Method for manufacturing hepatocytes

Abstract

The present invention provides a method of producing hepatocytes, the method comprising a step of culturing hepatoblasts in suspension in a culture medium comprising an agonist of the oncostatin M receptor, an ALK inhibitor, an agonist of cAMP, and an agonist of the adrenergic receptor.

Description

Technical Field

[0001] This invention relates to a method for manufacturing hepatocytes, hepatocytes manufactured by the method, and the uses of the hepatocytes. Background Technology

[0002] Every year, many patients die from liver failure, including chronic and acute liver failure caused by cirrhosis. The only radical cure for cirrhosis is orthotopic liver transplantation. However, the supply of liver grafts, the primary source of donors, remains significantly insufficient worldwide. Furthermore, due to transplant rejection and the requirement for at least partial HLA matching between donor and recipient, patients must wait until an HLA-matched donor becomes available from a limited pool of donors, leaving a large number of patients unable to receive transplants at the appropriate time.

[0003] As a solution to the shortage of donors, the use of pluripotent stem cells, such as human induced pluripotent stem cells (iPS cells), is anticipated. During mammalian development, the liver develops from the embryonic endoderm, an early embryonic tissue, and is formed via hepatocytes, which are embryonic hepatic progenitor cells that give rise to both hepatocyte and bile duct cell lineages. Several methods for inducing hepatocytes by differentiating human pluripotent stem cells by reproducing this developmental process have been reported (e.g., Non-Patent Documents 1 and 2). Furthermore, the present inventors have also reported a method for inducing hepatocytes by differentiating pluripotent stem cells (Patent Document 1).

[0004] Existing technical documents Patent documents Patent document 1: International Publication No. 2016 / 104717.

[0005] Non-patent literature Non-patent literature 1: Lay Teng Ang et al., Cell Rep. 2018 Feb 20;22(8):2190-2205, Non-patent literature 2: Maki Kotaka et al., Sci Rep. 2017 Dec 1;7(1):16734. Summary of the Invention

[0006] The problem that the invention aims to solve However, hepatocytes induced by the existing differentiation induction methods described above are not capable of fully exerting their therapeutic effect in terms of albumin production capacity. Therefore, the objective of this invention is to provide a method for inducing hepatocytes that enhances albumin production capacity by modifying existing hepatocyte induction methods, and to provide hepatocytes obtained using this method, etc.

[0007] Methods for solving problems The inventors conceived of whether, by using compounds in various combinations in existing methods for inducing hepatocytes from pluripotent stem cells, it was possible to efficiently induce hepatocytes with increased albumin production. Therefore, a combination of hepatocyte growth factor (HGF), OSM (an OSM receptor agonist), A83-01 (an ALK inhibitor), DAPT (a NOTCH inhibitor), phenylephrine (an adrenergic receptor agonist), and forskolin (a cAMP agonist) was tested. The results showed that, unexpectedly, HGF had no significant effect on albumin production capacity in the method of inducing hepatocyte differentiation from hepatocytes via suspension culture using OSM. Furthermore, it was demonstrated that combining OSM with A83-01, DAPT, and phenylephrine (ADPF) yielded hepatocyte clusters with increased albumin production capacity, and these clusters exhibited significantly different morphology compared to combinations of other compounds (OSM, OSM+ADP), leading to the conclusion that this morphological difference was due to forskolin. Furthermore, further research using OSM and foscorine as a pivotal point revealed that DAPT had no effect on albumin production capacity. Therefore, it is evident that a combination of oncostatin M receptor agonists, ALK inhibitors, cAMP agonists, and adrenaline receptor agonists is crucial for inducing hepatocytes with high albumin production capacity through differentiation from hepatoblasts. Based on these insights, the inventors conducted further and repeated research, resulting in this invention.

[0008] That is, the present invention is as follows.

[0009] [1] A method for producing hepatocytes, the method comprising suspending and culturing hepatocytes in a culture medium containing a tumor suppressor M receptor agonist, an ALK inhibitor, a cAMP agonist and an adrenaline receptor agonist.

[0010] [2] According to the method described in [1], the hepatocytes are in the form of cell aggregates.

[0011] [3] According to the method of [1] or [2], the method includes a step of performing an oscillating culture.

[0012] [4] The method according to any one of [1] to [3] includes a step of culturing in a culture bag having a plurality of pores.

[0013] [5-1] The method according to any one of [1] to [4], wherein the suspension culture is a culture in a medium that does not contain hepatocyte growth factor and / or DAPT.

[0014] [5-2] The method according to any one of [1] to [4], wherein the suspension culture is cultured in a medium that does not contain hepatocyte growth factor and DAPT.

[0015] [6] The method according to any one of [1] to [5-2], wherein at least one of the tumor suppressor M receptor agonists is tumor suppressor M.

[0016] [7] The method according to any one of [1] to [6], wherein at least one of the ALK inhibitors is A83-01.

[0017] [8] The method according to any one of [1] to [7], wherein at least one of the cAMP agonists is forscorin.

[0018] [9] The method according to any one of [1] to [8], wherein at least one of the adrenergic receptor agonists is phenylephrine.

[0019] [10-1] The method according to any one of [1] to [9], wherein the hepatoblasts are derived from pluripotent stem cells.

[0020] [10-2] The method according to any one of [1] to [10-1], wherein the hepatoblasts are derived from humans.

[0021]

[11] The method according to any one of [2] to [10-2], wherein the aggregates of hepatoblasts are obtained by suspension culture in a culture medium containing a tumor suppressor M receptor agonist, an ALK inhibitor, a cAMP agonist and an adrenaline receptor agonist.

[0022]

[12] Hepatocytes, which are obtained by any one of [1] to

[11] .

[0023]

[13] A cell transplantation therapy agent comprising hepatocytes as described in

[12] .

[0024]

[14] The agent according to

[13] is used to treat or prevent liver disease.

[0025]

[15] A method for evaluating the metabolic extent of a test sample, the method comprising: (I) The process of bringing the test sample into contact with hepatocytes as described in

[12] ; (II) Procedures for determining the activity levels of drug-metabolizing enzymes in the hepatocytes; and (III) A process for evaluating the extent to which the test sample is metabolized by drug-metabolizing enzymes based on the activity level of drug-metabolizing enzymes in step (II).

[0026]

[16] A method for the treatment or prevention of liver disease, wherein an effective amount of hepatocytes according to

[12] are transplanted into a mammal.

[0027]

[17] The hepatocytes described in

[12] are used to treat or prevent liver disease.

[0028]

[18] The use of hepatocytes as described in

[12] in the manufacture of medicines for the treatment or prevention of liver disease.

[0029] The effects of the invention According to the present invention, hepatocytes with excellent albumin-producing capacity can be manufactured in vitro. Cells manufactured in this way can possess characteristics more closely resembling those of biological hepatocytes, and therefore are expected to be used for hepatocyte transplantation. Attached Figure Description

[0030] [ Figure 1 This diagram illustrates the differentiation branches from hepatoblasts to bile duct epithelial cells or hepatocytes.

[0031] [ Figure 2 [Image showing the results of hepatocytes suspending in a culture medium containing hemostatin M and various compounds under stirred conditions, differentiating into hepatocyte aggregates (HLCs). (a) Culture protocol. (b) Bright-field image of HLC aggregates on day 15 of induction. Scale bar, 500] μ m. (c) Amount of albumin secreted into the culture medium every 24 hours by HLC aggregates on day 15 of induction. Technical replicates, n=4; error bars, standard deviation. OSM, 20 ng / ml hepatocyte growth factor M; HGF, 20 ng / ml hepatocyte growth factor (Peprotech, Cat# 100-39); DEX, 100 nM dexamethasone (Merck, Cat# D4902-25 MG); Phe, 1 μ M-benzylphosphonic acid.

[0032] [ Figure 3 [Image showing the results of hepatocytes suspending in a culture medium containing cytosine M and various compounds under stirred conditions to differentiate into HLC aggregates. (a) Culture protocol. (b) Bright-field image of HLC aggregates on day 15 of induction. Scale bar, 500] μ m. (c) Amount of albumin secreted into the culture medium every 24 hours by HLC aggregates on day 15 of induction. OSM, 20 ng / ml oncogene M; HGF, 20 ng / ml hepatocyte growth factor; A, 1 μ M A83-01 (TGF β Inhibitor); D, 10 μMDAPT (NOTCH inhibitor, R&D, Cat# #2634 / 10); P, 1 μ M-benfural ( α 1-Adrenergic agonists); F, 10 μ M-Forsocrine (PKA agonist). Technical repeatability, n=2; error bars, standard deviation.

[0033] [ Figure 4 [Image showing the results of hepatocytes suspending in a culture medium containing cytosine M and various compounds under stirred conditions to differentiate into HLC aggregates. (a) Culture protocol. (b) Bright-field image of HLC aggregates on day 15 of induction. Scale bar, 500] μ m. (c) Amount of albumin secreted into the culture medium every 24 hours by HLC aggregates induced on day 15. Technical replicates, n=2; error bars, standard deviation. (d) Bright-field image of HLC aggregates induced on day 15. Scale bar, 500. μ m. (e) Amount of albumin secreted into the culture medium every 24 hours by HLC aggregates on day 15 of induction. Biological replicates, n=2. OSM, 20 ng / ml cytosolic acid M; A, 1 μ M A83-01; D, 10 μ M DAPT; P, 1 μ M-benzylphosphonium; F, 10 μ M-Foscoline.

