JP7510133B2 - Method for producing liver organoids, medium for producing liver organoids, liver organoids, cell preparations, and method for evaluating test substances - Google Patents

Description

The present invention relates to a method for producing liver organoids, a medium for producing liver organoids, liver organoids, cell preparations, and a method for evaluating test substances.

In pharmacokinetic testing for pharmaceutical development, in vivo testing using rodents and in vitro testing using primary (frozen) hepatocytes derived from rodents are conducted. However, due to species differences, it is difficult to predict toxicity that will occur specifically in humans. On the other hand, there is a large difference between lots of primary (frozen) human hepatocytes, making it difficult to consistently obtain high-quality hepatocytes. In addition, human-derived hepatoma cell lines such as HepG2 cells have low activity of cytochrome P450 (CYP), making it impossible to evaluate toxicity related to metabolism by CYP. For this reason, there is a need for human-derived hepatocytes that can be used more stably.

Liver organoids were established by Hans Clevers et al. in 2013. These liver organoids are expected to serve as a stable source of liver cells for pharmaceutical development (see Patent Documents 1 to 4).

JP 2013-535201 A International Publication No. 2013/183571 US Patent Application Publication No. 2017/0191031 US Patent Application Publication No. 2016/0266096

Liao J. K., et al., Rho Kinase (ROCK) Inhibitors, J Cardiovasc Pharmacol., 50 (1), 17-24, 2007. Dupont S., et al., Role of YAP/TAZ in mechanotransduction, Nature, 474(7350), 179-183, 2011.

The objective of the present invention is to provide a method for producing liver organoids with excellent albumin expression levels.

The present invention includes the following embodiments.
[1] A method for producing a liver organoid, comprising a differentiation step of culturing a liver cell aggregate in a differentiation medium containing at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide, to obtain a liver organoid.
[2] The method for producing a liver organoid described in [1], wherein in the differentiation step, the liver cell aggregate is contacted with an extracellular matrix and cultured.
[3] The method for producing a liver organoid according to [1] or [2], wherein the volume fraction of the dimethyl sulfoxide contained in the differentiation medium is 0.1 volume% or more.
[4] The method for producing liver organoids according to any one of [1] to [3], wherein the differentiation medium further contains at least one growth factor selected from the group consisting of EGF, FGF, HGF, and IGF.
[5] The differentiation medium further contains at least one TGF-β inhibitor selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY364947, SD-208, and SJN2511. [1] to [4] The method for producing liver organoids.
[6] The method for producing liver organoids according to any one of [1] to [5], wherein the differentiation medium further contains at least one Notch inhibitor selected from the group consisting of DAPT, dibenzazepine, benzodiazepine, and LY-411575.
[7] The method for producing a liver organoid according to any one of [1] to [6], wherein the differentiation medium further contains dexamethasone.
[8] The method for producing liver organoids according to any one of [1] to [7], further comprising a proliferation step of culturing epithelial hepatic stem cells in a proliferation medium containing at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide, prior to the differentiation step, to obtain the liver cell aggregate.
[9] The method for producing liver organoids described in [8], wherein the volume fraction of dimethyl sulfoxide contained in the growth medium is 0.1 volume% or more.
[10] The method for producing liver organoids described in [8] or [9], wherein the proliferation medium further contains at least one growth factor selected from the group consisting of EGF, FGF, HGF, and IGF.
[11] The method for producing a liver organoid according to any one of [8] to [10], wherein the growth medium further contains nicotinamide.
[12] The method for producing liver organoid according to any one of [8] to [11], wherein the growth medium further contains at least one Wnt agonist selected from the group consisting of Wnt family members, R-spondin 1 to 4, Norrin, and GSK inhibitors.
[13] The method for producing liver organoids according to any one of [8] to [12], wherein the growth medium further contains at least one TGF-β inhibitor selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY364947, SD-208, and SJN2511.
[14] The method for producing liver organoids according to any one of [8] to [13], wherein the growth medium further contains at least one BMP inhibitor selected from the group consisting of Noggin, DAN, and DAN-like proteins including Cerberg and Gremlin.
[15] A medium for producing liver organoids, comprising at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide.
[16] A liver organoid produced by the production method according to any one of [1] to [14].
[17] A liver organoid obtained by culturing epithelial hepatic stem cells, wherein the expression level of albumin is 2 to 1000 times higher than the expression level of albumin in a tissue fragment containing the epithelial hepatic stem cells.
[18] The liver organoid according to [17], wherein the expression level of CYP2C19 is 50% to 200% of the expression level of CYP2C19 in the tissue fragment containing the epithelial hepatic stem cells.
[19] A cell preparation containing the liver organoid according to any one of [16] to [18].
[20] A method for evaluating a test substance comprising the steps of contacting a test substance with the liver organoid according to any one of [16] to [18], and evaluating the metabolic ability of the liver organoid for the test substance.

