited to these examples.

[0125] Example 1 The outline of the experimental protocol of Example 1 is shown in FIG.

[0126] [Methods] 1. Primary human hepatocytes Cryopreserved floating primary human hepatocytes (single donor-derived hepatocytes and pooled hepatocytes derived from 20 donors) were purchased from Sekisui Xeno Tech.

[0127] 2. Seeding of HPHs using Medium 1 and induction of adhesion. 3x10 HPHs were seeded using Medium 1. 4 cells / cm 2 The cells were seeded onto Matrigel-coated plates at 100°C. They were then cultured overnight (3-24 hours) in a normoxic (5% CO2) incubator at 37°C. Medium 1 consisted of DMEM / F12 supplemented with 4% fetal bovine serum (FBS), 1x100 penicillin-streptomycin solution (PS), 1x100 GlutaMAX (GlutaMAX), 1x100 N2 supplement, 10 μg / mL ascorbic acid, 20 ng / mL hepatocyte growth factor (HGF), 20 ng / mL epidermal growth factor (EGF), 1 μM sphingosine-1-phosphate (S1P), 5 μM lysophosphatidic acid (LPA), and 10 μM Y27632, with or without 10 μM Y27632.

[0128] 3. 2D culture system: (New culture system for HPHs) After seeding, HPHs were evaluated for cell adhesion and cultured for 7–12 days based on the lot and cell morphology using Medium 2, consisting of DMEM / F12 supplemented with 4% FBS, ×1 PS, ×1 GlutaMAX (×100), 10 μg / mL ascorbic acid, 20 ng / mL HGF, 20 ng / mL EGF, 1 μM S1P, 5 μM LPA, 1 μM A83-01, 3 μM CHIR99021, and 10 μM Y27632. The medium was changed daily. To improve the maturation and function of cultured HPHs, the medium was then replaced with Medium 3, consisting of DMEM / F12 supplemented with 4% FBS, 1× B27 supplement without vitamin A, 1× PS, 1× GlutaMAX (100×), 1× N2 supplement, 10 μg / mL ascorbic acid, 20 ng / mL HGF, 20 ng / mL EGF, 1 μM S1P, 5 μM LPA, 1 μM A83-01, 3 μM CHIR99021, 10 μM Y27632, 10 μM DAPT, 20 ng / mL oncostatin M (OSM), 10 μM dexamethasone, 10 μM SB431542, and 10 μM forskolin, for an additional 2 days. SB431542 and forskolin improve the maturation and function of HPHs. Furthermore, adding 0.25% DMSO to Medium 3 can improve cell morphology. Mature HPHs were then overlaid with Matrigel and cultured in a sandwich culture, with or without the need for sandwich culture. Finally, the cells were washed with phosphate-buffered saline (PBS) and used for drug-metabolizing enzyme induction assays and other assays.

[0129] Assay 4. Imaging by Phase Contrast Microscopy The morphology of HPHs was observed and evaluated daily by phase contrast microscopy. Images of HPHs were recorded before and after medium change on day 0 and before and after the induction assay on day 12.

[0130] 5. Cytochrome 450 (CYP) Induction Assay and mRNA Expression. After confirming the morphology of HPHs on day 10 of culture, omeprazole, phenobarbital, or rifampicin, known inducers of CYP1A2, CYP2B6, or CYP3A4, were added to commercially available opti incubate medium at final concentrations of 50 μM, 750 μM, or 25 μM, respectively. 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), an inducer of CYP2B6, was added to a final concentration of 1 nM. Controls contained less than 0.1% DMSO. Cells were incubated for 48 hours. Fresh CYP induction medium was prepared and replaced daily. Cells were then harvested and subjected to quantitative real-time PCR (qPCR).

