WO2025211311A1 - Method for producing organoid and culture medium for producing organoid - Google Patents

Abstract

Provided are: a method for producing an organoid, the method comprising a step for culturing somatic stem cells in a culture medium containing an agonist of the interferon-γ receptor; and a culture medium for producing an organoid, the culture medium containing an agonist of the interferon-γ receptor.

Description

Method for producing organoids and culture medium for producing organoids

The present invention relates to a method for producing organoids and a culture medium for producing organoids. This application claims priority to Japanese Patent Application No. 2024-059202, filed April 1, 2024, the contents of which are incorporated herein by reference.

Organoids are cultured cells formed by the accumulation of cells, and have a structure and function similar to that of organs in the body. In recent years, there has been active research into creating various organoids from stem cells, such as somatic stem cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells). Organoids are produced from stem cells by controlling signaling pathways to induce stem cell proliferation, differentiation, etc.

To date, for example, intestinal organoids, liver organoids, kidney organoids, stomach organoids, lung organoids, ovarian cancer organoids, biliary tract cancer organoids, and brain organoids have been created.

The application of organoids in regenerative medicine is also being considered. For this reason, there is a need to establish technology for efficiently expanding and culturing organoids.

Interferon-gamma (IFN-γ) is a type of inflammatory cytokine. For example, Non-Patent Document 1 describes how IFN-γ regulates the homeostatic functions of cell proliferation and cell death via the serine-threonine protein kinase AKT-β-catenin signaling pathway and the Wingless-Int (Wnt)-β-catenin signaling pathway. Furthermore, Non-Patent Document 2 describes how intestinal organoids were exposed to IFN-γ to recreate an inflammatory state in order to create an evaluation system for therapeutic drugs for Crohn's disease.

Nava P, et al., Interferon-gamma regulates intestinal epithelial homeostasis through converging beta-catenin signaling pathways, Immunity, 32 (3), 392-402, 2010. Osaki L. H., et al., Interferon-gamma directly induces gastric epithelial cell death and is required for progression to metaplasia, J Pathol., 247 (4), 513-523, 2019.

The present invention aims to provide a technology for efficiently expanding and culturing organoids.

The present invention includes the following embodiments.
[1] A method for producing organoids, comprising culturing somatic stem cells in a medium containing an agonist of the interferon-γ receptor.
[2] The method for producing organoids described in [1], wherein the culture medium further contains an agonist of the hepatocyte growth factor (HGF) receptor.
[3] The method for producing an organoid described in [1] or [2], wherein the culture medium further contains a vitamin D receptor agonist.
[4] The method for producing an organoid according to any one of [1] to [3], wherein the culture medium further contains a Hippo signaling pathway inhibitor.
[5] The method for producing an organoid described in [4], wherein the Hippo signaling pathway inhibitor is a LATS1/2 inhibitor.
[6] The method for producing an organoid according to [5], wherein the culture medium contains 0.1 μM to 50 μM of the LATS1/2 inhibitor.
[7] The method for producing an organoid according to any one of [1] to [6], wherein the medium contains 0.1 pM to 500 pM of an agonist of the interferon-γ receptor.
[8] A method for producing an organoid according to any one of [1] to [7], wherein the somatic stem cells are cultured in the culture medium for 24 hours or more from the start of culture.
[9] The method for producing organoids according to any one of [1] to [8], wherein the culture medium further comprises one or a combination of two or more selected from the group consisting of epidermal growth factor (EGF) receptor agonists, bone morphogenetic protein (BMP) signaling pathway inhibitors, p38 inhibitors, insulin-like growth factor 1 (IGF1) receptor agonists, fibroblast growth factor 2 (bFGF, FGF2) receptor agonists, Wnt signaling pathway activators and Rho-binding kinase inhibitors.
[10] The method for producing an organoid according to any one of [1] to [9], wherein the somatic stem cells are intestinal epithelial stem cells.
[11] A medium for producing organoids, comprising an agonist of interferon-γ receptor.
[12] The organoid production medium described in [11], further containing an agonist of the hepatocyte growth factor (HGF) receptor.
[13] The organoid production medium according to [11] or [12], further comprising a vitamin D receptor agonist.
[14] The organoid production medium according to any one of [11] to [13], further comprising a Hippo signaling pathway inhibitor.
[15] The organoid production medium according to any one of [11] to [14], further comprising one or a combination of two or more selected from the group consisting of epidermal growth factor (EGF) receptor agonists, bone morphogenetic protein (BMP) signaling pathway inhibitors, p38 inhibitors, insulin-like growth factor 1 (IGF1) receptor agonists, fibroblast growth factor 2 (bFGF, FGF2) receptor agonists, Wnt signaling pathway activators and Rho-binding kinase inhibitors.