It can also be said that the present invention includes the following embodiments.
[P1] A method for producing a liver cell cluster, comprising a differentiation step of differentiating a liver organoid in a differentiation medium to obtain a liver cell cluster, the differentiation medium comprising at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide.
[P2] The method according to [P1], wherein in the differentiation step, the liver organoid is cultured in contact with an extracellular matrix.
[P3] The manufacturing method described in [P1] or [P2], wherein the differentiation medium contains 0.1 vol.% or more of dimethyl sulfoxide, based on 100 vol.% of the differentiation medium.
[P4] The manufacturing method according to any one of [P1] to [P3], wherein the differentiation medium further contains at least one selected from the group consisting of a growth factor selected from the group consisting of EGF, FGF, HGF, and IGF; a TGF-β inhibitor selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY364947, SD-208, and SJN2511; a Notch inhibitor selected from the group consisting of DAPT, dibenzazepine (DBZ), benzodiazepine (BZ), and LY-411575; and dexamethasone.
[P5] The manufacturing method described in any one of [P1] to [P4], wherein the differentiation medium further contains at least one selected from the group consisting of gastrin, B27 supplement, N2 supplement, and N-acetylcysteine.
[P6] The method according to any one of [P1] to [P5], further comprising a proliferation step of proliferating liver organoids in a proliferation medium prior to the differentiation step, wherein the proliferation medium contains at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide.
[P7] The manufacturing method described in [P6], in which the liver organoid is contacted with an extracellular matrix and cultured in the proliferation step.
[P8] The method according to [P6] or [P7], wherein the growth medium contains 0.1% by volume or more of dimethyl sulfoxide, based on 100% by volume of the growth medium.
[P9] The method according to any one of [P6] to [P8], further comprising at least one selected from the group consisting of growth factors selected from the group consisting of EGF, FGF, HGF and IGF; nicotinamide; Wnt agonists selected from the group consisting of Wnt family members, R-spondin 1 to 4, Norrin and GSK inhibitors; TGF-β inhibitors selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY364947, SD-208 and SJN2511; and BMP inhibitors selected from the group consisting of proteins selected from the group consisting of Noggin, DAN, and DAN-like proteins including Cerberug and Gremlin.
[P10] The production method described in any one of [P6] to [P9], wherein the growth medium further contains at least one selected from the group consisting of gastrin, B27 supplement, N2 supplement, and N-acetylcysteine.
[P11] The method according to any one of [P6] to [P10], in which the differentiation step is initiated at least one day after the initiation of the proliferation step.
[P12] The ROCK inhibitor is selected from the group consisting of Y-27632, Fasudil, Y39983, Wf-536, SLx-2119, Azabenzimidazole-aminofurazans, DE-104, H-1152P, ROKα inhibitor, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, Rhostain, BA-210, BA-207, Ki-23095 and VAS-012, and the YAP inhibitor is selected from the group consisting of Verteporfin, C3, CA3, (R)-PFI. The method for producing the compound according to any one of [P1] to [P11], wherein the compound is selected from the group consisting of YAP-TEAD Inhibitor 1, YAP-TEAD Inhibitor 2, Super-TDU, and YAP-TEAD Inhibitor 1.
[P13] A differentiation medium for inducing differentiation of a liver organoid into a liver cell mass, comprising at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide.
[P14] A growth medium for growing liver organoids, comprising at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide.
[P15] A liver cell mass produced by the production method according to any one of [P1] to [P12].
[P16] A liver cell mass contained in a container at a density of 200,000 cells/mL or more.
[P17] A cell preparation comprising the liver cell mass described in [P15] or [P16].
[P18] A method for evaluating a test substance, comprising the steps of culturing the liver cell cluster according to [P15] or [P16] in the presence of the test substance, and evaluating the response of the liver cell cluster.
[P19] A method for evaluating a test substance, comprising the steps of: acting a test substance on the liver cell cluster according to [P15] or [P16]; and evaluating the response of the liver cell cluster.

According to an embodiment of the present invention, liver organoids with excellent albumin expression levels can be produced.

The present invention will be described in more detail below with reference to the following embodiments, but the present invention is not limited to these embodiments.

Each component exemplified in this specification, for example, the components contained in the medium or the components used in each step, can be used alone or in combination of two or more types, unless otherwise specified.

In this specification, expressions expressing a numerical range such as "A to B" are synonymous with "A or more, B or less." Furthermore, expressions expressing a numerical range such as "A to B, preferably a to b" are 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."

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

[Method for producing liver organoids]
The method for producing a liver organoid of this embodiment includes a differentiation step of culturing a liver cell aggregate in a differentiation medium containing at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide.

The method for producing liver organoids of this embodiment can further include a proliferation step, prior to the differentiation step, in which epithelial hepatic stem cells are cultured in a proliferation medium containing at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide, to obtain a liver cell aggregate. By carrying out the proliferation step, a liver cell aggregate to be used in the differentiation step can be obtained.

The liver cell aggregates may be commercially available, for example, products under the trade name "Mouse Hepatic Organoids" (catalog number "#70932", Stem Cell Technologies, Inc.).

A cell aggregate refers to an aggregate formed by adhesion of two or more cells. The liver organoid of this embodiment is also one form of a cell aggregate. In addition, a liver organoid refers to a culture having a structure anatomically similar to liver tissue.

As used herein, "differentiate" and "induce differentiation" refer to inducing differentiation along a specific cell lineage. In the manufacturing method of this embodiment, liver cell aggregates are induced to differentiate into liver organoids.