[0034] [ Figure 5 [Image showing the results of hepatocytes suspending in a culture medium containing cytosine M and various compounds under stirred conditions to differentiate into HLC aggregates. (a) Culture protocol. (b) Bright-field image of HLC aggregates on day 15 of induction. Scale bar, 500] μ m. (c) Amount of albumin secreted into the culture medium every 24 hours by HLC aggregates on day 15 of induction. Biological replicates, n=2. OSM, 20 ng / ml serotonin M; A, 1 μ M A83-01; D, 10 μ M DAPT; P, 1 μ M-benzylphosphonium; F, 10 μ M-Foscoline.

[0035] [ Figure 6[Image showing the results of hepatocytes forming hepatocyte clusters through suspension culture in a medium containing hemostatin M, A83-01, phenylephrine, and forscolin, and differentiating into HLC aggregates under stirred conditions. (a) Culture protocol. Forced aggregation refers to cell clusters formed by forced aggregation of cells into wells on the bottom of a cell-low adhesion microporous culture bag. (b) Cell-low adhesion microporous culture bag used to form hepatocyte clusters from hepatocytes. (c) Bright-field image of HLC aggregates on day 17 of induction. Scale bar, 500 μ m. (d) Diameter distribution of HLC aggregates on day 17 of induction. (e) HE-stained image of HLC aggregates. Scale bar, 50 μ m. (f) Immunostaining image of HLC aggregates. Scale bar, 100 μ m.

[0036] [ Figure 7 [Image showing results of differentiation of hepatocytes into HLC aggregates via planar culture (2D) or suspension culture under stirred conditions. (a) Culture protocol. (b) RT-qPCR. Technical replicates, n=5; error bars, standard deviation.]

[0037] [ Figure 8 [Image showing results of hepatocyte differentiation into HLC aggregates under planar or suspension culture conditions. (Top) Culture protocol. 3D indicates the existing method (if cell suspension is cultured in a 12-well plate with shaking, cell clusters naturally form. The method of culturing them directly by shaking is used. The same applies below.). 3D, apparatus, and 3D_d indicate the culture bag method (the method of creating cell clusters using microwell culture bags with low cell adhesion, then removing the cell clusters from the culture bags and transferring them to 12-well plates for shaking culture is used. The same applies below.). (Bottom) RT-qPCR. Technical replicates, n=3; error bars, standard deviation.]

[0038] [ Figure 9 [Results showing differentiation of hepatocytes into HLC aggregates under planar or suspension culture conditions. (Top) Culture protocol. (Bottom) RT-qPCR. Technical replicates, n=3; error bars, standard deviation.]

[0039] [ Figure 10 [Results showing differentiation of hepatocytes into HLC aggregates under planar or suspension culture conditions. (Top) Culture protocol. (Bottom) RT-qPCR. Technical replicates, n=3; error bars, standard deviation.]

[0040] [ Figure 11[Image showing results of hepatocyte differentiation into HLC aggregates under planar or suspension culture conditions. In the figure, Agi- indicates no oscillation, and Agi+ indicates oscillation. (Top) Culture protocol. (Bottom) RT-qPCR. Technical replicates, n=3; error bars, standard deviation.]

[0041] [ Figure 12 [Image showing results of hepatocyte differentiation into HLC aggregates under planar or suspension culture conditions. In the figure, Agi- indicates no oscillation, and Agi+ indicates oscillation. (Top) Culture protocol. (Bottom) RT-qPCR. Technical replicates, n=3; error bars, standard deviation.] Detailed Implementation

[0042] 1. Preparation of hepatocytes This invention provides a method for producing hepatocytes from hepatoblasts. Specifically, this invention provides a method for producing hepatocytes (hereinafter sometimes referred to as "the method of this invention"), the method comprising the step of suspending hepatoblasts in a culture medium containing an OSMR agonist, an ALK inhibitor, a cAMP agonist, and an adrenergic receptor agonist (hereinafter sometimes referred to collectively as "hepatocyte induction-promoting substances") (in other words, suspension culture in the presence of hepatocyte induction-promoting substances).

[0043] In this specification, "hepatoblast" means a cell capable of differentiating into hepatocytes (and typically, also into bile duct epithelial cells) and expressing at least one marker gene selected from AFP, Dlk, E-cadherin, Liv2, CD13, and CD133. Hepatoblasts are preferably cells expressing AFP.

[0044] In this specification, "hepatocyte" refers to a cell that expresses albumin and is typically capable of producing albumin. Hepatocytes typically express albumin selected from AFP and HNF4. α Hepatocytes are cells of at least one (typically all) of FOXA2, PROX1, and A1AT. In addition, hepatocytes may have one or more of the functions known to hepatocytes, such as glycogen accumulation, low-density lipoprotein (LDL) uptake, albumin secretion, ammonia metabolism and urea synthesis, cytochrome P450 activity, lipid metabolism, and drug metabolism.

[0045] In this specification, unless otherwise specified, "cell" includes "cell population". Additionally, unless otherwise specified, "cell" refers to cells obtained through cell culture. A cell population may consist of one type of cell or two or more types of cells. Furthermore, unless otherwise specified, "cell population" also includes "cell aggregate" (also called "cell cluster").

[0046] Therefore, the term "hepatocyte" also includes cell populations containing hepatocytes and cell aggregates containing hepatocytes (also called "hepatocyte clusters"). Similarly, the term "hepatocyte" also includes cell populations containing hepatocytes and cell aggregates containing hepatocytes (also called "hepatocyte clusters"). In one aspect of the invention, hepatocytes are provided for culture in the form of cell aggregates. Alternatively, in another aspect of the invention, hepatocytes are in the form of cell aggregates. The proportion of hepatocytes or hepatocytes contained in the cell population or cell aggregate (number of hepatocytes or stem cells / total number of cells) is not particularly limited, but is preferably 20% or more, and can be 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0047] In this specification, the terms "expressed" gene or "positive" are used, unless otherwise specified, to at least include the meaning of "producing mRNA encoded by the gene," and preferably to further include the meaning of "producing protein encoded by the mRNA." Therefore, a gene can be said to be expressed if the production of mRNA encoded by the gene is detected at least by RT-qPCR.

[0048] In this specification, "OSMR receptor agonist" (hereinafter also referred to as "OSMR agonist") means a substance that can directly bind to the OSMR receptor (OSMR) to exert its effect. OSMR is a cell surface receptor that binds to OSM to transduce signals to downstream factors, forming an OSM-specific OSMR receptor. β OSMR β OSM is a heterodimer of gp130, which is shared by IL-6 family receptors. OSM can bind with low affinity to either OSMRβ or gp130, but it cannot transduce signals intracellularly even when bound alone. β These are single-transmembrane proteins. Examples of OSMR agonists include OSM. Commercially available OSM products, such as those from Peprotech, can be used. Specifically, examples of human OSM include OSM isotype 1 (an example of the base sequence encoding this isotype is shown as Serial No. 1) as shown in NCBI accession number NP_065391.1 (Serial No. 2), and OSM isotype 2 (an example of the base sequence encoding this isotype is shown as Serial No. 3) as shown in NCBI accession number NP_001306037.1 (Serial No. 4). Multiple OSMR agonists can be used in combination.

[0049] When using OSM as an OSMR agonist, its concentration in the culture medium is typically 0.1 ng / ml to 500 ng / ml, preferably 1 ng / ml to 100 ng / ml, and more preferably 10 ng / ml to 50 ng / ml (in one regimen, 20 ng / ml). When using OSMR agonists other than OSM, the concentration of the OSMR agonist in the culture medium can be appropriately selected.

[0050] Examples of ALK inhibitors used in this invention include SB431542 (4-(5-benzo[1,3]dioxacyclopenten-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide, 4-[4-(1,3-benzodioxacyclopenten-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide, 4-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide), A83-01 (3-(6-methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinoline-4-ylpyrazole), and LDN193189. (4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), GW788388 (4-[4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl]-N-(tetrahydro-2H-pyran-4-yl)benzamide), SM16 (4-[4-(1,3-benzodioxacyclopenten-5-yl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-2-yl]-bicyclo[2.2.2]octane-1-carboxamide), IN-1130 (3-[[5-(6-methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]-benzamide), GW6604 (2-Phenylacetyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine), SB505124 (2-(5-benzo[1,3]dioxacyclopenten-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine), etc., preferably A83-01. ALK inhibitors can also be used in combination.

[0051] When using A83-01 as an ALK inhibitor, its concentration in the culture medium is typically 0.01. μ M~10 μ M, preferably 0.05 μ M~5 μ M, more preferably 0.1 μ M~2.5 μ M (in one scheme, it is 1) μWhen using ALK inhibitors other than A83-01, the concentration of the ALK inhibitor in the culture medium can be appropriately selected.

