Method for culturing human hepatocytes, cultured human hepatocytes, culture medium and method for producing the same, method for producing culture supernatant of human hepatocytes (non-parenchymal cells)

Abstract

A method for culturing a human hepatic parenchymal cell, the method comprising culturing the human hepatic parenchymal cell in a culture medium comprising a culture supernatant of a human hepatic nonparenchymal cell, a Wnt signaling promoter and a cell-division-promoting growth factor.

Description

Technical Field

[0001] The present invention relates to a method for culturing human hepatocytes, cultured human hepatocytes obtained by the method, a medium used in the method and a method for producing the same, and a method for producing a culture supernatant of human non-parenchymal hepatocytes.

Background Art

[0002] In pharmacokinetic studies for drug development, in vivo tests using rodents and in vitro tests using rodent-derived hepatocytes (parenchymal hepatocytes) are being conducted. However, in tests using rodents and their cells, it is difficult to predict human-specific toxicity due to species differences. On the other hand, it is difficult to stably obtain the amount of primary human hepatocytes required for pharmacokinetic studies in drug development. Therefore, it is required to establish a hepatocyte strain (master cell line) with excellent growth properties that can be cultured for a long period from primary human hepatocytes.

[0003] Hepatic organoids are hepatocyte cultures cultured from hepatocytes and are expected as a stable source of hepatocytes for drug development. An improved method for constructing hepatic organoids has been developed. Patent Document 1 discloses a method for producing human hepatic organoids using a medium containing epidermal growth factor (EGF), a Notch inhibitor, and a transforming growth factor β (TGF-β) inhibitor, or a medium containing EGF, a BMP inhibitor, and a Wnt agonist.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] This invention provides a method for culturing human hepatocytes with excellent proliferative properties that can be cultured for a long period of time, cultured human hepatocytes produced by this method, and a culture medium used in this method. Furthermore, this invention provides a method for producing the culture supernatant of human hepatocytes. [Means for solving the problem]

[0006] The present invention includes the following embodiments. [1] A method for culturing human hepatic parenchymal cells, comprising culturing human hepatic parenchymal cells in a culture medium containing the culture supernatant of human hepatic non-parenchymal cells, a Wnt signaling promoter, and a mitogenic growth factor. [2] The method according to [1], wherein the Wnt signaling promoter contains a Wnt family member protein. [3] The method according to [2], wherein the Wnt signaling promoter further contains an R-Spondin superfamily protein. [4] The method according to any one of [1] to [3], wherein the mitotic growth factor contains one or more selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). [5] The method according to any one of [1] to [4], wherein the culture medium further contains one or more selected from Rho kinase (ROCK) signaling inhibitors, transforming growth factor β (TGF-β) signaling inhibitors, and interleukin-6 (IL-6) family cytokines. [6] The method according to [5], wherein the culture medium contains a ROCK signaling inhibitor. [7] The method according to [5] or [6], wherein the culture medium contains a TGF-β signaling inhibitor. [8] The method according to any one of [5] to [7], wherein the culture medium contains IL-6 family cytokines. [9] The method according to any one of [1] to [8], wherein the culture medium further contains one or more selected from retinoic acid, nicotinamide, cAMP activator, gastrin, neurobiological supplements, and antioxidants.

[10] The method according to any one of [1] to [9], wherein the culture supernatant of human non-parenchymal hepatic cells is produced by culturing human non-parenchymal hepatic cells in a culture medium for human non-parenchymal hepatic cells containing a Wnt signaling promoter and a mitogenic growth factor, and separating the supernatant from the culture medium after the culture.

[11] The method according to

[10] , wherein the culture medium for human liver nonparenchymal cells further contains a ROCK signaling inhibitor.

[12] The method according to

[10] or

[11] , wherein the culture medium for human liver nonparenchymal cells further contains one or more selected from IL-6 family cytokines, TGF-β signaling inhibitors, retinoic acid, nicotinamide, cAMP activators, gastrin, neurobiological supplements, and antioxidants.

[13] Cultured human hepatocytes cultured by any one of the methods described in [1] to

[12] above.

[14] A culture medium containing the culture supernatant of human non-parenchymal hepatocytes, a Wnt signaling promoter, and a mitogenic growth factor.

[15] Human non-parenchymal hepatic cells are cultured in a medium containing a Wnt signaling promoter and a mitogenic growth factor. Separating the supernatant from the culture medium after the culture. A method for producing the culture supernatant of human non-parenchymal liver cells, including the above.

