Method for assessing excretion of test substances by human hepatocyte-like cells

Abstract

A method for evaluating the excretion of a substance of interest by a human hepatocyte-like cell comprises: providing a bladderlike-material-containing solution which contains a bladderlike material produced in vitro and having a membrane of a human hepatocyte-like cell and 0 to 10% by volume of a suspended extracellular matrix; adding the substance of interest to the bladderlike-material-containing solution to bring the substance of interest into contact with the bladderlike material; and removing the bladderlike material from the bladderlike-material-containing solution and measuring the concentration of the substance of interest or a metabolite thereof excreted into an inner cavity of the bladderlike material.

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the excretion of a test substance by human hepatocyte-like cells, and a method for producing sacs having a membrane of human hepatocyte-like cells. This application claims priority based on Japanese Patent Application No. 2020-195476, filed on November 25, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] The liver is composed of hepatocytes, which are responsible for the essential functions of the liver, and non-parenchymal cells, which support the proliferation and survival of hepatocytes. Hepatocytes are also called hepatocytes. Non-parenchymal cells include hepatic stellate cells, sinusoidal endothelial cells, Kupffer cells, and bile duct epithelial cells.

[0003] Hepatocytes convert exogenous drugs and substances produced during metabolism into less toxic substances and excrete them through the following two excretion pathways. The first pathway excretes the converted, less toxic substances into the bile canaliculi along with bile, then passes through the gallbladder and intestine and is excreted from the body in feces. The second pathway excretes the converted, less toxic substances into the sinusoids, reaches the kidney, and is excreted from the body in urine. It is known that the transporters that take up exogenous drugs and substances produced during metabolism into hepatocytes and the transporters that excrete the converted, less toxic substances are different in these two excretion pathways.

[0004] In drug discovery, many of the candidate drugs identified in initial screening are eliminated in the preclinical trial stage. The large number of rejected candidate drugs leads to the loss of treatment opportunities for patients. Preclinical trials consist of screening the efficacy of candidate drugs and testing their safety, and it is known that the use of in vitro preclinical models is useful for these screenings (see, for example, Non-Patent Documents 1 to 3). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Oshikata MA et al., “Development of an oxygenation culture method for activating the liver-specific functions of HepG2 cells utilizing a collagen vitrigel membrane chamber.”, Cytotechnology, Vol. 68, pp. 1801-1811, 2016. [Non-patent document 2] Jang KJ et al., “Reproducing human and cross-species drug toxicities using a Liver-Chip”, Sci. Transl. Med., Vol. 11, Issue 517, eaax5516, 2019, doi: 10.1126 / scitranslmed.aax5516. [Non-patent document 3] Swift B et al., “Sandwich-Cultured Hepatocytes: An In Vitro Model to Evaluate Hepatobiliary Transporter-Based Drug Interactions and Hepatotoxicity”, Vol. 42, Issue 3, pp. 446-471, 2010. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made in consideration of the above circumstances, and provides a method for evaluating the excretion of a test substance by human hepatocyte-like cells using an evaluation model for evaluating excretion by human hepatocyte-like cells, and a method for producing the evaluation model. [Means for solving the problem]

[0007] That is, the present invention includes the following aspects. (1) A method for evaluating the excretion of a test substance by human hepatocyte-like cells, comprising: providing a sac-containing solution containing sacs having a membrane of in vitro generated human hepatocyte-like cells and 0 to 10% by volume of suspended extracellular matrix; placing a test substance in the sac-containing liquid to contact the test substance with the sac-like substance; and removing the sac-like substance from the sac-like substance-containing liquid and measuring the concentration of the test substance or its metabolites excreted into the lumen of the sac-like substance; A method comprising: (2) The method according to (1), wherein the human hepatocyte-like cells are cells that can be cultured as organoids. (3) The method according to (1) or (2), wherein the human hepatocyte-like cells have at least one transporter selected from the group consisting of BSEP, MRP2, P-gp, MATE, and BCRP. (4) The method according to any one of (1) to (3), wherein in step 3, the concentration of the test substance or its metabolites excreted into the lumen of the sac-like substance is measured by decomposing the sac-like substance. (5) The method according to any one of (1) to (4), wherein in step 3, the concentration of the test substance or its metabolites excreted into the lumen of the sac-like substance is measured by liquid chromatography-mass spectrometry. (6) The method according to any one of (1) to (5), wherein the sac-like structure contains bile as a content in its lumen. (7) The method according to any one of (1) to (6), wherein the membrane of the human hepatocyte-like cells is a monolayer membrane. (8) A method for producing a sac-like substance having a membrane of human hepatocyte-like cells, comprising: Culturing human hepatic progenitor cells or human hepatic cells embedded in an extracellular matrix to form a culture; dispersing the culture to form a dispersion; and the dispersion is subjected to suspension culture in a medium in which an extracellular matrix is ​​suspended in an amount of 0 to 10% by volume relative to the total volume of the medium, thereby forming the cysts; The manufacturing method of the present invention. [Effects of the Invention]

