Method for producing metabolism-activated liver organ, composition, and method for evaluating test substance

Abstract

A method for producing a metabolism-activated liver organ, comprising a step for culturing primary hepatocytes or hepatocyte-like cells in a differential medium to obtain a metabolism-activated liver organ, the differential medium is a medium containing a glucocorticoid receptor agonist and one or two selected from the group consisting of a growth hormone receptor agonist and a prolactin receptor agonist.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for producing a metabolically-activated liver organoid, a composition, and an evaluation method for a test substance. This application claims priority based on Japanese Patent Application No. 2023-074944 filed in Japan on April 28, 2023, the contents of which are incorporated into the present application. BACKGROUND

[0002] Human hepatocytes are important experimental tools for drug development. Human primary hepatocytes, while maintaining liver functions to some extent, have problems in terms of ethics, problems in terms of acquisition difficulty, problems in terms of difficulty in ensuring the amount required for experimental tools, problems in terms of functional decline, and problems in terms of large batch-to-batch variation, and are difficult to use for drug development. Therefore, as a supply source of human hepatocytes, attempts have been made to use human liver organoids instead of human primary hepatocytes.

[0003] As the main functions of hepatocytes, the following can be listed: (1) drug metabolism function, (2) transporter function, (3) bile production function, (4) protein production function, (5) lipid metabolism function, (6) gluconeogenesis function, and (7) detoxification of ammonia and the like (urea production function). However, so far, a human liver organoid that sufficiently exhibits the gluconeogenesis function has not been known.

[0004] In Non-Patent Literature 1, a human liver organoid obtained by culturing a human hepatocellular carcinoma cell line G2 (HepG2) in a culture medium containing glucagon and its gluconeogenesis are described.

[0005] PRIOR ART DOCUMENTS

[0006] NON-PATENT LITERATURE

[0007] Non-Patent Literature 1: Gamboa C.M., et al., Optimized 3D Culture of Hepatic Cells for Liver Organoid Metabolic Assays, Cells, 10(12), 3280, 2021. SUMMARY

[0008] PROBLEMS TO BE SOLVED BY THE INVENTION

[0009] An object of the present application is to provide a production technology of a metabolically-activated liver organoid in which gluconeogenesis and urea production have been activated.

[0010] Technical means for solving the problem

[0011] The present application includes the following embodiments.

[0012] [1] A method for producing a metabolically activated liver organoid, comprising: a step (a) of culturing primary hepatocytes or hepatocyte-like cells in a differentiation medium to obtain a metabolically activated liver organoid, the differentiation medium being a medium containing one or both selected from the group consisting of a growth hormone receptor agonist and a prolactin receptor agonist, and a glucocorticoid receptor agonist.

[0013] [2] The method for producing a metabolically activated liver organoid according to [1], wherein the differentiation medium contains a growth hormone receptor agonist and a prolactin receptor agonist.

[0014] [3] The method for producing a metabolically activated liver organoid according to [1] or [2], wherein the differentiation medium further contains one or both selected from the group consisting of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and fibroblast growth factor (FGF).

[0015] [4] The method for producing a metabolically activated liver organoid according to any one of [1] to [3], wherein the differentiation medium further contains a transforming growth factor β receptor antagonist.

[0016] [5] The method for producing a metabolically activated liver organoid according to any one of [1] to [4], wherein the differentiation medium further contains a γ-secretase inhibitor.

[0017] [6] The method for producing a metabolically activated liver organoid according to any one of [1] to [5], wherein the differentiation medium further contains one or more selected from the group consisting of estradiol, dehydroepiandrosterone, and glucagon.

[0018] [7] The method for producing a metabolically activated liver organoid according to any one of [1] to [6], wherein, after the step (a), further sequentially comprising: a step (b) of subjecting the metabolically activated liver organoid to fasting; and a step (c) of culturing the metabolically activated liver organoid in a gluconeogenesis medium, the gluconeogenesis medium being a medium containing a gluconeogenesis substrate.

[0019] [8] The method for producing a metabolically activated liver organoid according to [7], wherein the gluconeogenesis medium further contains a glucagon receptor agonist.

[0020] [9] The method for producing a metabolically-activated liver organoid according to [7] or [8], wherein the gluconeogenic medium further comprises one or both selected from the group consisting of a growth hormone receptor agonist and a prolactin receptor agonist, and a glucocorticoid receptor agonist.

[0021]

[10] A composition comprising one or both selected from the group consisting of a growth hormone receptor agonist and a prolactin receptor agonist, and a glucocorticoid receptor agonist.

[0022]

[11] A method for evaluating a test substance, comprising: a step of bringing a metabolically-activated liver organoid produced by the method for producing a metabolically-activated liver organoid according to any one of [1] to [9] into contact with a test substance; and a step of evaluating an influence of the test substance on the metabolically-activated liver organoid.

[0023] Effects of the Invention

[0024] By the present invention, a technique for producing a metabolically-activated liver organoid in which gluconeogenesis and urea production have been activated can be provided. BRIEF DESCRIPTION OF DRAWINGS

[0025] [ Figure 1 ] Figure 1 is a graph showing the flow of Experimental Example 1.

[0026] [ Figure 2 ] Figure 2 is a graph showing the flow of Experimental Example 2.

[0027] [ Figure 3 ] Figure 3 is a graph showing the flow of Experimental Example 3.

[0028] [ Figure 4 ] Figure 4 is a graph showing the amount of albumin production measured in Experimental Examples 1 to 3.

[0029] [ Figure 5 ] Figure 5 is a graph showing the amount of bile acid production measured in Experimental Examples 1 to 3.

[0030] [ Figure 6 ] Figure 6 is a graph showing the concentration of bile acid in general human liver tissue and in human serum.

[0031] [ Figure 7 ] Figure 7 is a graph showing the quantitative results of glucose in Experimental Example 5.

[0032] [ Figure 8 ] Figure 8is a graph showing the quantitative results of glucose in Experimental Example 5.

[0033] [ Figure 9 ] Figure 9 is a graph showing the quantitative results of glucose in Experimental Example 6.

[0034] [ Figure 10 ] Figure 10 is a graph showing the measurement results of glucose in Experimental Example 7.

[0035] [ Figure 11 ] Figure 11 is a graph showing the measurement results of urea in Experimental Example 7.

[0036] [ Figure 12 ] Figure 12 is a graph showing the flow of Experimental Example 8.

[0037] [ Figure 13 ] Figure 13 is a graph showing the measurement results of CYP metabolites in Experimental Example 8.

[0038] [ Figure 14 ] Figure 14 is a graph showing the flow of Experimental Example 9.

[0039] [ Figure 15 ] Figure 15 is a graph showing the measurement results of CYP metabolites in Experimental Example 9.

[0040] [ Figure 16 ] Figure 16 is a heat map showing the results of Experimental Example 10.

[0041] [ Figure 17 ] Figure 17 is a graph showing the flow of Experimental Example 11.

[0042] [ Figure 18 ] Figure 18 is a graph showing the measurement results of urea and glucose in Experimental Example 11.

[0043] [ Figure 19 ] Figure 19 is a graph showing the flow of Experimental Example 12.

[0044] [ Figure 20 ] Figure 20 is a graph showing the measurement results of urea and glucose in Experimental Example 12.

[0045] [ Figure 21 ] Figure 21 is a graph showing the measurement results of urea and glucose in Experimental Example 12.

[0046] [ Figure 22 ] Figure 22 is a graph showing the measurement results of glycogen in Experimental Example 13.

[0047] [ Figure 23 ] Figure 23 is a table showing the results of quantitative real-time PCR in Experimental Example 14.

[0048] [ Figure 24 ] Figure 24 is a graph showing the measurement results of glucose in Experimental Example 15.

[0049] [ Figure 25 ] Figure 25 is a graph showing the measurement results of glucose in Experimental Example 16.

[0050] [ Figure 26 ] Figure 26 is a graph showing the results of quantitative real-time PCR in Experimental Example 17.

[0051] [ Figure 27 ] Figure 27 is a graph showing the measurement results of the amounts of secreted proteins in Experimental Example 18.

[0052] [ Figure 28 ] Figure 28 is an image showing the results of lipid staining of the liver organoids in Experimental Example 19.

[0053] [ Figure 29 ] Figure 29 is a graph showing the measurement results of the amounts of lipids in Experimental Example 19.

[0054] [ Figure 30 ] Figure 30 is a graph showing the results of lipoprotein profiling of triglycerides in Experimental Example 19.

[0055] [ Figure 31 ] Figure 31 is a graph showing the results of lipoprotein profiling of cholesterol in Experimental Example 19.

[0056] [ Figure 32 ] Figure 32 is an image showing the results of periodic acid-Schiff (PAS) staining of the liver organoids in Experimental Example 20.

[0057] [ Figure 33 ] Figure 33 is a graph showing the measurement results of the amounts of ATP in Experimental Example 21. Figure 33 is a graph showing the measurement results of the amounts of LDH in Experimental Example 21.

[0058] [ Figure 34 ]Figure 34 Fig. 22 is a fluorescence image of a liver organoid taken in Experimental Example 22. DETAILED DESCRIPTION

[0059] Each component exemplified in the present specification, such as a component contained in a medium or a component used in each step, can be used singly, or two or more kinds can be used in combination, as long as not particularly mentioned.