[16] A method for producing a culture medium, comprising mixing the culture supernatant of human non-parenchymal hepatic cells produced by the method described in

[15] , a Wnt signaling promoter, and a mitogenic growth factor. [Effects of the Invention]

[0007] This invention provides a method for culturing human hepatocytes with excellent proliferative properties that can be cultured for a long period of time. According to this invention, it becomes possible to stably supply human hepatocytes useful for drug development and other applications. [Brief explanation of the drawing]

[0008] [Figure 1] Proliferative capacity of human hepatocytes in Experimental Examples 2 to 4. [Figure 2-1] Activated pathways in cultured human hepatocytes from Experimental Examples 2 and 4. [Figure 2-2] Continuation of Fig. 2-1. [Figure 2-3] Continuation of Fig. 2-2. [Figure 2-4] Continuation of Fig. 2-3. [Figure 3] Correlation of gene expression among primary human hepatocytes (PHHs), human hepatocytes in Experimental Example 2 (Proli-Orgs), mature human hepatocytes in Experimental Example 6 (Differ-Orgs and Differ-Orgs (long-term)), and human cholangiocytes in Experimental Example 7 (Chol). The numerical values in the figure are correlation coefficients. [Figure 4] Microscopic images and gene expression levels of PHHs, Proli-Orgs, and Differ-Orgs (long-term). [Figure 5-1] Microscopic image of cultured human hepatocytes in Experimental Example 2. [Figure 5-2] Microscopic image of cultured human hepatocytes in Experimental Example 9. [Figure 6] Microscopic image of cultured human hepatocytes in Experimental Example 10.

Mode for Carrying Out the Invention

[0009] Hereinafter, the present invention will be described in detail by showing embodiments. However, the present invention is not limited to the following embodiments.

[0010] Each component exemplified in this specification can be used alone or in combination of two or more, unless otherwise specified.

[0011] In this specification, the notation representing a numerical range such as "A to B" is synonymous with "A or more and B or less", and A and B are included in the numerical range.

[0012] The proteins exemplified in this specification include mutant proteins with substitutions, duplications, deletions, insertions, or frameshift mutations in their amino acid sequences or the nucleotide sequences encoding them, provided that the mutant proteins have the same function as before the mutation, and such mutant proteins are included in the proteins before the mutation.

[0013] In this specification, "a medium containing substance X" and "in the presence of substance X" mean a medium to which exogenous substance X has been added, a medium containing exogenous substance X, or in the presence of exogenous substance X. That is, when cells or tissues present in the medium endogenously express, secrete, or produce substance X, the endogenous substance X is distinguished from the exogenous substance X, and a medium that does not contain exogenous substance X, even if it contains endogenous substance X, is not considered to fall within the category of "a medium containing substance X".

[0014] <Method for culturing human hepatocytes> In one embodiment of the present invention, a method for culturing human hepatocytes is provided. The culturing method of this embodiment includes culturing human hepatocytes in a medium containing the culture supernatant of human non-parenchymal liver cells, a Wnt signaling promoter, and a mitogenic growth factor (hereinafter also referred to as "step 1"). By the culturing method of this embodiment, cultured human hepatocytes of one embodiment of the present invention are produced.

[0015] The liver is composed of hepatic parenchymal cells (hepatic parenchymal cell or hepatocyte) that are responsible for the essence of liver function and hepatic non-parenchymal cells (hepatic non parenchymal cell) that support the growth and survival of these hepatic parenchymal cells. Examples of hepatic non-parenchymal cells include hepatic stellate cells, sinusoidal endothelial cells, and Kupffer cells. The population of hepatic parenchymal cells usually includes hepatic stem cells, preferably epithelial hepatic stem cells.

[0016] In this embodiment, the human hepatocytes cultured in step 1 can be isolated from a liver sample taken from a human organism by a known method such as collagenase perfusion, or primary human hepatocytes or passaged human hepatocytes can be prepared from the isolated human hepatocytes and used as the human hepatocytes cultured in step 1. Alternatively, commercially available primary human hepatocytes or passaged human hepatocytes can be used as the human hepatocytes cultured in step 1. The human hepatocytes cultured in step 1 are preferably isolated human hepatocytes that substantially do not contain non-parenchymal human hepatocytes, more preferably primary human hepatocytes and passaged human hepatocytes, and even more preferably primary human hepatocytes.

[0017] Methods for culturing human hepatocytes in Step 1 include, for example, suspension culture and adherent culture. Suspension culture is a method of culturing cells while keeping them suspended in the culture medium without adhering them to the surface of the culture vessel, and contacting the extracellular matrix (ECM) as needed. Adherent culture is a method of culturing cells while adhering them to the surface of the culture vessel, via the ECM or other means as needed.

[0018] During culture, it is preferable to culture human hepatocytes in contact with the extracellular matrix (ECM). For example, human hepatocytes can be brought into contact with the ECM by embedding them in the ECM, adding them to a culture vessel coated with the ECM, or adding donor human hepatocytes to a culture medium in which the ECM is suspended. In a preferred embodiment, human hepatocytes embedded in the ECM are prepared by bringing the ECM precursor into contact with the human hepatocytes during the gelling process, and these are then cultured.

[0019] Examples of ECM or ECM precursors (hereinafter referred to as "ECM, etc.") include components contained in the basement membrane such as type IV collagen, laminin, heparan sulfate proteoglycan, and entactin; and glycoproteins present in the intercellular space such as collagen, laminin, entactin, fibronectin, and heparin sulfate. Such ECM, etc. can be prepared according to conventional methods, or commercially available products can be used. Examples of commercially available ECM, etc. for cell culture include Matrigel® (Corning Corporation product name) and human-type laminin (Sigma-Industrial Chemicals product name).