[0008] According to the method of the above aspect, a method for evaluating the efflux of a test substance by human hepatocyte-like cells can be provided, using an evaluation model for evaluating efflux by human hepatocyte-like cells. According to the production method of the above aspect, an evaluation model for evaluating efflux by human hepatocyte-like cells can be provided. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a bright-field image of the evaluation model in Experimental Example 1. [Figure 2] Figure 2 shows images of the evaluation model in which Rho123 was incorporated into the cysts in Experimental Example 1. (a) is a bright-field image, and (b) is a fluorescent image. [Figure 3] 3 shows images of a culture in which Rho123 was incorporated into the lumen of the culture in Experimental Example 5. (a) is a bright-field image, and (b) is a fluorescent image. [Figure 4] FIG. 4 is a light microscope image of the cysts. [Figure 5] FIG. 5 shows an image of a culture in Experimental Example 6 in which Rho123 was incorporated into the lumen of the culture. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will be described in more detail below by showing embodiments, but the present invention is not limited to the following embodiments in any way.

[0011] Unless otherwise specified, each of the components exemplified in this specification, for example, components contained in the culture medium and components used in each step, can be used alone or in combination of two or more types.

[0012] In this specification, the expression "A to B" or the like representing a numerical range is synonymous with "A or more, B or less." Furthermore, in this specification, the expression "A to B, preferably a to b" or the like representing a numerical range is synonymous with "A or more, B or less," "A or more, b or less," "a or more, B or less," and "a or more, b or less."

[0013] As used herein, "a medium containing substance X" or "in the presence of substance X" means a medium to which exogenous substance X has been added, a medium containing exogenous substance X, or the presence of exogenous substance X. In other words, if cells or tissues present in the medium endogenously express, secrete, or produce substance X, endogenous substance X is distinguished from exogenous substance X, and a medium that does not contain exogenous substance X does not fall under the category of "a medium containing substance X," even if it contains endogenous substance X.

[0014] [Method for assessing the excretion of test substances by human hepatocyte-like cells] The method of this embodiment is a method for evaluating the excretion of a test substance (hereinafter also referred to as a "substrate") by human hepatocyte-like cells (hereinafter also referred to as the "evaluation method of this embodiment"), and includes the following steps 1 to 3. preparing a sac-containing solution containing a sac-like structure having a membrane of human hepatocyte-like cells produced in vitro (hereinafter also referred to as "the present evaluation model") and 0 to 10% by volume of a suspended extracellular matrix (hereinafter also referred to as "Step 1"); adding a test substance to the sac-containing liquid to bring the test substance into contact with the sac-like substance (hereinafter also referred to as "step 2"); Removing the sac-like substance from the sac-like substance-containing liquid and measuring the concentration of the test substance or its metabolites excreted in the sac-like substance (hereinafter also referred to as "step 3").

[0015] This evaluation model is suitable for use as a biomimetic model (Micro Physiological System, MPS) to evaluate the excretion of test substances by human hepatocyte-like cells. To mimic the structure of human liver tissue in vivo, the biomimetic model requires that human hepatocytes be oriented and arranged in a membranous configuration. To achieve this, human hepatic progenitor cells and human hepatocytes have traditionally been cultured embedded in an extracellular matrix. The resulting cysts are embedded in an extracellular matrix.

[0016] In order to quantify the concentration of a test substance taken up into a cystic structure by excretion, the extracellular matrix must be removed physically or chemically using enzymes or other methods when removing the test substance. However, removing the extracellular matrix may disrupt the human hepatocyte-like cell membrane of the cystic structure, potentially resulting in leakage of the test substance. On the other hand, if the test substance is removed from the cystic structure without removing the extracellular matrix and the concentration of the test substance is quantified using LC-MS or other methods, the extracellular matrix becomes noise, making it difficult to accurately measure the amount of the test substance. For these reasons, it has been difficult to quantify the amount of test substance excreted into the cystic structure using conventional cystic structures embedded in an extracellular matrix.

[0017] In contrast, in the evaluation method of the present embodiment, although 0 to 10% by volume of extracellular matrix is ​​present in the cystic material-containing liquid, the cystic material is not embedded in the extracellular matrix, and therefore the cystic material can be removed from the cystic material-containing liquid without breaking it, and the concentration of the test substance incorporated into the cystic material can be easily quantified.