[0060] In the present specification, the expression "A to B" or the like indicating a numerical range has the same meaning as "A or more and B or less". In addition, in the present specification, the expression "A to B, preferably a to b" or the like indicating a numerical range has the same meaning as "A or more and B or less", "A or more and b or less", "a or more and B or less", and "a or more and b or less".

[0061] In the present specification, the phrase "a medium containing a substance X", "in the presence of a substance X" means a medium to which an exogenous substance X is added, a medium containing an exogenous substance X, or in the presence of an exogenous substance X. That is, in the case where a substance X is expressed, secreted, or produced endogenously by a cell or a tissue present in the medium, the endogenous substance X is distinguished from the exogenous substance X, and a medium not containing the exogenous substance X does not fall within the category of "a medium containing a substance X", even if it contains the endogenous substance X.

[0062] [Method for producing metabolically-activated liver organoid]

[0063] In one embodiment, the present application provides a production method, which is a method for producing a metabolically-activated liver organoid, and includes: a step (a) of obtaining a metabolically-activated liver organoid by culturing a primary hepatocyte or a hepatocyte-like cell in a differentiation medium, the differentiation medium being a medium containing one or both selected from the group consisting of a growth hormone receptor agonist and a prolactin receptor agonist, and a glucocorticoid receptor agonist.

[0064] In the present specification, the term "organoid" means a self-organizing cell tissue body obtained by cell culture. Furthermore, the term "liver organoid" means an organoid formed from a primary hepatocyte, a pluripotent stem cell, or a liver precursor cell. In addition, the term "metabolically-activated liver organoid" means a liver organoid having gluconeogenesis ability and urea production ability equivalent to or higher than those of a primary hepatocyte.

[0065] By the production method of the present embodiment, a metabolically-activated liver organoid can be produced. As described later in the examples, the metabolically-activated liver organoid obtained by the production method of the present embodiment has improved gluconeogenesis and urea production compared to a conventional liver organoid, and thus has a function similar to that of a human primary hepatocyte.

[0066] The production method of the present embodiment includes a step of culturing primary hepatocytes or hepatocyte-like cells in a differentiation medium to obtain metabolically activated liver organoids.

[0067] In the production method of the present embodiment, the primary hepatocytes are cells directly obtained from liver tissue. As the primary hepatocytes, Primary Human Hepatocytes (PHHs) are preferable. The hepatocyte-like cells mean cells having functions or forms similar to those of the primary hepatocytes. As the hepatocyte-like cells, cells contained in liver organoids, etc. can be exemplified. In the production method of the present embodiment, it is preferable to use hepatocyte-like cells, more preferable to use liver organoids, and further preferable to use human liver organoids.

[0068] The primary hepatocytes or hepatocyte-like cells can also be cells that have been subcultured in a suitable medium. The medium used in subculturing is not particularly limited as long as it is a medium that is generally used in subculturing of primary hepatocytes or hepatocyte-like cells, and for example, the following can be used: Gamboa C. M. et al., Optimized 3D Culture of Hepatic Cells for Liver Organoid Metabolic Assays, Cells, 10, 3280, 2021 (describes a culture method for organoids derived from HepG2 cells, and the culture medium contains B27 Supplement (without vitamin A), 1:100 N2 Supplement, 1 mM N-acetylcysteine, 10% (v / v) Rspondin 1 conditioned medium, 10 mM Nicotinamide, 10 nM recombinant human [Leu15]-Gastrin-I, 50 ng / mL recombinant human EGF, 100 ng / mL recombinant human FGF 10, 25 ng / mL recombinant human HGF); Mitani et al., Hepatocyte-like cells derived from human iPS cells can produce functional bile, Hepatology, 58, 1, 2013 (describes a culture method for hepatocyte-like cells derived from human iPS cells, and the culture medium contains B27 Supplement (without vitamin A), 1:100 N2 Supplement, 1 mM N-acetylcysteine, 10% (v / v) Rspondin 1 conditioned medium, 10 mM Nicotinamide, 10 nM recombinant human [Leu15]-Gastrin-I, 50 ng / mL recombinant human EGF, 100 ng / mL recombinant human FGF 10, 25 ng / mL recombinant human HGF); and the like.) "Human ESC / iPSC-Derived Hepatocyte-like Cells Achieve Zone-Specific Hepatic Properties by Modulation of WNT Signaling" (Molecular Therapy, 25(6), 2017) (described a differentiation induction method to various hepatocyte-like cells having zone-specific properties within a liver lobule, for example, in a method of differentiation induction to hepatocytes, in a Roswell Park Memorial Institute (RPMI) 1640 medium used, 1 × B27 Supplement (without vitamin A) (Thermo Fisher Scientific), 1% GlutaMAX (Thermo Fisher Scientific), 20 ng / mL HGF (R&D Systems) were contained; Peng W.C., et al. "Inflammatory Cytokine TNFa Promotes the Long-Term Expansion of Primary Hepatocytes in 3D Culture" (Cells 175, 1607-1619, 1028) (described an expansion culture method of a liver cell-derived organoid, in a medium, 3 μM CHIR99021 (PeproTech), 25 ng / mL EGF (PeproTech), 50 ng / mL HGF (PeproTech), 100 ng / mL tumor necrosis factor alpha (TNFα) (PeproTech), 50 ng / mL noggin (PeproTech) were contained); Hu H., et al.Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids (Cells 175, 1591-1606, 2018) (describing an expansion culture method of organoids derived from primary hepatocytes, for example, in an expansion culture medium of organoids derived from primary hepatocytes, containing: AdDMEM / F12 (Thermo Fisher Scientific, added with hydroxyethylpiperazine ethanesulfonic acid (HEPES), propylvaline dipeptide, and penicillin-streptomycin) + 15% RSP01 conditioned medium (manufactured in Japan), B27 (without vitamin A), 50 ng / mL EGF (PeproTech), 1.25 mM N-acetyl cysteine (Sigma), 10 nM gastrin (Sigma), 3 μM CHIR 99021 (Sigma), 50 ng / mL HGF (PeproTech), 100 ng / mL FGF 7 (PeproTech), 100 ng / mL FGF10 (PeproTech), 2 μM A83-01 (Tocris), 10 mM nicotinamide (Sigma), 10 μM Rho Inhibitor γ-27632 (Calbiochem), and 20 ng / mL TGFa), and the like, or an expansion culture medium (effective microorganisms (EM) culture medium) used in the following examples, and the like.

[0069] In the production method of the present embodiment, the differentiation medium contains one or both selected from the group consisting of a growth hormone receptor agonist and a prolactin receptor agonist, and a glucocorticoid receptor agonist.

[0070] In the present specification, a receptor agonist includes a factor that mediates signal transduction of the receptor and transcription factors downstream thereof.

[0071] Growth hormone is a hormone secreted from growth hormone-secreting cells of the anterior lobe of the pituitary. It is known to activate signal transducer and activator of transcription (STAT) 5. As a growth hormone receptor agonist, for example, a ligand having agonistic action on the growth hormone receptor can be cited. As such a ligand, growth hormone, growth hormone mimetics, and the like can be cited. As growth hormone mimetics, agonistic antibody, antibody fragment, peptide, and the like against the growth hormone receptor can be cited. As the accession number of the complementary deoxyribonucleic acid (cDNA) of human growth hormone in the National Center of Biotechnology Information (NCBI), NM_000515.5, NM_022559.4, NM_022560.4, NM_022561.2, NM_022562.2, and the like can be cited.

[0072] The concentration of the growth hormone receptor agonist contained in the differentiation medium is preferably 0.01 ng / mL to 1,000 ng / mL, for example, 0.1 ng / mL to 500 ng / mL, for example, 1 ng / mL to 100 ng / mL.

[0073] Prolactin is a hormone secreted mainly from prolactin-secreting cells of the anterior lobe of the pituitary. Regarding prolactin, it is considered that the gene constitution, amino acid sequence, and the like are similar to those of growth hormone, and the gene of an ancestor is duplicated and the function is differentiated.

[0074] As a prolactin receptor agonist, for example, a ligand having agonistic action on the prolactin receptor can be cited. As such a ligand, prolactin, prolactin mimetics, and the like can be cited. As prolactin mimetics, agonistic antibody, antibody fragment, peptide, and the like against the prolactin receptor can be cited. As the accession number of the cDNA of human prolactin in the NCBI, NM_000948.6, NM_001163558.3, and the like can be cited. In the production method of the present embodiment, a prolactin receptor agonist can be added to the medium instead of or together with a growth hormone receptor agonist.

[0075] The concentration of the prolactin receptor agonist contained in the differentiation medium is preferably 0.01 ng / mL to 1,000 ng / mL, for example, 0.1 ng / mL to 500 ng / mL, for example, 1 ng / mL to 100 ng / mL.

[0076] The so-called glucocorticoid, also referred to as glucocorticoid, is one of the adrenocortical hormones produced by the fasciculate zone of the adrenal cortex. As a glucocorticoid receptor agonist, for example, a ligand having agonistic action on the glucocorticoid receptor can be exemplified. As such a ligand, a glucocorticoid, a glucocorticoid mimetic, and the like can be exemplified. As a glucocorticoid mimetic, a synthetic steroid such as prednisolone (Chemical Abstracts Service (CAS) No.: 50-24-8), dexamethasone (CAS No.: 50-02-2), betamethasone (CAS No.: 378-44-9), and the like; an agonistic antibody, an antibody fragment, a peptide, and the like against the glucocorticoid receptor can be exemplified. As a glucocorticoid, cortisone (CAS No.: 50-23-7), cortexolone (CAS No.: 50-22-6), cortisone (CAS No.: 53-06-5), and the like can be exemplified.