[0020] For suspension culture, it is preferable to use a culture vessel with a cell-non-adherent surface. Examples of culture vessels with cell-non-adherent surfaces include vessels with surfaces treated with cell-non-adherent materials such as MPC polymer, and vessels with surfaces shaped with irregularities. For adherent culture, it is preferable to use a culture vessel with a surface coated with ECM.

[0021] The culture in step 1 is usually carried out under conditions of 30°C to 50°C, preferably 32°C to 48°C, and more preferably 34°C to 46°C. Furthermore, the culture in step 1 is carried out in an atmosphere with a carbon dioxide content of 1% to 15% by volume, preferably 2% to 14% by volume, and more preferably 3% to 13% by volume. The above culture conditions can be achieved by adjusting the temperature and carbon dioxide content of the container (incubator, etc.) in which the culture vessels are placed.

[0022] During culture, components in the culture medium become inactive or disappear, so the medium is usually replaced every 1 to 5 days. Furthermore, human liver parenchymal cells obtained through culture can be removed from the medium, dispersed as needed, and then subcultured again under the same conditions.

[0023] In the culture method used in this embodiment, human hepatocytes exhibit excellent proliferative capacity and can be cultured for at least 30 days, preferably at least 90 days, thus enabling long-term culture of human hepatocytes.

[0024] <Culture medium for human hepatocytes> The culture medium used in step 1 is a medium containing the culture supernatant of human nonparenchymal liver cells, a Wnt signaling promoter, and a mitogenic growth factor. The culture medium used in step 1 may be referred to as "Culture Medium 1" in this specification.

[0025] As shown in the RNA-seq results of the examples described later, the culture supernatant of human non-parenchymal liver cells may contain factors related to the pathways listed in Figures 2-1 to 2-4 (hereinafter collectively referred to as "specific factors"). It is preferable that the culture supernatant of human non-parenchymal liver cells used in Step 1 contains at least one, preferably two or more, and more preferably all of the specific factors selected from among them.

[0026] Culture medium 1 is typically prepared by adding the culture supernatant of human non-parenchymal hepatic cells, a Wnt signaling promoter, and mitogenic growth factors, as well as other components as described below, to the basal medium.

[0027] Examples of basal media contained in medium 1 include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham medium, RPMI 1640 medium, and Fischer's medium, as well as mixed media of these.

[0028] The culture supernatant of human non-parenchymal hepatic cells contained in medium 1 can be prepared by separating the supernatant from the culture medium after culturing human non-parenchymal hepatic cells by centrifugation or the like. The method for preparing the culture supernatant of human non-parenchymal hepatic cells will be described in detail later. The content ratio of the culture supernatant of human non-parenchymal hepatic cells contained in medium 1 is usually 2.5 volume(v / v)% to 50 volume(v / v)%, preferably 5 volume(v / v)% to 30 volume(v / v)%, and more preferably 10 volume(v / v)% to 20 volume(v / v)%, of the total volume of medium 1, when the total concentration of the specified factors contained in the culture supernatant of human non-parenchymal hepatic cells is 1 ng / mL to 1 mg / mL.

[0029] Wnt signaling functions as a transcription factor, primarily regulating cell proliferation and differentiation by modulating β-catenin protein levels. Wnt signaling promoters (including Wnt agonists) are agonists that activate the Wnt signaling pathway. Examples of Wnt signaling promoters include Wnt family member proteins, R-spongin family proteins, Norrin, and glycogen synthase (GSK) inhibitors.

[0030] Examples of Wnt family member proteins include 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. Amino acid sequence information for each of these Wnt family proteins can be obtained from the NCBI database ([www.ncbi.nlm.nih.gov / ]).

[0031] Afamin is known to contribute to the stabilization and solubilization of Wnt family member proteins. Therefore, it is preferable to use Wnt family member proteins in complex with afamin. Afamin refers to glycoproteins belonging to the albumin family. Examples of afamin include human afamin (GenBank accession number: AAA21612.1) and bovine afamin (GenBank accession number: DAA28569.1).

[0032] Wnt family member proteins, or complexes of Wnt family member proteins and afamin, can be used as a maturation culture medium (conditioning medium) containing them. The concentration of Wnt family member proteins in the conditioning medium is preferably 18 ng / mL to 900 ng / mL. When using a conditioning medium in which the concentration of Wnt family member proteins is within the above range, the proportion of the conditioning medium in medium 1 is usually 1 volume (v / v)% to 50 volume (v / v)%, preferably 10 volume (v / v)% to 30 volume (v / v)%, and more preferably 15 volume (v / v)% to 25 volume (v / v)%, in the total volume of medium 1.

[0033] Examples of R-spongin family proteins include R-spongin 1, R-spongin 2, R-spongin 3, and R-spongin 4. Among these, R-spongin 1 is preferred. Amino acid sequence information for each protein of the R-spongin family can be obtained from the NCBI database. When R-spongin family proteins bind to Lgr5 in the cell membrane, they are removed from the cell membrane by autoubiquitination, and as a result, Frizzled, which induces activation of the Wnt signaling pathway, activates the β-catenin pathway in the cell membrane.