[0131] 6. CYP metabolic activity after induction assay. After confirming the morphology of cultured HPHs on day 10, induction assays of CYP1A2, CYP2B6, or CYP3A4 were performed by adding 50 μM omeprazole, 750 μM phenobarbital, or 25 μM rifampicin to commercially available opti incubate medium. Cells were incubated for 48 hours with less than 0.1% DMSO as a control. Fresh CYP induction medium was prepared and replaced daily, after which the cells were harvested. Then, on day 12, CYP activity assays were performed using a cocktail of CYP substrates as follows: Cells were incubated in medium supplemented with the substrates at 37°C for 30 minutes to 4 hours. The substrates used were 50 μM phenacetin for CYP1A2, 500 μM bupropion for CYP2B6, 5 μM diclofenac for CYP2C9, 50 μM S-mephenytoin for CYP2C19, and 5 μM midazolam for CYP3A4. The culture supernatant was then collected and metabolites were quantified by LC-MS / MS.

[0132] [Results] Morphological images of HPHs from lot 1 before and after medium change on day 0 (Figure 2) showed that the cells adhered to the plate. Morphological images of the 2D culture system (without passage, before the induction test) of HPHs from each of the three lots on day 10 (Figure 3) also showed that the cells were adherent. Morphological images of the 2D culture system (without passage) of HPHs from lot 1 after the induction test (Figure 4) are shown. These results demonstrate that the HPHs obtained by the method of the present invention can be cultured in a 2D manner, converting suspension cells into adherent cells.

[0133] The results of measuring the mRNA expression of each metabolic enzyme in the induction test are shown in Figure 5. CYP1A2 mRNA expression was induced approximately 80-fold by omeprazole (Figure 5A). CYP2B6 mRNA expression was induced by phenobarbital but not by rifampicin (Figure 5B). CYP3A4 mRNA expression was induced approximately 4-fold and approximately 8-fold by rifampicin and phenobarbital, respectively (Figure 5C). These results demonstrate that the HPHs obtained by the method of the present invention can be used in induction tests.

[0134] Example 2 The outline of the experimental protocol of Example 2 is shown in FIG.

[0135] [Methods] 7. Subculture of HPHs, CYP induction assay, and mRNA expression. To verify whether HPHs could be subcultured in vitro as adherent HPHs, cells were cultured for 6-8 days for each lot, and after confirming their morphology, the cells were harvested using Trypsin-EDTA (TE), a commercially available cell detachment solution. Subcultured sHPHs were harvested using 0.25% TE. These cells were then replated or cryopreserved. When replated, 3 x 10 cells were placed on a plate pre-coated with Matrigel. 4 cells / cm 2The cells were replated at a density of 1000 x g. Medium 2 was used for two days, with daily changes. After confirming morphology, medium 3 was used for two more days, without overlaying Matrigel. On day 5, induction assays of CYP1A2, CYP2B6, and CYP3A4 were performed using the same method as in Example 1. Cell morphology was confirmed on day 7 of the induction test. Finally, cultured HPHs were harvested and quantitative real-time PCR (qPCR) was performed. For cell cryopreservation, cells were suspended in commercially available cell cryopreservation solutions, Stem Cell Banker or Cell Banker Plus 1, according to the manufacturer's instructions.

[0136] 8. CYP metabolic activity after induction assay After confirming the morphology of the cultured HPHs on day 7, a CYP metabolic activity assay was performed. The culture supernatant was collected, and metabolites were quantified by LC-MS / MS in the same manner as in Example 1.

[0137] [Results] Morphological images of Lot 1 (after induction test, subcultured, without Matrigel overlay) on day 7 (Figure 7) demonstrated that HPHs obtained by the method of the present invention can be subcultured. Furthermore, while it has previously been impossible to maintain HPHs alive for more than 4-5 hours, this was demonstrated to be possible with the HPHs obtained by the method of the present invention. Furthermore, the results of measuring the metabolic activity of CYP3A4 using these cells (Figure 8) demonstrated that induction assays and metabolic activity assays were possible using adherent HPHs obtained by the method of the present invention, even after subculture.

[0138] For cryopreservation, each lot was cultured for 6–8 days, and after confirming morphology, cells were harvested using the commercially available cell detachment solution described in the Methods section. The harvested HPHs were then cryopreserved. The cryopreserved HPHs were then cultured for 2 days using Medium 2 and replated onto Matrigel-coated plates. After confirming morphology, the medium was replaced with Medium 3 and cultured for 2 days. The cells were overlaid with 30x diluted Matrigel, and morphological images were confirmed using a phase-contrast microscope. The results are shown in Figure 9.

