Proliferative liver organoids, metabolically activated liver organoids, and their use

Abstract

To provide a proliferative liver organoid having excellent proliferation and a method for producing the organoid, and a metabolism activating liver organoid with excellent metabolic activity which is differentiated from the proliferative liver organoid, and a method for producing the metabolism activating liver organoid.SOLUTION: A method for producing proliferative liver organoid includes culturing a liver stem cell or a tissue slice including the liver stem cell in a medium for proliferation to obtain a proliferative liver organoid, and the medium for proliferation contains an interleukin-6 family cytokine. A method for producing a metabolism activating liver organoid includes culturing the proliferative liver organoid produced by the method for producing proliferative liver organoid in a medium for differentiation to obtain a metabolism activating liver organoid, and the medium for differentiation does not substantially contain the interleukin-6 family cytokine.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to proliferative liver organoids, metabolically activated liver organoids, and their uses. This application claims priority based on Japanese Patent Application No. 2019-226717 filed in Japan on December 16, 2019, the content of which is incorporated herein by reference.

Background Art

[0002] In pharmacokinetic tests for pharmaceutical development, in vivo tests using rodents and in vitro tests using primary (frozen) liver cells (hepatocytes) derived from rodents are conducted. However, due to species differences, it is difficult to predict human-specific toxicity. On the other hand, in human primary (frozen) liver cells, it is difficult to stably obtain high-quality liver cells because of the limited quantity.

[0003] In pharmacokinetic studies, research and development of drugs is progressing, with particular focus on cytochrome P450 (CYP), which is abundant in hepatocytes. CYP is one of the major enzymes that metabolize xenobiotics present in the human body. When analyzing drug metabolism by CYP, a human liver tumor-derived cell line called HepaRG (registered trademark; hereafter, the term "registered trademark" will be omitted) developed at the French National Institute of Health and Medical Research (INSERM) is used. HepaRG cells are thought to have average CYP activity for human hepatocytes. However, HepaRG cells require culture time to restore CYP activity and are also costly to purchase. Furthermore, human-derived liver cancer cell lines such as HepG2 cells have low CYP activity, making it impossible to evaluate toxicity related to CYP metabolism. In addition, the use of hepatocytes derived from pluripotent stem cells such as human iPS cells (induced pluripotent stem cells) is being considered from the perspective of ensuring a stable number of cells. However, human iPS cell-derived hepatocytes, like human-derived hepatocellular carcinoma cell lines, exhibit low CYP activity, and their cell maturity is inferior to that of primary (frozen) hepatocytes. For these reasons, there is a need for more stable in vitro-produced human-derived hepatocytes (liver organoids).

[0004] In 2013, Hans Clevers et al. established a method for culturing liver organoids from mouse-derived hepatocytes, and subsequently, in 2015, the same group established liver organoids from human-derived hepatic stem cells. Furthermore, in 2018, the same group established liver organoids from hepatic stem cells of a different cell origin than those used for the aforementioned liver organoids, and these are expected to be a new source of hepatocytes (see Patent Document 1 and Non-Patent Documents 1-2). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japan Special Publication No. 2013-535201 [Non-patent literature]

[0006] [Non-Patent Document 1] Huch M et al., “Long-term culture of genome-stable bipotent stem cells from adult human liver.”, Cell, Vol. 160, Issue 1, p299-312, 2015. [Non-Patent Document 2] Hu H et al, “Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids.”, Cell, Vol. 175, Issue 6, p1591-1606, 2018. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention has been made in view of the above circumstances, and provides a proliferative liver organoid with excellent proliferative properties and a method for producing the same, as well as a metabolically activated liver organoid with excellent metabolic activity, separated from the proliferative liver organoid, and a method for producing the same. [Means for solving the problem]