Differentiation process
In the differentiation step, the liver cell aggregates are cultured in a differentiation medium containing at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide, to differentiate into liver organoids. The culture is usually preferably performed in a suspension culture. The culture period in the differentiation step is usually 5 to 15 days, preferably 7 to 12 days.

In the differentiation step, it is preferable to culture the liver cell aggregates in contact with an extracellular matrix (hereinafter also referred to as "ECM"). The liver organoids may be mixed with dissolved ECM, the ECM may be gelled, and then the gelled ECM may be overlaid with a differentiation medium for culture. In this specification, the culturing of liver organoids in a differentiation medium is expressed as "culturing in a differentiation medium", including the embodiment in which a differentiation medium is overlaid on polymerized ECM containing liver organoids for culture.

The ECM preferably contains at least two types of ECM. For example, an ECM containing collagen and laminin can be used. The ECM can be a synthetic ECM or a natural ECM. The ECM is preferably Matrigel (registered trademark) (BD Biosciences), which contains laminin, entactin, and collagen IV. The extracellular matrix material can be in a state coated on a cell culture vessel or in a dissolved state.

Differentiation medium
The differentiation medium contains at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide. The differentiation medium may further contain an extracellular matrix. The differentiation medium is usually prepared by adding various components to a basal medium.

Examples of basal media include Dulbecco's modified Eagle's medium (DMEM), basal medium (MEM), knockout-DMEM (KO-DMEM), Glasgow basal medium (G-MEM), Basal Eagle's medium (BME), DMEM/Ham's F12, Advanced DMEM/Ham's F12, Iscove's modified Dulbecco's medium, Ham's F-10, Ham's F-12, 199 medium, and RPMI 1640 medium.

As the basal medium, DMEM/F12, RPMI1640, which contains glutamine, insulin, penicillin/streptomycin, and transferrin; Advanced DMEM/F12, which is optimized for serum-free culture and already contains insulin; and Advanced RPMI; are preferred. It is preferable to add glutamine and penicillin/streptomycin to Advanced DMEM/F12 and Advanced RPMI media.

ROCK inhibitors include Y-27632 (CAS number: 146986-50-7), Fasudil (CAS number: 105628-07-7), Y39983 (CAS number: 203911-26-6), Wf-536 (CAS number: 539857-64-2), SLx-2119 (CAS number: 911417-87-3), Azabenzimidazole-aminofurazans (CAS number: 850664-21-0), DE-104 (see Non-Patent Document 1), H-1152P (see Non-Patent Document 1), and ROKα inhibitor (see Non-Patent Document 1), XD-4000 (see Non-Patent Document 1), HMN-1152 (see Non-Patent Document 1), 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides (see Non-Patent Document 1), Rhostain (see Non-Patent Document 1), BA-210 (see Non-Patent Document 1), BA-207 (see Non-Patent Document 1), Ki-23095 (see Non-Patent Document 1), and VAS-012 (see Non-Patent Document 1). Among these, Y-27632 is preferred. The final concentration of the ROCK inhibitor contained in the differentiation medium is usually 0.1 μM to 20 μM. The ROCK inhibitor may be used alone or in a mixture of two or more types. The final concentration refers to the concentration of a specific component contained in the medium as a result of adding the specific component to the medium.

Examples of YAP inhibitors include Verteporfin (CAS number: 129497-78-5), C3 toxin produced by Clostridium botulinum (see Non-Patent Document 2), CA3 (CAS number: 300802-28-2), (R)-PFI 2 hydrochloride (CAS number: 1627607-87-7), Super-TDU (CAS number: 1599441-71-0), and YAP-TEAD Inhibitor 1 (catalog number "S8164", Selleck Chemicals, etc.). The final concentration of the YAP inhibitor contained in the differentiation medium is usually 0.1 μM to 10 μM. One type of YAP inhibitor may be used alone, or two or more types may be mixed and used.

The volume fraction of dimethyl sulfoxide contained in the differentiation medium is preferably 0.1% by volume or more, more preferably 0.3% by volume or more, and even more preferably 0.5% by volume or more. The volume fraction of dimethyl sulfoxide contained in the differentiation medium is usually 10% by volume or less, preferably 5% by volume or less, and more preferably 3% by volume or less. If it is within the above numerical range, liver organoids with enhanced albumin production ability can be obtained.

The differentiation medium may further contain at least one growth factor selected from the group consisting of EGF (epidermal growth factor), FGF (fibroblast growth factor), hepatocyte growth factor (HGF) and insulin-like growth factor (IGF).

The final concentration of EGF contained in the differentiation medium is usually 5 ng/mL to 500 ng/mL, preferably 25 ng/mL to 300 ng/mL, and more preferably 50 to 100 ng/mL.

FGFs include FGF2, FGF4, FGF7, and FGF10, with FGF10 being preferred. The final concentration of FGF contained in the differentiation medium is usually 20 ng/mL to 500 ng/mL, preferably 50 ng/mL to 500 ng/mL, and more preferably 100 to 500 ng/mL.

The final concentration of HGF contained in the differentiation medium is usually 1 ng/mL to 100 ng/mL, preferably 10 ng/mL to 100 ng/mL, more preferably 20 ng/mL to 100 ng/mL, and even more preferably 40 ng/mL to 100 ng/mL.