[0139] Example 3 The outline of the experimental protocol of Example 3 is shown in FIG.

[0140] [Methods] 9. Sandwich Culture of Passaged HPHs, CYP Induction Assay, and mRNA Expression Measurement. To verify the feasibility of sandwich culture of HPHs as adherent HPHs, trypsinized HPHs were seeded onto Matrigel-precoated plates. Cells were cultured in Medium 2 for 2 days, with daily changes of medium. Morphology was then confirmed, and the cells were changed to Medium 3 and cultured for an additional 2 days. On Day 4, 30-fold diluted Matrigel was overlaid and incubated in a normoxic environment (5% CO2 incubator) for sandwich culture. On Day 5, induction assays and other assays were performed using sandwich-cultured HPHs. CYP1A2, CYP2B6, and CYP3A4 induction assays were performed using the same methods as in Examples 1 and 2, and cell morphology after the induction assay was confirmed by imaging. Finally, the cultured HPHs were harvested and quantitative real-time PCR (qPCR) was performed.

[0141] 10. CYP metabolic activity after induction assay On day 7, the morphology of HPHs was confirmed, and then a CYP metabolic activity assay was performed. The culture supernatant was collected, and metabolites were quantified by LC-MS / MS in the same manner as in Example 2.

[0142] [Results] Figure 11 shows the morphological images of sandwich-cultured HPHs from Lot 1 on day 7 (passaged after induction). Figure 12 also shows the results of measuring the mRNA expression of each metabolic enzyme in the induction test using sandwich-cultured HPHs from Lot 1 on day 7 (passaged after induction). CYP1A2 mRNA expression was induced approximately 300-fold by omeprazole (Figure 12A). CYP2B6 mRNA expression was induced approximately 5-fold and 17-fold by rifampicin and phenobarbital, respectively (Figure 12B). CYP2C9 mRNA expression was induced approximately 2.5-fold by both rifampicin and phenobarbital (Figure 12C). CYP2C19 mRNA expression was induced approximately 2.5-fold by both rifampicin and phenobarbital (Figure 12D). CYP3A4 mRNA expression was induced approximately 70-fold and 40-fold by rifampicin and phenobarbital, respectively (Fig. 12E). Furthermore, the metabolic activity of CYP3A4 was measured using sandwich-cultured HPHs of Lot 1 on day 7 (passaged after the induction test) and the results are shown in Fig. 13.

[0143] Figure 14 shows the morphology of sandwich-cultured HPHs from Lot 2 on day 7 (passaged after induction). Figure 15 also shows the results of measuring mRNA expression in an induction test using sandwich-cultured HPHs from Lot 2 on day 7 (passaged after induction). CYP1A2 mRNA expression was induced approximately 14-fold by omeprazole (Figure 15A). CYP2B6 mRNA expression was induced approximately 5-fold by both rifampicin and phenobarbital (Figure 15B). CYP2C9 mRNA expression was induced more than 2-fold by rifampicin but not by phenobarbital (Figure 15C). CYP2C19 mRNA expression was induced 2-fold and 1.5-fold by rifampicin and phenobarbital, respectively (Figure 15D). CYP3A4 mRNA expression was induced 30-fold and 20-fold by rifampicin and phenobarbital, respectively (Fig. 15E). Furthermore, the metabolic activity of CYP3A4 was measured using sandwich-cultured HPHs of Lot 2 on day 7 (passaged after the induction test) and the results are shown in Fig. 16.

[0144] These results demonstrate that the HPHs obtained by the method of the present invention can be sandwich cultured. Furthermore, when the adherent HPHs obtained by the method of the present invention were used in sandwich culture, induction assays and CYP metabolic activity assays were possible.

[0145] Example 4 An outline of the experimental protocol of Example 4 is shown in FIG.