[0008] In other words, the present invention includes the following embodiments. (1) A method for producing proliferative liver organoids, This includes culturing hepatic stem cells or tissue fragments containing hepatic stem cells in a growth medium to obtain proliferative liver organoids. The aforementioned growth medium contains interleukin-6 family cytokines, and is manufactured using a method for this purpose. (2) The method for producing the product according to (1), wherein the interleukin-6 family cytokine is at least one selected from the group consisting of interleukin-6, interleukin-11, oncostatin M, leukemia suppressor, cardiotropin-1, and ciliary neurotrophic factor. (3) The method for producing a culture medium according to (1) or (2) above, wherein the culture medium for growth substantially does not contain nicotinamide. (4) The method for producing a product according to any one of (1) to (3) above, wherein the growth medium further contains a growth factor. (5) The method for producing the product according to (4), wherein the growth factor is at least one selected from the group consisting of epidermal growth factor, fibroblast growth factor, hepatocyte growth factor, amphiregulin, and heparin-binding EGF-like growth factor. (6) The method for producing a product according to any one of (1) to (5), wherein the growth medium further comprises a Wnt agonist. (6) The method for producing the product according to (6), wherein the Wnt agonist is at least one selected from the group consisting of Wnt family members, R-spongin 1, R-spongin 2, R-spongin 3, R-spongin 4, noline, and glycogen synthase inhibitors. (8) The method for producing a product according to any one of (1) to (7), wherein the growth medium further comprises a Rho kinase inhibitor. (9) The method for producing the product according to (8), wherein the Rho kinase inhibitor is at least one selected from the group consisting of Y-27632, fasudil, Y39983, Wf-536, SLx-2119, azabenzimidazole-aminoflazan, DE-104, H-1152P, Rho kinase α inhibitor, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamide, Rho statin, BA-210, BA-207, Ki-23095, and VAS-012. (10) The method for producing a product according to any one of (1) to (9), wherein the growth medium further comprises a transforming growth factor-β inhibitor. (11) The method for producing the transforming growth factor-β inhibitor according to (10), wherein the transforming growth factor-β inhibitor is at least one selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY364947, SD-208, and SJN2511. (12) The method for producing a product according to any one of (1) to (11), wherein the growth medium further comprises a bone morphogenetic protein inhibitor. (13) The method for producing bone morphogenetic protein according to (12), wherein the bone morphogenetic protein inhibitor is at least one selected from the group consisting of noggin, Differential screening-selected gene Aberrative in Neuroblastoma, Cerberus, and gremlin. (14) The method for producing a product according to any one of (1) to (13), wherein the growth medium further comprises forskolin. (15) The method for producing a culture medium according to any one of (1) to (14), wherein the culture medium further comprises at least one selected from the group consisting of gastrin, neurobiological supplements, and N-acetylcysteine. (16) The method for producing the product according to any one of (1) to (15), wherein, in the culture in the growth medium, the hepatic stem cells or tissue fragments containing the hepatic stem cells are brought into contact with the extracellular matrix during the culture. (17) The method for producing the product according to (16), wherein in the culture in the growth medium, the extracellular matrix is ​​a mixture of collagen and Matrigel. (18) The method for producing a culture in the growth medium, wherein the culture is carried out for at least two weeks, according to any one of (1) to (17) above. (19) A method for producing metabolically activated liver organoids, The method comprises culturing a proliferative liver organoid produced by any one of the methods described in (1) to (18) above in a differentiation medium to obtain a metabolically activated liver organoid, A method for producing the differentiation medium, wherein the differentiation medium is substantially free of interleukin-6 family cytokines. (20) The method for producing the product according to (19), wherein the differentiation medium substantially does not contain nicotinamide. (21) The manufacturing method according to (19) or (20), wherein the differentiation medium further comprises a growth factor. (22) The method for producing the product according to (21), wherein the growth factor is at least one selected from the group consisting of epidermal growth factor, fibroblast growth factor, and hepatocyte growth factor. (23) The manufacturing method according to any one of (19) to (22), wherein the differentiation medium further comprises a Wnt agonist. (24) The method for producing the product according to (23), wherein the Wnt agonist is at least one selected from the group consisting of Wnt family members, R-spongin 1, R-spongin 2, R-spongin 3, R-spongin 4, noline, and glycogen synthase inhibitors. (25) The method for producing a product according to any one of (19) to (24), wherein the differentiation medium further comprises a Rho kinase inhibitor. (26) The method for producing the product according to (25), wherein the Rho kinase inhibitor is at least one selected from the group consisting of Y-27632, fasudil, Y39983, Wf-536, SLx-2119, azabenzimidazole-aminoflazan, DE-104, H-1152P, Rho kinase α inhibitor, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamide, Rho statin, BA-210, BA-207, Ki-23095, and VAS-012. (27) The method for producing a product according to any one of (19) to (26), wherein the differentiation medium further comprises a transforming growth factor-β inhibitor. (28) The method for producing the transforming growth factor-β inhibitor according to (27), wherein the transforming growth factor-β inhibitor is at least one selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY364947, SD-208, and SJN2511. (29) The method for producing a product according to any one of (19) to (28), wherein the differentiation medium further comprises a bone morphogenetic protein inhibitor. (30) The method for production according to (29) above, wherein the bone morphogenetic protein inhibitor is at least one selected from the group consisting of noggin, Differential screening-selected gene Aberrative in Neuroblastoma, Cerberus, and gremlin. (31) The method for production according to any one of (19) to (30) above, wherein the differentiation medium further contains forskolin. (32) The method for production according to any one of (19) to (31) above, wherein the differentiation medium further contains at least one selected from the group consisting of gastrin, neurobiological supplement, and N-acetylcysteine. (33) The method for production according to any one of (19) to (32) above, wherein the differentiation medium further contains vitamin D. (34) The method for production according to any one of (19) to (33) above, wherein the differentiation medium further contains a Notch inhibitor. (35) A method for inducing a metabolically activated liver organoid into a proliferative liver organoid, comprising culturing the metabolically activated liver organoid produced by the method for production according to any one of (19) to (34) above in an induction medium to induce the metabolically activated liver organoid into a proliferative liver organoid, wherein the induction medium contains an interleukin-6 family cytokine. (36) A proliferative liver organoid produced by the method for production according to any one of (1) to (18) above. (37) A metabolically activated liver organoid produced by the method for production according to any one of (19) to (34) above. (38) A growth medium for culturing a proliferative liver organoid, which contains an interleukin-6 family cytokine. (39) A method for evaluating a test substance, comprising bringing the metabolically activated liver organoid according to (37) above into contact with the test substance, and evaluating the response of the metabolically activated liver organoid. [Advantages of the Invention]

[0009] According to the method for producing a proliferative liver organoid of the above aspect, a proliferative liver organoid excellent in proliferation can be provided. According to the method for producing a metabolically activated liver organoid of the above aspect, a metabolically activated liver organoid excellent in metabolic activity, which is differentiated from the proliferative liver organoid, can be provided. [Brief Description of the Drawings]

[0010] [Figure 1] FIG. 1 is a microscopic image of a proliferative liver organoid in Experimental Example 1. The scale bar is 100 μm. [Figure 2] FIG. 2 is a microscopic image of a liver organoid in Experimental Example 2. The scale bar is 100 μm. [Figure 3] FIG. 3 is a microscopic image of a metabolically activated liver organoid in Experimental Example 5. [Figure 4A] FIG. 4A is a graph showing a comparison of the number of passages when Matrigel, a mixture of collagen and Matrigel, and collagen are used as the extracellular matrix in Experimental Example 13. In FIG. 4A, P indicates the number of passages. [Figure 4B] FIG. 4B is a microscopic image of a proliferative liver organoid 14 passages after culturing and 190 days after culturing when a mixture of collagen and Matrigel is used as the extracellular matrix in Experimental Example 13. [Figure 4C] FIG. 4C is an enlarged image of FIG. 4B. The scale bar is 100 μm. [Figure 4D] FIG. 4D is a graph showing a comparison of the proliferative ability when Matrigel, a mixture of collagen and Matrigel, and collagen are used as the extracellular matrix in Experimental Example 13. [Modes for Carrying Out the Invention]

[0011] The present invention will be described in more detail below with reference to embodiments, but the present invention is not limited in any way to the following embodiments.

[0012] Each component exemplified herein, for example, each component in the growth medium or the differentiation medium, may be contained individually or in combination of two or more, unless otherwise specified.

[0013] In this specification, notations representing numerical ranges such as "A to B" are synonymous with "greater than or equal to A, less than or equal to B," and A and B are included within that numerical range.

[0014] <Method for producing proliferative liver organoids> In one embodiment, the present invention provides a method for producing proliferative liver organoids, comprising culturing liver stem cells or tissue fragments containing liver stem cells in a growth medium to obtain proliferative liver organoids (hereinafter also referred to as "step A"), wherein the growth medium contains interleukin-6 (IL-6) family cytokines.

[0015] According to the method for producing proliferative liver organoids of this embodiment, proliferative liver organoids with excellent proliferative properties can be obtained.

[0016] With conventional human primary (frozen) hepatocytes, the limited number of available cells made it difficult to consistently obtain high-quality hepatocytes.

[0017] In contrast, the method for producing proliferative liver organoids according to this embodiment allows for the production of proliferative liver organoids with proliferative capacity from hepatic stem cells such as human primary (frozen) hepatocytes or tissue fragments containing hepatic stem cells. Therefore, it is possible to stably supply high-quality hepatocytes necessary for pharmacokinetic studies.

[0018] Furthermore, by differentiating the proliferative liver organoids produced by the method for producing proliferative liver organoids of this embodiment, metabolically activated liver organoids with excellent metabolic activity can be obtained. In this specification, "metabolic activated liver organoids" refers to a group of cells that have characteristics similar to hepatocytes, which constitute the liver tissue of living organisms. Examples of metabolic activated liver organoids include liver organoids in which the expression level of albumin is 50% or higher, the expression level of CYP2E1 is 300% or higher, the expression level of UGT1A1 is 300% or higher, and the expression level of NRP2 is 500% or higher compared to the expression levels of human primary frozen suspension hepatocytes. Furthermore, in this specification, "to differentiate" and "to induce differentiation" refer to actions that cause at least one of the following to occur: complication or isomerization. In the method for producing metabolically activated liver organoids of this embodiment, which will be described later, proliferative liver organoids are induced to differentiate into metabolically activated liver organoids.