[0018] The evaluation method of this embodiment enables highly accurate quantitative evaluation of the metabolism of a test substance, drug interactions, hepatotoxicity, transporter activity, etc. by human hepatocyte-like cells using liquid chromatography-mass spectrometry, instead of the low-precision evaluation that has traditionally been performed based on fluorescence intensity using fluorescently labeled substrates or bile.

[0019] <Process 1> In step 1, a cyst-containing solution containing the in vitro-produced evaluation model and 0 to 10% by volume of suspended extracellular matrix is ​​prepared.

[0020] The sac-like substance in this evaluation model is also called a cyst, and as shown in FIG. 4, has a bag-like structure formed by the membrane of human hepatocyte-like cells.

[0021] In this evaluation model, it is preferable that bile canaliculi-like structures are constructed between the human hepatocyte-like cells. The presence of bile canaliculi-like structures between the human hepatocyte-like cells allows the cells to have tight junctions.

[0022] The lumen of this evaluation model preferably contains bile. Furthermore, the human hepatocyte-like cells of this evaluation model preferably have at least one transporter selected from the group consisting of bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp), multidrug and toxin extrusion (MATE), and breast cancer resistance protein (BCRP). These transporters are known to act as bile efflux transporters for different substances.

[0023] Since this evaluation model has a bile canaliculus-like structure and the human hepatocyte-like cells of this evaluation model have the above-mentioned transporter, the test substance or its metabolites taken up into the human hepatocyte-like cells can be excreted from the human hepatocyte-like cells through the bile canaliculus-like structure by the above-mentioned transporter, to the outside of the human hepatocyte-like cells, and accumulated in the lumen of the sac-like structure together with bile.

[0024] In addition to the transporters described above, the human hepatocyte-like cells of this evaluation model can also have uptake transporters such as NTCP (Na+-taurocholate cotransporting polypeptide), OATP (organic anion transporting polypeptide) family transporters, and OCT1 (organic cation transporter 1).

[0025] The human hepatocyte-like cells of this evaluation model can further express genes for metabolic enzymes such as CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4.

[0026] The human hepatocyte-like cells of this evaluation model preferably have a monolayer membrane, which allows the evaluation of the effect of a test substance per cell.

[0027] The volume of this evaluation model is 4e -6 ~3e -2 mm 3 , preferably 3e -5 ~1e -2 mm 3 , more preferably 1e -4 ~4e -3 mm 3 is.

[0028] From the viewpoint of functional and property stability, the human hepatocyte-like cells of this evaluation model are preferably cells that can be cultured as organoids. Organoid culture is a three-dimensional self-organizing culture of stem cells, and the cells obtained by organoid culture are also called organoids.

[0029] The content of saccular structures in the saccular structure-containing liquid is 10 to 2,000 structures / mL, preferably 50 to 1,000 structures / mL, and more preferably 100 to 500 structures / mL.

[0030] The extracellular matrix includes components contained in basement membranes, glycoproteins present in intercellular spaces, etc. Components contained in basement membranes include type IV collagen, laminin, heparan sulfate proteoglycans, and entactin.

[0031] The content of extracellular matrix in the sac-containing liquid is 0 to 10% by volume, preferably 0.001 to 5% by volume, more preferably 0.1 to 4% by volume, and even more preferably 1 to 3% by volume, relative to the total volume of the sac-containing liquid.

[0032] The saccular-shaped substance-containing liquid is preferably a liquid containing a medium used for suspension culture, an extracellular matrix, and saccular-shaped substances having membranes of human hepatocyte-like cells in the "Method for producing saccular-shaped substances having membranes of human hepatocyte-like cells" described below.

[0033] <Process 2> In step 2, a test substance is placed in the sac-containing liquid prepared in step 1, and the test substance is brought into contact with the present evaluation model.

[0034] Test substances include natural compound libraries, synthetic compound libraries, existing drug libraries, and metabolite libraries. Organic or inorganic compounds of various molecular sizes can be used as test substances. 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.).

[0035] Test substances include components contained in pharmaceuticals, nutritional foods, etc. Test substances include plant extracts, cell extracts, and culture supernatants. By adding two or more test substances simultaneously, interactions and synergistic effects between the test substances can be investigated. Test substances may be derived from natural products or may be artificially synthesized.

[0036] The period for contacting the test substance with the present evaluation model is usually from 10 minutes to 3 days, preferably from 30 minutes to 1 day. The contacting of the test substance with the present evaluation model can be carried out in multiple sessions.

[0037] The test substance is taken up by the human hepatocyte-like cells of this evaluation model, and then the taken up test substance is either excreted into the lumen of this evaluation model, or the taken up test substance is metabolized by metabolic enzymes in the human hepatocyte-like cells of this evaluation model and excreted into the lumen of this evaluation model.