[0146] [Methods] 11. 3D culture system of HPHs and 2D culture system on cell culture inserts. Each lot was cultured for 6-8 days, and after confirming the morphology, the cells were harvested using 0.25% TE. They were then seeded on EZSPHERE plates and cultured for 1-2 days to produce uniform spheres. After sphere formation, the cells were cultured in suspension for 6-12 days in Medium 2 containing 30x diluted Matrigel, depending on the lot and morphology. Then, TrypLE Select TM The cells were then cryopreserved or replated. Cryopreservation was achieved by suspending them in a commercially available cryopreservation solution, as described in Example 2. For replated cells, they were cultured in Medium 2 on Matrigel-precoated cell culture inserts for approximately two days. After confirming their morphology, the medium was replaced. To promote HPH maturation, the cells were cultured for an additional two days in Medium 3. The resulting cells were overlaid with Matrigel. The cells were then washed with PBS and used for induction assays and other assays.

[0147] 12. CYP induction assay and mRNA expression measurement On day 18, mRNA expression was evaluated based on cell morphology using the same method as in Example 3.

[0148] 13. CYP metabolic activity after induction assay On day 18, metabolic activity was evaluated based on cell morphology in the same manner as in Example 3.

[0149] 14. Immunofluorescence staining. Cultured HPHs cells were fixed and permeabilized with cold 100% methanol for 5 minutes at room temperature. Then, they were washed with PBS. The cells were blocked with 5% fetal bovine serum (FBS) for 20 minutes at room temperature. Then, the cells were treated with primary antibodies overnight at 4°C. The next day, the cells were rinsed with PBS and then incubated with secondary antibodies for 1 hour at room temperature. Nuclei were then stained with DAPI and observed under a KEYENCE fluorescence microscope.

[0150] [Results] The morphological images taken on day 20 of culture (Fig. 18) demonstrated that three-dimensional spheroids could be produced using HPHs obtained by the method of the present invention.

[0151] Figures 19A and 19B show morphological images of 2D culture on cell culture inserts on day 18 (lot 2, after induction test). Figure 20 shows the results of measuring mRNA expression of each metabolic enzyme in an induction test using 2D culture on cell culture inserts on day 18 (lot 2, after induction test). It was demonstrated that 3D spheroids could be prepared from HPHs obtained by the method of the present invention and then further cultured in 2D. While conventional primary hepatocytes could only be cultured for about 4–5 days, HPHs obtained by the method of the present invention could be cultured for approximately one month, demonstrating that induction assays and other procedures were possible even after such long-term culture.

[0152] Immunofluorescent staining of tight junctions and transporters on day 18 of 2D-cultured spheroids is shown in Figure 21A and B. Cell-cell adhesion was observed in spheroids prepared from the adherent HPHs obtained by the present invention and then cultured in 2D. Expression of tight junctions and transporter proteins was also observed in these cells.

[0153] Example 5 An outline of the experimental protocol for Example 5 is shown in Figure 22. We investigated whether suspension-type HPHs could be cultured to produce spheroids, and then passaged in vitro as adherent-type HPHs. We also investigated whether shortening the culture time and improving function could be achieved.

[0154] [Method] 15. Cultivation of Suspension Human Primary Hepatocytes (sHPHs) A single lot of cryopreserved suspension human primary hepatocytes (sHPHs) were plated onto EZSPHERE® plates at a density of 3 × 10 4 cells / cm 2 Uniform spheres were formed based on the cell morphology over 1-2 days. The cells were then incubated at 37°C under normoxic conditions (5% CO2 incubator). After sphere formation, the cells were cultured in 30-fold diluted Matrigel-containing Medium 2 as suspension culture in low-adhesion plates for an additional 4 days. The cells were then collected in a tube and washed with PBS(-). TrypLE Select TM The cells were then incubated at 37°C for 15 minutes and reconfirmed. After single-cell formation, 10% FBS-containing medium was added and centrifuged at 1000 rpm for approximately 3 minutes. Subsequently, the cells were either cryopreserved or reseeded. For reseeding, the passaged sHPHs were plated onto a Transwell plate precoated with Matrigel and cultured in Medium 2 for approximately 1 day. After confirming their morphology, the medium was changed to Medium 3 and cultured for an additional 2 days. The resulting cells were either overlaid with Matrigel (30-fold diluted) and sandwich-cultured for 1 day, or cultured without sandwich formation. The cells were then washed with PBS and used for QPCR assays. For cryopreservation, the cells were suspended in commercially available cryopreservation medium according to the manufacturer's instructions.