[0052] In this specification, "cAMP agonist" refers to adenosine 3',5'-cyclic monophosphate (cAMP) or a substance having the same function as cAMP, or a substance that can increase the intracellular cAMP concentration upon contact with cells. Examples of cAMP agonists used in this invention include adenylate cyclase activators and cAMP analogs. Examples of cAMP analogs include 8-Br-cAMP, dibutyryl-cAMP (bradiclofenac), N6-benzoylcAMP, and 8-thiomethylcAMP. Examples of adenylate cyclase activators include forsocolin, forsocolin derivatives (e.g., NKH477 (cofosindaropterin), PACAP-27, PACAP-38, and SKF83822), with forsocolin being preferred. Multiple cAMP agonists can be used in combination.

[0053] When using forscolin as a cAMP agonist, its concentration in the culture medium is typically 0.1 μM to 100 μM, preferably 0.5 μM to 50 μM, and more preferably 1 μM to 20 μM (10 μM in one formulation). When using cAMP agonists other than forscolin, the concentration of the cAMP agonist in the culture medium can be appropriately selected.

[0054] In this specification, "adrenergic receptor agonist" means a substance that can directly bind to adrenergic receptors to exert its effect. Preferably, it binds at least to adrenergic receptors. α 1. Adrenergic receptor-binding substances, such as adrenaline, noradrenaline, etifolin, naphazoline, phenformin ((R)-3-(1-hydroxy-2-(methylamino)ethyl)phenol), methoxamine, midodrine, etc. In this invention, preferred adrenaline receptor agonists are etifolin, phenformin, and methoxamine, with phenformin being the most preferred. Multiple adrenaline receptor agonists can be used in combination.

[0055] When using phenylephrine as an adrenergic receptor agonist, the concentration in the culture medium is typically 0.01. μ M~10 μ M, preferably 0.05 μ M~5 μ M, more preferably 0.1 μ M~2.5 μ M (in one scheme, it is 1) μ M). When using adrenergic receptor agonists other than phenylephrine, the concentration of the adrenergic receptor agonist in the culture medium can be appropriately selected.

[0056] On the other hand, as shown in the following examples, in the preparation method of the present invention, hepatocyte growth factor (HGF) and DAPT (N-[N-(3,5-difluorophenylacetyl-L-alanyl)]-(S)-phenylglycine tert-butyl ester), which are widely used in existing methods, are not essential. Therefore, in one aspect of the present invention, the suspension culture in the preparation method of the present invention is a culture in a medium that does not contain HGF and / or DAPT (in other words, a culture in the absence of HGF and / or DAPT).

[0057] When hepatoblasts are provided in a state of adhesion to the culture vessel, the preparation method of the present invention can be implemented by replacing the culture medium containing hepatoblasts with a culture medium containing hepatoblast induction and promotion substances, and culturing under suspension culture conditions. Alternatively, when hepatoblasts are provided in a culture medium in a suspension state, the culture medium containing hepatoblasts can also be replaced with a culture medium containing hepatoblast induction and promotion substances, and cultured directly under suspension culture conditions.

[0058] When hepatocytes are provided as a cell conglomerate, they can be cultured after being separated into single cells by any method of substantially separation (or dissociation). Examples of separation methods include mechanical separation, or the use of separation solutions with protease and collagenase activity (e.g., solutions containing trypsin and collagenase, such as Accutase). TM Accumax TM Separation can be achieved using methods such as (Nacalai Tesque) or separation solutions with only collagenase activity. Separated or dissociated cells can also be further separated using surface antigens as indicators, employing methods such as flow cytometry or mass cytometry, magnetic cell separation, or affinity columns immobilizing the desired antigen.

[0059] In this specification, "suspension culture" refers to culture performed under conditions in which cells or cell aggregates are maintained in suspension within the culture medium, i.e., culture where a strong cell-substratum junction is not formed between the cells or cell aggregates and the culture vessel. Conversely, in this specification, "adhesion culture" (also known as "planar culture") refers to culture performed under conditions in which a strong cell-substratum junction is formed between the cells or cell aggregates and the culture device, etc.

[0060] There are no particular limitations on the culture vessel used for suspension culture. Examples include flasks, tissue culture flasks, dishes, Piper dishes, tissue culture dishes, multi-well dishes, microplates, microwell plates, microwells, multilayer plates, multi-well plates, cavity slides, culture dishes, tubes, trays, culture bags, roller flasks, etc. However, for the shaking culture (also known as "stirred culture") described later, a sealed culture vessel is preferred. Examples of such culture vessels include tissue culture flasks, culture bags, roller flasks, etc. In addition, in order to enable culture under non-adhesive conditions, the culture vessel is preferably cell-non-adhesive. As a cell-non-adhesive culture container, containers whose surfaces have not been artificially treated to improve cell adhesion (e.g., coating treatment using extracellular matrix, etc.) or containers that have undergone artificial adhesion inhibition treatment (e.g., coating treatment using poly-HEMA), etc., can be used.

[0061] Furthermore, as shown in the following embodiments, it is demonstrated that uniform hepatocyte clusters can be effectively produced by using a culture bag with multiple pores (also referred to as a "porous device"). Therefore, in one embodiment, the method of preparation of the present invention includes a step of cell culture within a porous device. From the viewpoint of reducing the risk of contamination during culture, the porous device is preferably a device capable of culture in a closed system. The porous device has multiple pores of uniform size that have undergone cell non-adhesion treatment, thereby allowing hepatocyte clusters to form in each pore. Thus, a large quantity of uniformly sized hepatocyte clusters can be produced through a single culture.

[0062] The porous device can be made using commercially available products (e.g., products manufactured by Toyo Seikan Group Holdings, Ltd.) or by known methods (e.g., the method described in Japanese Patent Application Publication No. 2017-184716). The porous device used in this invention is not particularly limited as long as it has multiple holes, but the porous device described in Japanese Patent Application Publication No. 2017-184716 is preferred. In one embodiment, the porous device used in this invention is characterized by having a container body made of a breathable plastic film and injection / outflow holes, the periphery of the container body being sealed, the top surface of the container having a bulging shape, and multiple recesses (holes) forming cell culture sections provided on the bottom surface of the container body. In such a porous device, typically, the opening diameter of the recesses is 0.3 to 10 mm (0.5 mm in one embodiment), the depth is 0.1 mm or more (0.2 mm in one embodiment), and the area occupied by the recesses on the bottom surface is 30% to 90% of the area of ​​the bottom surface. The ratio d / D of the depth d to the diameter D of the recess is preferably 0.05 to 1. Furthermore, the oxygen permeability of the aforementioned plastic film is typically 5000 mL / (m²). 2(Above 1000 meters)

[0063] The material used for the container body in manufacturing the aforementioned porous device is not particularly limited as long as it has the desired air permeability. Examples include polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, silicone elastomers, polystyrene elastomers, and thermoplastic resins such as tetrafluoroethylene-hexafluoropropylene copolymer (FEP). The number of pores can also be appropriately selected according to the desired number of cell clusters, typically from 100 to 30,000 (18,000 in one embodiment).

[0064] As shown in the examples described later, culturing hepatocytes with shaking can induce hepatocytes with superior albumin production capacity compared to the case without shaking. Without being bound by any theory, it is hypothesized that the physical agitation of the culture medium caused by shaking culture can achieve the supply of oxygen and nutrients and the effective exchange of excreted waste, thereby inducing hepatocytes with superior albumin production capacity. Therefore, the shaking culture used in this invention is not limited to any method that allows for physical agitation of the culture medium. Shaking culture can be performed, for example, by placing the culture vessel for culturing hepatocytes on a shaker, rotator, or the like. Alternatively, it can be performed by placing the hepatocytes in a rotating environment such as a stirrer or a mixing paddle.

[0065] Shaking culture can be performed throughout the entire suspension culture period in the preparation method of the present invention, or only during a portion of the suspension culture period. For example, static culture can also be performed in a medium containing hepatocyte induction-promoting substances before shaking culture, typically for 6 hours to 3 days, preferably 0.5 days to 2 days, and more preferably 0.5 days to 1 day. Static culture enables the production of homogeneous hepatocyte clusters. In one aspect of the invention, the hepatocyte clusters used in the preparation method of the present invention are obtained by suspension culture (static culture) in the presence of an OSMR agonist, an ALK inhibitor, a cAMP agonist, and an adrenaline receptor agonist. Alternatively, in another aspect, the preparation method of the present invention includes a step of suspension culture (static culture) of hepatocytes in the presence of an OSMR agonist, an ALK inhibitor, a cAMP agonist, and an adrenaline receptor agonist before the step of suspension culture of hepatocyte clusters in a medium containing hepatocyte induction-promoting substances.