[0038] <Process 3> In step 3, the present evaluation model is removed from the sac-like fluid-containing fluid after step 2, and the concentration of the test substance or its metabolites excreted into the lumen of the present evaluation model is measured.

[0039] Before measuring the concentration, it is preferable to wash the evaluation model removed from the sac-containing fluid with phosphate-buffered saline (PBS) or similar, which can reduce the introduction of extracellular matrix into the concentration measurement environment.

[0040] The test substance in the lumen of the extracted evaluation model is preferably extracted by decomposing the membrane of the human hepatocyte-like cells. Methods for decomposing the membrane of human hepatocyte-like cells include, for example, chemical methods using methanol or physical methods such as homogenization.

[0041] The concentration of a test substance or its metabolites can be measured by mass spectrometry, liquid chromatography, immunological techniques, etc., depending on the type of test substance or its metabolite. Immunological techniques include, for example, fluorescence immunoassay (FIA) and enzyme immunoassay (EIA). Among these, liquid chromatography mass spectrometry (LC-MS) is preferred for measuring the concentration of a test substance or its metabolites because of its excellent quantitative properties.

[0042] Step 3 allows the toxicity of the test substance to be evaluated. For example, the state of the human hepatocyte-like cells after contact with the test substance is examined to evaluate the toxicity of the test substance. The human hepatocyte-like cells can be evaluated based on the viability, cell morphology, and the amount of liver damage markers (e.g., GOT, GPT) present in the culture medium.

[0043] [Method of manufacturing a sac-shaped substance having a membrane of human hepatocyte-like cells] The method for producing a sac-shaped material having a membrane of human hepatocyte-like cells of this embodiment (hereinafter also referred to as "the production method of this embodiment") includes the following steps A to C. Embedding human hepatic progenitor cells or human hepatic cells in an extracellular matrix and culturing them to form a culture (hereinafter also referred to as "Step A"); dispersing the culture to form a dispersion (hereinafter also referred to as "Step B"); The dispersion is cultured in suspension in a medium in which an extracellular matrix is ​​suspended in an amount of 0 to 10% by volume relative to the total volume of the medium, to form sac-like structures having a membrane of human hepatocyte-like cells (hereinafter also referred to as "Step C").

[0044] The sac-like structures having a membrane of human hepatocyte-like cells produced by the production method of this embodiment can be used as the evaluation model for the evaluation method of this embodiment. Each step of the manufacturing method of this embodiment will be described in detail below.

[0045] <Process A> In step A, human hepatic progenitor cells or human liver cells are embedded in an extracellular matrix and cultured to form a culture. The culture thus formed contains human hepatocyte-like cells.

[0046] Human hepatic progenitor cells can be differentiated into definitive endoderm by culturing human pluripotent stem cells in a medium containing factors such as TGFβ signaling promoters (Wnt, Nodal, activin A, etc.), or any combination thereof. Human hepatic progenitor cells can then be obtained by culturing the stem cells in a medium containing factors such as FGF, BMP, or any combination thereof. Examples of human pluripotent stem cells include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).

[0047] Human liver cells typically include hepatic parenchymal cells and non-parenchymal liver cells such as bile duct cells, sinusoidal endothelial cells, Kupffer cells, mesothelial cells, and hepatic stellate cells. Among these, non-parenchymal liver cells are preferred. It is preferable to use human hepatocytes in step A, since human hepatocyte-like cells with a genetic phenotype similar to that of wild-type human hepatocytes can be obtained.

[0048] Human hepatic progenitor cells or human liver cells are embedded in an extracellular matrix and cultured to proliferate and mature into human hepatocyte-like cells. Examples of the extracellular matrix include those exemplified in Step 1.

[0049] Examples of methods for embedding human hepatic progenitor cells or human liver cells in an extracellular matrix and culturing them include a method in which human hepatic progenitor cells or human liver cells are mixed with extracellular matrix precursors, the extracellular matrix precursors are polymerized to form extracellular matrix, and then a culture medium is layered on top to culture the cells.

[0050] The culture conditions in step A are typically a temperature of 30° C. to 40° C., preferably 37° C. Other culture conditions include typically an atmosphere with a CO 2 concentration of about 5% by volume.

[0051] The culture time can be adjusted appropriately depending on the number of cells, the state of the cells, etc. The medium can be changed every 1 to 3 days. Furthermore, in step A, by performing subculture multiple times, a culture with stable functions and properties can be obtained.

[0052] The culture in step A is preferably an organoid culture. The organoids obtained by organoid culture have high expression levels of transporters and metabolic enzyme genes, and the membranes of the human hepatocyte-like cells in the sac-like structures have high transepithelial electrical resistance and can form tight junctions.