[0155] 16. Phase-contrast microscopic imaging. Morphological photographs of sHPHs were obtained using a ZEISS Primovert inverted microscope (Carl ZEISS, Oberkochen, Germany).

[0156] 17. qPCR Total RNA was extracted using the RNeasy Mini Kit and QIAshredder (QIAGEN, Venlo, Netherlands) according to the manufacturer's instructions. RNA concentration was calculated by measuring absorbance at 260 nm using a Nanodrop (Thermo Fisher Scientific). cDNA was then generated from the extracted RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover according to the manufacturer's instructions. Real-time PCR was performed using the KAPA SYBR Fast qPCR Kit Master Mix (2X) on an ABI Prism. Samples were reacted using a Light Cycler 96 System (Roche, Basel, Switzerland). After a 3-minute preincubation at 95°C, the qPCR process consisted of 40 cycles of 95°C for 10 seconds, 60°C for 20 seconds, and 72°C for 1 second. Real-time PCR results were calculated using ΔΔCt values ​​using hypoxanthine phosphoribosyltransferase as a housekeeping gene.

[0157] 18. Fluorescence Efflux Assay (CDFDA Assay) CDFDA (carboxy-DCFDA (5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate), C369, Invitrogen) is cleaved by intracellular esterases to produce fluorescent (CDF) 5-(and-6)-carboxy-2',7'-dichlorofluorescein, which is then excreted into the bile duct by the MRP2 efflux transporter. Cells were treated with 10 μM CDFDA for 15 minutes. After this, cells were washed twice with Hank's balanced salt solution (HBSS) and incubated in HBSS for 15 minutes. After incubation, cells were washed twice more with HBSS, and finally fresh HBSS was added. Accumulation of CDF (green) in the bile duct was observed using a LEICA DMi8 fluorescence microscope (Leica Microsystem GmbH, Germany).

[0158] [Results] Cell morphology (3D culture, cryopreservation, and subculture) and CDFDA assay results are shown in Figure 23. The method of this example demonstrated the elimination of the Medium 1 procedure and the shortening of the culture period. Furthermore, it was demonstrated that spheroid formation was possible directly from floating HPHs (Figure 23, 1 and 2), and that cryopreservation after dissociation was also possible (Figure 23, 5). Furthermore, the confirmation of drug excretion into the bile canaliculi via MRP2 (Figure 23, 3) demonstrated the feasibility of using the cells obtained in this example to test for cholestatic liver injury.

[0159] The analysis results of mRNA expression of metabolic enzymes and drug transporters in sandwich-cultured HPHs on day 3 after subculture are shown in Figure 24. The mRNA expression of each CYP and transporter was significantly higher than that of commercially available adherent HPHs (represented as pHPHs in Figure 24) after 48 hours of culture.

[0160] Example 6 [Method] 19. Two-dimensional culture system: (A two-dimensional culture system of sHPHs was used to examine factors) sHPHs were divided into two groups: one using Medium 2, and the other receiving only one of the factors contained in Medium 2. 3x10 sHPHs were cultured on a two-dimensional Transwell plate pre-coated with Matrigel matrix. 4 cells / cm 2 The cells were seeded at 1000 kJ / well. They were then cultured at 37°C under normoxic conditions (5% CO2 incubator) for 3 hours. Next, we assessed mRNA expression using the method described above to examine the expression of caspase 3, which has anti-apoptotic properties and may promote the longevity, adhesion, and growth of sHPHs (Figure 25A). All of these factors may directly or indirectly affect apoptosis and adhesion, but these were not examined in sHPHs.

[0161] After coating the Transwell plates with Matrigel, the cells were divided into the following 11 groups (n=1) (Figure 25B). 1: 0-hr sHPHs: sHPHs were thawed and directly harvested as a 0-hr control. 2: M2: Medium 2 containing factors likely involved in adhesion, apoptosis, and culture (FBS, Y27632, vitamin C, lysophosphatidic acid, D-erythro-sphingosine-1-phosphate, EGF, HGF, A83-01, and CHIR99021) was used. 3: FBS only was added. 4: Y27632 only was added. 5: Vitamin C only was added. 6: Lysophosphatidic acid only was added. 7: D-erythro-sphingosine-1-phosphate only was added. 8: EGF only was added. 9: HGF only was added. 10: A83-01 only was added. 11: CHIR99021 only was added. sHPHs were then harvested and subjected to QPCR to measure caspase 3 gene expression.