[0077] The concentration of the glucocorticoid receptor agonist contained in the differentiation medium is preferably 10 ng / mL to 1,000 ng / mL, for example, can be 10 ng / mL to 500 ng / mL, for example, can be 10 ng / mL to 200 ng / mL.

[0078] The differentiation medium can contain a growth hormone receptor agonist and a glucocorticoid receptor agonist, can contain a prolactin receptor agonist and a glucocorticoid receptor agonist, and can contain a growth hormone receptor agonist, a prolactin receptor agonist, and a glucocorticoid receptor agonist.

[0079] The differentiation medium preferably further contains one or two growth factors selected from the group consisting of an epithelial growth factor (EGF), a hepatocyte growth factor (HGF), and a fibroblast growth factor (FGF).

[0080] EGF is a growth factor that activates an epithelial growth factor receptor (EGFR or ErbB1). The activated EGFR mainly activates a mitogen-activated protein kinase (MAPK) signal transduction pathway, in addition to a phosphatidylinositol 3-kinase (PI3K) signal transduction pathway, and a Janus kinase / signal transduction and activator of transcription (Jak / stat) signal transduction pathway.

[0081] The concentration of EGF contained in the differentiation medium is preferably 10 ng / mL to 1,000 ng / mL, and can be, for example, 10 ng / mL to 500 ng / mL, or 10 ng / mL to 200 ng / mL.

[0082] HGF is a growth factor that activates the mesenchymal epithelial transition factor (Met) receptor, and the activated Met receptor activates the HGF-Met signal transduction pathway. Activation of the HGF-Met signal transduction pathway promotes activation of the β-catenin pathway, and promotes angiogenesis or production of metalloproteases.

[0083] The concentration of HGF contained in the differentiation medium is preferably 10 ng / mL to 1,000 ng / mL, and can be, for example, 10 ng / mL to 500 ng / mL, or 10 ng / mL to 200 ng / mL.

[0084] As the FGF, one that binds to FGF receptor 2 (FGFR2) or FGF receptor 4 (FGFR4) is preferred, and FGF2, FGF4, FGF7, or FGF10 is preferred, and FGF10 is particularly preferred.

[0085] The concentration of FGF contained in the differentiation medium is preferably 20 ng / mL to 500 ng / mL, and can be, for example, 20 ng / mL to 300 ng / mL, or 20 ng / mL to 150 ng / mL.

[0086] The differentiation medium preferably further contains a Transforming Growth Factor (TGF) β receptor antagonist.

[0087] The TGFβ signal contributes to cell proliferation inhibition, or cell differentiation, induction of apoptosis, and the like. The TGFβ receptor antagonist is a substance that down-regulates control of the TGFβ signal, and can also be referred to as a TGF-β signal transduction pathway inhibitor.

[0088] In the present specification, the TGFβ receptor antagonist is an inhibitor that inhibits activation of the type I receptor or the type II receptor of the serine / threonine kinase type receptor, and refers to a TGFβ receptor antagonist with which phosphorylation inhibition of small mother against decapentaplegic (Smad) 2 / 3 is accompanied.

[0089] As a TGFβ receptor antagonist with accompanying inhibition of phosphorylation of Smad2 / 3, for example, A83-01 (CAS No. 909910-43-6), SB-431542 (CAS No. 301836-41-9), SB-505124 (CAS No. 694433-59-5), SB-525334 (CAS No. 356559-20-1), LY364947 (CAS No. 396129-53-6), SD-208 (CAS No. 627536-09-8), and SJN2511 (CAS No. 446859-33-2) can be listed, of which A83-01 is preferred.

[0090] The concentration of the TGFβ receptor antagonist contained in the differentiation medium is preferably 0.05 μM to 50 μM, for example, 0.5 μM to 30 μM, for example, 1 μM to 15 μM.

[0091] The differentiation medium preferably further contains a γ-secretase inhibitor. As a γ-secretase inhibitor, (3,5-Difluorobenzoylamido)-L-alanyl-L-2-phenylglycine tert-butyl ester (DAPT) (CAS No. 208255-80-5), dibenzazepine (CAS No. 256-96-2), and the like can be listed. These γ-secretase inhibitors function as inhibitors of Notch signaling by inhibiting the cleavage of Notch.

[0092] The concentration of the γ-secretase inhibitor contained in the differentiation medium is preferably 0.05 μM to 50 μM, for example, 0.5 μM to 30 μM, for example, 1 μM to 15 μM.

[0093] The differentiation medium preferably further contains one or two or more selected from the group consisting of estradiol, dehydroepiandrosterone, and glucagon. Estradiol and dehydroepiandrosterone are a kind of sex steroid hormones that are biosynthesized in vivo using cholesterol as a raw material. Estradiol is a kind of female hormone. Dehydroepiandrosterone is a kind of male hormone. As described later in the examples, in the case where the differentiation medium contains these substances, gluconeogenesis and urea production in the liver organoid are promoted.

[0094] The concentration of estradiol contained in the differentiation medium is preferably 10 nM to 50 μM, for example, 10 nM to 30 μM, for example, 10 nM to 15 μM.

[0095] The concentration of dehydroepiandrosterone contained in the differentiation medium is preferably 0.05 μM to 50 μM, for example, it can be 0.5 μM to 30 μM, for example, it can be 1 μM to 15 μM.

[0096] The concentration of glucagon contained in the differentiation medium is preferably 0.01 ng / mL to 1,000 ng / mL, for example, it can be 0.1 ng / mL to 500 ng / mL, for example, it can be 1 ng / mL to 100 ng / mL.

[0097] In addition to these, as components which can be contained in the differentiation medium, the following can be listed: gastrin (or an appropriate substitute such as Leu15-gastrin I); antibacterial agents such as penicillin antibiotics, cephem antibiotics, macrolide antibiotics, tetracycline antibiotics, and the like; cyclic adenosine monophosphate (cAMP) inducers such as forskolin, and the like.

[0098] The differentiation medium can contain basic culture components such as amino acids, vitamins, inorganic salts, and a carbon source such as glucose. The basic culture components can use basic components contained in commercially available basic medium or medium additives. As the basic medium, there can be mentioned Basal Medium Eagle (BME), BGJb medium, Connaught Medical Research Laboratories (CMRL) 1066 medium, Glasgow Minimum Essential Medium (GMEM), Improved MEM Zinc Option medium, Iscove's Modified Dulbecco's Medium (IMDM), Medium 199 medium, Eagle MEM medium, aMEM medium, Dulbecco's Modified Eagle Medium (DMEM), F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, serum mimicking medium, and Fischer's medium; and a medium as a mixed medium of these, and the like. As the commercially available medium additives, there can be mentioned B27 supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), and the like containing insulin.

[0099] The differentiation medium is generally prepared by adding the above components to a basic medium and adding a medium additive as necessary.

[0100] As the components that are preferably not contained in the differentiation medium, there can be mentioned components that up-regulate control of Wnt signaling such as Wingless / Integrated (Wnt) protein or R-Spondin, interleukin 6, IL-6 family cytokines such as oncostatin and leukemia inhibitory factor (LIF); and Bone Morphogenetic Protein (BMP) receptor antagonists with inhibition of phosphorylation of Smad1 / 5 / 9.

[0101] The production method of the present embodiment can further include, after the step (a), a step (b) of subjecting the metabolically-activated liver organoid to fasting, and a step (c) of culturing the metabolically-activated liver organoid in a gluconeogenic medium. Here, the gluconeogenic medium is a medium containing a gluconeogenic substrate.

[0102] Gluconeogenesis refers to a method / pathway in which a human or non-human animal produces glucose from substances other than sugars such as pyruvic acid, lactic acid, glycogenic amino acids, propionic acid, and glycerol, using the secretion of glucagon as a signal.

[0103] The inventors have confirmed that, by the production method of the present embodiment further including the step (b) and the step (c), gluconeogenesis and urea production of the metabolically-activated liver organoid are enhanced.

[0104] Fasting refers to a state in which a cell is maintained without being supplied with a carbon source such as glucose or glutamine. As a method of fasting, for example, a method of culturing using a fasting medium can be exemplified. As the fasting medium, for example, a medium not containing or containing only a small amount of a carbon source such as glucose or glutamine among the above-described basic components of the medium can be exemplified. In the fasting medium, it is preferable not to contain a component that adjusts sugar metabolism such as insulin.

[0105] The production method of the present embodiment preferably removes a component produced from a hepatocyte contained in the culture supernatant of the differentiation culture before fasting after the culturing in the differentiation medium. As a method thereof, a method of replacing the differentiation medium after washing the differentiation medium with a fasting medium or the like can be exemplified.

[0106] The gluconeogenic medium is a medium containing a gluconeogenic substrate. As the gluconeogenic substrate, pyruvic acid, lactic acid, glycogenic amino acids, propionic acid, and glycerol can be exemplified. One or two or more of the substrates among the gluconeogenic substrates are contained in the gluconeogenic medium.

[0107] The gluconeogenic medium preferably further contains a glucagon receptor agonist. Glucagon is associated with a cAMP circuit, and is a hormone that causes an increase in blood glucose level by glucose production through glycogenolysis and gluconeogenesis in the liver.