[0066] The parameters such as the shaking culture speed can be appropriately set by those skilled in the art. For example, when using a 3D shaking shaker (e.g., Mini-Shaker 3D, Biosan, etc.) for shaking culture, the shaking speed range is typically set to 5-60 rpm. When using a reciprocating shaker (e.g., NS-LR, AS ONE, etc.) for shaking culture, the shaking rate range is typically set to 15-60 rpm. When using a seesaw shaker (e.g., NS-S, AS ONE, etc.) for shaking culture, the shaking rate range is typically set to 50-50 rpm. Alternatively, for example, a rotary bottle (e.g., 3152, Corning, etc.) can be placed on a magnetic stirrer, and the culture can be performed at a rotation speed that visually prevents the hepatocyte clusters from settling. A three-dimensional rotating suspension culture device (e.g., CellPet CUBE, J-Tech; Clinostar, Celvivo, etc.) can also be used for culture. When using a three-dimensional rotating suspension culture device for oscillation culture, the rotation speed range can typically be set within the range of 15 to 60 rpm. Furthermore, the oscillation culture can be performed by rotating perpendicularly to the direction of gravity (lateral rotation) or parallel to the direction of gravity (longitudinal rotation), with lateral rotation being preferred.

[0067] The culture medium used in this invention can typically be prepared by adding hepatocyte induction-promoting substances to a basal culture medium. Examples of such basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, etc. α MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and Lonza's HBM medium. TM Basal Medium, HCM TM SingleQuots TM Kit, HMM TM Basal Medium or HMM TM SingleQuots TMKit, PromoCell's Hepatocyte Growth Medium or Hepatocyte Maintenance Medium, Sigma-Aldrich's Hepatocyte Medium, and their mixed culture media, etc.

[0068] These basal culture media may contain serum (e.g., fetal bovine serum (FBS), horse serum, human serum, etc.) or may be serum-free. In the case of serum-free media, depending on requirements, one or more serum substitutes may be included, such as albumin, transferrin, KnockOut Serum Replacement (KSR) (Invitrogen) (serum substitute for ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, etc. The culture medium may also contain one or more substances selected from lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins (e.g., nicotinamide, ascorbic acid), growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, glucagon, hydrocortisone, EGF (epidermal growth factor), dexamethasone, and their equivalents.

[0069] The duration of the process for manufacturing hepatocytes from hepatoblasts according to the present invention has no upper limit, as it is presumed that long-term culture does not have a significant impact on the efficiency of hepatocyte manufacturing; typically, it is 20 days or less (e.g., 15 days or less, 10 days or less, 9 days or less, 8 days or less, 7 days or less). Alternatively, for the aforementioned culture period, examples include 3 days or more, 4 days or more, 5 days or more, 6 days or more, and 7 days or more. Typically, it is 3 to 9 days, preferably 5 to 8 days, and more preferably 5 to 7 days.

[0070] In the preparation method of the present invention, cells can be cultured under conditions of no feeder layer and / or under conditions of xeno-free. In the preparation method of the present invention, the entire process can be carried out under conditions of no feeder layer and no xeno-free. In this specification, "no feeder layer" means a culture medium or culture condition that does not contain other cell species (i.e., feeder cells) used to regulate the culture conditions of the target cells. Furthermore, "no xeno-free" means a culture medium or culture condition that does not contain components from organisms different from the biological species of the target cells.

[0071] The hepatoblasts used in this invention can be obtained by known methods. Examples include methods for isolating hepatoblasts from biological tissues (e.g., embryonic liver), methods for differentiating and inducing endoderm cells (also called "embryonic endoderm cells"), and methods obtained from companies such as ATCC. For example, hepatoblasts can be isolated from biological tissues using surface antigens as indicators, employing methods such as flow cytometry or mass spectrometry, magnetic cell separation, or affinity columns immobilizing the desired antigen. The hepatoblasts used in this invention are preferably obtained by differentiating and inducing endoderm cells. Therefore, in one embodiment, the method of this invention includes a step of culturing endoderm cells to manufacture (or "differentiate-induced") hepatoblasts (hereinafter sometimes also referred to as the "hepatoblast manufacturing step").

[0072] The process of manufacturing hepatocytes can be carried out by known methods (e.g., methods described in Patent Document 1, Non-Patent Document 1, Non-Patent Document 2, etc.). Specifically, examples include methods for culturing endoderm cells in a medium containing FGF2 and BMP4; methods for inducing hepatocytes by culturing endoderm cells in a medium containing dimethyl sulfoxide (DMSO) (optionally further containing HGF); methods for inducing foregut cells by culturing endoderm cells in a medium containing FGF7 and SB431542 or a medium containing BMP4, TTNPB, A83-01, and FGF2; and methods for inducing hepatocytes by culturing the cells in a medium containing FGF7, BMP4, and BMP3 or a medium containing forscorin, BMP4, activin A, and C59 or CHIR99201, etc.

[0073] Endoderm cells are cells that differentiate during mammalian development to form tissues that form organs such as the digestive tract, lungs, thyroid gland, pancreas, and liver. In this specification, "endoderm cell" refers to a cell expressing at least one of SOX17, FOXA2, and CXCR4, preferably a cell expressing at least SOX17.

[0074] When culturing endoderm cells, if the endoderm cells are provided as cell masses, they can be cultured as single cells by substantially separating or dissociating them using any method, or they can be cultured as cell masses. Examples of separation methods include mechanical separation, or the use of separation solutions with protease and collagenase activity (e.g., solutions containing trypsin and collagenase, such as Accutase). TM Accumax TM Separation of solutions containing (NacalaiTesque) or only collagenase activity, etc.

[0075] For example, commercially available products such as those from Fujifilm and Koichi Chemical Co., Ltd. can be used for FGF2. When using the FGF2 used in this process, the concentration of FGF2 in the culture medium is typically 0.1 ng / ml to 100 ng / ml, preferably 1 ng / ml to 50 ng / ml, and more preferably 5 ng / ml to 20 ng / ml (in one embodiment, 10 ng / ml).

[0076] BMP4 can be commercially available products such as those from Peprotech. When using the BMP4 used in this process, the concentration of BMP4 in the culture medium is typically 0.2 ng / ml to 200 ng / ml, preferably 2 ng / ml to 100 ng / ml, and more preferably 10 ng / ml to 40 ng / ml (in one embodiment, 20 ng / ml).

[0077] The duration of the hepatoblast production process has no upper limit, as it is presumed that long-term culture does not significantly affect the induction efficiency of hepatoblasts; typically, it is less than 20 days (e.g., less than 15 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days). Alternatively, the aforementioned culture period can be 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. Typically, it is 3 to 9 days, preferably 3 to 8 days, and more preferably 4 to 7 days (in one embodiment, 5 days).

[0078] The endoderm cells used in this invention can be obtained by known methods. Examples include methods for isolating cells from biological tissues (e.g., embryonic tissues), methods for differentiating and inducing pluripotent stem cells, and methods obtained from companies such as ATCC. For instance, endoderm cells can be isolated from biological tissues using surface antigens as indicators, employing methods such as flow cytometry or mass spectrometry, magnetic cell separation, or affinity columns immobilizing the desired antigen. The endoderm cells used in this invention are preferably obtained by methods for differentiating and inducing pluripotent stem cells. Therefore, in one embodiment, the method of this invention includes a step of culturing pluripotent stem cells to manufacture (or, alternatively, "differentiation-induced") endoderm cells (hereinafter sometimes referred to as the "endoderm cell manufacturing step").

[0079] The process of manufacturing endoderm cells can be carried out by known methods (e.g., methods described in Patent Document 1, Non-Patent Document 1, Non-Patent Document 2, etc.). Specifically, examples include methods for activating activin A and Wnt signaling pathways in pluripotent stem cells, methods for activating Wnt signaling pathways in pluripotent stem cells under adhesion culture conditions (WO2007 / 050043), and methods for co-culturing pluripotent stem cells and supporting cells (e.g., M15 cells) (WO2006 / 126574), etc.

[0080] Preferably, the endoderm cell manufacturing process involves using cells containing activin A and GSK-3. β The process of culturing pluripotent stem cells in a culture medium containing inhibitors. In such a process, the culture medium may further contain histone deacetylase (HDAC) inhibitors.

[0081] In addition, the culture medium used in the endoderm cell manufacturing process may also contain ROCK inhibitors. Examples of such ROCK inhibitors include Y-27632 (e.g., see Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), fasudil / HA1077 (e.g., see Uenata et al., Nature 389: 990-994 (1997)), SR3677 (e.g., see Feng Y et al., J Med Chem. 51: 6642-6645 (2008)), GSK269962 (e.g., see Stavenger RA et al., J MedChem. 50: 2-5 (2007) or WO2005 / 037197), GSK429286A, and H1152 (e.g., see Sasaki et al., Pharmacol. Ther.). 93: 225-232 (2002)), Wf-536 (e.g., see Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2003)), thiazovivin, and their derivatives, etc. Among them, Y-27632 is preferred. The concentration of Y-27632 in the culture medium when using it as a ROCK inhibitor is typically 1 μ M~20 μ M, preferably 5 μ M~15 μ M (in one scheme, it is 10) μ M).

[0082] "Pluripotent stem cell" refers to a stem cell that can differentiate into various tissues or cells with different morphologies or functions in an organism, and has the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm). Examples of pluripotent stem cells used in this invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained through nuclear transfer (nuclear transfer embryonic stem cells: ntES cells), multipotent germline stem cells ("mGS cells"), embryonic germline stem cells (EG cells), etc., preferably iPS cells (more preferably human iPS cells). When the aforementioned pluripotent stem cells are ES cells or any cells derived from a human embryo, the cells can be cells created by destroying the embryo or cells created without destroying the embryo; from an ethical point of view, cells created without destroying the embryo are preferred.