The present invention provides a technology for efficiently expanding and culturing organoids.

FIG. 1 is a graph showing the results of evaluating cell proliferation in Experimental Example 1. FIG. 2 shows bright-field observation images and fluorescence microscope images of small intestinal organoids in Experimental Example 2. FIG. 3 is a graph showing the results of evaluating cell proliferation in Experimental Example 3. FIG. 4 is a graph showing the results of evaluating cell proliferation in Experimental Example 4. FIG. 5 is a bright-field observation image of small intestinal organoids in Experimental Example 4. FIG. 6 is a graph showing the results of evaluating cell proliferation in Experimental Example 5. FIG. 7 is a graph showing the results of evaluating cell proliferation in Experimental Example 6. FIG. 8 shows bright-field observation images and fluorescence microscope images of small intestinal organoids in Experimental Example 6. FIG. 9 is a graph showing the results of evaluating cell proliferation in Experimental Example 7. FIG. 10 shows bright-field observation images and fluorescence microscope images of small intestinal organoids in Experimental Example 7. FIG. 11 is a graph showing the results of evaluating cell proliferation in Experimental Example 8. FIG. 12 shows bright-field observation images and fluorescence microscope images of small intestinal organoids in Experimental Example 8. FIG. 13 is a bright-field observation image of intestine-derived organoids in Experimental Example 9. FIG. 14 is a graph showing the results of evaluating cell proliferation in Experimental Example 9. FIG. 15 is a bright-field observation image of the liver organoid in Experimental Example 10. FIG. 16 is a graph showing the results of evaluating cell proliferation in Experimental Example 10. FIG. 17 is a graph showing the results of evaluating cell proliferation in Experimental Example 11. FIG. 18 is a bright-field observation image of small intestinal organoids in Experimental Example 11. FIG. 19 is a bright-field observation image of small intestinal organoids in Experimental Example 11. FIG. 20 is a graph showing the results of evaluating cell proliferation in Experimental Example 12. FIG. 21 is a bright-field observation image of small intestinal organoids in Experimental Example 12. FIG. 22 is a graph showing the evaluation results of cell yield in Experimental Example 13. FIG. 23 is a graph showing the results of evaluating cell proliferation in Experimental Example 14. FIG. 24 shows bright-field observation images and fluorescence microscope images of small intestinal organoids in Experimental Example 14. FIG. 25 is a graph showing the results of evaluating cell proliferation in Experimental Example 15. FIG. 26 is a bright-field observation image of small intestinal organoids in Experimental Example 15.

Unless otherwise specified, each component exemplified in this specification, such as a component contained in the culture medium or a component used in each step, can be used alone or in combination of two or more types.

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

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

In this specification, human genes and human proteins will be represented by capital letters. Mouse genes will be represented by an initial capital letter followed by lowercase letters. Mouse proteins will be represented by capital letters. However, in some cases, human genes, mouse genes, human proteins, and mouse proteins may be represented without making a strict distinction.

[Method for producing organoids]
In one embodiment, the present invention provides a method for producing organoids, comprising culturing somatic stem cells in a medium containing an agonist of interferon-gamma (IFN-γ) receptor.