As an IGF, IGF1 is preferred. The final concentration of IGF contained in the differentiation medium is usually 5 ng/mL to 1000 ng/mL, preferably 10 ng/mL to 1000 ng/mL, more preferably 50 ng/mL to 1000 ng/mL.

The differentiation medium may further contain at least one TGF-β inhibitor (transforming factor β inhibitor) selected from the group consisting of A83-01 (CAS number: 909910-43-6), SB-431542 (CAS number: 301836-41-9), SB-505124 (CAS number: 694433-59-5), SB-525334 (CAS number: 356559-20-1), LY364947 (CAS number: 396129-53-6), SD-208 (CAS number: 627536-09-8), and SJN2511 (CAS number: 446859-33-2).

A83-01 is preferably used as a TGF-β inhibitor. The final concentration of the TGF-β inhibitor contained in the differentiation medium is usually 0.01 nM to 600 nM.

The differentiation medium may further contain at least one Notch inhibitor selected from the group consisting of DAPT (CAS number: 208255-80-5), dibenzazepine (DBZ) (CAS number: 209984-56-5), benzodiazepine (BZ) (CAS number: 209986-17-4), and LY-411575 (CAS number: 209984-57-6).

DAPT is preferably used as a Notch inhibitor. The final concentration of the Notch inhibitor contained in the differentiation medium is usually 1 μM to 50 μM, preferably 5 μM to 30 μM, and more preferably 5 μM to 20 μM.

The differentiation medium may further contain dexamethasone (CAS number: 50-02-2). The final concentration of dexamethasone contained in the differentiation medium may be 1 μM to 50 μM, 1 μM to 40 μM, or 1 μM to 35 μM.

The differentiation medium may further contain neurobiological supplements such as gastrin, B27 supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), and N-acetylcysteine.

The final concentration of gastrin contained in the differentiation medium is usually 5 nM to 15 nM, preferably 7 nM to 10 nM.

B27 supplement is a composition containing biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin, and transferrin, and is commercially available as a 50x liquid concentrate. Of these ingredients, at least linoleic acid, retinol, retinyl acetate, and triiodothyronine (T3) are nuclear hormone receptor agonists.

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

The final concentration of N-acetylcysteine contained in the differentiation medium is usually 150 ng/mL to 250 ng/mL, preferably 170 ng/mL to 200 ng/mL.

<Proliferation process>
In the proliferation step, the epithelial hepatic stem cells are cultured in a proliferation medium containing at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide, to obtain the liver cell aggregate. By having the proliferation step, liver organoids with high albumin production ability can be produced.

In the proliferation step, it is preferable to culture the liver organoids in contact with an extracellular matrix (ECM). For example, as described later in the Examples, the liver organoids may be mixed with the ECM, the ECM may be polymerized, and then the polymerized ECM may be overlaid with a proliferation medium for culture. In this specification, the cultivation of liver organoids in a proliferation medium is expressed as "culturing in a proliferation medium", including the embodiment in which the proliferation medium is overlaid on the polymerized ECM containing the liver organoids for culture. The ECM used in the proliferation step may be the same as the ECM used in the differentiation step described above.

Epithelial hepatic stem cells are isolated from liver fragments or hepatic bile ducts. Methods for isolating these cells are known. For example, as described in the Examples herein, a cell population containing epithelial hepatic stem cells can be obtained by dissociating adult liver tissue. Dissociation of the tissue can be performed by enzymatic dissociation, and enzymes such as collagenase, dispase, etc. can be used.

Growth medium
The proliferation medium contains at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide. In the proliferation medium, the ROCK inhibitor and the YAP inhibitor can be the same as those in the differentiation medium described above at the same concentrations. In addition, the proliferation medium preferably contains 0.1% by volume or more of dimethyl sulfoxide (CAS number: 67-68-5) based on the proliferation medium being 100% by volume, more preferably 0.3% by volume or more, and even more preferably 0.5% by volume or more. In addition, the proliferation medium preferably contains 10% by volume or less of dimethyl sulfoxide (CAS number: 67-68-5) based on the proliferation medium being 100% by volume, more preferably 5% by volume or less, and even more preferably 3% by volume or less. If the above numerical values are within the range, a liver cell mass with enhanced albumin production ability can be obtained.

Other Additives
The growth medium contains a growth factor selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF) and insulin-like growth factor (IGF); nicotinamide (CAS number: 98-92-0); a Wnt agonist selected from the group consisting of Wnt family members, R-spondin 1 to 4, Norrin and a GSK inhibitor; A83-01 (CAS number: 909910-43-6), SB-431542 (CAS number: 301836-41-9), SB-505124 (CAS number: 694433-59-5), SB-5 and a bone morphogenetic protein (BMP) inhibitor selected from the group consisting of proteins selected from the group consisting of Noggin, DAN, and DAN-like proteins including Cerberug and Gremlin.

The EGF content in the proliferation medium is the same as the EGF content in the differentiation medium described above.

The proliferation medium may contain the same FGF as the differentiation medium described above in the same amount.

The HGF content in the proliferation medium is the same as the HGF content in the differentiation medium described above.

The proliferation medium may contain an IGF similar to that in the differentiation medium described above, in a similar amount.