[0162] [Results] The results are shown in Figure 25B. All factors were shown to reduce caspase 3 compared to sHPHs at time 0, i.e., reduce apoptosis, and promote the lifespan, adhesion, and culture of sHPHs.

[0163] Example 7 An outline of the experimental protocol of Example 7 is shown in FIG.

[0164] [Method] 20. Two-dimensional culture system: (To examine the factors, a two-dimensional culture system of sHPHs was used.) sHPHs were cultured using a standard three-step protocol. Briefly, 3 × 10 4 cells / cm 2sHPHs were seeded onto a matrix 2D Transwell plate precoated with Matrigel and cultured for 3 hours. Medium 1 was then replaced with Medium 2 and cultured for 8 days. Medium 2 was then replaced with Medium 3 and cultured for an additional 2 days. Other groups were treated with the standard protocol, except that factors were omitted from Medium 1, Medium 2, and Medium 3 as indicated below (Figure 26). After removing all factors, the cells were cultured for 1 day and then sandwich-cultured with 30x diluted Matrigel. Cells were then washed with phosphate-buffered saline (PBS) and prepared for mRNA expression assessment as described above. Expression of albumin, CYP1A2, and CYP3A4 was examined.

[0165] The usual protocol is a three-step protocol in which wells are coated with Matrigel, seeded using medium 1, medium 2 is added after 3 to 4 hours, and then medium 3 is used on the 8th day until the 10th day.Medium 1: Ingredients (FBS, Y27632, vitamin C, lysophosphatidic acid (L), D-erythro-sphingosine-1-phosphate (S), EGF, and HGF) Medium 2: (FBS, Y27632, vitamin C, lysophosphatidic acid (L), D-erythro-sphingosine-1-phosphate (S), EGF, and HGF, A83-01, and CHIR99021) Medium 3: (FBS, Y27632, vitamin C, lysophosphatidic acid (L), D-erythro-sphingosine-1-phosphate (S), EGF, and HGF, A83-01, CHIR99021, DAPT, dexamethasone, OSM, B27, forskolin, and SB431542) 1. Regular protocol 2. 1. AC (A83-01 and CHIR99021 were omitted from all steps of the standard protocol) 2. Y (Y27632 was omitted from all steps of the standard protocol) 4. A (A83-01 was omitted from all steps of the standard protocol) 5. CH (CHIR99021 was omitted from all steps of the standard protocol) 6. HGF (HGF was omitted from all steps of the standard protocol) 7. S (D-erythro-sphingosine-1-phosphate was omitted from all steps of the standard protocol) 8. L (Lysophosphatidic acid was omitted from all steps of the standard protocol) 9. VitC (Vitamin C was omitted from all steps of the standard protocol) 10. EGF (EGF was omitted from all steps of the standard protocol) 11. 1. FBS (standard protocol, but without FBS at all steps) 2. (-FBS)+B27 (standard protocol, but without FBS, and with B27 minus vitamin A added as a serum replacement) 3. iMatrix matrix (standard protocol) (laminin (~60%), one of the four major basement membrane ECM proteins that is the main component of Matrigel) 4. Collagen matrix (standard protocol) 5. sHPHs were then harvested on day 11 and QPCR was performed to measure gene expression of albumin, CYP1A2, and CYP3A4.

[0166] 21. Phase-contrast microscopic imaging Morphological photographs of sHPHs were obtained using a ZEISS Primovert inverted microscope (Carl ZEISS, Oberkochen, Germany).

[0167] [Results] The standard protocol using Medium 1 and Medium 2 resulted in faster cell-cell adhesion than without some factors (Figures 27-29). While serum or serum substitutes (FBS, KSR, B27-vitamin A) are essential for culturing sHPHs, pHPHs could be cultured without serum (Figure 28). Laminin was found to be one of the Matrigel components that can be used to culture and adhere sHPHs (Figure 29). Matrigel and laminin 511 were found to be superior to collagen.