[0034] R-spongin family proteins can be used as a conditioning medium containing them at a concentration of 0.13 μg / mL to 6.5 μg / mL. When using a conditioning medium in which the concentration of R-spongin family proteins is within the above range, the proportion of the conditioning medium in medium 1 is typically 1 volume (v / v)% to 50 volume (v / v)%, preferably 5 volume (v / v)% to 25 volume (v / v)%, and more preferably 8 volume (v / v)% to 20 volume (v / v)%, in the total volume of medium 1.

[0035] GSK inhibitors are inhibitors of glycogen synthase 3β (GSK3β). Since GSK3β inhibits Wnt signaling by phosphorylating β-catenin and promoting its degradation, GSK inhibitors act as Wnt signaling promoters. 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).

[0036] As a Wnt signaling promoter, it is preferable to use a combination of Wnt family proteins and R-spondin family proteins, more preferably a combination of Wnt3a and R-spondin 1, and even more preferably a combination of a complex of Wnt3a and afamin and R-spondin 1.

[0037] Mitozoal growth factors (Mitioprostimulants) initiate signaling pathways involving mitogenic factor-activated protein kinase (MAPK) and are involved in cell proliferation. Examples of Mitioprostimulants include epidermal growth factor (EGF), fibroblast growth factors (FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGF), and vascular endothelial growth factor (VEGF). Among these, one or more selected from EGF, FGF, and HGF are preferred, and a combination of EGF, FGF, and HGF is more preferred. Mitioprostimulant growth factors are preferably human proteins.

[0038] EGF is a growth factor that activates the epidermal growth factor receptor (EGFR or ErbB1). Activated EGFR primarily activates the MAPK signaling pathway, as well as the PI3K signaling pathway and the Jak / Stat signaling pathway. The concentration of EGF in medium 1 is typically 0.5 ng / mL to 1,000 ng / mL, preferably 1 ng / mL to 500 ng / mL, and more preferably 2 ng / mL to 200 ng / mL.

[0039] Preferably, the FGF is one that can bind to any of the FGF receptors 1 (FGFR1), 2 (FGFR2), 3 (FGFR3), and 4 (FGFR4). Preferred examples include FGF1, FGF2, FGF3, FGF4, FGF7, and FGF10. In addition, as FGF, a chimeric form of heterologous FGF can be used, such as a chimeric FGF containing a portion of the region of FGF1 and a portion of the region of FGF2 (hereinafter also referred to as "FGFC"). The concentration of FGF in medium 1 is usually 0.5 ng / mL to 1,000 ng / mL, preferably 1 ng / mL to 500 ng / mL, and more preferably 2 ng / mL to 200 ng / mL.

[0040] HGF is a growth factor that activates Met receptors, and activated Met receptors activate the HGF-Met signaling pathway. Activation of the HGF-Met signaling pathway promotes activation of the β-catenin pathway, which in turn promotes angiogenesis and metalloproteinase production. The concentration of HGF in medium 1 is typically 0.5 ng / mL to 1,000 ng / mL, preferably 1 ng / mL to 500 ng / mL, and more preferably 2 ng / mL to 200 ng / mL.

[0041] In addition to the components listed above, culture medium 1 may further contain one or more selected from Rho kinase (ROCK) signaling inhibitors, transforming growth factor β (TGF-β) signaling inhibitors, and interleukin-6 (IL-6) family cytokines.

[0042] Culture medium 1 preferably contains a ROCK signaling inhibitor from the viewpoint of inhibiting apoptosis. ROCK signaling inhibitors act as antagonists of IGF-1 signaling. Examples of ROCK signaling inhibitors include Y-27632 (CAS number: 146986-50-7), Fasudil (CAS number: 105628-07-7), Y39983 (CAS number: 203911-26-6), WF-536 (CAS number: 539857-64-2), SLx-2119 (CAS number: 911417-87-3), Azabenzimidazole-aminoflazan (GSK269962) (CAS number: 850664-21-0), DE-104, H-1152P (CAS number: 872543-07-6), and Rho kinase α inhibitors (ROKα Examples include ROCK signaling inhibitors, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, Rho statin, BA-210, BA-207, Ki-23095, and VAS-012. Among these, Y-27632 is preferred. The concentration of ROCK signaling inhibitor in medium 1 is usually 1 μM to 20 μM, preferably 5 μM to 15 μM, and more preferably 8 μM to 12 μM.

[0043] Culture medium 1 preferably contains a TGF-β signaling inhibitor from the viewpoint of promoting the proliferation of human hepatocytes. TGF-β signaling contributes to the suppression of cell proliferation, cell differentiation, and induction of apoptosis. TGF-β signaling inhibitors downregulate TGF-β signaling. Examples of TGF-β signaling inhibitors include inhibitors that inhibit the activation of type I or type II serine / threonine kinase receptors, such as TGF-β inhibitors that inhibit the phosphorylation of Smad2 / 3 and BMP inhibitors that inhibit the phosphorylation of Smad1 / 5 / 9.