The nicotinamide content in the growth medium may be 5-15 mM, or 8-12 mM.

The Wnt agonist may be at least one selected from the group consisting of Wnt family members, R-spondin1, R-spondin2, R-spondin3, R-spondin4, Norrin, and GSK inhibitors. Examples of Wnt family members include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. As the R-spondin, R-spondin1 or R-spondin4 is preferred, and R-spondin1 is particularly preferred.

The Wnt agonist preferably contains R-spondin 1. The content of the Wnt agonist in the growth medium may be 200 ng/mL or more, 300 ng/mL or more, 400 ng/mL or more, or 500 ng/mL or more. The upper limit of the content of the Wnt agonist is not particularly limited, but may be, for example, about 600 ng/mL.

When the liver organoids are derived from humans, it is preferable that the growth medium contains a TGF-β inhibitor. A83-01 is preferably used as the TGF-β inhibitor. The content of the TGF-β inhibitor in the growth medium may be 0 to 20 μM, or 0 to 10 μM.

The BMP inhibitor is preferably Noggin. The content of the BMP inhibitor in the differentiation medium may be 10 to 100 ng/mL, 20 to 100 ng/mL, or 50 to 100 ng/mL. There is no particular upper limit to the content of the BMP inhibitor, but it may be, for example, about 150 ng/mL.

The proliferation medium preferably further contains at least one selected from the group consisting of gastrin, B27 supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), and N-acetylcysteine. In the proliferation medium, the gastrin, B27 supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), and N-acetylcysteine can be the same as those in the differentiation medium described above, and can be used at the same concentrations.

In the method for producing liver organoids of this embodiment, when a proliferation step is performed, it is preferable to start the differentiation step at least one day after the proliferation step is started. The period of the proliferation step (culture period) may be, for example, 1 to 15 days, for example, 1 to 5 days, or for example, 2 to 4 days.

The proliferation of liver organoids can be evaluated by measuring the expression level of the Lgr5 gene or Lgr5 protein. Liver organoids express Lgr5 protein.

In addition, in the method for producing liver organoids of this embodiment, differentiation of liver cell aggregates into liver organoids can be determined or evaluated using the expression of liver cell markers, drug metabolism activity, and the like as indicators. Examples of liver cell markers include albumin (ALB), α-fetoprotein (AFP), tyrosine aminotransferase (TAT), pregnane X receptor (PXR), and the like. The expression level of the liver cell marker may be measured at the gene level or the protein level.

One method for obtaining a population consisting only of liver organoids from a population of liver cell aggregates is, for example, a method for selecting and separating liver cell aggregates using the presence of a cell surface marker characteristic of liver organoids as an indicator.

Drug metabolism activity can be evaluated by detecting the expression of drug metabolism enzymes or by drug metabolism assays. Examples of drug metabolism enzymes include cytochrome P450 2C19 (CYP2C19), cytochrome P450 3A4 (CYP3A4), cytochrome P450 3A7 (CYP3A7), cytochrome P450 1A (CYP1A), cytochrome P450 3A (CYP3A), cytochrome P450 2B (CYP2B), cytochrome P450 2D (CYP2D), uridine diphosphate-glucuronosyltransferase (UGT), sulfotransferase (SULT), etc.

The conditions such as culture temperature in the proliferation step and differentiation step may be the conditions generally adopted in the culture of animal cells, specifically, for example, culture may be performed in an environment of 37°C and 5% _CO2 .

[Liver organoids]
The liver organoid of this embodiment can be produced by the above-mentioned liver organoid production method.The liver organoid of this embodiment can be suitably used for in vitro evaluation of the metabolism of test substance, drug interaction, hepatotoxicity, transporter activity, etc.In addition, it can also be used as a cell preparation for transplanting into a patient with liver disease to treat liver disease.

The expression level of albumin in the liver organoid of this embodiment is preferably 2 to 1000 times, more preferably 2.5 to 100 times, and even more preferably 3 to 50 times, the expression level of albumin in the tissue fragment containing epithelial hepatic stem cells used in the culture.

The expression level of CYP2C19 in the liver organoid of this embodiment is preferably 50% to 200%, more preferably 80% to 180%, and even more preferably 100% to 150% of the expression level of CYP2C19 in the tissue fragment containing epithelial hepatic stem cells used in the culture.

The density of the liver organoids in this embodiment is preferably 200,000 cells/mL or more.

[Cell preparations]
The cell preparation of the present embodiment contains the above-mentioned organoid.The epithelial hepatic stem cell that is the origin of the liver organoid contained in the cell preparation of the present embodiment can be derived from the liver tissue that immunogenicity is reduced by genetic engineering method or the like, can be derived from the liver tissue of patient, or can be derived from the liver tissue of a third person that is immunologically compatible with patient.

The cell preparation of this embodiment can be used to treat various liver diseases. For example, it is expected to be used as a material for regenerating and rebuilding damaged or dysfunctional liver tissue. Therefore, the cell preparation of this embodiment can be used for regenerative medicine.

The cell preparation of this embodiment can be produced, for example, by suspending the above-mentioned liver organoids in physiological saline or a buffer solution (e.g., a phosphate buffer solution), etc. In order to administer a therapeutically effective amount of cells, it is preferable to contain, for example, 1 x 10 ⁵ to 1 x 10 ¹⁰ cells per dose.