[0053] (Culture medium) The medium used in step A preferably contains at least one selected from the group consisting of proliferation-related factors such as mitogenic growth factors, Wnt signaling promoters, Rho kinase (ROCK) signaling inhibitors, IL-6 family cytokines, and nicotinamide; differentiation-suppressing factors such as transforming growth factor β (TGF-β) signaling inhibitors; maturation-related factors such as retinoic acid, DAPT (γ-secretase inhibitor), dimethyl sulfoxide (DMSO), and dexamethasone (Dex); and cell receptor activation factors such as forskolin.

[0054] When culturing human hepatic progenitor cells, the medium preferably contains a factor related to maturation, and preferably contains at least one factor selected from DAPT, DMSO, and Dex.

[0055] When culturing human liver cells, the medium preferably contains a maturation factor, a proliferation factor, a differentiation-suppressing factor, and a factor related to cell receptor activation. The maturation factor preferably contains DAPT, and the proliferation factors preferably contain an IL-6 family cytokine, a mitogenic growth factor, a Wnt signaling promoter, and a ROCK signaling inhibitor.

[0056] (1) Mitogenic growth factors Mitogenic growth factors are factors that induce the initiation of cell division. Examples of mitogenic growth factors include epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), etc. FGFs that can bind to FGF receptor 2 (FGFR2) or FGF receptor 4 (FGFR4) are preferred, and FGF2, FGF4, FGF7, or FGF10 are preferred, with FGF10 being particularly preferred. Among these, EGF, FGF10, or HGF are preferred, and it is more preferred to include all of EGF, FGF10, and HGF.

[0057] The concentrations of EGF and HGF contained in the medium are generally 10 ng / mL to 500 ng / mL, preferably 15 ng / mL to 100 ng / mL, more preferably 20 ng / mL to 80 ng / mL, and even more preferably 25 ng / mL to 50 ng / mL. The concentration of FGF contained in the medium is usually 20 ng / mL to 500 ng / mL, preferably 50 ng / mL to 300 ng / mL, more preferably 80 ng / mL to 150 ng / mL, and even more preferably 90 ng / mL to 110 ng / mL.

[0058] (2) Wnt signaling promoter Wnt signaling is involved in the proliferation of stem cells and the maintenance of undifferentiated state. Wnt signaling can be upregulated by Wnt signaling promoters. Examples of Wnt signaling promoters include Wnt agonists, Lgr5 agonists, and glycogen synthase (GSK) inhibitors.

[0059] Wnt agonists include Wnt family members such as Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. Among these, Wnt3a is preferred.

[0060] Afamin is known to be involved in the stabilization and solubilization of Wnt family members, and a complex of a Wnt family member with afamin can be used as a Wnt agonist. The content of the Wnt agonist in the medium is generally 1% to 50% by volume, preferably 10% to 30% by volume, and more preferably 15% to 25% by volume, relative to the total volume of the medium.

[0061] Examples of Lgr5 agonists include R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4. Among these, R-spondin 1 is preferred. The content of the Lgr5 agonist in the medium is generally 1% to 50% by volume, preferably 5% to 30% by volume, and more preferably 5% to 15% by volume, relative to the total volume of the medium.

[0062] 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). Among these, CHIR99021 is preferred. The concentration of the GSK inhibitor contained in the medium is usually 0.1 μM to 10 μM. Here, "M" means mol / L, and the same applies hereinafter.

[0063] As the Wnt signaling promoter, it is preferable to use a Wnt agonist and an Lgt5 agonist in combination.

[0064] (3) ROCK signaling inhibitors Downregulation of ROCK signaling allows hepatocytes to acquire proliferative potential. ROCK signaling inhibitors include Y-27632 (CAS No.: 146986-50-7), Fasudil (CAS No.: 105628-07-7), Y39983 (CAS No.: 203911-26-6), Wf-536 (CAS No.: 539857-64-2), SLx-2119 (CAS No.: 911417-87-3), azabenzimidazole-aminofurazans (CAS No.: 850664-21-0), DE-104, H-1152P (CAS No.: 872543-07-6), and Rho kinase α inhibitors (ROKα). inhibitor), XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, Rhostain, BA-210, BA-207, Ki-23095, and VAS-012. Among these, Y-27632 is preferred.

[0065] The concentration of the ROCK signaling inhibitor contained in the medium is usually 1 μM to 20 μM, and preferably 5 μM to 15 μM.

[0066] (4) TGF-β signaling inhibitors Downregulation of TGF-β signaling can suppress differentiation. TGF-β signaling inhibitors include TGF-β inhibitors, which induce phosphorylation of Smad2 / 3, and BMP inhibitors, which induce phosphorylation of Smad1 / 5 / 9.