[0108] As the glucagon receptor agonist, for example, a ligand having an agonistic action on a glucagon receptor can be exemplified. As such a ligand, glucagon, a glucagon mimetic, and the like can be exemplified. As the glucagon mimetic, an agonistic antibody, an antibody fragment, a peptide, and the like against a glucagon receptor can be exemplified. As the NCBI accession number of the cDNA of glucagon, NM_002054.5 and the like can be exemplified.

[0109] The gluconeogenic medium preferably further contains one or both of a growth hormone receptor agonist and a prolactin receptor agonist, and a glucocorticoid receptor agonist. The growth hormone receptor agonist, the prolactin receptor agonist, and the glucocorticoid receptor agonist are the same as described above.

[0110] The gluconeogenic medium can generally contain, as the basal medium component, one that does not contain or contains only a small amount of glucose or glutamine or the like as a carbon source, like the fasting medium.

[0111] In the production method of the present embodiment, it is preferable to suspend the primary hepatocytes or hepatocyte-like cells in an extracellular matrix, and then to culture the cells on the polymerized extracellular matrix with the gluconeogenic medium layered thereon.

[0112] The extracellular matrix is a substance that becomes a scaffold for cells in cell culture. As components of the extracellular matrix, for example, components included in a basement membrane, glycoproteins present in the intercellular space can be listed. As components included in the basement membrane, for example, collagen type IV, laminin, heparan sulfate proteoglycan, entactin, and the like can be listed. As glycoproteins present in the intercellular space, collagen, laminin, entactin, fibronectin, fibrinogen, heparin sulfate, and the like can be listed. As components of the extracellular matrix, one of them can be used alone, or two or more of them can be used in combination. Among them, the extracellular matrix preferably contains laminin, and more preferably contains laminin-111.

[0113] As commercially available products of the extracellular matrix, for example, Matrigel (registered trademark), Cultrex (Bio-Techne), Geltrex (ThermoFisher Scientific), EHS (Engelbreth-Holm-Swarm) gel basement membrane matrix (Fuji Photo Film and Wako Pure Chemical, Sigma), Collagen I (Nitta Gelatin), and the like can be listed.

[0114] As a substance that becomes a scaffold for cells, a hydrogel containing an artificial high molecule can also be used. As commercially available products of the hydrogel containing an artificial high molecule, for example, Mebiol Gel (Mebiol), VitroGel (The Well Bioscience), GrowDex (UPM Biomedicals), and the like can be listed.

[0115] [COMPOSITION]

[0116] In one embodiment, the present application provides a composition comprising one or both selected from the group consisting of a growth hormone receptor agonist and a prolactin receptor agonist, and a glucocorticoid receptor agonist.

[0117] The composition of the present embodiment can be preferably used as a culture medium for manufacturing a metabolically active liver organoid. In the composition of the present embodiment, as for the growth hormone receptor agonist, the prolactin receptor agonist, and the glucocorticoid receptor agonist, the same as described above applies.

[0118] [Method for evaluating a test substance]

[0119] In one embodiment, the present application provides a method for evaluating a test substance, comprising: a step of bringing a metabolically active liver organoid manufactured by the manufacturing method into contact with a test substance; and a step of evaluating an influence of the test substance on the metabolically active liver organoid.

[0120] There is no particular limitation on the test substance, and for example, a natural compound library, a synthetic compound library, a library of existing drugs, or the like can be used.

[0121] As described later in the examples, the metabolically active liver organoid manufactured by the manufacturing method exhibits the same behavior as human hepatocytes. Therefore, by the evaluation method of the present embodiment, it is possible to evaluate an influence of a test substance on human hepatocytes in vivo in vitro.

[0122] The influence of a test substance on the metabolically active liver organoid can be evaluated, for example, by measuring gluconeogenesis, urea production, albumin production, bile acid production, cytochrome P450 (CYP) activity, or the like.

[0123] Examples

[0124] Hereinafter, the present embodiment will be described more specifically based on examples, but the present embodiment is not limited to these examples.

[0125] [Experimental Example 1]

[0126] (Evaluation of gluconeogenesis, urea production, albumin production, and bile acid production of human primary hepatocytes)

[0127] Figure 1is a diagram showing an evaluation procedure for glycolysis, urea production, albumin production, and bile acid production of human primary hepatocytes (hereinafter, sometimes referred to as "PHHs"). Frozen human primary hepatocytes were thawed and suspended in Primary Hepatocyte Thawing and Plating Medium (Thermo Fisher Scientific, hereinafter sometimes referred to as "PM medium"). The donor information of the human primary hepatocytes used is shown in Table 1 below.

[0128] [Table 1]

[0129]

[0130] Next, the suspension was suspended with Matrigel (registered trademark, Corning) in the suspension of human primary hepatocytes. The suspension was seeded into a 24-well plate so as to be 1 x 105cells / well. After the Matrigel (registered trademark) gelled, 500 μL / well of PM medium was added to the wells to perform layering, and incubation was performed for 3 hours in an environment of 37°C, 1 atm, and CO2 concentration 5 v / v%. 4

[0131] Next, the medium was changed to 500 μL / well of Primary Hepatocyte Maintenance Medium (Thermo Fisher Scientific, hereinafter sometimes referred to as "MM medium"), and incubation was performed for 24 hours in an environment of 37°C, 1 atm, and CO2 concentration 5 v / v%. The supernatant of the medium after incubation was recovered, and the albumin and bile acid contained in this medium were quantified. The results of the measurement will be described later.

[0132] For human albumin (hereinafter, sometimes referred to as "albumin"), a Human Albumin ELISA Kit (Bethyl Laboratories, Catalog No. E88-129) was used and measurement was performed in accordance with the manufacturer's procedure manual.

[0133] ​For bile acids, a system in which an electrospray ionization (ESI) device, Nexera X2 (Shimadzu Corporation), and a triple quadrupole mass spectrometer, LCMS-8040 (Shimadzu Corporation), were connected to an ultra-high performance liquid chromatograph was used for measurement. As an eluent, a mixed solution of 0.1% acetic acid aqueous solution (A) and acetonitrile (B) was used, and the flow rate was set to 0.3 mL / min. As an analytical column, Waters XBridge C18 (particle size 3.5 μm, 2.1 mm x 50 mm, Waters) was used at 40°C. The gradient program of the eluent was set to 5%→55% B (0 min-12 min), 55%→95% B (12 min-12.5 min), 95% B (12.5 min-13.5 min), 95%→5% B (13.5 min-13.51 min), 5% B (13.51 min-17 min). For the eluate of the column, after electrospray ionization was performed at an applied voltage of 4.5 kV and -3.5 kV in the positive ionization mode and the negative ionization mode, respectively, the bile acids were detected by mass spectrometry based on multiple reaction monitoring (MRM). The ionization mode, the monitored ions, and the collision energy of each analysis object were shown in Table 2 below.

[0134] [Table 2]

[0135]

[0136] In the culture supernatant, 4 volumes of methanol were added to precipitate the proteins, and the bile acids in the supernatant were measured using the system. The bile acids were measured by using an absolute calibration curve method using the peak area. In addition, a calibration curve was prepared by using an internal standard method using a known sample.

[0137] In addition, the incubated human primary hepatocytes were washed 3 times with a fasting medium (Fasting Medium) (hereinafter, sometimes referred to as "FM medium") having the composition shown in Table 3 below, and 500 μL / well of the FM medium was added, and incubated for 18 hours at 37°C, 1 atmosphere, and a CO2 concentration of 5 v / v%.

[0138] [Table 3]

[0139]

[0140] The culture medium was then replaced with the gluconeogenesis assay medium (hereinafter sometimes referred to as "GM medium") with the composition shown in Table 4 below, and incubated for 24 hours at 37°C, 1 atm, and a CO2 concentration of 5 v / v%. The supernatant of the incubated medium was recovered, and the glucose and urea contained in the medium were quantified. The results will be described later.

[0141] [Table 4]

[0142]

[0143] For glucose, use glucose-serum globulin. TM Determination (Glucose-Glo TM Assay (Promega, catalog number: J6022) was performed according to the manufacturer's procedure manual. A mixture of 0.1% aqueous acetic acid (A) and acetonitrile (B) was used as the eluent, with a flow rate set at 0.3 mL / min.

[0144] For urea, use the Quantitative Chromium Urea Assay Kit (BioAssay Systems, catalog number: DIUR-100) and follow the manufacturer's procedure manual.

[0145] [Experimental Example 2]

[0146] (Evaluation of gluconeogenesis, urea production, albumin production and bile acid production in liver organoids 1)

[0147] Figure 2 This is a diagram illustrating the evaluation process of gluconeogenesis, urea production, albumin production, and bile acid production in liver organoids in this experimental example.

[0148] Production of Liver Organoids

[0149] The donor information of the human primary hepatocytes used is shown in Table 1 above. The frozen human primary hepatocytes were thawed, suspended in a culture medium prepared by adding HEPES, GLUTAMAX (Thermo Fisher Scientific), penicillin / streptomycin (Thermo Fisher Scientific) to Advanced DMEM / F12 (Thermo Fisher Scientific), and subjected to centrifugation. The supernatant was removed and suspended in Advanced DMEM / F12. Subsequently, the suspension was suspended in the presence of Matrigel (registered trademark) in the suspension of the human primary hepatocytes. The suspension was seeded into a 24-well plate at 2 x 10 4 The Matrigel (registered trademark) was gelled, and 500 μL / well of an expansion medium (Expansion Medium, hereinafter sometimes referred to as "EM medium") shown in Table 5 below was added to the wells to perform layering, and incubation was performed for 14 days in an environment of 37°C, 1 atmosphere, and a CO2 concentration of 5 v / v%. The obtained liver organoids were subjected to multiple subcultures.