[0083] ES cells are pluripotent and self-replicating stem cells constructed from the internal cell mass of early embryos (e.g., blastocysts) in mammals such as humans or mice. ES cells were first discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were also constructed in primates such as humans and monkeys (JA Thomson et al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55: 254-259; JA Thomson and VS Marshall (1998), Curr. Top.Dev. Biol., 38: 133-165). ES cells can be constructed by extracting the inner cell mass from the blastocyst of a fertilized egg in a test animal and culturing the inner cell mass on a fibroblast feeder. Alternatively, ES cells can be constructed using only single blastomeres from pre-blastocyst embryos at the cleavage stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or from embryos that have stopped developing (Zhang X. et al. (2006), Stem Cells 24: 2669-2676.).

[0084] As for the ES cell lines used in this invention, if they are mouse ES cells, various mouse ES cell lines constructed by companies such as inGenioustargeting Laboratory and RIKEN can be used. If they are human ES cell lines, various human ES cell lines constructed by companies such as the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child and Physical Medicine, and Cellartis can be used. Specifically, examples of human ES cell lines include CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 sold by ESIBio, H1 and H9 sold by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 sold by RIKEN.

[0085] iPS cells are cells obtained by introducing specific factors (nuclear initialization factors) into mammalian somatic cells or undifferentiated stem cells and reprogramming them. Currently, iPS cells encompass various cell types. Besides iPSCs constructed by Yamanaka et al. through the introduction of four factors—Oct3 / 4, Sox2, Klf4, and c-Myc—into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human cell-derived iPSCs can also be constructed by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al., Cell, (2007) 131: 861-872). Nanog-iPSCs, constructed by screening and using Nanog expression as an indicator after the introduction of the aforementioned four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), and iPSCs produced using a c-Myc-free method (Nakagawa M, Yamanaka S., et al., Nature). Biotechnology, (2008) 26, 101-106); iPSCs constructed by introducing 6 factors through a virus-free method (Okita K et al. Nat. Methods 2011 May;8(5): 409-12; Okita K et al. Stem Cells. 31(3): 458-66.) etc. In addition, induced pluripotent stem cells constructed by introducing four factors—OCT3 / 4, SOX2, NANOG, and LIN28—by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells constructed by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells constructed by Sakurada et al. (Japanese Patent Application Publication No. 2008-307007) can also be used.

[0086] In addition, all publicly available papers (e.g., Shi Y., Ding S. et al., Cell Stem Cell, (2008) Vol. 3, No. 5, 568-574; Kim JB., Scholer HR. et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. 7) Induced pluripotent stem cells known in the art as described in patent publications (e.g., Japanese Patent Application Publication No. 2008-307007, Japanese Patent Application Publication No. 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852) may be used.

[0087] As induced pluripotent stem cell lines, various iPSC lines constructed by institutions such as the NIH, RIKEN, and Kyoto University can be used. For example, human iPSC lines include RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 lines, and Kyoto University's 253G1, 253G4, 1201C1, 1205D1, 1210B2, 1383D2, 1383D6, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3, and FfI-01s04 lines.

[0088] The induced pluripotent stem cells used in this invention can be cells derived from patients with hereditary diseases (such as hereditary liver disease). Cells induced from the differentiation of pluripotent stem cells from patients with hereditary liver disease can serve as disease models reflecting the pathological state of the disease, and are therefore suitable for screening therapeutic or preventative drugs for the disease. Alternatively, pluripotent stem cells from patients with hereditary liver disease can also be used to repair genes through genome editing using systems such as CRISPR-Cas, and then differentiate into target cells, thereby using these cells as therapeutic agents for the disease.

[0089] There are no particular limitations on the source species of pluripotent stem cells. For example, they can be cells from rodents such as rats, mice, hamsters, and guinea pigs; lagomorphs such as rabbits; even-toed ungulates such as pigs, cattle, goats, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees. The preferred source species is humans.

[0090] Activator A can be a commercially available product, such as those from R&D Systems. The concentration of activator A in the culture medium used in this process is typically 1 ng / ml to 1000 ng / ml, preferably 10 ng / ml to 500 ng / ml, and more preferably 50 ng / ml to 200 ng / ml (in one embodiment, 100 ng / ml).

[0091] The GSK-3 used in this invention β Inhibitors are any substances that can inhibit GSK-3. β The function of the substance, such as kinase activity, is not particularly limited; for example, BIO (also known as GSK-3), a derivative of indirubin, can be listed. β Inhibitor IX, 6-bromoindorubin 3'-oxime; SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione) as a maleimide derivative; and phenyl α GSK-3, a compound of bromomethyl ketone β Inhibitor VII (4-dibromoacetophenone), L803-mts (also known as GSK-3), a cell membrane permeable phosphorylated peptide. β Peptide inhibitors, such as Myr-N-GKEAPPAPPQSpP-NH2, and the highly selective CHIR99021 (Nature (2008) 453: 519-523), are used. CHIR99021 is preferred. GSK-3 is used in this process. β The concentration of the inhibitor can be determined based on the GSK-3 used. β Inhibitors are appropriately selected by those skilled in the art, for example, when using CHIR99021 as a GSK-3 inhibitor. β In the case of inhibitors, the concentration in the culture medium is typically 0.01. μ M~10 μ M, preferably 0.05 μ M~5 μ M, more preferably 0.1 μ M~2.5 μ M (in one scheme, it is 1) μ M).

[0092] Examples of HDAC inhibitors used in this invention include valproic acid (VPA), trichostatin A, NaB, vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, apicidin, and MGCD-0103. The concentration of the HDAC inhibitor used in this process can be appropriately selected by those skilled in the art based on the specific HDAC inhibitor used.

[0093] The duration of the endoderm cell manufacturing process is not considered to have a significant impact on the manufacturing efficiency of endoderm cells due to speculation that long-term culture has no particular effect, and therefore has no upper limit, typically less than 20 days (e.g., less than 15 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days). Alternatively, the culture period can be more than 2 days, more than 3 days, more than 4 days, more than 5 days, or more than 6 days. Typically, it is 2 to 8 days, preferably 2 to 7 days, and more preferably 3 to 6 days (in one embodiment, 4 days).

[0094] When the substances used in this invention are proteins or peptides such as OSM or activator A, their source is not particularly limited, but is preferably mammalian (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.), with human origin being more preferred. In one embodiment, the proteins or peptides used in this invention are human or orthologs of humans from other mammalian species. Furthermore, the proteins or peptides used in the preparation method of this invention include not only wild-type proteins or peptides, but also variants thereof having the same function (e.g., agonist effect on OSMR). Examples of variants include proteins or peptides that exhibit high homology (e.g., 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) relative to the amino acid sequence of a specific wild-type protein or peptide (e.g., the sequence shown in sequence number 2 or 4). Also preferred are variants of the wild-type protein or peptide that consist of an amino acid sequence consisting of one or more (2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids that are missing, substituted, inserted and / or added to the amino acid sequence of a particular wild-type protein or peptide (e.g., the sequence shown in sequence number 2 or 4) and have the same function as the wild-type protein or peptide.

[0095] When the substance used in this invention is a compound, the compound includes not only the free form but also its pharmacologically acceptable salt. Pharmacologically acceptable salts vary depending on the type of compound. Examples include inorganic base salts such as alkali metal salts (sodium, potassium, etc.), alkaline earth metal salts (calcium, magnesium, etc.), aluminum salts, and ammonium salts; and organic base salts such as trimethylamine, triethylamine, pyridine, methylpyridine, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine, which are base addition salts; or inorganic acid salts such as methanesulfonates, hydrochlorides, hydrobromates, sulfates, hydroiodates, nitrates, and phosphates; and organic acid salts such as citrates, oxalates, acetates, formates, propionates, benzoates, trifluoroacetates, maleates, tartrates, methanesulfonates, benzenesulfonates, and p-toluenesulfonates, which are acid addition salts. For example, phenylephrine also includes salts such as phenylephrine hydrochloride.

[0096] When the compound used in this invention has isomers such as optical isomers, stereoisomers, positional isomers, and rotational isomers, any such isomer or mixture thereof is included in the compound. These isomers can be obtained individually using known synthetic methods, separation methods (e.g., concentration, solvent extraction, column chromatography, recrystallization, etc.), and optical fractionation methods (e.g., fractional recrystallization, chiral column chromatography, diastereomeric methods, etc.). The compound used in this invention can be crystalline, and both single-crystal and mixed-crystal forms are included in the compound. Crystallization can be achieved by crystallization using known crystallization methods. The compound used in this invention can be a solvate (e.g., hydrate, etc.) or a solvateless (e.g., non-hydrate, etc.), and both are included in the compound. Furthermore, the compound used in this invention also contains isotopes (e.g.,...). 3 H, 14 C 35 S, 125 Compounds labeled with I et al.