[0044] Examples of TGF-β inhibitors that inhibit the phosphorylation of Smad2 / 3 include A83-01 (CAS number: 909910-43-6), SB-431542 (CAS number: 301836-41-9), SB-505124 (CAS number: 694433-59-5), SB-525334 (CAS number: 356559-20-1), LY364947 (CAS number: 396129-53-6), SD208 (CAS number: 627536-09-8), and SJN2511 (CAS number: 446859-33-2). Among these, A83-01 is preferred. The concentration of the TGF-β inhibitor in culture medium 1 is typically 0.05 μM to 50 μM, preferably 0.5 μM to 30 μM, and more preferably 1 μM to 15 μM.

[0045] Examples of BMP inhibitors that inhibit the phosphorylation of Smad1 / 5 / 9 include noggin, differential screening-selected gene Aberrative in Neuroblastoma (DAN), and DAN-like proteins. Among these, noggin is preferred. The concentration of the BMP inhibitor in medium 1 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.

[0046] Culture medium 1 is preferably composed of IL-6 family cytokines from the viewpoint of promoting the proliferation of human hepatocytes. IL-6 family cytokines, which are inflammatory cytokines, are produced in vivo by Kupffer cells and are thought to contribute to the proliferation of human hepatocytes by activating Kupffer cells themselves and MAPK signaling. Examples of IL-6 family cytokines include interleukin-6 (IL-6), interleukin-11 (IL-11), oncostatin M (OSM), leukemia suppressor factor (LIF), cardiotropin-1 (CT-1), and ciliary neurotrophic factor (CNTF). Among these, IL-6 is preferred. The concentration of IL-6 family cytokines in culture medium 1 is usually 10 ng / mL to 1.0 μg / mL, preferably 50 ng / mL to 500 ng / mL, and more preferably 80 ng / mL to 200 ng / mL.

[0047] Furthermore, culture medium 1 may contain one or more selected from retinoic acid, nicotinamide, cAMP activator, gastrin, neurobiological supplements, and antioxidants.

[0048] Including retinoic acid in medium 1 can increase the orientation of human hepatocytes. The concentration of retinoic acid in medium 1 is typically 1 μM to 30 μM. Including nicotinamide in medium 1 can improve cell proliferation. The concentration of nicotinamide in medium 1 is typically 5 mM or less. Forskolin is an example of a cAMP activator. Including a cAMP activator in medium 1 can increase the intracellular concentration of adenosine monophosphate (AMP), thereby reactivating cell receptors. The concentration of cAMP activator in medium 1 is typically 0.1 μM to 100 μM. The concentration of gastrin in medium 1 is typically 5 nM to 15 nM. N-acetylcysteine ​​is an example of an antioxidant. The concentration of antioxidants in medium 1 is typically 0.1 mM to 10 mM. Examples of neurobiological supplements include insulin-containing supplements such as B27 supplement (Thermo Fisher Scientific) and N2 supplement (Thermo Fisher Scientific). The percentage of neurobiological supplements in medium 1 is typically 0.5(v / v)% to 10(v / v)% of the total volume of medium 1.

[0049] <Method for preparing the culture supernatant of human non-parenchymal hepatocytes> The culture supernatant of human non-parenchymal hepatic cells used in Step 1 is prepared by culturing human non-parenchymal hepatic cells in a culture medium for human non-parenchymal hepatic cells containing a Wnt signaling promoter and mitogenic growth factors (hereinafter also referred to as "Culture Medium 2"), and then separating the supernatant from the culture medium. Examples of human non-parenchymal hepatic cells subjected to this culture include hepatic stellate cells, sinusoidal endothelial cells, Kupffer cells, and mixtures of two or more of these.

[0050] Medium 2 can typically be prepared by adding a Wnt signaling promoter and a mitogenic growth factor, as well as other components as described below, to a basal medium. The basal medium contained in Medium 2 is the same as that used for Medium 1.

[0051] Preferably, medium 2 contains a Wnt signaling promoter and a mitogenic growth factor. Examples of Wnt signaling promoters and mitogenic growth factors that can be used in medium 2, and their respective proportions in medium 2, are as illustrated above with respect to medium 1.

[0052] Preferably, medium 2 further contains ROCK signaling inhibitors in addition to the components listed above. Examples of ROCK signaling inhibitors that can be used in medium 2, and their concentrations in medium 2, are as illustrated above with respect to medium 1. If necessary, medium 2 may contain, in addition to the components listed above, one or more other components selected from IL-6 family cytokines, TGF-β signaling inhibitors, retinoic acid, nicotinamide, cAMP activators such as forskolin, gastrin, neurobiological supplements, and antioxidants such as N-acetylcysteine. Examples of other components that can be used in medium 2, and their concentrations in medium 2, are as illustrated above with respect to medium 1.

[0053] As a method for culturing human non-parenchymal liver cells using medium 2, suspension culture is preferred. During culture, it is preferable to culture the human non-parenchymal liver cells while keeping them in contact with the ECM. For example, the human non-parenchymal liver cells can be brought into contact with the ECM by embedding them in the ECM or by adding them to a medium in which the ECM is suspended. In a preferred embodiment, the human non-parenchymal liver cells are brought into contact with the ECM during the gelation process of the ECM precursor according to a conventional method, thereby preparing human non-parenchymal liver cells embedded in the ECM. Examples of ECM or ECM precursors used for culturing human non-parenchymal liver cells include those exemplified above in relation to step 1, such as Matrigel®.