[0150] [Table 5]

[0151]

[0152] Expansion culture of liver organoids

[0153] The liver organoids obtained as described above were suspended in Advanced DMEM / F12 (Thermo Fisher Scientific). The suspension was suspended in the presence of Matrigel (registered trademark) in the suspension. The suspension was seeded into a 24-well plate at 2 x 10 4The liver organoids were inoculated into 24-well plates at 1 well / 1 well. After the Matrigel was gelled, 500 μL / well of a medium in which the Advanced DMEM / F12 was replaced with a plasma-like medium in the composition shown in Table 5 was added to the wells to perform layering, and incubation was performed for two weeks in an environment of 37°C, 1 atm, and a CO2 concentration of 5 v / v%. The medium was replaced every seven days. The plasma-like medium was prepared with reference to Cantor J.R., et al., Physiologic Medium Rewires Cellular Metabolism and Reveals Uric Acid as an Endogenous Inhibitor of UMP Synthase, Cells, 169(2), 258-272, 2017.

[0154] Evaluation of gluconeogenesis, urea production, albumin production, and bile acid production

[0155] The medium of the liver organoids was replaced with 500 μL / well of a primary hepatocyte maintenance medium (Thermo Fisher Scientific, hereinafter sometimes referred to as "MM medium"), and incubation was performed for 24 hours in an environment of 37°C, 1 atm, and a CO2 concentration of 5 v / v%. The supernatant of the medium after incubation was recovered, and albumin and bile acid contained in the medium were quantified. The results of the measurement will be described later.

[0156] In addition, the liver organoids after incubation were washed three times with a fasting medium (Fasting Medium) (hereinafter, sometimes referred to as "FM medium") in the composition shown in Table 3, and 500 μL / well of FM was added, and incubation was performed for 18 hours in an environment of 37°C, 1 atm, and a CO2 concentration of 5 v / v%.

[0157] Subsequently, the medium was changed to a gluconeogenesis assay medium (hereinafter, sometimes referred to as "GM medium") having the composition shown in Table 4 below, and incubation was performed for 24 hours in an environment of 37°C, 1 atm, and 5 v / v% CO2concentration. The supernatant of the medium after incubation was recovered, and the glucose and urea contained in the medium were quantified. The quantification of glucose and urea was performed in the same manner as in Experimental Example 1. The results of the assay will be described later.

[0158] [Experimental Example 3]

[0159] (Evaluation 2 of gluconeogenesis, urea production, albumin production, and bile acid production of liver organoids)

[0160] Figure 3 is a diagram showing the procedure of the evaluation of gluconeogenesis, urea production, albumin production, and bile acid production of liver organoids in this experimental example. This experimental example differs from Experimental Example 2 in that the liver organoids after the expansion culture were subjected to differentiation induction.

[0161] (Differentiation culture of liver organoids)

[0162] The medium of the liver organoids, which were prepared and subjected to the expansion culture for two weeks in the same manner as in Experimental Example 2, was changed to 500 μL / well of a differentiation medium (hereinafter, sometimes referred to as "DM medium") having the composition shown in Table 6 below, and incubation was performed for two weeks in an environment of 37°C, 1 atm, and 5 v / v% CO2concentration. The medium was changed every seven days.

[0163] [Table 6]

[0164]

[0165] (Evaluation of gluconeogenesis, urea production, albumin production, and bile acid production)

[0166] Subsequently, the gluconeogenesis, urea production, albumin production, and bile acid production of the liver organoids were evaluated in the same manner as in Experimental Example 2. The results of the glucose production amount and the urea production amount will be described later.

[0167] Figure 4 is a graph showing the albumin production amounts measured in Experimental Examples 1 to 3. In addition, the estimated values of the production amounts of albumin, urea, and glucose produced by human hepatocytes are shown in Table 7 below. Figure 4 In Table 7, "PHHs" indicates the measurement results of human primary hepatocytes in Experimental Example 1, "EM" indicates the measurement results of liver organoids in Experimental Example 2, and "DM" indicates the measurement results of liver organoids in Experimental Example 3.

[0168] [Table 7]

[0169]

[0170] Figure 5 is a graph showing the amounts of bile acids produced in Experimental Examples 1 to 3. In addition, Figure 6 is a graph showing the concentrations of bile acids in general human liver tissues and in human serum. It is generally considered that the daily bile acid synthesis amount of human liver (2.5 x 10 11 cells) is 200 mg to 500 mg. Figure 5 In Table 4, "PHHs" indicates the measurement results of human primary hepatocytes in Experimental Example 1, "EM" indicates the measurement results of liver organoids in Experimental Example 2, and "DM" indicates the measurement results of liver organoids in Experimental Example 3. Figure 5 and Figure 6 In Table 4, "CA" indicates cholic acid, "GCA" indicates glycolic acid, "TCA" indicates taurocholic acid, "CDCA" indicates chenodeoxycholic acid, "GCDCA" indicates glycochenodeoxycholic acid, "TCDCA" indicates taurochenodeoxycholic acid, "DCA" indicates deoxycholic acid, and "GDCA" indicates glycodeoxycholic acid.

[0171] [Experimental Example 4]

[0172] (Study on the composition of the differentiation medium)

[0173] The gluconeogenesis of liver organoids was evaluated in the same manner as in Experimental Example 3, except that the composition of the differentiation medium was changed. Under the conditions shown in Table 8 below, a medium in which growth hormone, prolactin, and / or corticosteroid were removed from the differentiation medium whose composition is shown in Table 6, and a medium to which glucagon was added were used. At the time of addition, the concentration of glucagon was 200 ng / mL. In addition, the donor of human primary hepatocytes used for making liver organoids was Donor 1. The quantitative values of glucose are also shown in Table 8 below.

[0174] [Table 8]

[0175]

[0176] As a result, it was shown that the gluconeogenesis rate was the most improved under the condition where growth hormone, prolactin, and corticosteroid were added to the differentiation medium. In addition, the gluconeogenesis was improved under the condition where growth hormone, prolactin, corticosteroid, and glucagon were added to the differentiation medium.

[0177] [Experimental Example 5]

[0178] (Study on the optimal concentration of the composition of the differentiation medium)

[0179] In addition to changing the composition of the differentiation medium, gluconeogenesis of the liver organoid was evaluated in the same manner as in Experimental Example 3. From the differentiation medium having the composition shown in Table 6, the addition concentration of cortisol, growth hormone, and prolactin was changed to the conditions shown in Table 9 below, and used for the culture. In addition, the donor of the human primary hepatocyte used for the production of the liver organoid was donor 9.

[0180] [Table 9]

[0181]

[0182] Figure 7 is a graph showing the quantitative results of glucose. Figure 7 The values shown are values corrected using the amount of adenosine triphosphate (ATP) measured by 3D-Cell Titer Glo Assay. Figure 7 In Table 9, “EM” indicates an expansion medium, and “DM” indicates a differentiation medium. Regarding the 3D-Cell Titer Glo Assay, CellTiter-Glo(R) 3D Cell Viability Assay (Promega Corporation, G9682) was used, and the measurement was performed in accordance with the manufacturer’s procedure manual.

[0183] Subsequently, using a differentiation medium in which the addition concentration of cortisol, growth hormone, and prolactin from the differentiation medium having the composition shown in Table 6 was changed to the conditions shown in Table 10 below, gluconeogenesis of the liver organoid was evaluated in the same manner as in Experimental Example 3, except for this. In addition, the donor of the human primary hepatocyte used for the production of the liver organoid was donor 9.

[0184] [Table 10]

[0185]

[0186] Figure 8 is a graph showing the quantitative results of glucose. Figure 8 The values shown are values corrected using the amount of ATP measured by 3D-Cell Titer Glo Assay. Figure 8 In Table 10, “EM” indicates an expansion medium, and “DM” indicates a differentiation medium. In addition, “GH” indicates growth hormone, “PR” indicates prolactin, and “-Hormone” indicates that cortisol, growth hormone, and prolactin are not included.

[0187] Figure 7 and Figure 8The results showed that gluconeogenesis in liver organoids was increased in a concentration-dependent manner by using a differentiation medium containing at least one of growth hormone, prolactin, and cortisol. This further indicates that gluconeogenesis in liver organoids was improved by using a differentiation medium containing at least one of growth hormone, prolactin, and cortisol compared to using a differentiation medium containing at least one of these substances.

[0188] [Experiment Example 6]

[0189] (Study on the composition of differentiation culture medium)

[0190] Except for altering the composition of the differentiation medium, gluconeogenesis of liver organoids was evaluated in the same manner as in Experimental Example 3. Medium from which A83-01 was removed, medium from which DAPT was removed, or medium from which recombinant human FGF10, recombinant human EGF, and recombinant human HGF were removed were used. Furthermore, donor 9 was used as the donor of the human primary hepatocytes used to produce the liver organoids.

[0191] exist Figure 9 The results show the quantitative results for glucose. Figure 9 The values ​​shown are the same as those in Experiment 5, and are corrected using the amount of ATP measured by 3D cell titration of serum globulin. Figure 9 In this text, "EM" represents amplification medium and "DM" represents differentiation medium. Additionally, "-A83-01" indicates the medium after removing A83-01, "-DAPT" indicates the medium after removing DAPT, and "-EHF" indicates the medium after removing recombinant human FGF10, recombinant human EGF, and recombinant human HGF.