[0097] The culture used in the hepatoblast production process and / or endoderm cell production process can be adhesion culture or suspension culture, preferably adhesion culture. Adhesion culture is typically performed by culturing cells in coated culture containers. Examples of coating agents include, for instance, BD Biosciences, Synthemax (Corning), collagen, gelatin, heparan sulfate proteoglycan, or nestin, and combinations thereof, preferably BD Biosciences, Synthemax, or gelatin.

[0098] Alternatively, from the viewpoint of having no heterologous components, laminin or fragments thereof are also preferred. Examples of laminin or fragments thereof include, for example, laminin-111 or a fragment containing its E8 region, laminin-211 or a fragment containing its E8 region (e.g., iMatrix-211), laminin-121 or a fragment containing its E8 region, laminin-221 or a fragment containing its E8 region, laminin-332 or a fragment containing its E8 region, laminin-3A11 or a fragment containing its E8 region, laminin-411 or a fragment containing its E8 region (e.g., iMatrix-411), laminin-421 or a fragment containing its E8 region, and laminin-511 or a fragment containing its E8 region (e.g., iMatrix-511, iMatrix-511). Laminin-521 or a fragment containing its E8 region, laminin-213 or a fragment containing its E8 region, laminin-423 or a fragment containing its E8 region, laminin-523 or a fragment containing its E8 region, laminin-212 / 222 or a fragment containing its E8 region, laminin-522 or a fragment containing its E8 region, etc.

[0099] The culture media used in the hepatoblast cell manufacturing process and the endoderm cell manufacturing process can typically be prepared by adding FGF2, BMP4, activin A, and GSK-3 to the basal medium. β Differentiation-inducing agents such as inhibitors are used to prepare the culture medium for the target cells. Examples of basal culture media include IMDM medium, Medium 199 medium, and Eagle's Minimum Essential Medium (EMEM). α MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, KnockOut TMDMEM medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and their mixed media are available. The basal medium may contain serum (e.g., fetal bovine serum (FBS), horse serum, human serum, etc.) or be serum-free. In the case of serum-free medium, depending on the requirements, it may contain one or more serum substitutes such as albumin, transferrin, KSR (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, etc. In addition, the basal medium may also contain one or more substances selected from lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and their equivalents. In one embodiment, the basal culture medium used in the hepatocyte manufacturing process is KnockOut, which contains KSR, L-glutamine, NEAA, 2-mercaptoethanol, and antibiotics. TM DMEM medium. Alternatively, in one embodiment, the medium used in the hepatoblast production process is RPMI 1640 medium containing B27 supplement and antibiotics.

[0100] The hepatocyte manufacturing process and / or endoderm cell manufacturing process can be a process of culturing cells under conditions without feeder layer and / or under conditions without heterologous components.

[0101] The culture temperature in each step of the preparation method of the present invention is not particularly limited, but is 30°C to 40°C, preferably 37°C, and is carried out in an air atmosphere containing CO2, with the CO2 concentration preferably being 2% to 5%.

[0102] The cell seeding density in each step of the manufacturing process of this invention is not particularly limited, as long as the cells can proliferate. Typically, it is 1.0 × 10⁻⁶. 2 ~1.0×10 7 cells / cm 2 Preferably 1.0×10 3 ~1.0×10 6 cells / cm 2 More preferably 1.0×10 4 ~1.0×10 5 cells / cm 2 .

[0103] The substances used in this invention can be synthesized using known methods or commercially available products. Furthermore, in the case of compounds that are proteins or peptides such as Wnt protein, the target protein or peptide can be obtained from cells that express them. Cells expressing the target protein or peptide can be created by inserting DNA encoding the target protein or peptide into a known expression vector and introducing the resulting expression vector into a suitable host cell.

[0104] The preparation method of this invention may include a step of recovering the target cells or tissues obtained in each step. The recovered cells may be cryopreserved using a cell cryopreservation solution. In addition, the various cells obtained may be counted using a cell counter, labeled with antibodies against cell surface markers, and screened or purified by flow cytometry, mass cytometry, magnetic cell separation, or the like.

[0105] 2. Uses of hepatocytes In another aspect of the invention, hepatocytes (hereinafter sometimes referred to as "hepatocytes of the invention") obtained by the preparation method of the invention are also provided ("obtained" may be appropriately interpreted as "has been obtained"). The hepatocytes of the invention may be in the form of hepatocyte clusters or single cells obtained by flow cytometry or mass spectrometry, magnetic cell separation, etc.

[0106] The hepatocytes of the present invention can be used in regenerative medicine and other treatments; therefore, in another embodiment, a transplantation therapy agent comprising the hepatocytes of the present invention is provided (hereinafter sometimes referred to as "the transplantation therapy agent of the present invention"). Furthermore, methods for treating or preventing liver diseases by transplanting an effective amount of the hepatocytes of the present invention into mammals (e.g., humans, mice, rats, monkeys, cattle, horses, pigs, dogs, etc.) that are the subjects of treatment or prevention are also included in the present invention. In this specification, unless otherwise stated, the medicine (or method of treatment or prevention) for the disease also includes a drug (or method) that can treat and prevent the disease.

[0107] Liver diseases that can be treated or prevented include, for example, hemorrhagic pigmentation disorders, Wilson's disease, glycogen storage disease, amino acid metabolism disorders, urea cycle metabolism disorders, porphyria, constitutional jaundice, fibrotic polycystic liver disease, non-alcoholic steatohepatitis, liver cancer, hepatitis B, liver fibrosis, cirrhosis, and hereditary ATTR amyloidosis (familial amyloid polyneuropathy (FAP)). α -1 Antitrypsin deficiency (AATD), etc. The hepatocytes of this invention exert therapeutic or preventative effects on liver diseases by transplanting them to the affected area or to sites removed for treatment of diseases.

[0108] The transplant therapy agent of this invention can be transplanted into the body of a recipient. The amount of liver cells transplanted can be as small as possible for therapeutic or preventative purposes, and can vary depending on factors such as the recipient's age, weight, size of the transplant site, and severity of the disease; there are no particular limitations. For example, the number of cells can be 10 × 10⁻⁶. 4 10 × 10 cells 11 Approximately one cell per day. More specifically, the transplantation of the transplant agent of the present invention to a patient can be exemplified by methods such as thinning and attaching the hepatocytes of the present invention to the patient's liver, directly transplanting the obtained hepatocytes into the patient's liver by suspending them in physiological saline or the like, and transplanting the obtained hepatocyte clusters by three-dimensional culture on a scaffold made of matrix gel or the like.

[0109] When using the hepatocytes of the present invention as a transplant therapy agent, from the viewpoint of avoiding rejection, it is preferable to use hepatocytes derived from iPS cells, which are constructed from somatic cells with the same or substantially the same HLA genotype as the target individual. Here, "substantially the same" means that the transplanted cells have a consistent HLA genotype to the extent that immunosuppressants can suppress the immune response, for example, somatic cells with the same HLA type having three loci (HLA-A, HLA-B, and HLA-DR) or four loci (HLA-C). If sufficient cells cannot be obtained due to age or physical condition, they can be encapsulated in capsules or porous containers such as polyethylene glycol or silicone, and transplantation can be performed without the risk of rejection.

[0110] The hepatocytes of the present invention can be mixed with pharmaceutically acceptable carriers using conventional methods to prepare non-oral formulations such as injections, suspensions, and infusions. Therefore, in one embodiment, a method for preparing a transplant therapy agent is also provided, the method including a step of formulation of the hepatocytes of the present invention. Such a method may also include a step of preparing the hepatocytes of the present invention. Furthermore, a step of preserving the hepatocytes of the present invention may also be included.

[0111] Pharmaceutically acceptable carriers that can be included in this non-oral formulation include, for example, physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), and other aqueous solutions for injection. The hepatocytes of the present invention can be combined with, for example, buffers (e.g., phosphate buffer, sodium acetate buffer), analgesics (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.

[0112] The transplantation therapy agent of the present invention can also be provided in a cryopreserved state under conditions typically used for cell cryopreservation, and thawed for use. In this case, it may further contain serum or a substitute thereof, an organic solvent (e.g., DMSO), etc. In this case, the concentration of serum or a substitute thereof is not particularly limited, and may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, and may be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0113] Furthermore, the hepatocytes of the present invention can also be used in methods for screening agents, i.e., candidate agents, that are useful for the treatment or prevention of liver diseases. Therefore, in yet another aspect of the present invention, a method for screening drugs for the treatment or prevention of liver diseases is provided, the screening method comprising the step of culturing the hepatocytes of the present invention in the presence or absence of a test sample. For example, as hepatocytes, when using a disease model reflecting a pathological state of liver disease that is improved in the presence of a test sample, the test sample can be selected as a candidate substance for the treatment or prevention of liver diseases. Examples of such liver diseases include those identical to the aforementioned liver diseases that are the targets of treatment or prevention for the transplant therapy agents of the present invention.

[0114] The hepatocytes of this invention can also be used for in vivo dynamic evaluation or toxicity evaluation of drugs. Specifically, using the hepatocytes of this invention, for example, one can test drug interactions of test samples, induction of drug-metabolizing enzymes, induction of drug transport proteins, toxicity, etc.