The dosage of the cell preparation of this embodiment can be adjusted appropriately taking into consideration the purpose of use, the target disease, the gender, age, weight, condition of the affected area, and condition of the cells of the recipient. In addition, the cell preparation of this embodiment can be administered by local administration to the liver, intradermal administration, intramuscular administration, intravenous administration, etc.

[Test substance evaluation method]
In one embodiment, the present invention provides a method for evaluating the test substance, comprising the steps of culturing the liver organoid produced by the above-mentioned production method in the presence of the test substance, and evaluating the metabolic ability of the liver organoid.In another embodiment, the present invention provides a method for evaluating the test substance, comprising the steps of making the liver organoid produced by the above-mentioned production method act on the test substance, and evaluating the response of the liver organoid.

The evaluation method of this embodiment allows the metabolism, drug interactions, hepatotoxicity, transporter activity, etc. of a test substance to be evaluated in vitro, and results consistent with in vivo evaluations can be obtained.

For example, the metabolism of a test substance can be tested using liver organoids. The test substance is not particularly limited, and examples thereof 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 the test substance. 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, vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.), and the like.

Preferred test substances include existing or candidate components of medicines, nutritional foods, etc. Plant extracts, cell extracts, culture supernatants, etc. may also be used as test substances. In addition, by adding two or more types of test substances simultaneously, it is possible to examine interactions, synergistic effects, etc. between the test substances. Test substances may be derived from natural products or may be artificially synthesized. In the latter case, an efficient assay system can be constructed, for example, by using combinatorial synthesis techniques.

In the evaluation method of this embodiment, the period for contacting the test substance with the liver organoids can be set arbitrarily. The contact period may be, for example, 10 minutes to 3 days, or 1 hour to 1 day. The test substance may be contacted with the liver organoids multiple times.

The response of liver organoids can be evaluated by an appropriate analytical method. For example, mass spectrometry, liquid chromatography, immunological techniques, etc. can be used depending on the metabolic products produced. Immunological techniques include, for example, fluorescent immunoassay (FIA) and enzyme immunoassay (EIA).

The metabolism of the test substance can also be measured using the expression of drug-metabolizing enzymes (e.g., cytochromes, UGTs, etc.) in liver organoids as an indicator. The expression of drug-metabolizing enzymes can be evaluated at the mRNA level or the protein level.

The toxicity of a test substance can also be tested using the evaluation method of this embodiment. For example, the state of liver organoids after contact with the test substance is examined, and the toxicity of the test substance is evaluated. Examples of the state of liver organoids include viability, cell morphology, and the amount of liver damage markers (e.g., GOT, GPT, etc.) present in the culture medium.

[Example 1]
(Formation of mouse liver cell aggregates)
The liver of the excised C57BL/6 mouse was washed with PBS and then minced finely with surgical scissors. The minced liver was reacted with a mixed solution of collagenase and dispase in a 50 mL tube at 37° C. for 2 hours while shaking. The liver suspension after the reaction was filtered with a cell strainer with a mesh size of 100 μm (catalog number "352360", cell strainer (mesh 100 μm), BD Falcon) to remove undispersed liver fragments. The liver suspension after filtration was centrifuged and washed with Advanced DMEM/F12. After this washing operation was performed twice, the cells were suspended in Advanced DMEM/F12 to prepare a hepatocyte suspension. 10,000 hepatocytes from this suspension were mixed with 50 μL of Matrigel (BD Biosciences), seeded on a 24-well tissue culture plate, and incubated at 37° C. for 5 to 10 minutes until the Matrigel was completely polymerized. After the Matrigel was polymerized, 500 μL of medium was overlaid and cultured. The mouse liver cell aggregates obtained by the culture were diluted by enzymatic dissociation and subcultured.

(Differentiation of mouse liver organoids)
After enzymatic dissociation of the formed mouse liver cell aggregates, 1,000 of the cells obtained were mixed with 50 μL of Matrigel (BD Biosciences), seeded on a 24-well tissue culture plate, and incubated at 37° C. for 5 to 10 minutes until the Matrigel was completely polymerized. After the Matrigel was polymerized, 500 μL of growth medium was layered and cultured for 4 days. The growth medium used had the composition shown in Table 1.

Next, on the fourth day after the proliferation medium was layered, the entire medium was replaced with differentiation medium. Thereafter, the medium was replaced with fresh differentiation medium every other day. The differentiation medium used had the composition shown in Table 1.

(Measurement of albumin expression at the protein level)
Nine days after the replacement of the differentiation medium, the medium was replaced with a new one, and the culture was continued for another three days without replacing the medium. The culture supernatant was then collected, and the albumin production level in the medium was measured by ELISA. The ELISA was performed using a commercially available kit (catalog number "E99-134", Betyl Laboratories).

After collecting the culture supernatant, the cells were subjected to measurement of ATP activity using a commercially available kit (product name "CellTiter-Glo", Promega).

Then, the absorbance at a wavelength of 450 nm measured by the above-mentioned ELISA method was divided by the measured value of the above-mentioned ATP activity, and the amount of albumin produced was determined by correcting for the cell mass. The measurement results of the amount of albumin produced are shown in Table 1.