[0054] Human non-parenchymal liver cells are typically cultured under temperature conditions of 30°C to 50°C, preferably 32°C to 48°C, and more preferably 34°C to 46°C. Furthermore, the culture of human non-parenchymal liver cells is carried out in an atmosphere with a carbon dioxide content of typically 1% to 15% by volume, preferably 2% to 14% by volume, and more preferably 3% to 13% by volume. The culture period is typically 1 day or more, preferably 1 to 20 days.

[0055] The culture supernatant of human non-parenchymal liver cells used in step 1 can be prepared by collecting the supernatant from the culture medium of the obtained human non-parenchymal liver cells using a standard procedure such as centrifugation.

[0056] The culture supernatant of the prepared human liver nonparenchymal cells preferably contains at least one, preferably two or more, and more preferably all of, specific factors related to the genes shown in Figures 2-1 to 2-4.

[0057] The total concentration of the specific factor contained in the culture supernatant of the human non-parenchymal liver cells is typically 1 ng / mL to 1 mg / mL, preferably 10 ng / mL to 100 μg / mL.

[0058] <Cultured human hepatocytes> Another embodiment of the present invention provides cultured human hepatocytes obtained by the human hepatocyte culture method described above.

[0059] The cultured human hepatocytes provided in this embodiment are proliferative human hepatocytes. These human hepatocytes possess drug-metabolizing activity. The drug-metabolizing activity of the cells can be evaluated by detecting the expression of drug-metabolizing enzymes or by drug-metabolizing assays. Examples of drug-metabolizing enzymes that can be expressed by human hepatocytes in this embodiment include cytochrome P450 1A2 (CYP1A2), cytochrome P450 2B (CYP2B), cytochrome P450 2C9 (CYP2C9), cytochrome P450 2C19 (CYP2C19), cytochrome P450 2D6 (CYP2D6), cytochrome P450 2E1 (CYP2E1), cytochrome P450 3A4 (CYP3A4), cytochrome P450 3A7 (CYP3A7), uridine diphosphate-glucuronosyltransferase (UGT), and sulfotransferase (SULT).

[0060] Preferably, the drug metabolism activity of cultured human hepatocytes in this embodiment is typically 20-200 for CYP1A2, 10-200 for CYP2B, 20-200 for CYP2C9, 20-200 for CYP2C19, 10-190 for CYP2D6, 1-150 for CYP2E1, 0.1-200 for CYP3A4, 0.1-120 for YP3A7, 10-300 for UGT, or 20-250 for SULT, when the expression level of drug-metabolizing enzymes in human hepatocytes before culture is set to 100.

[0061] By differentiating the cultured human hepatocytes of this embodiment, mature human hepatocytes with improved expression levels of drug-metabolizing enzymes can be obtained. Examples of differentiation culture methods include those that control TGF-β signaling, Wnt signaling, and Notch signaling, which contribute to the proliferation of human hepatocytes. Preferably, human hepatocytes are cultured in a medium containing DAPT ((3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl; CAS number: 208255-80-5). Differentiation into mature human hepatocytes can be confirmed by gene expression analysis of the cells. [Examples]

[0062] The present invention will be described in more detail below with reference to experimental examples. However, the present invention is not limited in any way by these experimental examples. In the following experimental examples, sterilized equipment was used in all experiments. Furthermore, all experiments were conducted in accordance with an ethical research plan approved by the Ethics Committee of Keio University School of Medicine.

[0063] [culture medium] The culture media A to E shown in Table 1 were prepared. Advanced DMEM / F12 was used as the basal medium. In the media, R-spondin 1 was used in the form of a conditioning medium containing R-spondin 1, and the concentration of R-spondin 1 relative to the total volume of the conditioning medium was 1.3 μg / mL. In addition, Wnt3a was used in the media in the form of a conditioning medium containing a complex of Wnt3a and afamin, and the concentration of Wnt3a relative to the total volume of the conditioning medium was 360 ng / mL. Culture supernatants of human liver nonparenchymal cells prepared in Experimental Example 1 described below were added to media B, C, and F.

[0064] [Table 1]

[0065] [Experimental Example 1] Preparation of culture supernatant of human non-parenchymal hepatocytes Frozen primary human hepatic nonparenchymal cells ("NPC-101", BIOREWDIC) were thawed in a 37°C water bath, suspended in a 50 mL tube with serum-free medium, and centrifuged. The serum-free medium was prepared by adding HEPES, GlutaMAX, and penicillin / streptomycin to Advanced DMEM / F12. After centrifugation, the supernatant was removed and then suspended in serum-free medium to prepare a suspension of human hepatic nonparenchymal cells. 10,000 human hepatic nonparenchymal cells were taken from this suspension, mixed with 50 μL of Matrigel® (BD Biosciences), seeded in each well of a 24-well tissue culture plate, and incubated at 37°C for 5 to 10 minutes until the Matrigel completely polymerized. After the Matrigel polymerized, a medium containing the components shown in "Medium A" in Table 1 was overlaid, and the human hepatic nonparenchymal cells were cultured for 5 days. After centrifugation of the medium, the culture supernatant of the human hepatic nonparenchymal cells was collected.