[0019] Furthermore, the "proliferative liver organoids" produced by the manufacturing method of this embodiment, and the "metabolic activated liver organoids" produced by differentiating from the aforementioned proliferative liver organoids by the manufacturing method of metabolic activated liver organoids of this embodiment, which will be described later, may collectively be referred to as "hepatocyte aggregates."

[0020] [Process A] In step A, hepatic stem cells or tissue fragments containing hepatic stem cells are cultured in a growth medium to obtain proliferative liver organoids.

[0021] The liver is composed of hepatocytes, which are responsible for the essential functions of the liver, and non-parenchymal hepatocytes, which support the proliferation and survival of these hepatocytes. Hepatocytes are also called hepatocytes. Non-parenchymal hepatocytes consist of hepatic stellate cells, sinusoidal endothelial cells, Kupffer cells, bile duct epithelial cells, etc.

[0022] Hepatic stem cells are cells that retain the amphotericity to differentiate into hepatocytes and bile duct epithelial cells. They are present in both hepatocytes and non-parenchymal cells within the liver and are a group of stem cells that play a role in liver regeneration when tissue is damaged. A tissue fragment containing hepatic stem cells is a tissue fragment of hepatocytes.

[0023] In step A, it is preferable to culture the hepatic stem cells or tissue fragments containing hepatic stem cells in contact with the extracellular matrix (ECM).

[0024] One method for culturing ECM in contact with hepatic stem cells or tissue fragments containing hepatic stem cells is to mix hepatic stem cells or tissue fragments containing hepatic stem cells with an extracellular matrix precursor, gel the extracellular matrix precursor to form ECM, and then immerse the ECM in a growth medium for cultivation.

[0025] In step A, the ECM used is preferably one containing at least two specific glycoproteins. For example, it may be an ECM containing two different types of collagen, or, for example, an ECM containing collagen and laminin. The ECM may be a synthetic hydrogel extracellular matrix or a natural ECM. It is preferable to use Matrigel® (BD Biosciences), which contains laminin, entactin, and collagen IV, as the ECM. Alternatively, a mixture of collagen I and Matrigel may be used, in which case the mixing ratio is preferably 1:1 by volume. The extracellular matrix may be in a coated state on the cell culture vessel or in a dissolved state.

[0026] The culture conditions in step A can be those commonly used in animal cell culture. For example, it can be carried out at a temperature of approximately 30°C to 40°C (preferably around 37°C) and under a CO2 concentration of approximately 5% by volume (1 atm).

[0027] The culture time can be adjusted as appropriate depending on the number of cells and the state of the cells. Proliferative liver organoids can be formed after a period of approximately one to two weeks from the start of culture. In particular, from the viewpoint of proliferation of proliferative liver organoids and organoid formation, it is preferable to culture for at least two weeks.

[0028] [Cultivation medium for growth] The growth medium is a culture medium for proliferative liver organoids and contains IL-6 family cytokines. The growth medium is preferably substantially free of nicotinamide. Furthermore, it is preferable that it further contains growth factors, Wnt agonists, and TGF-β inhibitors in addition to IL-6 family cytokines, and more preferably that it further contains ROCK inhibitors, BMP inhibitors, and forskolin.

[0029] Growth media can usually be prepared by adding various components to a basic medium. Examples of basic media include Dulbecco's modified Eagle medium (DMEM), basal medium (MEM), knockout-DMEM (KO-DMEM), Glasgow basic medium (G-MEM), Eagle basic medium (BME), DMEM / Ham F12, Advanced DMEM / Ham F12 (Advanced DMEM / F12), Iskov modified Dulbecco medium, Ham F-10, Ham F-12, 199 medium, and RPMI1640 medium.

[0030] Among these, DMEM / F12 and RPMI1640, which contain HEPES, glutamine, and penicillin / streptomycin, are preferred. Furthermore, Advanced DMEM / F12 or Advanced RPMI, which are optimized for serum-free culture and contain GlutaMAX (GIBCO, L-alanyl-L-glutamine) instead of glutamine, are preferred. It is preferable to add glutamine and penicillin / streptomycin to the Advanced DMEM / F12 or Advanced RPMI medium.

[0031] The inventors focused on IL-6 family cytokines, which are inflammatory cytokines, among various factors such as cytokines and growth factors involved in the transition from the G0 phase to the G1 phase of the cell cycle. They discovered that by using a growth medium containing IL-6 family cytokines, it is possible to obtain proliferative liver organoids that can be cultured for a long period of time while maintaining high proliferative capacity.

[0032] (1) IL-6 family cytokines Examples of IL-6 family cytokines include interleukin-6 (IL-6), interleukin-11 (IL-11), oncostatin M (OSM), leukemia suppressor (LIF), cardiotropin-1 (CT-1), and ciliary neurotrophic factor (CNTF), with IL-6 being preferred among them.

[0033] The origin of IL-6 family cytokines is not particularly limited, and those derived from various organisms can be used. Among these, those derived from mammals are preferred. Examples of mammals include humans, mice, rats, cattle, pigs, and rabbits, with humans being preferred.

[0034] The amino acid sequences of various components contained in growth media, including major mammalian IL-6 family cytokines, and the base sequences of the genes encoding them can be obtained from known databases such as GenBank. For example, in GenBank, the amino acid sequence of human IL-6 is registered under accession numbers XP_011513692 and XP_005249802.

[0035] The concentration of IL-6 family cytokines in the growth medium is typically 10 ng / mL to 1.0 μg / mL, preferably 50 ng / mL to 500 ng / mL, and more preferably 80 ng / mL to 200 ng / mL.

[0036] (2) Nicotinamide From the viewpoint of improving and maintaining cell proliferation ability during long-term culture, it is preferable that the growth medium is substantially free of nicotinamide. "Substantially free of nicotinamide" means that the growth medium is completely free of nicotinamide (0 mM relative to the total volume of the growth medium), or contains only a trace amount that does not hinder the improvement and maintenance of cell proliferation ability during long-term culture, for example, at a concentration of 9 mM or less, preferably 5 mM or less, and more preferably 1 mM or less.

[0037] (3) Growth factors The growth medium preferably contains growth factors to improve cell proliferation. Growth factors refer to diffusible signaling proteins that stimulate cell growth, differentiation, survival, inflammation, and tissue repair.

[0038] Growth factors include epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), amphiregulin, and heparin-bound EGF-like growth factor (HB-EGF).

[0039] EGF is a member of the EGF family and is a growth factor that activates the epidermal growth factor receptor (EGFR or ErbB1). Activated EGFR primarily activates the MAPK signaling pathway, as well as the PI3K signaling pathway and the Jak / stat signaling pathway.