[0067] Examples of TGF-β inhibitors include A83-01 (CAS No.: 909910-43-6), SB-431542 (CAS No.: 301836-41-9), SB-505124 (CAS No.: 694433-59-5), SB-525334 (CAS No.: 356559-20-1), LY364947 (CAS No.: 396129-53-6), SD-208 (CAS No.: 627536-09-8), and SJN2511 (CAS No.: 446859-33-2). Among these, A83-01 is preferred.

[0068] The concentration of the TGF-β inhibitor contained in the medium is usually 0.05 μM to 50 μM, preferably 0.5 μM to 30 μM, more preferably 1 μM to 15 μM, and more preferably 4 μM or more and 6 μM or less.

[0069] Examples of BMP inhibitors include Noggin, Differential screening-selected gene Aberrative in Neuroblastoma (DAN), DAN-like protein, etc. Among these, Noggin is preferred.

[0070] The concentration of the BMP inhibitor contained in the medium is usually 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.

[0071] (5) IL-6 family cytokines IL-6 family cytokines can enhance the proliferation of human hepatic progenitor cells. IL-6 family cytokines include interleukin-6 (IL-6), interleukin-11 (IL-11), oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotropin-1 (CT-1), and ciliary neurotrophic factor (CNTF). Among these, IL-6 is preferred.

[0072] The concentration of the IL-6 family cytokine contained in the culture medium is generally 10 ng / mL to 1.0 μg / mL, preferably 50 ng / mL to 500 ng / mL, more preferably 80 ng / mL to 200 ng / mL, and even more preferably 90 ng / mL to 110 ng / mL.

[0073] (6) Retinoic acid By including retinoic acid in the medium, it is possible to increase the number of bile canaliculi and enhance the orientation of cells. The concentration of retinoic acid contained in the medium is usually 1 μM to 30 μM.

[0074] (7) Nicotinamide By including nicotinamide in the medium, the cell proliferation ability can be improved. The concentration of nicotinamide contained in the medium is usually 5 mM or less.

[0075] (8) Forskolin By further including forskolin in the medium, the intracellular concentration of AMP can be increased, thereby reactivating the cell receptor. The concentration of forskolin contained in the medium is usually 0.1 μM to 100 μM, preferably 1 μM to 50 μM, and more preferably 5 μM to 15 μM.

[0076] (9) DAPT, DMSO, and Dex The inclusion of DAPT, DMSO, or Dex in the culture medium can enhance maturation into human hepatocyte-like cells.

[0077] (10) Other ingredients In addition to the above components, the medium may further contain gastrin, neurobiological supplements, N-acetylcysteine, and the like. Neurobiological supplements include insulin-containing supplements such as B27 supplement (Thermo Fisher Scientific) and N2 supplement (Thermo Fisher Scientific).

[0078] The medium can be prepared by adding the above-mentioned components to a basal medium, such as Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Essential Medium (G-MEM), Minimal Essential Medium (BME), DMEM / Ham's F12, Advanced DMEM / Ham's F12 (Advanced DMEM / F12), Iscove's Modified Dulbecco's Medium, Ham's F-10, Ham's F-12, 199 Medium, RPMI 1640 Medium, and human plasma mimicking medium.

[0079] <Process B> In step B, the culture formed in step A is dispersed to form a dispersion.

[0080] The culture formed in step A is embedded in an extracellular matrix. In step B, the extracellular matrix is ​​disrupted and the culture is extracted. Methods for disrupting the extracellular matrix include physical disruption of the extracellular matrix and chemical disruption using enzymes. Of these, physical disruption of the extracellular matrix is ​​preferred because it causes less damage to the cells.

[0081] The culture removed from the extracellular matrix is ​​then dispersed to form a dispersion. Dispersion refers to separating the cells into cell populations of 100 or less, preferably cell populations of 50 or less, and more preferably single cells, by a dispersion treatment such as enzymatic treatment or physical treatment. Dispersion treatments include mechanical dispersion treatment, cell dispersion treatment, and treatment with the addition of a cytoprotective agent. These treatments can be combined as appropriate. Among these, cell dispersion treatment is preferred.

[0082] Examples of cell dispersion solutions used in cell dispersion treatment include solutions containing enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, and papain; or chelating agents such as ethylenediaminetetraacetic acid. Commercially available cell dispersion solutions include TrypLE Select and TrypLE Express manufactured by Thermo Fisher Scientific. Mechanical dispersion treatments include pipetting or scraping with a scraper.

[0083] Before the dispersion process, the cells can be treated with a cytoprotective agent to prevent cell death, such as fibroblast growth factor (hereinafter also referred to as "FGF"), heparin, ROCK inhibitor, insulin-like growth factor (hereinafter also referred to as "IGF"), serum, or serum substitute.