[0066] [Experimental Example 2] Culture of human hepatocytes Frozen primary human hepatocytes ("HEP187-S", BIOPRRIDIC, containing trace amounts of human bile duct cells) were thawed in a 37°C water bath, suspended in a 50 mL tube with serum-free medium, and centrifuged. The serum-free medium was prepared by adding HEPES, GlutaMAX, and penicillin / streptomycin to Advanced DMEM / F12. After centrifugation, the supernatant was removed and the cells were then suspended in serum-free medium to prepare a human hepatocyte suspension (containing human bile duct cells). 10,000 cells were taken from this suspension, mixed with 50 μL of Matrigel® (BD Biosciences), seeded in each well of a 24-well tissue culture plate, and incubated at 37°C for 5-10 minutes until the Matrigel polymerized completely. After the Matrigel polymerized, a medium containing the components shown in "Medium B" in Table 1 was overlaid, and the human hepatocytes were cultured while changing the medium every 5 days. The culture was periodically collected, centrifuged to recover the cells, and the number of cells was counted. The increase in the number of cells since the start of culture was calculated to evaluate the cell proliferation rate.

[0067] [Experimental Example 3] Culture of human hepatocytes Human liver parenchymal cells were cultured using the same procedure as in Experimental Example 2, except that medium C shown in Table 1 was used instead of medium B. Cells were periodically collected and counted, and their proliferation was evaluated, as in Experimental Example 2.

[0068] [Experimental Example 4] Culture of human hepatocytes Human liver parenchymal cells were cultured using the same procedure as in Experimental Example 2, except that medium D shown in Table 1 was used instead of medium B. Cells were periodically collected and counted, and their proliferative capacity was evaluated, as in Experimental Example 2.

[0069] Figure 1 shows the increase in the number of cells in Experimental Examples 2 to 4.

[0070] [Experimental Example 5] Gene expression analysis of cultured human hepatocytes RNA-seq analysis of human hepatocytes from Experimental Example 2 and Experimental Example 4 after 2 weeks of culture was performed to measure the expression levels of various genes in the cells of Experimental Example 2 and Experimental Example 4. From the gene expression analyzed by RNA-seq, pathways that were likely to be activated were estimated using the pathway analysis software IPA. The results are shown in Figures 2-1 to 2-4. In Figures 2-1 to 2-4, NPC(-) represents human hepatocytes from Experimental Example 4, and NPC(+) represents human hepatocytes from Experimental Example 2. Different activated pathways existed between NPC(-) and NPC(+). Factors related to these activated pathways may contribute to the proliferation of hepatocytes.

[0071] [Experimental Example 6] Mature human hepatocytes After two weeks of culture, human hepatocytes from Experimental Example 2 were subjected to mechanical dissociation to disperse and prepare a human hepatocyte suspension. 10,000 cells were taken from this suspension, mixed with 50 μL of Matrigel® (BD Biosciences), and seeded in each well of a 24-well tissue culture plate. The suspension was incubated at 37°C for 5 to 10 minutes until the Matrigel was completely polymerized. After the Matrigel polymerized, a culture medium containing the components shown in "Culture Medium E" in Table 1 was added, and the cultured human hepatocytes from Experimental Example 2 were cultured while changing the medium every 7 days. The resulting culture was centrifuged, and the cells were collected.

[0072] [Experimental Example 7] Culture of human bile duct cells Frozen primary human hepatocytes ("HEP187-S", BIOPRRIDIC, containing trace amounts of human bile duct cells) were thawed in a 37°C water bath, suspended in a 50 mL tube with serum-free medium, and centrifuged. The serum-free medium was prepared by adding HEPES, GlutaMAX, and penicillin / streptomycin to Advanced DMEM / F12. After centrifugation, the supernatant was removed and the cells were then suspended in serum-free medium to prepare a human hepatocyte suspension (containing human bile duct cells). 10,000 cells were taken from this suspension, mixed with 50 μL of Matrigel® (BD Biosciences), seeded in each well of a 24-well tissue culture plate, and incubated at 37°C for 5 to 10 minutes until the Matrigel was completely polymerized. After Matrigel polymerized, a culture medium containing the components shown in "Culture Medium B" in Table 1 was added as a top layer. Human bile duct cells were cultured while the culture medium was changed every 5 days, and the proliferated human hepatocytes were removed from the medium as appropriate.

[0073] [Experimental Example 8] Comparative Analysis of Gene Expression RNA-seq gene expression analysis was performed on primary human hepatocytes ("HEP187-S", BIOPRRIDIC, hereinafter referred to as "PHHs") as a control, cultured human hepatocytes from Experimental Example 2 after 10 days of culture (hereinafter referred to as "Proli-Orgs"), mature human hepatocytes from Experimental Example 6 after 10 days of culture (hereinafter referred to as "Differ-Orgs"), mature human hepatocytes from Experimental Example 6 after 100 days of culture (hereinafter referred to as "Differ-Orgs (long-term)"), and human bile duct cells from Experimental Example 7 after 30 days of culture (hereinafter referred to as "Chol").