(Measurement of albumin expression at the gene level)
After collecting the culture supernatant, total ribonucleic acid (RNA) was extracted from the cells using a commercially available kit (trade name "FastLane Cell cDNA Kit", Qiagen), cDNA was synthesized, and the expression level of the mRNA of the albumin gene was measured by real-time quantitative PCR. Real-time quantitative PCR was performed using a commercially available kit (trade name "SYBR(R) Premix Ex Taq(TM) (Perfect Real Time)", Takara Bio Inc.). In addition, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to correct the measurement results. Table 1 shows the measurement results of the albumin production amount.

[Examples 2 to 7]
The liver cell aggregates were induced to differentiate into liver organoids and the amount of albumin produced was measured in the same manner as in Example 1, except that the compositions of the proliferation medium and differentiation medium were changed to those shown in Table 1. Table 1 shows the results of the measurement of the amount of albumin produced.

[Example 8]
(Formation of human liver cell aggregates)
Human frozen floating hepatocytes HMCS1S (Thermo Fisher Scientific) were thawed, added to Advanced DMEM/F12 and centrifuged. After removing the supernatant, the cells were washed with Advanced DMEM/F12. After this washing procedure was performed twice, the cells were suspended in Advanced DMEM/F12 to prepare a hepatocyte suspension. From this suspension, 60,000 hepatocytes were mixed with 50 μL of Matrigel (BD Biosciences), seeded on a 24-well tissue culture plate, and incubated at 37° C. for 5 to 10 minutes until the Matrigel was completely polymerized. Subsequently, after the Matrigel was polymerized, 500 μL of growth medium was overlaid and cultured. The expanded human hepatocyte aggregates were diluted by enzymatic dissociation and passaged.

(Induction of differentiation of human liver cell aggregates)
After enzymatic dissociation of the formed human liver cell aggregates, the resulting 10,000 cells were mixed with 50 μL of Matrigel (BD Biosciences), seeded on a 24-well tissue culture plate, and incubated at 37 ° C for 5 to 10 minutes until the Matrigel was completely polymerized. After the Matrigel was polymerized, 500 μL of growth medium was layered and cultured for 10 days to obtain human liver organoids. As the growth medium, a medium having the composition described in Table 1 was used.

Next, on the 10th day after the proliferation medium was layered, the entire medium was replaced with differentiation medium. Thereafter, the medium was replaced with fresh differentiation medium every two days. The differentiation medium used had the composition shown in Table 1.

(Measurement of albumin expression at the gene level)
Ten days after the medium was replaced with the differentiation medium, total ribonucleic acid (RNA) was extracted from the cells using a commercially available kit (trade name "FastLane Cell cDNA Kit", Qiagen), cDNA was synthesized, and the expression level of the mRNA of the albumin gene was measured by real-time quantitative PCR. Real-time quantitative PCR was performed using a commercially available kit (trade name "SYBR(R) Premix Ex Taq(TM) (Perfect Real Time)", Takara Bio Inc.). In addition, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to correct the measurement results. Table 1 shows the measurement results of the albumin production amount.

[Comparative Example 1]
(Formation of mouse liver cell aggregates)
In the same manner as in Example 1, mouse liver cell aggregates were formed.

(Differentiation of mouse liver organoids)
After enzymatic dissociation of the formed mouse liver cell aggregates, 1,000 of the cells obtained were mixed with 50 μL of Matrigel (BD Biosciences), seeded on a 24-well tissue culture plate, and incubated at 37° C. for 5 to 10 minutes until the Matrigel was completely polymerized. After the Matrigel was polymerized, 500 μL of growth medium was layered and cultured for 3 days. The growth medium used had the composition shown in Table 2.

Next, on the third day after the proliferation medium was layered, the entire medium was replaced with differentiation medium. Thereafter, the medium was replaced with fresh differentiation medium every other day. The differentiation medium used had the composition shown in Table 2.

(Measurement of albumin expression at the protein level)
Nine days after the replacement of the differentiation medium, the medium was replaced with a new one, and the culture was continued for another three days without replacing the medium. The culture supernatant was then collected, and the albumin production level in the medium was measured by ELISA. The ELISA was performed using a commercially available kit (catalog number "E99-134", Betyl Laboratories).

After collecting the culture supernatant, the cells were subjected to measurement of ATP activity using a commercially available kit (product name "CellTiter-Glo", Promega).

Next, the absorbance at a wavelength of 450 nm measured by the above-mentioned ELISA method was divided by the measured value of the above-mentioned ATP activity, and the amount of albumin produced was determined by correcting for cell mass.

(Measurement of albumin expression at the gene level)
After collecting the culture supernatant, total ribonucleic acid (RNA) was extracted from the cells using a commercially available kit (trade name "FastLane Cell cDNA Kit", Qiagen), cDNA was synthesized, and the expression level of the mRNA of the albumin gene was measured by real-time quantitative PCR. Real-time quantitative PCR was performed using a commercially available kit (trade name "SYBR(R) Premix Ex Taq(TM) (Perfect Real Time)", Takara Bio Inc.). In addition, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to correct the measurement results. Table 2 shows the measurement results of the albumin production amount.