[0192] The results showed that gluconeogenesis based on liver organoids could be confirmed even when using a medium from the differentiation medium with the composition shown in Table 6 after removing A83-01, after removing DAPT, or after removing recombinant human FGF10, recombinant human EGF and recombinant human HGF.

[0193] [Experiment Example 7]

[0194] (Study on the differentiation culture period)

[0195] Except for changing the number of days of incubation in the amplification medium, gluconeogenesis and urea production of the liver organoids were evaluated in the same manner as in Experimental Example 2. Furthermore, except for changing the number of days of incubation in the differentiation medium, gluconeogenesis and urea production of the liver organoids were evaluated in the same manner as in Experimental Example 3.

[0196] Figure 10 This is a graph representing the results of glucose measurement. Additionally, Figure 11is a graph showing the results of measurement of urea. Figure 10 and Figure 11 In the above, "expansion" indicates the result of not being cultured in the differentiation medium, and "differentiation" indicates the result of being cultured in the differentiation medium. In addition, when not being cultured in the differentiation medium, the number of days indicates the number of days of culture in the expansion medium, and when being cultured in the differentiation medium, the number of days indicates the total number of days of culture in the expansion medium and the differentiation medium. "29 days" indicates the result of the liver organoid of Experimental Example 2 or Experimental Example 3.

[0197] As a result, it was shown that the liver organoid cultured in the differentiation medium had a high amount of glucose production.

[0198] [Experimental Example 8]

[0199] (Measurement of CYP activity)

[0200] The CYP activity of human primary hepatocytes, the liver organoid obtained in Experimental Example 2 by culturing in the expansion medium (EM medium), and the liver organoid obtained in Experimental Example 3 by culturing in the differentiation medium (DM medium) was measured.

[0201] The procedure for measurement of CYP activity is shown in Figure 12 As in Experimental Example 1, the frozen human primary hepatocytes were thawed and incubated in the PM medium. Then, the medium was changed to the MM medium and the cells were suspended. The suspension was suspended by adding Matrigel (registered trademark, Corning) in the suspension. The suspension was seeded into a 96-well collagen-coated plate (IWAKI, product catalog number: #4860-010) at 1 x 10 5 The medium was changed to a medium obtained by adding the MM medium and a cocktail substrate for CYP analysis to William's E medium, and left to stand for 1 hour. As the cocktail substrate for CYP analysis, a mixture of the substrates shown in Table 11 below was used.

[0202] The MM medium was changed to a medium obtained by adding the MM medium and a cocktail substrate for CYP analysis to William's E medium, and left to stand for 1 hour. As the cocktail substrate for CYP analysis, a mixture of the substrates shown in Table 11 below was used.

[0203] [Table 11]

[0204]

[0205] After standing for 1 hour, an internal standard solution (0.2 μM quinidine, 0.2 μM ibuprofen, and 0.1% formic acid in a 1:1 mixture of acetonitrile and methanol) in an amount equivalent to the culture medium was added to the wells, and the proteins were removed, and the supernatant was subjected to liquid chromatography-mass spectrometry (LC / MS) to measure the CYP metabolites. The metabolites were quantified by an internal standard calibration curve method using the peak area. Quinidine contained in the internal standard solution was used in the positive mode in the internal standard, and ibuprofen was used in the negative mode in the internal standard.

[0206] In LC / MS, a system in which a Nexera X2 (Shimadzu Corporation) ionization device and a triple quadrupole mass spectrometer LCMS-8040 (Shimadzu Corporation) were connected to an ultra-high performance liquid chromatograph was used for measurement. As the eluent, a mixture of 0.1% acetic acid aqueous solution (A) and acetonitrile (B) was used, and the flow rate was set to 0.3 mL / min. As the analytical column, YMC Triart C18 (particle size 3 μm, 2.0 mm x 50 mm, YMC) was used at 40°C. The gradient program of the eluent was set to 5%→95% B (0 min-4 min), 95% B (4 min-5 min), 95%→5% B (5 min-5.01 min), 5% B (5.01 min-8 min). The column eluate was subjected to mass spectrometry based on multiple reaction monitoring after being subjected to electrospray ionization in the positive ionization mode and the negative ionization mode at an applied voltage of 4.5 kV and -3.5 kV, respectively, to detect the CYP metabolites. The ionization mode, the monitored ions, and the collision energy of each of the analysis objects are shown in Table 12 below.

[0207] [Table 12]

[0208]

[0209] Figure 13 is a graph showing the results of measurement of CYP metabolites. The amount of production of CYP metabolites is high in the case of high CYP activity. In Figure 13 In Table 1, “PHHs” indicates the results of human primary hepatocytes, “Expanded” indicates the results of liver organoids obtained by culturing in an expansion medium (EM medium), and “Diff” indicates the results of liver organoids obtained by culturing in a differentiation medium (DM medium).

[0210] The results showed that liver organoids obtained by culturing in differentiation medium had significantly higher CYP activity compared to liver organoids obtained by culturing in expansion medium but not in differentiation medium.

[0211] [Experiment Example 9]

[0212] (CYP induction test)

[0213] The CYP activity of human primary hepatocytes and liver organoids obtained by culturing in differentiation medium (DM medium) in Experiment 3 was induced and analyzed.

[0214] exist Figure 14 The procedure for this experimental example is shown below. Similar to Example 1, frozen primary human hepatocytes were thawed and incubated in PM medium. The medium was then replaced with MM medium, and the cells were resuspended. 250 μg / mL Matrigel (registered trademark, Corning) was added to the suspension for resuspending. The suspension was then prepared at a concentration of 1 × 10⁻⁶. 5 Inoculate the cells per well into 96-well collagen-coated plates (IWAKI, catalog number: #4860-010) and incubate for 3 hours. Then, replace the medium with MM medium containing 250 μg / mL Matrigel. After 24 hours, replace the medium with MM medium supplemented with the drugs listed in Table 13 below.

[0215] Furthermore, rifampicin is a known CYP3A4 inducer. Additionally, phenobarbital is a known CYP2B6 and CYP3A4 inducer. Omeprazole is a known CYP1A2 inducer. This experimental example can also be considered an evaluation of drug interactions and combinations of administration.

[0216] [Table 13]

[0217]

[0218] Then, the culture medium was replaced every other day with the medium containing each drug, and each drug was exposed for 3 days. Then, CYP metabolites were measured in the same manner as in Experiment 9.

[0219] Figure 15 This is a graph representing the results of CYP metabolite assays. Higher levels of CYP metabolites indicate higher CYP activity. Figure 15In the present experiment, "PHHs" indicates the result of human primary hepatocytes, and "DM" indicates the result of liver organoids obtained by culturing in a differentiation medium (DM medium).

[0220] As a result, induction of CYP activity was confirmed by adding each drug to the medium. In addition, liver organoids obtained by culturing in a DM medium showed the same behavior as human hepatocytes.

[0221] [Experiment Example 10]

[0222] (Evaluation of expression of gluconeogenesis, urea production, albumin production, bile acid production-related genes, CYP genes, and transporter-related genes)

[0223] Ribonucleic Acid sequencing (RNA-seq) analysis was performed on human primary hepatocytes, liver organoids obtained by culturing in an expansion medium (EM medium) in Experiment Example 2, and liver organoids obtained by culturing in a differentiation medium (DM medium) in Experiment Example 3, and the expression of gluconeogenesis-related genes, urea production-related genes, albumin production-related genes, bile acid production-related genes, CYP genes, and transporter-related genes was evaluated.

[0224] Figure 16 is a heat map showing the expression of each gene. In Figure 16 In the present experiment, "EM" indicates the result of liver organoids obtained by culturing in an expansion medium (EM medium), and "DM" indicates the result of liver organoids obtained by culturing in a differentiation medium (DM medium). As a result, it was shown that the expression levels of these genes in liver organoids obtained by culturing in a DM medium had approached the expression levels in human primary hepatocytes.

[0225] [Experiment Example 11]

[0226] (Effect of metformin)

[0227] Metformin is known to have an effect of inhibiting gluconeogenesis in human hepatocytes. Therefore, in addition to adding metformin to MM medium, FM medium, and GM medium so as to be 1 mM or 10 mM, gluconeogenesis and urea production were evaluated in the same manner as in Experiment Example 3. In addition, for comparison, a group obtained by preparing liver organoids in the same manner as in Experiment Example 2 and a group to which no metformin was added were prepared. Figure 17 is a diagram showing the flow of the present experiment example.

[0228] Figure 18 is a graph showing the results of measuring urea and glucose. Figure 18In the present experiment, "Expansion" indicates the result of not culturing in a differentiation medium (DM medium), and "Diff" indicates the result of culturing in a DM medium.

[0229] As a result, it was shown that gluconeogenesis and urea production in the liver organoid were inhibited by adding metformin to the medium. This result indicates that the liver organoid obtained by culturing in a DM medium as in Experimental Example 3 showed the same behavior as human hepatocytes.

[0230] [Experimental Example 12]

[0231] (Investigation of differentiation culture conditions)

[0232] A liver organoid was produced as in Experimental Example 3 except that the factors shown in Table 14 below were added to a differentiation medium (DM medium), and gluconeogenesis and urea production were evaluated. Figure 19 is a graph showing the flow of the present experimental example.