[0040] The concentration of EGF in the growth medium is typically 10 ng / mL to 1,000 ng / mL, preferably 50 ng / mL to 500 ng / mL, and more preferably 80 ng / mL to 200 ng / mL.

[0041] HGF is a growth factor that activates Met receptors, and activated Met receptors activate the HGF-Met signaling pathway. Activation of the HGF-Met signaling pathway promotes activation of the β-catenin pathway, which in turn promotes angiogenesis and metalloproteinase production.

[0042] The concentration of HGF in the growth medium is typically 10 ng / mL to 1,000 ng / mL, preferably 50 ng / mL to 500 ng / mL, and more preferably 80 ng / mL to 200 ng / mL.

[0043] The FGF is preferably one that can bind to FGF receptor 2 (FGFR2) or FGF receptor 4 (FGFR4), and is preferably FGF2, FGF4, FGF7, or FGF10, with FGF10 being particularly preferred.

[0044] The concentration of FGF contained in the growth medium is typically 20 ng / mL to 500 ng / mL, preferably 50 ng / mL to 300 ng / mL, and more preferably 80 ng / mL to 150 ng / mL or less.

[0045] Amphiregulin and HB-EGF are members of the EGF family and, like EGF, activate EGFR, thereby activating the MAPK signaling pathway, the PI3K signaling pathway, or the Jak / stat signaling pathway.

[0046] The concentration of Amphiregulin in the growth medium is typically 10 ng / mL to 1,000 ng / mL, preferably 50 ng / mL to 500 ng / mL, and more preferably 80 ng / mL to 200 ng / mL.

[0047] The final concentration of HB-EGF contained in the growth medium is typically 10 ng / mL to 1,000 ng / mL, preferably 50 ng / mL to 500 ng / mL, and more preferably 80 ng / mL to 200 ng / mL.

[0048] (4) Wnt agonist The growth medium preferably further contains a Wnt agonist from the viewpoint of maintaining hepatic stem cells and improving cell proliferation. A Wnt agonist is an agonist that activates the Wnt signaling pathway. Examples of Wnt agonists include Wnt family members, R-spongin family, Norrin, and glycogen synthase (GSK) inhibitors.

[0049] Examples of Wnt family members include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16, with Wnt3a being the preferred choice.

[0050] Since afamin is known to contribute to the stabilization and solubilization of Wnt family members, it is more preferable to use a complex of a Wnt family member and afamin as a Wnt agonist. The complex of a Wnt family member and afamin can be used as a aged culture medium (conditioning medium) containing the complex with a Wnt family member concentration of 18 ng / mL to 900 ng / mL.

[0051] Afamine refers to a glycoprotein belonging to the albumin family. In GenBank, the amino acid sequence of human afamine is registered as AAA21612, and the amino acid sequence of bovine afamine is registered as DAA28569.

[0052] When using a condition medium in which the concentration of the Wnt family member is within the above range as a Wnt family member, the amount of condition medium contained in the growth medium is usually 1 volume(v / v)% to 50 volume(v / v)%, preferably 10 volume(v / v)% to 30 volume(v / v)%, or 15 volume(v / v)% to 25 volume(v / v)%, relative to the total volume of the growth medium.

[0053] Examples of the R-spongin family include R-spongin 1, R-spongin 2, R-spongin 3, and R-spongin 4, with R-spongin 1 being preferred. When the R-spongin family binds to Lgr5 in the cell membrane, it is removed from the cell membrane by autoubiquitination, resulting in the stabilization of Frezzled, which induces activation of the Wnt signaling pathway, and the activation of the β-catenin pathway. R-spongin family members can be used in a matured culture medium containing the complex at concentrations of 0.13 μg / mL to 6.5 μg / mL.

[0054] When using a conditioning medium in which the concentration of the R-spongin family member is within the above range, the amount of conditioning medium contained in the growth medium is usually 1 volume(v / v)% to 50 volume(v / v)%, preferably 5 volume(v / v)% to 25 volume(v / v)%, or 8 volume(v / v)% to 20 volume(v / v)%, relative to the total volume of the growth medium.

[0055] GSK inhibitors are inhibitors of glycogen synthase 3β (GSK3β). Since GSK3β phosphorylates β-catenin and promotes its degradation, GSK inhibitors act as Wnt agonists.

[0056] Examples of GSK inhibitors include CHIR99021 (CAS number: 252917-06-9), SB216763 (CAS number: 280744-09-4), SB415286 (CAS number: 264218-23-7), CHIR98014 (CAS number: 252935-94-7), AZD1080 (CAS number: 612487-72-6), and LY2090314 (CAS number: 603288-22-8), with CHIR99021 being the preferred choice.

[0057] As a Wnt agonist, it is preferable to use a combination of Wnt family members and R-spongin family members, more preferably a combination of Wnt3a and R-spongin 1, and even more preferably a combination of a complex of Wnt3a and afamin and R-spongin 1.

[0058] (5) Rho kinase inhibitors The growth medium preferably further contains a Rho kinase (ROCK) inhibitor from the viewpoint of inhibiting apoptosis. ROCK inhibitors act as antagonists of IGF-1 signaling.

[0059] Examples of ROCK inhibitors include Y-27632 (CAS number: 146986-50-7), Fasudil (CAS number: 105628-07-7), Y39983 (CAS number: 203911-26-6), Wf-536 (CAS number: 539857-64-2), SLx-2119 (CAS number: 911417-87-3), Azabenzimidazole-aminofurazans (CAS number: 850664-21-0), DE-104, H-1152P (CAS number: 872543-07-6), and Rho kinase α inhibitors (ROKα). Examples include inhibitors, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, Rho statins (Rhostain), BA-210, BA-207, Ki-23095, and VAS-012. Among these, Y-27632 is preferred.

[0060] The concentration of ROCK inhibitor in the growth medium is typically 1 μM to 20 μM, preferably 5 μM to 15 μM, and more preferably 8 μM to 12 μM. The unit "μM" represents a concentration of 1 / 1,000,000 of the molecular weight (mol / L) in 1 liter of growth medium, and the same concentration will be used hereafter.

[0061] (6) Transformation growth factor-β inhibitors From the viewpoint of maintaining hepatic stem cells, the growth medium preferably contains a transforming growth factor (TGF)-β inhibitor.

[0062] TGF-β inhibitors preferably have inhibitory activity of 50% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more, compared to the TGF-β activity level in the absence of the inhibitor. TGF-β inhibitory activity can be evaluated by methods known to those skilled in the art. Such evaluation systems include cell assays in which cells are stably transfected with a reporter construct containing a human PAI-1 promoter or Smad2 / 3 binding site that activates a luciferase reporter gene.