As will be described later in the Examples, the manufacturing method of this embodiment allows for efficient expansion and culture of organoids. The inventors have found that culturing intestinal epithelial stem cells by adding the inflammatory cytokine IFN-γ to the medium results in higher cell proliferation activity than with conventional culture methods. They speculate that this is because IFN-γ recreates the microenvironment created during tissue regeneration, promoting cell proliferation.

In the manufacturing method of this embodiment, somatic stem cells include epithelial stem cells. Examples of epithelial stem cells include intestinal epithelial stem cells, hepatic stem cells, kidney stem cells, gastric tissue stem cells, pulmonary epithelial stem cells, and neural stem cells. The somatic stem cells may be intestinal epithelial stem cells. Intestinal epithelial stem cells are preferably intestinal epithelial stem cells that express Lgr5. The intestinal tract is not particularly limited, and examples include the small intestine (duodenum, jejunum, ileum), large intestine (colon, rectum), etc.

Furthermore, somatic stem cells are preferably derived from mammals. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, and humans.

In the manufacturing method of this embodiment, it is preferable to suspend somatic stem cells in an extracellular matrix, polymerize the extracellular matrix, and then layer a culture medium on the polymerized extracellular matrix for culture.

The extracellular matrix is a substance that serves as a scaffold for cells in cell culture. Components of the extracellular matrix include, for example, components contained in basement membranes and glycoproteins present in the intercellular spaces. Components contained in basement membranes include, for example, type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. Glycoproteins present in the intercellular spaces include collagen, laminin, entactin, fibronectin, fibrinogen, and heparin sulfate. One of these components may be used alone, or two or more may be used in combination as the extracellular matrix component. It is particularly preferable for the extracellular matrix to contain laminin, and it is even more preferable for it to contain laminin-111.

Commercially available extracellular matrices include, for example, Matrigel (registered trademark), Cultrex (Bio-Techne), Geltrex (Thermo Fisher Scientific), EHS Gel Basement Membrane Matrix (Fujifilm Wako Pure Chemical Industries, Sigma), and Collagen I (Nitta Gelatin).

Hydrogels made from artificial polymers can also be used as scaffolding materials for cells. Commercially available hydrogels made from artificial polymers include Mebiol Gel (Mebiol), VitroGel (The Well Biosciences), and GrowDex (UPM Biomedicals).

As used herein, receptor agonists include factors that mediate signal transmission to the receptor and its downstream transcription factors. It is preferable to use receptor agonists derived from the same species as the somatic stem cells to be cultured.

The IFN-γ receptor is a heterodimeric receptor consisting of IFNGR1 and IFNGR2, and the binding of IFN-γ to the IFN-γ receptor activates the JAK-STAT pathway.

Agonists for the IFN-γ receptor are not particularly limited as long as they specifically bind to the IFN-γ receptor and activate the JAK-STAT pathway, and include endogenous agonists (i.e., IFN-γ) and exogenous agonists. For example, IFN-γ isolated and purified from a living organism can be used as an endogenous agonist for the IFN-γ receptor. Exogenous agonists for the IFN-γ receptor may be small organic compounds, inorganic compounds, peptides, polypeptides, peptidomimetics, oligonucleotides, aptamers, agonist antibodies against the IFN-γ receptor, or fragments thereof, etc.

NCBI accession numbers for human IFN-γ cDNA include NM_000619.3, etc. NCBI accession numbers for mouse IFN-γ cDNA include NM_008337.4, etc.

In the production method of this embodiment, the concentration of the IFN-γ receptor agonist in the culture medium is preferably 0.1 pM to 500 pM, more preferably 0.1 pM to 300 pM, even more preferably 0.5 pM to 100 pM, particularly preferably 1 pM to 50 pM, and most preferably 1 pM to 30 pM. As described below in the Examples, when the concentration of the IFN-γ receptor agonist in the culture medium is within the above range, the organoid proliferation-promoting effect tends to be high.