[0233] [Table 14]

[0234]

[0235] Figure 20 and Figure 21 is a graph showing the results of measurement of urea and glucose. In Figure 20 and Figure 21 In the present experiment, "Expansion" indicates the result of a liver organoid that was not cultured in a differentiation medium (DM medium), "Diff" indicates the result of a liver organoid that was cultured in a DM medium, and "-" indicates the result of a liver organoid to which no factor shown in Table 14 was added.

[0236] As a result, it was shown that gluconeogenesis and urea production in the liver organoid were promoted if the factors shown in Table 14 were added to a DM medium.

[0237] [Experimental Example 13]

[0238] (Evaluation of glycogen after fasting)

[0239] A liver organoid was subjected to differentiation induction as in Experimental Example 3 except that the composition of the FM medium was changed and the incubation time after addition of the FM medium was set to 18 hours or 24 hours. After culturing in the FM medium, glycogen in the cells was quantified.

[0240] For quantification of glycogen, a glycogen-Glo TM assay (Glycogen-Glo TMAssay) (Promega, J5052) and in accordance with the manufacturer's procedure manual. FM medium of the composition shown in Table 3, FM medium added with 200 ng / mL of glucagon, a mixed solution (also referred to as Hormone Mix) added with 10 ng / mL of growth hormone, 10 ng / mL of prolactin, and 100 ng / mL of cortisol, and FM medium of 200 ng / mL of glucagon were used. In addition, the donor of human primary hepatocytes used for making liver organoids was Donor 8.

[0241] In Figure 22 quantitative values of glycogen are shown. The results thereof show that glycogen is reduced from liver organoids by fasting.

[0242] [Experimental Example 14]

[0243] (Study 1 of optimal concentration of composition of gluconeogenic medium)

[0244] A GM medium was prepared from a gluconeogenic medium (GM medium) of the composition shown in Table 4, from which glucagon and cortisol were removed, and to which the components shown in Table 15 below were added. Liver organoids were made in the same manner as in Experimental Example 3, except that incubation was performed for 6 hours after the addition of FM medium, and for 4 hours after the addition of the GM medium. In addition, the donor of human primary hepatocytes used for making liver organoids was Donor 2.

[0245] [Table 15]

[0246]

[0247] Next, the expression of various genes in each of the liver organoids produced was analyzed by quantitative real-time PCR (qPCR). Specifically, total RNA was extracted from the cells using a Direct-zol RNA Kit micro (Zymo Research, Catalog No. #R2063), and a reverse transcription reaction was performed using a PrimeScript RT Master Mix (Takara Bio, Catalog No. RR036A) to obtain cDNA. Next, TB Green Premix Ex Taq II (Takara Bio, Catalog No. #RR820A) was used, and quantitative real-time PCR was performed using a LightCycler 480 system (Roche). The cDNA of human primary hepatocytes was diluted and a calibration curve was prepared. The base sequences of the primers used are shown in Table 16 below.

[0248] [Table 16]

[0249]

[0250] Figure 23 is a table showing the results of qPCR. Figure 23 In Table 16, Nos. 1 to 11 correspond to Nos. 1 to 11 of Table 15. Figure 23 No. 12 of Table 16 is the results of human primary hepatocytes used in the calibration curve of qPCR. The results thereof indicate that the expression of gluconeogenesis-related genes and urea production-related genes increased the most in the GM medium containing 200 ng / mL of glucagon and 100 ng / mL of cortisol.

[0251] In addition, it was shown that the expression of gluconeogenesis-related genes and urea production-related genes increased in the GM medium containing 200 ng / mL of glucagon and 400 ng / mL of cortisol. In addition, it was found that insulin inhibits the expression of gluconeogenesis-related genes.

[0252] [Experiment Example 15]

[0253] (Study 2 of the Optimal Concentration of the Composition of the Gluconeogenesis Medium)

[0254] Regarding the composition of the gluconeogenesis medium (GM medium), the concentrations of various added factors were varied, and the gluconeogenesis of liver organoids was evaluated in the same manner as in Experimental Example 3. GM medium with the added concentrations of growth hormone and prolactin changed from those shown in Table 4 to those shown in Table 17 was used for culturing. Furthermore, the human primary hepatocytes used to produce liver organoids were donor 9.

[0255] [Table 17]

[0256]

[0257] Figure 24 This is a graph representing the quantitative results of glucose. In Figure 24 In this context, "DM" represents differentiation medium. Figure 24 The values ​​shown are corrected using the amount of ATP measured by 3D cell titration of serum globulin.

[0258] [Experimental Example 16]

[0259] (Study on the optimal concentration of gluconeogenic culture medium composition 3)

[0260] Regarding the composition of the gluconeogenesis medium (GM medium), the concentrations of various added factors were varied, and the gluconeogenesis of liver organoids was evaluated in the same manner as in Experimental Example 3. GM medium was prepared by removing growth hormone, prolactin, cortisol, and glucagon from the gluconeogenesis medium (GM medium) with the composition shown in Table 4, and then adding the components shown in Table 18 below. Except for using the GM medium, the gluconeogenesis of liver organoids was evaluated in the same manner as in Experimental Example 3. Furthermore, the donor for the human primary hepatocytes used to produce liver organoids was donor 9.

[0261] [Table 18]

[0262]

[0263] Figure 25 It is a graph showing the quantitative results of glucose. Figure 25 The values ​​shown are corrected using the amount of ATP measured by 3D cell titration of serum globulin. Figure 25 In this context, "GH" represents growth hormone, "PR" represents prolactin, and "-Hormone" indicates that it does not contain growth hormone, prolactin, cortisol, or glucagon.

[0264] Figure 24 and Figure 25 The results showed that gluconeogenesis in liver organoids was enhanced by using a gluconeogenesis medium containing at least one of growth hormone, prolactin, cortisol, or glucagon.

[0265] [Experimental Example 17]

[0266] Investigation of Transforming Growth Factor Beta Receptor Antagonist in Differentiation Induction of Liver Organoids

[0267] Liver organoids were produced in the same manner as in Experimental Example 3, except that transforming growth factor beta receptor antagonist (A83-01) was removed from the differentiation medium (DM medium), and the expression of gluconeogenesis-related genes, urea production-related genes, and CYP genes was investigated by quantitative real-time PCR.

[0268] Total RNA was extracted from the cells using Direct-zol RNA Kit micro (Zymo Research, Catalog No.: #R2063), and reverse transcription reaction was performed using PrimeScript RT Master Mix (Takara Bio, Catalog No.: RR036A) to obtain cDNA. Subsequently, TB Green Premix Ex Taq II (Takara Bio, Catalog No.: #RR820A) was used, and quantitative real-time PCR was performed using LightCycler 480 System (Roche). The cDNA of human primary hepatocytes was diluted and a calibration curve was prepared. The base sequences of the primers used are shown in Table 19 below.

[0269] [Table 19]

[0270]

[0271] Figure 26 is a graph showing the results of quantitative real-time PCR. Figure 26 In the graph, “Diff” indicates the results of liver organoids cultured in DM medium, and “Diff-A8301” indicates the results of liver organoids cultured in DM medium after removal of A83-01. In addition, the vertical axis of the graph indicates the relative value when the expression amount in human primary hepatocytes (PHHs) is set to 1.

[0272] The results showed that the expression of gluconeogenesis-related genes, urea production-related genes, and CYP genes was reduced in liver organoids cultured in DM medium after removal of A83-01.

[0273] [Experimental Example 18]

[0274] (Evaluation of Secreted Proteins)

[0275] In Experimental Examples 1 to 3, the amounts of albumin, complement C3, α1-antitrypsin, and coagulation factor IX, which are secreted proteins contained in the culture supernatant after culture in the MM medium, were measured using a Human Albumin ELISA Quantitation kit (Bethyl Laboratories), a Human alpha-1-antitrypsin AssayMax ELISA Kit (AssayPro), a Human Complement C3 ELISA Kit (Abcam), and a Human Factor IX ELISA Kit (Abcam), respectively. The measurement results are shown in Table 1. Figure 20

[0276] Figure 27 In Table 1, "PHHs" indicates the measurement results of human primary hepatocytes in Experimental Example 1, "eHHO" indicates the measurement results of liver organoids in Experimental Example 2, and "dHHO" indicates the measurement results of liver organoids in Experimental Example 3. In addition, with respect to the donors of human primary hepatocytes used in the production of liver organoids, "PY53" indicates Donor 2, "PY61" indicates Donor 3, and "PY39" indicates Donor 5. The data are shown as mean ± standard deviation.

[0277] Liver organoids obtained by culture in the EM medium were shown to secrete complement C3, which is a complement system protein known to play an important role in immune response, and α1-antitrypsin, which has an action of inhibiting proteolytic enzymes. Furthermore, it was shown that only liver organoids obtained by culture in the DM medium can secrete coagulation factor IX, which has an action of stabilizing fibrin formation.

[0278] [Experimental Example 19]

[0279] (Evaluation of the amount of lipid production)

[0280] The liver organoids in Experimental Example 2 and Experimental Example 3 were cultured in the MM medium for two days, and the amount of lipid production (the amount of lipoprotein production) based on the liver organoids was measured. In Experimental Example 3, the same measurement was also performed on liver organoids cultured in the DM medium supplemented with Wnt3a and R-Spondinl.