[Comparative Examples 2 to 4]
The liver cell aggregates were induced to differentiate into liver organoids in the same manner as in Comparative Example 1, except that the compositions of the proliferation medium and differentiation medium were changed to those shown in Table 2, and the amount of albumin produced was measured. Table 2 shows the results of the measurement of the amount of albumin produced.

[Comparative Example 5]
(Formation of human liver cell aggregates)
In the same manner as in Example 8, human liver cell aggregates were formed.

(Differentiation of human liver organoids)
After enzymatic dissociation of the formed human liver cell aggregates, 10,000 cells obtained were mixed with 50 μL of Matrigel (BD Biosciences), seeded on a 24-well tissue culture plate, and incubated at 37° C. for 5 to 10 minutes until the Matrigel was completely polymerized. After the Matrigel was polymerized, 500 μL of growth medium was layered and cultured for 10 days. The growth medium used had the composition shown in Table 2.

Next, on the 10th day after the proliferation medium was layered, the entire medium was replaced with differentiation medium. Thereafter, the medium was replaced with fresh differentiation medium every two days. The differentiation medium used had the composition shown in Table 2.

(Measurement of albumin expression at the gene level)
Ten days after the medium was replaced with the differentiation medium, total ribonucleic acid (RNA) was extracted from the cells using a commercially available kit (trade name "FastLane Cell cDNA Kit", Qiagen), cDNA was synthesized, and the expression level of the mRNA of the albumin gene was measured by real-time quantitative PCR. Real-time quantitative PCR was performed using a commercially available kit (trade name "SYBR(R) Premix Ex Taq(TM) (Perfect Real Time)", Takara Bio Inc.). In addition, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to correct the measurement results. Table 2 shows the measurement results of the albumin production amount.

(Measurement of expression level of each CYP at gene level)
In Example 8 and Comparative Example 5, 10 days after the medium was replaced with the differentiation medium, total ribonucleic acid (RNA) was extracted from the cells using a commercially available kit (trade name "FastLane Cell cDNA Kit", Qiagen), cDNA was synthesized, and the expression level of mRNA of each CYP gene was measured by real-time quantitative PCR. Real-time quantitative PCR was performed using a commercially available kit (trade name "SYBR(R) Premix Ex Taq(TM) (Perfect Real Time)", Takara Bio Inc.). In addition, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to correct the measurement results. Table 3 shows the measurement results of each CYP expression level relative to each CYP expression level in frozen hepatocytes. The values in Table 3 are the percentages (%) when the GAPDH expression level is set to 100%.

The present invention provides a method for producing liver organoids with excellent albumin expression levels.

Claims (10)

The method includes a differentiation step of culturing a liver cell aggregate in a differentiation medium containing at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide, to obtain a liver organoid;
In the differentiation step, the liver cell aggregate is contacted with an extracellular matrix and cultured,
The differentiation medium further contains at least one growth factor selected from the group consisting of EGF, FGF, HGF, and IGF; at least one TGF-β inhibitor selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY364947, SD-208, and SJN2511; at least one Notch inhibitor selected from the group consisting of DAPT, dibenzazepine, benzodiazepine, and LY-411575; and dexamethasone ,
A method for producing a liver organoid, wherein the volume fraction of dimethyl sulfoxide contained in the differentiation medium is 0.5 volume% or more and 3 volume% or less. The method for producing liver organoids according to claim 1, wherein the differentiation step is carried out for 5 to 15 days. The method for producing liver organoids according to claim 1 or 2, further comprising, prior to the differentiation step, a proliferation step of culturing epithelial hepatic stem cells in a proliferation medium containing at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide, to obtain the liver cell aggregates. The method for producing liver organoids according to claim 3, wherein the volume fraction of dimethyl sulfoxide contained in the growth medium is 0.1% by volume or more. The method for producing liver organoids according to claim 3 or 4, wherein the growth medium further contains at least one growth factor selected from the group consisting of EGF, FGF, HGF, and IGF. The method for producing liver organoids according to any one of claims 3 to 5, wherein the growth medium further contains nicotinamide. The method for producing liver organoids according to any one of claims 3 to 6, wherein the growth medium further contains at least one Wnt agonist selected from the group consisting of Wnt family members, R-spondins 1 to 4, Norrin, and GSK inhibitors. The method for producing liver organoids according to any one of claims 3 to 7, wherein the growth medium further contains at least one TGF-β inhibitor selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY364947, SD-208, and SJN2511. The method for producing liver organoids according to any one of claims 3 to 8, wherein the growth medium further contains at least one BMP inhibitor selected from the group consisting of Noggin, DAN, and DAN-like proteins including Cerberg and Gremlin. The composition comprises at least one selected from the group consisting of a ROCK inhibitor and a YAP inhibitor, and dimethyl sulfoxide;
at least one growth factor selected from the group consisting of EGF, FGF, HGF, and IGF; at least one TGF-β inhibitor selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY364947, SD-208, and SJN2511; at least one Notch inhibitor selected from the group consisting of DAPT, dibenzazepines, benzodiazepines, and LY-411575; and dexamethasone,
A culture medium for hepatic organoid differentiation , wherein the volume fraction of dimethyl sulfoxide is 0.5% by volume or more and 3% by volume or less .