[0063] Examples of TGF-β inhibitors include A83-01 (CAS number: 909910-43-6), SB-431542 (CAS number: 301836-41-9), SB-505124 (CAS number: 694433-59-5), SB-525334 (CAS number: 356559-20-1), LY364947 (CAS number: 396129-53-6), SD-208 (CAS number: 627536-09-8), and SJN2511 (CAS number: 446859-33-2), with A83-01 being the preferred choice among them.

[0064] The concentration of the TGF-β inhibitor in the growth medium is typically 0.05 μM to 50 μM, preferably 0.5 μM to 30 μM, and more preferably 1 μM to 15 μM or less.

[0065] (7) Osteogenesis imperfecta inhibitors The growth medium preferably further contains a bone morphogenetic protein (BMP) inhibitor, from the viewpoint of regulating the amount of hepatic stem cells contained within the organoids.

[0066] BMPs, as a dimeric ligand, bind to receptor complexes consisting of two different receptor serine / threonine kinases, type I and type II receptors. The type II receptor phosphorylates the type I receptor, thereby activating this receptor kinase. This type I receptor then phosphorylates specific receptor substrates (Smad1 / 5 / 9), resulting in transcriptional activity via a signaling pathway.

[0067] Examples of BMP inhibitors include noggin, differential screening-selected gene aberrative in neuroblastoma (DAN), and DAN-like proteins. Examples of DAN-like proteins include cerberus and gremlin. Among these, noggin is preferred.

[0068] The concentration of the BMP inhibitor in the growth medium is typically 10 ng / mL to 100 ng / mL, preferably 15 ng / mL to 50 ng / mL, and more preferably 20 ng / mL to 30 ng / mL.

[0069] (8) Forskolin From the viewpoint of improving cell proliferation, the growth medium preferably further contains forskolin.

[0070] The concentration of forskolin in the growth medium is typically 0.1 μM to 100 μM, preferably 1 μM to 50 μM, and more preferably 5 μM to 15 μM.

[0071] (9) Other ingredients In addition to the above components, the growth medium may further contain at least one selected from the group consisting of gastrin, neurobiological supplements, and N-acetylcysteine. Examples of neurobiological supplements include insulin-containing supplements such as B27 supplement (Thermo Fisher Scientific) and N2 supplement (Thermo Fisher Scientific).

[0072] The gastrin content in the growth medium is typically between 5 nM and 15 nM. The unit "nM" represents a concentration of 1 / 100,000,000 of the molecular weight (mol / L) in 1 liter of growth medium, and this concentration will be used hereafter.

[0073] B27 supplement is a composition containing biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin, and transferrin, and is commercially available as a 50x liquid concentrate.

[0074] N2 supplement is a composition containing 500 μg / mL human transferrin, 500 μg / mL bovine insulin, 0.63 μg / mL progesterone, 161 μg / mL putrescine, and 0.52 μg / mL sodium selenite, and is commercially available as a 100x liquid concentrate.

[0075] The concentration of N-acetylcysteine ​​in the growth medium is typically 150 ng / mL to 250 ng / mL.

[0076] <Method for producing metabolically activated liver organoids> In one embodiment, the present invention provides a manufacturing method comprising culturing proliferative liver organoids produced by the above-described method for producing proliferative liver organoids in a differentiation medium to obtain metabolically activated liver organoids (hereinafter also referred to as "step B"), wherein the differentiation medium substantially does not contain IL-6 family cytokines.

[0077] According to the method for producing metabolically activated liver organoids of this embodiment, metabolically activated liver organoids with excellent metabolic activity can be obtained by differentiating them from proliferative liver organoids produced by the above-mentioned method for producing proliferative liver organoids. As shown in the examples described later, the expression of various metabolic enzymes in these metabolically activated liver organoids is improved to a degree that makes them suitable for use in pharmacokinetic studies.

[0078] [Process B] In step B, the proliferative liver organoids produced by the above manufacturing method are differentiated into metabolically activated liver organoids in a differentiation medium.

[0079] As for the proliferative liver organoids used in step B, those that have been cultured for two weeks or more in step A are preferable from the viewpoint of proliferation of the proliferative liver organoids and organoid formation.

[0080] The incubation period in step B is typically 5 to 15 days, preferably 7 to 12 days.

[0081] In step B, it is preferable to culture the proliferative liver organoids in contact with the extracellular matrix (ECM). For example, as shown in the examples described later, in proliferative liver organoids cultured by layering a growth medium on polymerized ECM, if necessary, the organoids may be physically dissociated to contain an appropriate number of proliferative liver organoids, and then cultured by layering a differentiation medium instead of the growth medium.

[0082] The ECM used in process B is the same as the ECM used in process A, as described above.

[0083] The culture conditions in step B are the same as those in step A.

[0084] In the method for producing metabolically activated liver organoids according to this embodiment, differentiation of proliferative liver organoids into metabolically activated liver organoids can be determined or evaluated using indicators such as the expression of hepatocyte markers and drug metabolism activity. Examples of hepatocyte markers include albumin (ALB), α-fetoprotein (AFP), tyrosine aminotransferase (TAT), and pregnane X receptor (PXR). The expression levels of hepatocyte markers may be measured at the gene level or at the protein level.

[0085] One method for obtaining cells consisting solely of metabolically activated liver organoids from a cell population containing metabolically activated liver organoids is to select and isolate metabolically activated liver organoids using the presence of the aforementioned liver cell marker as an indicator.

[0086] Drug metabolism activity can be evaluated by detecting the expression of drug-metabolizing enzymes or by drug metabolism assays. Examples of drug-metabolizing enzymes include cytochrome P450 1A2 (CYP1A2), cytochrome P450 2B (CYP2B), cytochrome P450 2C9 (CYP2C9), cytochrome P450 2C19 (CYP2C19), cytochrome P450 2D6 (CYP2D6), cytochrome P450 2E1 (CYP2E1), cytochrome P450 3A4 (CYP3A4), cytochrome P450 3A7 (CYP3A7), uridine diphosphate-glucuronosyltransferase (UGT), and sulfotransferase (SULT).

[0087] [Differentiation medium] The differentiation medium is substantially free of IL-6 family cytokines. This allows for the differentiation of proliferative liver organoids into hepatocytes. The differentiation medium is preferably substantially free of nicotinamide, and preferably contains growth factors, Wnt agonists, and TGF-β inhibitors. In addition to these, it is preferable to contain ROCK inhibitors, BMP inhibitors, and forskolin.

[0088] Differentiation media can usually be prepared by adding various components to a basic medium. Basic media include the same basal media used for growth media.

[0089] (1) IL-6 family cytokines The differentiation medium is substantially free of IL-6 family cytokines. "Substantially free of IL-6 family cytokines" means that the concentration of IL-6 family cytokines in the differentiation medium is 0 ng / mL or very low, specifically, the concentration of IL-6 family cytokines in the differentiation medium is less than 10 ng / mL, preferably 1 ng / mL or less. Examples of IL-6 family cytokines are the same as those exemplified in the growth medium described above.