[0084] <Process C> In step C, the dispersion formed in step B is cultured in suspension in a medium in which an extracellular matrix is ​​suspended at 0 to 10% by volume relative to the total volume of the medium, to form sac-like structures containing human hepatocyte-like cells.

[0085] The extracellular matrix in step C may be the same as the extracellular matrix exemplified in step 1 above. The content of extracellular matrix in the medium in step C is 0 to 10% by volume, preferably 0.001 to 5% by volume, more preferably 0.1 to 4% by volume, and even more preferably 1 to 3% by volume, relative to the total volume of the medium.

[0086] Methods for suspending the extracellular matrix include mixing the medium with an extracellular matrix precursor and then gelling the extracellular matrix precursor to form the extracellular matrix. Methods for mixing the extracellular matrix precursor include pipetting on an ice bath. "Mixing" refers to a state in which no extracellular matrix is ​​visible in the medium.

[0087] Examples of the medium used in step C include the media exemplified in step A above.

[0088] The culture conditions in step C can be those generally used for culturing animal cells, such as a temperature of 30°C to 40°C, preferably 37°C, in an atmosphere with a CO2 concentration of 5% by volume.

[0089] The culture time can be adjusted appropriately depending on the number of cells, the state of the cells, etc. It is usually between one day and one week from the start of culture.

[0090] When sac-like bodies having a membrane of human hepatocyte-like cells produced by the production method of this embodiment are used as the evaluation model of the evaluation method of this embodiment, the medium after culture in step C contains the evaluation model and 0 to 10 volume % of suspended extracellular matrix, and therefore this medium can be used as the sac-like body-containing liquid in step 1 of the evaluation method of this embodiment. [Example]

[0091] The present invention will be described below with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[0092] [Experimental Example 1] (Preparation of human liver cell cultures) Human primary frozen suspension hepatocytes (BIOPR EHuman liver cells (HEP187-S, manufactured by DIC) were thawed in a 37°C water bath and suspended in a 50 mL tube containing serum-free medium (Advanced DMEM / F12 supplemented with HEPES, Glutamax, and Penicillin / Stereputomycin). The suspension was then centrifuged. After centrifugation, the supernatant was removed and the cells were suspended in serum-free medium to prepare a human liver cell suspension. From this suspension, 20,000 human liver cells were mixed with 50 μL of Matrigel (BD Biosciences), seeded into a 24-well tissue culture plate, and incubated at 37°C for 10 minutes until the Matrigel polymerized completely. After polymerization, 500 μL of the medium shown in Table 1 below was added and cultured at 37°C in the presence of 5% CO2 (volume). This resulted in a human liver cell culture. The resulting culture formed a membrane-enclosed lumen composed of human hepatocyte-like cells. The resulting culture was then subcultured repeatedly using the same procedure.

[0093] [Table 1]

[0094] [Experimental Example 2] (Production of the sac-like object (this evaluation model)) The culture of human liver cells after passage culture in Experimental Example 1, the medium shown in Table 1, and 2% by volume of Matrigel relative to the medium were added to a 6-well culture plate (Sumitomo Bakelite Co., Ltd., MS-8006R), and the cells were cultured in suspension at 37°C for 3 days in the presence of 5% by volume of CO2 to form sacs. A bright-field image of the sacs taken with an optical microscope (magnification: 400x (KEYENCE Corporation, BZ-X700)) of this evaluation model is shown in Figure 1.

[0095] [Experimental Example 3] (Rho123 excretion in this evaluation model) In Experimental Example 2, Rho123 (10 μM), a substrate for P-glycoprotein (P-gp), was added to the culture medium after suspension culture. Rho123 uptake into the lumen of this evaluation model was assessed for 30 minutes at 37°C in the presence of 5% CO2 (volume). The culture medium containing this evaluation model was pipetted into a 15 mL low-adsorption tube and centrifuged. After centrifugation, the supernatant was removed, the tube was washed with PBS, and then an optical microscope (magnification: 400x (KEYENCE, BZ-X700)) confirmed that Rho123 had been released into the lumen of this evaluation model. A bright-field image (Figure 2(a)) and a fluorescent image (Figure 2(b)) are shown in Figure 2. As shown in Figure 2, it was confirmed that Rho123 was excreted into the lumen of this evaluation model.

[0096] [Experimental Example 4] (Quantification of Rho123 incorporated into the lumen of this evaluation model) The human hepatocyte-like cells were killed by mixing the Rho123-incorporated evaluation model of Experimental Example 3 with methanol. After mixing, the mixture was centrifuged and the supernatant was extracted. The supernatant was analyzed using a liquid chromatography mass spectrometry (LC / MS) analyzer (Shimadzu Corporation, LCMSTM-8040). The concentration and amount of Rho123 incorporated into the lumen and the proportion transported into the lumen of the evaluation model were calculated from the Rho123 concentration in the culture medium. The LC / MS analysis conditions are shown below, and the results are shown in Table 3.