[0281] ​Figure 28 is an image showing the result of lipid staining of the liver organoid. Figure 28 In the table, "eHHO" indicates the measurement result of the liver organoid in Experimental Example 2, and "dHHO" indicates the measurement result of the liver organoid in Experimental Example 3. In addition, "+WR" indicates the result of the liver organoid cultured in the DM medium supplemented with Wnt3a and R-Spondinl.

[0282] The lipid staining was performed using a high-content screening lipid TOX TM Deep Red Neutral Lipid Stain, for cellular imaging (Thermo Fisher Scientific). TM Deep Red Neutral Lipid Stain, for cellular imaging (Thermo Fisher Scientific).

[0283] The amount of lipid accumulated in the cells was measured from the image of the lipid staining of the section. An image processing software (Image-J) was used in the measurement. The measurement was performed as the volume per unit number of cells (nuclei) based on the three-dimensional reconstruction image obtained by the histological stereological method.

[0284] The measurement result of the amount of lipid accumulated in the cells is shown in Figure 29 . Figure 29 In the table, "+PPAR-a / ga" indicates the result after addition of WY-16463, which is a low-molecular compound as a PPAR-a / g agonist. WY-16463 is a ligand of PPAR-a / g (peroxisome proliferator-activated receptor (PPAR)), and is known to lead to a decrease in the concentration of triglyceride in blood, etc.

[0285] Figure 30 is a graph showing the result obtained by performing lipoprotein profiling of the culture supernatant by gel permeation high-performance liquid chromatography (HPLC) of triglyceride. Figure 31 is a graph showing the result obtained by performing lipoprotein profiling of the culture supernatant by gel permeation HPLC of cholesterol.

[0286] Figure 30 , Figure 31In the present specification, "HDL" means high density lipoprotein, "LDL" means low density lipoprotein, and "VLDL" means very low density lipoprotein. In addition, "+MTPi" means that Lomitapide, a low molecular compound, is added. Lomitapide is a microsomal triglyceride transfer protein (MTP) inhibitor, and is known to selectively inhibit MTP associated with the synthesis and secretion of apolipoprotein B-containing lipoprotein, and to inhibit the production of VLDL and the synthesis of LDL to reduce LDL cholesterol in the liver and small intestine.

[0287] [Experiment Example 20]

[0288] (Evaluation of glycogen)

[0289] Figure 32 is an image showing the result of periodic acid Schiff (PAS (Sigma-Aldrich)) staining of the liver organoid before and after fasting in Experiment Example 3. By PAS staining, glycogen can be stained. Figure 32 In the present specification, "+ α-amylase" means the result after adding amylase. By adding amylase, the staining intensity is reduced, and thus it is confirmed that glycogen is stained by PAS staining.

[0290] The result thereof shows that glycogen of the liver organoid has been reduced by fasting.

[0291] [Experiment Example 21]

[0292] (Evaluation of toxicity of acetaminophen)

[0293] Using the liver organoid produced in the same manner as in Experiment Example 3, the toxicity of acetaminophen was evaluated. The donor of the human primary hepatocyte used to produce the liver organoid was donor 10.

[0294] Paracetamol was added to the differentiation medium (DM medium) at a final concentration of 0 mM, 0.3 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, and the liver organoids were cultured for 24 hours. Then, using the cultured cells, ATP amount measurement was performed by 3D Cell Titration-Glo Serum Globulin Assay. In addition, using the culture supernatant, quantification of lactic dehydrogenase (LDH) was performed based on LDH Serum Globulin Assay. Regarding the 3D Cell Titration-Glo Serum Globulin Assay, Cell Titration-Glo(R) 3D Cell Viability Assay (Promega, G9682) was used, and the measurement was performed in accordance with the manufacturer's procedure manual. Regarding the LDH Serum Globulin Assay, LDH-Glo TM Cytotoxicity Assay (Promega, J2381) was used, and the measurement was performed in accordance with the manufacturer's procedure manual. TM Cytotoxicity Assay (Promega, J2381) was used, and the measurement was performed in accordance with the manufacturer's procedure manual.

[0295] Figure 33 The upper panel is a graph showing the measurement results of the ATP amount. The vertical axis of the graph indicates the ATP amount (relative value), and the horizontal axis of the graph indicates the concentration of paracetamol added. Figure 33 The lower panel is a graph showing the measurement results of the LDH amount. The vertical axis of the graph indicates the LDH amount, and the horizontal axis of the graph indicates the concentration of paracetamol added. In the graph, Figure 33 " + WR" indicates the measurement results of the liver organoids produced in the same manner as in Experimental Example 3 except that 20 v / v% Wnt3a conditioned medium and 5 v / v% R-Spondinl conditioned medium were supplemented in the DM medium. In addition, "-NAC" indicates the results of the liver organoids cultured without adding N-acetyl-L-cysteine (NAC) in "dHHO" or "+ WR", and "+NAC (10 mM)" indicates the results of the liver organoids cultured by adding 10 mM of NAC in "dHHO" or "+ WR". In addition, "dHHO" indicates the measurement results of the liver organoids produced in the same manner as in Experimental Example 3, and "+WR" indicates the measurement results of the liver organoids produced in the same manner as in Experimental Example 3 except that 20 v / v% Wnt3a conditioned medium and 5 v / v% R-Spondinl conditioned medium were supplemented in the DM medium. In addition, "-NAC" indicates the results of the liver organoids cultured without adding N-acetyl-L-cysteine (NAC) in "dHHO" or "+ WR", and "+NAC (10 mM)" indicates the results of the liver organoids cultured by adding 10 mM of NAC in "dHHO" or "+ WR". " indicates a significant difference at p < 0.05, and " indicates a significant difference at p < 0.001.

[0296] As a result, it was shown that, by exposing paracetamol to the produced liver organoids, the ATP decreased in dependence on the concentration of paracetamol, and the LDH increased.

[0297] [Experimental Example 22]

[0298] (Functional evaluation of bile acid transporters)

[0299] Figure 34 The left is a fluorescence image showing the result obtained by adding 1 pg / mL of cholyl-lysyl-fluorescein (CLF) (Corning, 451041), which is a fluorescent substrate of bile acid salt export pump (BSEP) as a bile acid transporter, to the medium of the liver organoid produced in the same manner as in Experimental Example 3 and observing after 60 minutes. The scale bar is 50 pm. The donor of the human primary hepatocytes used for producing the liver organoid was Donor 11.

[0300] Figure 34 The right is a fluorescence image showing the result obtained by adding 10 pM of 5(6)-carboxy-2',7'-dichlorofluorescein diacetate (CDFDA) (Sigma-Aldrich, 21884), which is a fluorescent substrate of multidrug resistance-related protein 2 (MRP2) as a bile acid transporter, to the medium of the liver organoid produced in the same manner as in Experimental Example 3 and observing after 60 minutes. The scale bar is 50 pm. The donor of the human primary hepatocytes used for producing the liver organoid was Donor 11.

[0301] The result thereof shows that the liver organoid produced in the same manner as in Experimental Example 3 has the activities of BSEP and MRP2.

[0302] Industrial applicability

[0303] By the present application, a technique for producing a glycolysis- and urea production-activated metabolically activated liver organoid can be provided.

Claims

1. A method for manufacturing metabolically activated liver organoids, comprising: Step (a) involves culturing primary hepatocytes or hepatocyte-like cells in a differentiation medium to obtain metabolically activated liver organoids. The differentiation medium is a medium containing one or two of the group consisting of growth hormone receptor agonists and prolactin receptor agonists, as well as glucocorticoid receptor agonists.

2. The method for manufacturing metabolically activated liver organoids according to claim 1, wherein, The differentiation culture medium contains growth hormone receptor agonists and prolactin receptor agonists.

3. The method for manufacturing metabolically activated liver organoids according to claim 1 or 2, wherein, The differentiation medium also contains one or two growth factors selected from the group consisting of epithelial growth factor (EGF), hepatocyte growth factor (HGF), and fibroblast growth factor (FGF).

4. The method for manufacturing metabolically activated liver organoids according to claim 1 or 2, wherein, The differentiation medium also contains a transforming growth factor β receptor antagonist.

5. The method for manufacturing metabolically activated liver organoids according to claim 1 or 2, wherein, The differentiation medium also contains γ-secretase inhibitors.

6. The method for manufacturing metabolically activated liver organoids according to claim 1 or 2, wherein, The differentiation culture medium also contains one or more of the group consisting of estradiol, dehydroepiandrosterone, and glucagon.

7. The method for manufacturing metabolically activated liver organoids according to claim 1 or 2, wherein, Following step (a), the following are also included in sequence: Step (b) involves fasting the metabolically activated liver organoids; and Step (c): The metabolically activated liver organoids are cultured in gluconeogenesis medium. The gluconeogenic medium is a medium containing gluconeogenic substrates.

8. The method for manufacturing metabolically activated liver organoids according to claim 7, wherein, The gluconeogenic culture medium also contains a glucagon receptor agonist.

9. The method for manufacturing metabolically activated liver organoids according to claim 7, wherein, The gluconeogenic culture medium also contains one or two of the group consisting of growth hormone receptor agonists and prolactin receptor agonists, as well as glucocorticoid receptor agonists.

10. A composition comprising one or two selected from the group consisting of growth hormone receptor agonists and prolactin receptor agonists, and a glucocorticoid receptor agonist.

11. A method for evaluating a substance under test, comprising: The step of contacting the metabolically activated liver organoid manufactured by the manufacturing method as described in claim 1 or 2 with the test substance; as well as The step of evaluating the effect of the tested substance on the metabolically activated liver organoid.

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