[0090] (2) Nicotinamide From the viewpoint of improving and maintaining cell proliferation ability during long-term culture, the differentiation medium is preferably substantially free of nicotinamide. "Substantially free of nicotinamide" means that the concentration of nicotinamide in the differentiation medium is 0 mM or a trace amount, specifically, the concentration of nicotinamide in the differentiation medium is 9 mM or less, preferably 5 mM or less, and more preferably 1 mM or less.

[0091] (3) Growth factors From the viewpoint of improving cell proliferation, the differentiation medium preferably contains further growth factors. The types of growth factors and the concentrations of growth factors contained in the differentiation medium are the same as those for the proliferation medium described above.

[0092] (4) Wnt agonist The differentiation medium preferably further contains a Wnt agonist from the viewpoint of maintaining hepatic stem cells and improving cell proliferation. The type of Wnt agonist and the concentration of the Wnt agonist contained in the differentiation medium are the same as those for the proliferation medium described above.

[0093] (5) ROCK inhibitors The differentiation medium preferably further contains a ROCK inhibitor from the viewpoint of suppressing cell apoptosis. The type of ROCK inhibitor and the concentration of the ROCK inhibitor contained in the differentiation medium are the same as those for the growth medium described above.

[0094] (6) TGF-β inhibitors From the viewpoint of maintaining hepatic stem cells, the differentiation medium preferably further contains a TGF-β inhibitor. The type of TGF-β inhibitor and the concentration of the TGF-β inhibitor contained in the differentiation medium are the same as those for the proliferation medium described above.

[0095] (7) BMP inhibitors The differentiation medium preferably further contains a BMP inhibitor from the viewpoint of regulating the amount of hepatic stem cells contained in the organoids. The type of BMP inhibitor and the concentration of the BMP inhibitor contained in the differentiation medium are the same as those for the proliferation medium described above.

[0096] (8) Forskolin From the viewpoint of improving cell proliferation, the differentiation medium preferably contains forskolin. The concentration of forskolin in the differentiation medium is the same as that in the proliferation medium described above.

[0097] (9) Notch signaling inhibitors The differentiation medium may further contain Notch signaling inhibitors, which can increase the expression level of CYP3A4 in metabolically activated liver organoids. Notch signaling is responsible for intercellular communication and is a signal that controls cell differentiation.

[0098] Examples of Notch signaling inhibitors include γ-secretase inhibitors such as L-685458 (CAS number: 292632-98-5), DAPT (CAS number: 208255-80-5), DBZ (CAS number: 209984-56-5), MRK560 (CAS number: 677772-84-8), 3,5-bis(4-nitrophenoxy)benzoic acid, MRK003 (CAS number: 623165-93-5), MK0752 (CAS number: 471905-41-6), flurbiprofen, and JLK6 (CAS number: 62252-26-0).

[0099] The concentration of Notch signaling inhibitors in the differentiation medium is typically 10 ng / mL to 1,000 ng / mL, preferably 20 ng / mL to 500 ng / mL, and more preferably 30 ng / mL to 300 ng / mL.

[0100] (10) Vitamin D The differentiation medium can further contain vitamin D, which can increase albumin production in metabolically activated liver organoids. Activation of the vitamin D receptor induces the expression of P21 and P27 proteins, arresting the G0 / G1 phase in the cell cycle.

[0101] Vitamin D is synthesized and metabolized in the body. Therefore, vitamin D includes vitamin D precursors, vitamin D metabolites, and vitamin D sinusoids. Examples of vitamin D include vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), vitamin D precursors such as 7-dehydrocholesterol, vitamin D metabolites such as calcifediol and calcitriol, and vitamin D sinusoids such as calcipotriol, 24,25-dihydroxyvitamin D3, ZK191784, and ZK2032788.

[0102] The concentration of vitamin D in the differentiation medium is typically 10 nM to 1,000 nM, preferably 50 nM to 800 nM, and more preferably 100 nM to 500 nM.

[0103] (11) DNA demethylating agents The differentiation medium may further contain a DNA demethylating agent, which can increase the expression level of CYP3A4 in metabolically activated liver organoids.

[0104] Examples of DNA demethylating agents include cytidine analogs such as 5-aza-2-deoxycytidine, 5-azacitidine (azacitidine), zebralin, pseudoisocytidine, 5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacitidine, 2'-deoxy-5,6-dihydro-5-azacitidine, 6-azacitidine, 2',2'-difluoro-deoxycytidine, and cytosine-beta-D-arabinofuranoside.

[0105] The concentration of the DNA demethylating agent in the differentiation medium is typically 0.1 μM to 100 μM, preferably 1 μM to 50 μM, and more preferably 5 μM to 15 μM.

[0106] (12) Other additives In addition to the above components, the differentiation medium may contain gastrin, B27 supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), or N-acetylcysteine. In the differentiation medium, the same gastrin, B27 supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), and N-acetylcysteine ​​as those used in the growth medium described above can be used in the same concentrations.

[0107] <Method for inducing metabolically activated liver organoids into proliferative liver organoids> In one embodiment, the present invention provides an induction method comprising culturing metabolically activated liver organoids produced by the above-described method for producing metabolically activated liver organoids in an induction medium to induce the metabolically activated liver organoids into proliferative liver organoids (hereinafter also referred to as "step C"), wherein the induction medium contains IL-6 family cytokines.

[0108] According to the induction method of this embodiment, metabolically activated liver organoids can be converted back into proliferative liver organoids, thereby restoring the proliferative capacity of the cells.

[0109] The culture conditions in step C are the same as the culture conditions in step A of the above-described method for producing proliferative liver organoids. Furthermore, the induction medium used has the same composition as the growth medium described in the above-described method for producing proliferative liver organoids.

[0110] <Method for evaluating test substances> In one embodiment, the present invention provides a method for evaluating a test substance, comprising contacting a metabolically activated liver organoid produced by a method for producing metabolically activated liver organoids with the test substance, and evaluating the response of the metabolically activated liver organoid.

[0111] The evaluation method of this embodiment allows for the in vitro evaluation of the metabolism, drug interactions, hepatotoxicity, transporter activity, etc., of a test substance, and enables obtaining results that are close to those obtained in vivo.

[0112] Examples of test substances include libraries of natural compounds, synthetic compounds, existing drugs, and metabolites. Test substances can include 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.).

[0113] Existing or candidate components of pharmaceuticals and nutritional foods can also be used as test substances. Plant extracts, cell extracts, and culture supernatants can also be used as test substances. Furthermore, by adding two or more test substances simultaneously, it is possible to investigate the interactions and synergistic effects between them.

[0114] The contact period between the test substance and the metabolically activated liver organoid is typically 10 minutes to 3 days, preferably 1 hour to 1 day. The contact between the test substance and the metabolically activated liver organoid can be performed in multiple stages.

[0115] The response of metabolically activated liver organoids can be evaluated, for example, by mass spectrometry, liquid chromatography, or immunological methods, depending on the metabolites produced. Immunological methods include, for example, fluorescence immunoassay (FIA) and enzyme immunoassay (EIA).