[0115] Since the hepatocytes of this invention can express drug-metabolizing enzymes, the activity of these enzymes can be used as an indicator to evaluate the extent of drug (test sample) metabolism. By evaluating the extent of drug metabolism, the efficacy and toxicity of the drug in vivo can also be assessed. Furthermore, by analyzing (e.g., detection, quantification, identification, etc.) the drug metabolites generated from the enzymatic reaction using a drug as a substrate with drug-metabolizing enzymes, the efficacy and toxicity of the drug in vivo can also be evaluated. Therefore, in another aspect of this invention, a method for evaluating the extent of metabolism of a test sample is provided, the method comprising: (I) The step of bringing the test sample into contact with the hepatocytes of the present invention. (II) Procedures for determining the activity levels of drug-metabolizing enzymes in these hepatocytes, and (III) A process for evaluating the extent to which the test sample is metabolized by drug-metabolizing enzymes based on the activity level of drug-metabolizing enzymes in step (II).

[0116] Step (II) typically involves determining the expression levels of drug-metabolizing enzymes. The expression levels of drug-metabolizing enzymes can be evaluated at the mRNA or protein level. For example, if the mRNA level of a drug-metabolizing enzyme is increased compared to before exposure to the test sample or compared to exposure to a negative control, it can be determined that at least a portion of the test sample has been metabolized. Alternatively, the extent of drug metabolism (e.g., the proportion of drug metabolized, the rate of metabolism, etc.) can also be evaluated by the expression level. Examples of drug metabolism include chemical modifications of the test sample such as hydrolysis of esters, oxidation reactions (especially oxidation caused by cytochrome P450), and reduction reactions.

[0117] Additionally, in step (I), the hepatocytes of the present invention can also be reacted with a substance known to be metabolized by drug-metabolizing enzymes (e.g., triazolam (metabolite: α The drug can be reacted with substances such as 4-hydroxytriazolam, diclofenac (metabolite: 4-hydroxydiclofenac), and 7-hydroxycoumarin (metabolite: 7-hydroxycoumarin glucoside), and then the amount of the metabolite is measured. Alternatively, a commercially available drug-metabolizing enzyme activity assay kit (e.g., P450-Glo) can be used. TM The assay was performed using CYP3A4 Assay, etc.

[0118] Examples of drug-metabolizing enzymes used in this invention include cytochrome P450 (e.g., CYP3A4, CYP2C9, etc.), uridine diphosphate glucuronide transferases (e.g., UGT1A1, UGT1A8, UGT1A10, etc.), and sulfate transferases (e.g., SULT1A3, etc.). Multiple drug-metabolizing enzymes can be used in combination.

[0119] In yet another embodiment, according to the present invention, a method for evaluating the effects or toxicity of a drug in vivo is provided, the method comprising: (i) The process of contacting the test sample with the hepatocytes of the present invention, and (ii) Procedures for analyzing the metabolites of the test sample.

[0120] For example, if the amount of metabolites of the test sample is low, it can be determined that the test sample is unlikely to be affected by metabolism in the liver and has a high efficacy in vivo. Conversely, if the metabolites are toxic, it can be determined that the test sample is toxic to the organism.

[0121] As for the analysis of metabolites, as long as the appropriate analytical method is selected according to the test sample and the predicted metabolites, such as mass spectrometry, liquid chromatography, and immunological methods (e.g., fluorescence immunoassay (FIA) and enzyme immunoassay (EIA)) can be used.

[0122] Furthermore, a method for evaluating the toxicity of a drug in vivo is provided, the method comprising a step of contacting the test sample with the hepatocytes of the present invention, and a step of evaluating the state of the hepatocytes. The state of such hepatocytes can be evaluated by measuring survival rate, observing cell morphology, and measuring liver disease markers (GOT, GPT, etc.) in the culture medium. For example, when a decrease in survival rate is observed due to contact with the test sample, the test sample can be determined to be hepatotoxic. Similarly, when abnormal cell morphology is observed due to contact with the test sample, or when the amount of liver disease markers in the culture medium increases, the test sample can also be determined to be hepatotoxic. Quantitative determination can also be made based on the degree of decrease in survival rate or the amount of liver disease markers.

[0123] The process of bringing the test sample into contact with the hepatocytes of the present invention can typically be performed by adding the test sample to the culture medium in which the hepatocytes of the present invention are cultured, or by transferring the hepatocytes of the present invention to a culture medium in which the test sample has been added.

[0124] The duration of the process of contacting the test sample with the hepatocytes of the present invention is not particularly limited, but typically ranges from 1 minute to 5 days, preferably from 1 hour to 1 day.

[0125] Examples of test samples used in this invention include cell extracts, cell culture supernatants, microbial fermentation products, extracts from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low-molecular-weight compounds, and natural compounds. Test samples can be existing or candidate ingredients of pharmaceuticals or nutritional foods. Furthermore, by simultaneously contacting two or more test samples with cells, interactions and synergistic effects between the test samples can be verified.

[0126] The following examples illustrate the invention in more detail, but the invention is not limited to these examples. Example

[0127] Hepatocyte clusters were prepared following these steps. In this embodiment, human pluripotent stem cells (line 1231A3) were maintained and cultured using a feeder-free method as starting cells for subsequent differentiation induction experiments.

[0128] Phase 1 Human pluripotent stem cells were washed in each well with 0.5 mM EDTA (UltraPure 0.5 M EDTA, Thermo Fisher Scientific, Cat# 15575020) diluted with PBS(-) (Nacalai Tesque, Cat# 14249-24), and then dispersed using TrypLE Select CTS (Thermo Fisher Scientific, Cat# A1285901) diluted 2-fold with 0.5 mM EDTA. Cell counts were performed, and the necessary cell suspensions were suspended in a solution containing 10... μ Phase 1 culture medium for MY-27632 (Fujifilm-Wako, Cat# 253-00513) (48.5 ml RPMI 1640 (Nacalai Tesque, Cat# 30264-50), 1 ml B-27 supplement (Thermo Fisher Scientific, Cat# 17504044), 500 ml... μ 1 penicillin-streptomycin (Thermo Fisher Scientific, cat# 15140122), 100 ng / ml activator A (R&D, cat# 338-AC-01M), 1 μ M CHIR99021 (Axon Medchem, Cat# Axon 1386) was used, and then on 12-well (Greiner) plates coated with BD Matrigel Matrix Growth Factor Reduced (BD Biosciences, Cat# 354230) at 4 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well. The medium was changed several times with Stage 1 medium until day 4 of induction.

[0129] Phase 2 Starting from day 4 of induction, change the medium with Stage 2 medium. Use Stage 2 medium (44 ml KnockOut DMEM (Thermo Fisher Scientific, Cat# 10829018), 5 ml KnockOut serum supplement (Thermo Fisher Scientific, Cat# 10828028), 500 ml... μ l L-glutamine 200mM (Thermo Fisher Scientific, CAT# 25030081), 500 μl MEM non-essential amino acid solution x100 (Thermo Fisher Scientific, CAT# 11140050), 100 μ l 2-mercaptoethanol (Thermo Fisher Scientific, CAT#21985023), 250 μ The culture medium was changed every 2 days with 1 penicillin-streptomycin (Thermo Fisher Scientific, cat# 15140122), 20 ng / ml BMP4 (Peprotech, cat# AF-120-05ET), and 10 ng / ml FGF2 (Fujifilm-Wako, cat# 060-04543).

[0130] Phase 3 Cells were dispersed on day 9 of induction using Accumax (Nacalai Tesque, Cat# 1708754) or TrypLE SelectCTS and suspended in a solution containing 10 μ MY-27632 was cultured in stage 3 medium (HCM Bullet Kit (Lonza, Cat# CC-3198, without using the EGF included with the kit), candidate compound) with stirring.

[0131] (1) Case where microporous culture bags are not used (existing method) Cell suspensions were seeded into uncoated 12-well plates. The seeding density could be adjusted appropriately, for example, dispersing cells recovered from one well into one well, or dispersing cells recovered from two wells into one well. The 12-well plates were placed on a shaker in a 37°C, 5% CO2 incubator and cultured with agitation. On the second day, large cell clusters formed, and the medium was replaced with Stage 3 medium for stirred culture without Y-27632. Thereafter, the medium was replaced every two days using the same medium. HLC aggregates obtained on day 6 of stirred culture were used for analysis.

[0132] (2) Use of microporous culture bags (culture bag method) Two 12-well plates containing induced cells were suspended in a 10 mL container. μThe MY-27632-containing stirred culture stage 3 medium was injected into one 18,000-well microporous culture bag (Toyo Seikan) and incubated overnight at 37°C with 5% CO2. The resulting cell clusters were replaced with stirred culture stage 3 medium without MY-27632, and then seeded into two 12-well plates for stirred culture. The medium was replaced on day 6 of stirred culture, and the HLC aggregates obtained on day 7 were used for assays.