[0168] QPCR was performed after culturing in Medium 3 (Figure 30), which may be necessary to allow the cells to become more mature.

[0169] Example 8 The experimental protocol for Example 8 is outlined in Figure 31. The effect of removing culture in Medium 3 was investigated.

[0170] [Method] After coating the wells, the cells were seeded using medium 1. After 3-4 hours, the cells were cultured in medium 2 for 8 days, and then cultured in medium 2 until the 10th day, and morphological photographs were taken.

[0171] [Results] Cultivation in Medium 3 enhanced the function of the cells, but even without culturing in Medium 3, the cells adhered and could be cultured in a viable state (Figure 32).

[0172] Example 9 [Method] 22. Two-dimensional culture system: (Factors of sHPHs were examined using a two-dimensional culture system) Using a standard protocol consisting of culture in medium 1, medium 2, and medium 3, sHPHs were cultured at 3 × 10 cells / well on a matrix two-dimensional Transwell plate pre-coated with Matrigel. 4 cells / cm 2The cells were seeded in Medium 1 at 100°C for 3 hours and cultured in a 5% CO2 incubator under normoxic conditions. The cells were then cultured in Medium 2 for approximately 8 days, with daily medium changes. To improve the maturation function of the cultured cells, the medium was changed and the cells were cultured in Medium 3 or Medium 3 minus the factors listed below for an additional 2 days. All factors were then removed, and the cells were sandwich-cultured with Matrigel for 1 day. The cells were then washed with phosphate-buffered saline (PBS) and prepared for induction assays to assess CYP1A2 and CYP3A4 function.

[0173] 1. All factors (standard protocol including all steps for Medium 1, Medium 2, and Medium 3). Then, induction assays were performed using DMSO and RIF (rifampicin). 2. Medium 1, Medium 2, and Medium 3. Without FBS (including B27 without vitamin A). Then, induction assays were performed. 3. Medium 1, Medium 2, and Medium 3. Without forskolin in Medium 3. Then, induction assays were performed. 4. Medium 1, Medium 2, and Medium 3. Without dexamethasone in Medium 3. Then, induction assays were performed. 5. Medium 1, Medium 2, and Medium 3. Without oncostatin (onc) in Medium 3. Then, induction assays were performed. 6. Medium 1, Medium 2, and Medium 3. Without SB431542 (Sb) in Medium 3. Then, induction assays were performed. 7. Medium 1, Medium 2, and Medium 3. Without DAPT in Medium 3. Then, induction assays were performed. 8. The usual protocol was followed, including all steps, except that collagen was substituted for Matrigel. The induction assay was then performed.

[0174] CYP induction assay. CYP1A2 and CYP3A4 were induced by treatment with 50 μM omeprazole (Omep) and 25 μM rifampicin (RIF), respectively. The same volume of DMSO (0.1%) was used as a vehicle control, and cells were incubated in a 5% CO2, 37°C incubator for 48 hours. The CYP induction medium was replaced with freshly prepared medium every day, and cells were harvested for real-time PCR.

[0175] [Results] Removal of only one factor from Medium 3 did not affect the CYP1A2 and CYP3A4 induction functions (Figs. 33 and 34). Further experiments are needed to investigate the function of the remaining CYPs and other transporters after removal of Medium 3 factors.

[0176] Conclusions: 1. sHPHs remain viable even after the removal of the A83-01 (A) and CHIR99021 (CH) factors from the culture medium. Therefore, the mechanisms underlying sHPH adhesion, anti-apoptosis, and growth may be distinct from those underlying proliferation, and further investigation is needed. 2. Serum or serum replacement components, such as FBS, KSR, or B27 (0.5%–25%), are essential, but pHPHs can be cultured without serum. 3. While multiple factors are required for sHPH adhesion and growth, good morphology was achieved even when one factor was removed from the culture medium. Further experiments are needed to identify the underlying factors (removal of one factor may reveal the presence of another factor with a similar effect). 4. Based on this and previous experiments, the components of Medium 1 and Medium 2 reduced caspases and promoted sHPH adhesion and growth. The promotion of adhesion and growth may be due to partial reprogramming, resulting in blastogenesis (immaturity), but further experiments are needed to clarify the underlying mechanism. 5. Cells can survive without culture in Medium 3, but Medium 3 enhances the maturation and function of sHPHs. 6. Examples of extracellular matrices that can be used include Matrigel, iMatrix-511, laminin 511 (60% of the Matrigel component), and collagen.