[0116] The metabolism of a test substance can also be measured using the expression of drug-metabolizing enzymes (e.g., cytochrome, UGT, etc.) in metabolically activated liver organoids as an indicator. The expression of drug-metabolizing enzymes can be measured at the mRNA level or the protein level.

[0117] The evaluation method of this embodiment can also be used to test the toxicity of the test substance. For example, the toxicity of the test substance can be evaluated by examining the state of metabolically activated liver organoids after contact with the test substance. The state of metabolically activated liver organoids can be assessed by examining their viability, cell morphology, and the amount of liver damage markers (e.g., GOT, GPT, etc.) present in the culture medium.

[0118] <Other embodiments> In one embodiment, the present invention provides proliferative liver organoids produced by the above-described method for producing proliferative liver organoids and metabolically activated liver organoids produced by the method for producing metabolically activated liver organoids. The metabolically activated liver organoids of this embodiment can be suitably used for in vitro evaluation of the metabolism of test substances, drug interactions, hepatotoxicity, and transporter activity.

[0119] There may be differences in gene expression patterns, etc., between proliferative liver organoids and metabolically activated liver organoids and, for example, naturally occurring liver cells in the body. However, it is uncertain whether such differences exist, and identifying such differences and identifying the cells of this embodiment based on gene expression patterns, etc., would require an extremely large amount of trial and error, making it practically impossible. Therefore, it is practical to identify the cells of this embodiment by the manufacturing method described above.

[0120] In one embodiment, the present invention provides a growth medium and a differentiation medium. The growth medium and the differentiation medium are as described in the growth medium described in the method for producing proliferative liver organoids and the differentiation medium described in the method for producing metabolically active liver organoids, respectively. [Examples]

[0121] The present invention will be explained below with reference to experimental examples, but the present invention is not limited to these experimental examples. Furthermore, all experiments were conducted in accordance with an ethical research plan approved by the Ethics Committee of Keio University School of Medicine.

[0122] [Experimental Example 1] Production of human proliferative liver organoids Human primary frozen suspension hepatocytes (BIOPRRIDIC, HEP187-S) were thawed in a 37°C water bath, suspended in a 50 mL tube with serum-free medium, and centrifuged. The serum-free medium was Advanced DMEM / F12 with HEPES, GlutaMAX, and penicillin / streptomycin added. After centrifugation, the supernatant was removed, and the cells were then suspended in serum-free medium to prepare a hepatocyte suspension. From this suspension, 40,000 hepatocytes were mixed with 50 μL of Matrigel (BD Biosciences), seeded in a 24-well tissue culture plate, and incubated at 37°C for 5 to 10 minutes until the Matrigel was completely polymerized. Subsequently, after the Matrigel polymerized, the growth medium shown in Table 1 was overlaid and cultured for 12 weeks to produce proliferative liver organoids for Experimental Example 1.

[0123] R-Spongin 1 is used in the form of a conditioning medium containing R-Spongin 1, and the concentration of R-Spongin 1 relative to the total volume of the conditioning medium is 1.3 μg / mL. Similar to R-Spongin 1, Wnt3a is used in the form of a conditioning medium containing a complex of Wnt3a and afamin, with a concentration of Wnt3a relative to the total volume of the conditioning medium being 360 ng / mL.

[0124] The proliferation rate of proliferative liver organoids (2 weeks after the start of culture) from primary frozen suspension hepatocytes was visually determined. The results are shown in Table 1. The criteria for evaluation were (+++, ++, +, -) from highest to lowest proliferation rate.

[0125] The morphology of proliferative liver organoids was observed using a fluorescence microscope (KEYENCE, model "BZ-X710") two weeks after the start of culture. Organoids with a hollow interior were classified as "hollow," while those filled with cells were classified as "solid." The results are shown in Table 1. Microscopic images are also shown in Figure 1.

[0126] The feasibility of maintenance subculturing of proliferative liver organoids was determined. The determination was made by assessing the cell proliferation after subculturing using microscopic images. The results are shown in Table 1.

[0127] Two weeks after the start of culture, total ribonucleic acid (RNA) was extracted from proliferative liver organoids using a commercially available kit (product name "FastLane Cell cDNA Kit," Qiagen Corporation) to synthesize cDNA. The mRNA expression levels of albumin, metabolic enzymes, and transporter genes were measured by real-time quantitative PCR. Real-time quantitative PCR was performed using a commercially available kit (product name "SYBR® Premix Ex Taq (Perfect Real Time)," Takara Bio Inc.). In addition, glyceraldehyde triphosphate dehydrogenase (GAPDH) was used as an endogenous control to correct the measurement results. The results are shown in Table 3. In Table 3, the values ​​are expressed as relative values ​​with the expression level in human primary frozen suspension hepatocytes (BIOPRRIDIC, HEP187-S) set to 100.

[0128] [Experimental Examples 2-4] Production of liver organoids Liver organoids were prepared using the same method as in Experimental Example 1, except that growth media with the compositions shown in Table 1 were used. The growth rate, cell morphology, maintenance and expansion culture, and the expression levels of metabolic enzymes, transporters, and albumin at the gene level were also measured using the same method as in Experimental Example 1. The results are shown in Tables 1 and 3. Microscopic images of the liver organoids from Experimental Example 2 are shown in Figure 2.

[0129] [Experimental Example 5] Production of metabolically activated liver organoids Using the method of Experimental Example 1, human proliferative liver organoids cultured in growth medium for two weeks were diluted by mechanical dissociation and subcultured. During this process, the culture medium was changed from growth medium to differentiation medium, and the cells were cultured for one week to produce metabolically activated liver organoids. The differentiation medium used was a serum-free medium without IL-6, with the composition shown in Table 2. Cell morphology, maintenance and expansion culture, and the expression levels of metabolic enzymes, transporters, and albumin at the gene level were measured using the same methods as in Experimental Example 1. The results are shown in Tables 2 and 3. Microscopic images of the obtained liver organoids are shown in Figure 3.

[0130] [Reference Example 1] Manufacturing of human liver organoids Human liver organoids were produced using the method described in Non-Patent Document 2. Metabolic enzyme, transporter, and albumin expression levels were measured at the gene level using the same method as in Experimental Example 1. The results are shown in Table 3.

[0131] [Table 1]

[0132] [Table 2]

[0133] [Table 3]

[0134] Table 1 shows that the proliferative liver organoids obtained using a growth medium containing IL-6 (Experimental Example 1) had a hollow cell morphology, a high proliferation rate, and were suitable for maintenance and expansion culture. Furthermore, the microscopic image in Figure 1 revealed the presence of a red component inside the organoid.

[0135] In contrast, liver organoids obtained using a growth medium that did not contain IL-6 (Experimental Example 2) had a solid cell morphology and a high proliferation rate, but could not be maintained through expansion culture. Furthermore, liver organoids obtained using a growth medium that did not contain IL-6 but contained nicotinamide (Experimental Examples 3 and 4) had a solid cell morphology, a low proliferation rate, and could not be maintained through expansion culture.