[0133] Example 1: Verification of the role of a mixture of compounds in hepatocyte differentiation According to reports, NOTCH signal and TGF β The signal promotes differentiation from hepatoblasts into bile duct epithelial cells, but on the other hand, it hinders differentiation into hepatocytes. Furthermore, it has been reported that PKA agonists promote induced functional maturation of hepatocytes (Lay TengAng et al., Cell Rep. 22(8): 2190-2205 (2018)). Figure 1 Based on these reports of planar culture, combinations of compounds considered useful for inducing differentiation into hepatocytes were investigated. Unless otherwise stated, the abbreviations of the compounds have the following meanings.

[0134] OSM: 20 ng / ml Antitumor M HGF: 20 ng / ml hepatocyte growth factor DEX: 100nM Dexamethasone Phe or P: 1 μ M-benfural A: 1 μ M A83-01 D: 10 μ M DAPT (N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester) F: 10 μ M-Foscoline Figure 2 (a) shows the culture protocol of Example 1. Hepatocytes were cultured in Stage 3 medium with agitation from day 9 of induction. Candidate compounds (HGF+OSM+DEX, HGF+OSM, OSM, or OSM+Phe) were added to the Stage 3 medium, and evaluation was performed on day 15 of induction. The results showed no significant differences in the morphology of the HLC aggregates. Figure 2 (b)). In addition, the amount of albumin secreted into the culture medium by each HLC aggregate every 24 hours was determined by ELISA. Figure 2(c)). As a result, no additive effect of HGF, DEX, and Phe on the maturation of HLC aggregates was observed with OSM. In the following examples, the evaluation of the combination of candidate compounds in the stage 3 medium during stirred culture was performed by assessing whether or not OSM had an additive effect.

[0135] Example 2: Verification of the effect of the combination of OSM and ADPF mixture Figure 3 (a) shows the culture protocol of Example 2. Similar to Example 1, hepatocytes were cultured in Stage 3 medium with agitation from day 9 of induction. Candidate compounds (HGF+OSM, OSM, OSM+ADP, or OSM+ADPF) were added to the Stage 3 medium, and evaluation was performed on day 15 of induction. As a result, the morphology of HLC aggregates showed a clear change by adding the ADPF mixture to the medium. Figure 3 (b)). In addition, the amount of albumin secreted into the culture medium by each HLC aggregate every 24 hours was determined by ELISA. Figure 3 (c) As a result, the addition of ADPF mixture to tumor suppressin M significantly increased albumin secretion.

[0136] Example 3: Minus-one analysis and plus-one analysis of ADPF mixtures and OSM mixtures Figure 4 (a) shows the culture protocol of Example 3. Similar to Example 1, hepatocytes were cultured in Stage 3 medium with agitation from day 9 of induction. A mixture of OSM+ADPF or a mixture obtained by removing one compound (OSM+ADPF, OSM+DPF, OSM+APF, OSM+ADF, or OSM+ADP) was added to the Stage 3 medium, and evaluation was performed on day 15 of induction. The results showed that no morphological changes in HLC aggregates induced by the ADPF mixture were observed if forscorin was removed. Figure 4 (b)). In addition, the amount of albumin secreted into the culture medium by each HLC aggregate every 24 hours was determined by ELISA. Figure 4 (c) As a result, the removal of forscolin from the ADPF mixture significantly reduced albumin secretion. Therefore, it can be said that forscolin is a major factor in the maturation of HLC aggregates in the compound mixture.

[0137] Furthermore, a mixture of OSM containing one compound (OSM+A, OSM+D, OSM+P, OSM+F, OSM+ADPF, or OSM+APF) was added to the Stage 3 medium, and the results were evaluated on day 15 of induction. The results showed that the addition of forscoringin to OSM resulted in morphological changes in HLC aggregates. Figure 4 (d) Additionally, the amount of albumin secreted into the culture medium by each HLC aggregate every 24 hours was determined by ELISA. Figure 4 (e)). As a result, the additive effect of forscorin on OSM was significant. On the other hand, no additive effect of DAPT on OSM was observed; instead, albumin secretion increased by removing DAPT from the ADPF mixture, thus DAPT is ineffective in HLC aggregate maturation.

[0138] Example 4: Verification of the effect of the combination of OSM and APF mixture Figure 5 (a) illustrates the culture protocol of Example 4. Similar to Example 1, hepatocytes were cultured in Stage 3 medium with agitation from day 9 of induction. Candidate compounds (OSM+F, OSM+AF, OSM+DF, OSM+PF, or OSM+APF) were added to the Stage 3 medium, and evaluation was performed on day 15 of induction. The results showed that, although cell clusters were produced from approximately the same number of cells under all conditions, HLC aggregates were obtained most efficiently under the OSM+APF condition. Figure 5 (b)). In addition, the amount of albumin secreted into the culture medium by each HLC aggregate every 24 hours was determined by ELISA. Figure 5 (c)). As a result, albumin secretion was highest under OSM+APF. This indicates that the most suitable combination of compounds for stirring hepatocytes to obtain hepatocyte clusters is OSM+APF.

[0139] Example 5: Verification of the fabrication of HLC aggregates using a porous device It was verified that uniform HLC aggregates can be produced by using porous devices (also known as microporous culture bags). Figure 6 (a) illustrates the culture protocol of Example 5. On day 9 of induction, hepatocytes suspended in stage 3 medium were injected into microporous culture bags (…). Figure 6 In (b)), the cells were suspended in culture (static culture) overnight to form cell clusters (forced aggregation), and then the cell clusters were re-seeded into 12-well plates for stirred culture. OSM+APF was added to the stage 3 medium, and evaluation was performed on day 17 of induction. The results showed that homogeneous HLC aggregates could be effectively produced by using a porous device. Figure 6 (b)-(f)).

[0140] Example 6: Comparison of stirred culture and planar culture using OSM+APF The maturity of HLC in stirred culture and planar culture was compared. Figure 7(a) illustrates the culture protocol of Example 6. In planar culture, after day 9 of induction, 20 ng / ml OSM and 20 ng / ml HGF were added to the culture medium to prepare HLCs. In stirred culture, HLC aggregates were prepared according to the method of Example 5. Evaluation was performed on day 14 of induction in planar culture and on day 17 of induction in stirred culture. As a result, in terms of gene expression, the HLC aggregates prepared by OSM+APF and stirred culture were more mature than those obtained by conventional planar culture. Figure 7 (b)).

[0141] Example 7: Evaluation of HLC prepared under various culture conditions HLC was evaluated by varying the timing of RNA recovery, culture type (suspension or planar culture), and conditions with or without agitation. Culture protocols and results are shown in [Table / Insert Table ... Figures 8-12 These results indicate that, regardless of whether oscillation is present, more mature HLCs can be obtained in suspension culture compared to planar culture, but more mature HLCs can be obtained by oscillation.

[0142] Practicality in industry According to the present invention, hepatocytes can be manufactured in vitro. Since the cells manufactured in this way have characteristics more closely resembling those of living hepatocytes, they can be used for screening innovative drugs, evaluating drug toxicity, and regenerative medicine for liver diseases, and therefore are expected to have applications in the medical field, particularly in regenerative medicine.

[0143] This application is based on Japanese Special Application No. 2024-038486 (application date: March 12, 2024), the contents of which are included in this specification.

Claims

1. A method for producing hepatocytes, the method comprising the step of suspending and culturing hepatocytes in a culture medium containing a tumor suppressor M receptor agonist, an ALK inhibitor, a cAMP agonist and an adrenaline receptor agonist.

2. The method according to claim 1, wherein, Hepatocytes are morphological aggregates.

3. The method according to claim 1 or 2, wherein the method includes a step of performing an oscillating culture.

4. The method according to any one of claims 1 to 3, wherein the method includes a step of culturing in a culture bag having a plurality of pores.

5. The method according to any one of claims 1 to 4, wherein, Suspension culture is cultured in a medium that does not contain hepatocyte growth factor and / or DAPT.

6. The method according to any one of claims 1 to 5, wherein, At least one of the tumor suppressor M receptor agonists is tumor suppressor M.

7. The method according to any one of claims 1 to 6, wherein, At least one of the ALK inhibitors is A83-01.

8. The method according to any one of claims 1 to 7, wherein, At least one of the cAMP agonists is foscorine.

9. The method according to any one of claims 1 to 8, wherein, At least one of the adrenergic receptor agonists is phenylephrine.

10. The method according to any one of claims 1 to 9, wherein, Hepatoblasts originate from pluripotent stem cells.

11. The method according to any one of claims 2 to 10, wherein, The hepatoblast aggregates were obtained by suspension culture in a medium containing a tumor suppressor M receptor agonist, an ALK inhibitor, a cAMP agonist, and an adrenaline receptor agonist.

12. Hepatocytes, said hepatocytes being obtained by the method according to any one of claims 1 to 11.

13. A cell transplantation therapy agent comprising hepatocytes according to claim 12.

14. The pharmaceutical preparation according to claim 13, wherein the pharmaceutical preparation is used to treat or prevent liver disease.

15. A method for evaluating the metabolic extent of a test sample, the method comprising: (I) The process of bringing the test sample into contact with the hepatocytes according to claim 12; (II) Procedures for determining the activity levels of drug-metabolizing enzymes in the hepatocytes; and (III) A process for evaluating the extent to which the test sample is metabolized by drug-metabolizing enzymes based on the activity level of drug-metabolizing enzymes in step (II).

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