Claims

1. A method for producing adherent hepatocytes from floating hepatocytes, comprising: (ii) culturing the floating hepatocytes in the presence of at least five selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, epidermal growth factor (EGF), hepatocyte growth factor (HGF), sphingophospholipids, and glycerophospholipids.

2. The method of claim 1, comprising (i) culturing the floating hepatocytes in the presence of at least four selected from a ROCK inhibitor, epidermal growth factor, HGF, sphingophospholipids, and glycerophospholipids, prior to the culturing of (ii).

3. The method of claim 1, wherein the culturing in (ii) comprises forming spheroids from suspended hepatocytes.

4. The method according to claim 1 or 2, wherein the culturing of (i) and (ii) is carried out on a culture surface coated with a basement membrane component.

5. The method for producing the cell of any one of claims 1 to 3, comprising: (iii) culturing the cell in the presence of at least nine selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, a cAMP signal activator, EGF, HGF, sphingophospholipids, glycerophospholipids, a γ-secretase inhibitor, oncostatin M and dexamethasone, following the culturing of (ii).

6. The method of claim 1, further comprising a subculture procedure following the culture in (ii).

7. The method of claim 1, further comprising freezing and thawing the hepatocytes after culturing (ii).

8. The method according to claim 1 or 2, wherein the sphingophospholipid is sphingosine-1-phosphate or a salt thereof, and the glycerophospholipid is lysophosphatidic acid or a salt thereof.

9. The method of claim 1, wherein the ROCK inhibitor is Y27632, the TGFβ receptor inhibitor is A83-01, and the GSK-3β inhibitor is CHIR99021.

10. The method of claim 1, wherein the floating hepatocytes are floating primary hepatocytes.

11. The method according to claim 1, wherein the floating hepatocytes are human floating primary hepatocytes.

12. A method for measuring the metabolism of a test substance, comprising the following steps (1) to (3): (1) obtaining adherent hepatocytes by the method according to any one of claims 1 to 3; (2) contacting the adherent hepatocytes obtained in step (1) with a test substance; and (3) measuring the metabolism of the test substance.

13. A method for inducing metabolic enzymes in hepatocytes, comprising the following steps (1) and (2): (1) obtaining adherent hepatocytes by the method according to any one of claims 1 to 3; and (2) contacting the adherent hepatocytes obtained in step (1) with a metabolic enzyme inducer.

14. The method according to claim 14, which comprises sandwich culturing the adherent hepatocytes obtained in step (1).

15. A method for producing hepatocyte spheroids, comprising the following steps (1) and (2): (1) obtaining adherent hepatocytes by the method according to any one of claims 1 to 3; and (2) culturing the adherent hepatocytes obtained in step (1) to form hepatocyte spheroids.

16. A method for two-dimensional culture on a cell culture insert, comprising the following steps (1) to (3): (1) obtaining adherent hepatocytes by the production method described in any one of claims 1 to 3; (2) converting the adherent hepatocytes obtained in step (1) into single cells; and (3) two-dimensionally culturing the hepatocytes converted into single cells in step (2) on a cell culture insert.

17. The method according to claim 16, wherein the culture in step (3) is carried out on a culture surface coated with a basement membrane component.

18. A medium for use in the method according to claim 2, comprising at least four selected from a ROCK inhibitor, EGF, HGF, sphingophospholipid, and glycerophospholipid.

19. A medium for use in the method according to any one of claims 1, comprising at least five selected from a ROCK inhibitor, a TGFβ receptor inhibitor, a GSK-3β inhibitor, EGF, HGF, sphingophospholipids, and glycerophospholipids.

20. A kit comprising the medium according to claim 18, the medium according to claim 19, and suspended hepatocytes.

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