[0074] RNA-seq analysis was used to measure the expression levels of 57,095 genes, including metabolic enzymes, transporter genes, and hepatocyte markers. Read count data obtained from RNA-seq were corrected for total read count, followed by correction by transcript length (FRKM).

[0075] Figure 3 shows the correlation coefficients of linear regression of gene expression levels obtained by RNA-seq analysis between PHHs, Proli-Orgs, Differ-Orgs, Differ-Orgs (long-term), and Chol. As shown in Figure 3, the gene expression patterns of Proli-Orgs in Experimental Example 2, and Differ-Orgs and Differ-Orgs (long-term) in Experimental Example 6, all showed relatively high correlations with PHHs and were not similar to Chol, indicating that these cells were not altered into human non-parenchymal hepatic cells or other cells other than hepatic parenchymal cells.

[0076] Figure 4 shows microscopic images of Proli-Orgs and Differ-Orgs (long-term) and their gene expression levels. In Figure 4, gene expression levels are relative, with PHHs expression levels set to +++; a higher number of "+" signs indicates higher gene expression. From the microscopic images, it was clear that the lumen components of Proli-Orgs were red, and the lumen components of Differ-Orgs were yellow, indicating the presence of bile in the lumen. The results from Figures 3 and 4 demonstrate that the Differ-Orgs (long-term) from Example 3 have a high degree of similarity in gene expression patterns to PHHs, and therefore can be used in pharmacokinetic studies for drug development. Furthermore, these results indicate that the cultured human hepatocytes from Experimental Example 2 retain the ability to differentiate into mature human hepatocytes.

[0077] [Experimental Example 9] Human hepatocytes were cultured using the same procedure as in Experimental Example 2, except that medium F shown in Table 1 was used instead of medium B. Cell proliferation was confirmed by microscopic imaging. Microscopic images of cultured human hepatocytes from Experimental Example 2 and Experimental Example 9 are shown in Figures 5-1 and 5-2, respectively.

[0078] [Experimental Example 10] Human hepatocytes were cultured using the same procedure as in Experimental Example 2, except that collagen-coated plates were used instead of Matrigel-embedded plates. Microscopic images are shown in Figure 6. From the results of Experimental Example 10, it was confirmed that human hepatocytes proliferate in two dimensions on collagen-coated plates.

Claims

1. A method for culturing human hepatic parenchymal cells, comprising culturing human hepatic parenchymal cells in a culture medium containing the culture supernatant of human hepatic non-parenchymal cells, a Wnt signaling promoter, and a mitogenic growth factor.

2. The method according to claim 1, wherein the Wnt signaling promoter contains a Wnt family member protein.

3. The method according to claim 2, wherein the Wnt signaling promoter further contains an R-Spondin superfamily protein.

4. The method according to any one of claims 1 to 3, wherein the mitotic growth factor contains one or more selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF).

5. The method according to any one of claims 1 to 4, wherein the culture medium further contains one or more selected from Rho kinase (ROCK) signaling inhibitors, transforming growth factor β (TGF-β) signaling inhibitors, and interleukin-6 (IL-6) family cytokines.

6. The method according to claim 5, wherein the culture medium contains a ROCK signaling inhibitor.

7. The method according to claim 5 or 6, wherein the culture medium contains a TGF-β signaling inhibitor.

8. The method according to any one of claims 5 to 7, wherein the culture medium contains IL-6 family cytokines.

9. The method according to any one of claims 1 to 8, wherein the culture medium further contains one or more selected from retinoic acid, nicotinamide, cAMP activator, gastrin, neurobiological supplements, and antioxidants.

10. The method according to any one of claims 1 to 9, wherein the culture supernatant of human non-parenchymal hepatic cells is produced by culturing human non-parenchymal hepatic cells in a culture medium for human non-parenchymal hepatic cells containing a Wnt signaling promoter and a mitogenic growth factor, and then separating the supernatant from the culture medium.

11. The method according to claim 10, wherein the culture medium for human liver nonparenchymal cells further contains a ROCK signaling inhibitor.

12. The method according to claim 10 or 11, wherein the culture medium for human liver nonparenchymal cells further contains one or more selected from IL-6 family cytokines, TGF-β signaling inhibitors, retinoic acid, nicotinamide, cAMP activators, gastrin, neurobiological supplements, and antioxidants.

13. A culture medium containing the culture supernatant of human non-parenchymal hepatic cells, a Wnt signaling promoter, and a mitogenic growth factor.

14. A method for producing a culture medium, comprising mixing the culture supernatant of human nonparenchymal hepatocytes, a Wnt signaling promoter, and a mitogenic growth factor, The culture supernatant of the human non-parenchymal cells is Human non-parenchymal hepatocytes are cultured in a medium containing a Wnt signaling promoter and a mitogenic growth factor, and Separating the supernatant from the culture medium after the culture, It was manufactured by method.

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