[0136] Tables 2 and 3 show that the metabolically activated liver organoids of Experimental Example 5, differentiated from the proliferative liver organoids of Experimental Example 1 using a differentiation medium that does not contain IL-6, had a hollow cell morphology and could not be maintained or expanded. However, the expression levels of metabolic enzymes, transporters, and albumin at the gene level were uniformly improved, making them suitable for pharmacokinetic studies. Furthermore, microscopic images in Figure 3 revealed the presence of a yellow component, presumably bilirubin, inside the organoids.

[0137] [Experimental Examples 6-7] Production of Proliferative Liver Organoids Proliferative liver organoids were produced using the same method as in Experimental Example 1, except that the growth medium shown in Table 4 was used. The growth rate and maintenance culture were also measured using the same method as in Experimental Example 1. The results are shown in Table 4.

[0138] [Table 4]

[0139] [Experimental Examples 8-12] Production of metabolically active liver organoids Metabolic-active liver organoids were produced using the same method as in Experimental Example 5, except that the differentiation medium shown in Table 4 was used. Albumin expression levels and CYP3A4 expression levels were also measured using the same method as in Experimental Example 5. The results are shown in Table 5.

[0140] [Table 5]

[0141] [Experimental Example 13] Production of human proliferative liver organoids on collagen-Matrigel Human primary frozen suspension hepatocytes (BIOPRRIDIC, HEP187-S) were thawed in a 37°C water bath, suspended in a 50 mL tube with serum-free medium, and centrifuged. The serum-free medium was Advanced DMEM / F12 with HEPES, GlutaMAX, and penicillin / streptomycin added. After centrifugation, the supernatant was removed, and the cells were then suspended in serum-free medium to prepare a hepatocyte suspension. 50,000 hepatocytes from this suspension were mixed with 12.5 μL of Matrigel (BD Biosciences) and 12.5 μL of Collagen I (Nitta Gelatin Co., Ltd.), seeded in a 48-well tissue culture plate, and incubated at 37°C for 5 to 10 minutes until Matrigel and Collagen I were completely polymerized. Control samples using Matrigel alone or Collagen alone were also prepared. Next, after Matrigel and Collagen I polymerized, the growth medium shown in Table 1 above was layered and cultured to produce the proliferative liver organoids of Experimental Example 13.

[0142] As shown in Figures 4A to 4D, when an ECM containing a mixture of collagen and Matrigel was used, it was possible to culture for a greater number of passages, and enhanced growth was observed. [Industrial applicability]

[0143] The method for producing proliferative liver organoids of this embodiment yields proliferative liver organoids with excellent proliferative properties. The method for producing metabolically activated liver organoids of this embodiment yields metabolically activated liver organoids, which are differentiated from the proliferative liver organoids and have excellent metabolic activity.

Claims

1. A method for producing proliferative liver organoids, This includes culturing human liver-derived hepatocytes or human primary frozen hepatocytes in a growth medium to obtain proliferative liver organoids. The growth medium comprises interleukin-6 family cytokines, growth factors, Wnt agonists, transforming growth factor-β inhibitors, bone morphogenetic protein inhibitors, and forskolin. A method for producing the interleukin-6 family cytokine, wherein the interleukin-6 family cytokine is at least one selected from the group consisting of interleukin-6, interleukin-11, oncostatin M, leukemia suppressor, cardiotropin-1, and ciliary neurotrophic factor.

2. The manufacturing method according to claim 1, wherein the concentration of nicotinamide contained in the growth medium is 0 mM or 9 mM or less.

3. The method for producing the desired product according to claim 1 or 2, wherein the growth factor is at least one selected from the group consisting of epidermal growth factor, fibroblast growth factor, hepatocyte growth factor, amphiregulin, and heparin-binding EGF-like growth factor.

4. The manufacturing method according to any one of claims 1 to 3, wherein the Wnt agonist is at least one selected from the group consisting of Wnt family members, R-spongin 1, R-spongin 2, R-spongin 3, R-spongin 4, nolin, and glycogen synthase inhibitors.

5. The method for producing a product according to any one of claims 1 to 4, wherein the growth medium further comprises a Rho kinase inhibitor.

6. The method for producing the product according to claim 5, wherein the Rho kinase inhibitor is at least one selected from the group consisting of Y-27632, fasudil, Y39983, Wf-536, SLx-2119, azabenzimidazole-aminoflazan, DE-104, H-1152P, Rho kinase α inhibitor, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamide, Rho statin, BA-210, BA-207, Ki-23095, and VAS-012.

7. The manufacturing method according to any one of claims 1 to 6, wherein the transforming growth factor-β inhibitor is at least one selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY364947, SD-208, and SJN2511.

8. The method for producing bone morphogenetic protein according to any one of claims 1 to 7, wherein the bone morphogenetic protein inhibitor is at least one selected from the group consisting of noggin, Differential screening-selected gene Aberrative in Neuroblastoma, Cerberus, and gremlin.

9. The method for producing a culture medium according to any one of claims 1 to 8, wherein the culture medium further comprises at least one selected from the group consisting of gastrin, neurobiological supplements, and N-acetylcysteine.

10. The method for culturing in the growth medium, wherein the human liver-derived hepatocytes or the human primary frozen hepatocytes are brought into contact with the extracellular matrix during cultivation, as described in any one of claims 1 to 9.

11. The method for producing a cell culture according to claim 10, wherein the extracellular matrix in the culture medium is a mixture of collagen and Matrigel.

12. The manufacturing method according to any one of claims 1 to 11, wherein the culture in the growth medium is carried out for at least two weeks.

13. A method for producing metabolically activated liver organoids, The method comprises culturing proliferative liver organoids produced by the manufacturing method described in any one of claims 1 to 12 in a differentiation medium to obtain metabolically activated liver organoids, The differentiation medium comprises a growth factor, a transforming growth factor-β inhibitor, and forskolin. A method for producing the differentiation medium, wherein the concentration of interleukin-6 family cytokines contained in the differentiation medium is 0 ng / mL or less than 10 ng / mL, and the interleukin-6 family cytokines are at least one selected from the group consisting of interleukin-6, interleukin-11, oncostatin M, leukemia suppressor, cardiotropin-1, and ciliary neurotrophic factor.

14. A proliferative liver organoid produced by the manufacturing method described in any one of claims 1 to 12.

15. It contains interleukin-6 family cytokines, growth factors, Wnt agonists, transforming growth factor-β inhibitors, bone morphogenetic protein inhibitors, and forskolin. The aforementioned interleukin-6 family cytokine is at least one selected from the group consisting of interleukin-6, interleukin-11, oncostatin M, leukemia suppressor, cardiotropin-1, and ciliary neurotrophic factor, and is a culture medium for culturing proliferative liver organoids.

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