[0097] (LC conditions) Column: Xbridge C18 3.5 μm, 2.1 mm x 50 mm Column temperature: 40℃ Internal standard: Tolbutamine Elution solvent: Solution A (0.1% by mass formic acid solution), Solution B (acetonitrile) Gradient conditions: see Table 2 Runtime: 7 minutes Flow rate: 0.4mL / min Injection volume: 3 μL

[0098] [Table 2]

[0099] (MS conditions) Mode: Positive (standard substance is negative)

[0100] [Table 3]

[0101] As shown in Table 3, the amount of Rho123 taken up into the lumen of this evaluation model could be quantified, and the proportion of Rho123 in the culture medium that was transported into the lumen of this evaluation model could be calculated.

[0102] [Experimental Example 5] Rho123 (10 μM) was added to the culture medium containing the cultured Matrigel-embedded cells prepared in Experimental Example 1, and Rho123 was incorporated into the lumen of the cells in the presence of 5% CO2 at 37°C for 30 minutes. The incorporation of Rho123 into the lumen of the cells was confirmed using an optical microscope (magnification: 400x (KEYENCE, BZ-X700)). A bright-field image (Figure 3(a)) and a fluorescent image (Figure 3(b)) are shown in Figure 3. To measure the amount of internalized Rho123, the polymerized Matrigel was degraded by pipetting and enzymatic treatment (Corning® Cell Recovery Solution). However, as the Matrigel degraded, the internalized Rho123 from the liver organoids leaked out, making it impossible to measure the amount of internalized Rho123.

[0103] [Experimental Example 6] (CDFDA emissions in this evaluation model) In Experimental Example 2, 2 μM, 5 μM, or 10 μM of CDFDA (Carboxydichlorofluorescein Diacetate), a substrate for MRP2, was added to the culture medium after suspension culture. CDFDA uptake into the lumen of this evaluation model was assessed for 30 minutes at 37°C in the presence of 5% CO2 by volume. The culture medium containing this evaluation model was pipetted into a 15 mL low-adsorption tube and centrifuged. After centrifugation, the supernatant was removed, the tube was washed with PBS, and then the release of CDFDA into the lumen of this evaluation model was confirmed using an optical microscope (magnification: 400x (KEYENCE, BZ-X700)). The results are shown in Figure 5. [Industrial Applicability]

[0104] According to the method of this embodiment, a method for evaluating the metabolism or excretion of a test substance by human hepatocyte-like cells can be provided, using an evaluation model for evaluating excretion by human hepatocyte-like cells.

Claims

1. A method for evaluating the excretion of a test substance by human hepatocyte-like cells, comprising: providing a sac-containing solution containing sacs having a membrane of in vitro generated human hepatocyte-like cells and 0.001 to 5% by volume of suspended extracellular matrix; placing the test substance in the sac-containing liquid to contact the test substance with the sac-like substance; and removing the sac-like substance from the sac-like substance-containing liquid and measuring the concentration of the test substance or its metabolites excreted into the lumen of the sac-like substance; and The method, wherein the human hepatocyte-like cells are cells obtained by embedding human hepatic progenitor cells or human liver cells in an extracellular matrix and culturing them.

2. The method of claim 1, wherein the human hepatocyte-like cells are cells that can be cultured as organoids.

3. The method according to claim 1 or 2, wherein the human hepatocyte-like cells have at least one transporter selected from the group consisting of BSEP, MRP2, P-gp, MATE, and BCRP.

4. The method according to any one of claims 1 to 3, wherein the concentration of the test substance or its metabolite excreted into the lumen of the sac-like material is measured by disassembling the sac-like material.

5. The method according to any one of claims 1 to 4, wherein the concentration of the test substance or its metabolites excreted into the lumen of the sac-like substance is measured by liquid chromatography mass spectrometry.

6. The method according to any one of claims 1 to 5, wherein the lumen of the sac contains bile as a content.

7. The method according to any one of claims 1 to 6, wherein the membrane of the human hepatocyte-like cells is a monolayer membrane.

8. 1. A method for producing sacs having a membrane of human hepatocyte-like cells, comprising: Culturing human hepatic progenitor cells or human hepatic cells embedded in an extracellular matrix to form a culture; dispersing the culture to form a dispersion; and culturing the dispersion in a suspension culture medium in which an extracellular matrix is ​​suspended in an amount of 0.001 to 5% by volume relative to the total volume of the medium, thereby forming the cysts; The manufacturing method of the present invention.

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