In the manufacturing method of this embodiment, it is preferable that the culture medium further contains an agonist of the hepatocyte growth factor (HGF) receptor. An "agonist of the HGF receptor" is a substance that specifically binds to the HGF receptor and exhibits the physiological action of the HGF receptor.

The HGF receptor is a single-pass transmembrane receptor tyrosine kinase called MET (also known as c-Met). MET signaling begins when HGF binds to MET, causing it to dimerize and become activated, inducing its kinase catalytic activity.

HGF receptor agonists are not particularly limited as long as they specifically bind to the HGF receptor and promote MET signaling, and include endogenous agonists (i.e., HGF) and exogenous agonists.

As an endogenous agonist of the HGF receptor, for example, HGF isolated and purified from a living organism can be used.

Exogenous agonists for the HGF receptor may be organic small molecule compounds, inorganic compounds, peptides, polypeptides, peptidomimetics, oligonucleotides, aptamers, etc. Examples of the above peptides include HGF surrogate peptides (c-Met agonists), which are commercially available.

NCBI accession numbers for human HGF cDNA include NM_000601.6, NM_001010931.3, NM_001010932.3, NM_001010933.3, and NM_001010934.3. NCBI accession numbers for mouse HGF cDNA include NM_001289458.1, NM_001289459.1, NM_001289460.2, NM_001289461.1, and NM_010427.5.

In the production method of this embodiment, the concentration of the HGF receptor agonist in the culture medium is preferably 0.01 nM to 10 nM, but may also be 0.1 nM to 5 nM, or 0.1 nM to 1 nM. As described below in the Examples, when the concentration of the HGF receptor agonist in the culture medium is within the above range, the organoid proliferation-promoting effect tends to be high.

In the production method of this embodiment, the medium may contain a vitamin D receptor agonist. As described below in the Examples, when the medium contains a combination of an IFN-γ receptor agonist, an HGF receptor agonist, and a Hippo signaling pathway inhibitor described below, it is preferable that the medium further contains a vitamin D receptor agonist.

A "vitamin D receptor agonist" is a substance that specifically binds to the vitamin D receptor and exerts the physiological effects of the vitamin D receptor. The vitamin D receptor is a nuclear receptor (vitamin D receptor: VDR) that is activated by the binding of calcitriol and controls the transcription of target genes.

Vitamin D receptor agonists are not particularly limited as long as they specifically bind to vitamin D receptors and regulate the transcription of target genes, and include endogenous agonists (i.e., calcitriol, CAS number: 32222-06-3) and exogenous agonists.

As an endogenous agonist of vitamin D receptors, for example, calcitriol isolated and purified from living organisms can be used.

The exogenous agonist of the vitamin D receptor may be a small organic compound, an inorganic compound, a peptide, a polypeptide, a peptidomimetic, an oligonucleotide, an aptamer, etc. Examples of the exogenous agonist of the vitamin D receptor include vitamin _D2 (ergocalciferol, CAS number: 50-14-6), vitamin _D3 (cholecalciferol, CAS number: 67-97-0), the vitamin _D3 analog calcipotriol (CAS number: 112965-21-6), the vitamin _D3 analog calcipotriol hydrate (CAS number: 147657-22-5), and the vitamin _D2 analog doxercalciferol (CAS number: 54573-75-0).

In the production method of this embodiment, the concentration of the vitamin D receptor agonist in the culture medium is preferably greater than 0 nM and less than 100 nM, more preferably 1 nM to 50 nM, and even more preferably 1 nM to 30 nM. As described below in the Examples, when the concentration of the vitamin D receptor agonist in the culture medium is within the above range, it tends to have a high effect of promoting organoid proliferation.

As described below in the Examples, the medium preferably contains a combination of an IFN-γ receptor agonist and an HGF receptor agonist. The medium may further contain a vitamin D receptor agonist. This tends to further enhance the organoid proliferation-promoting effect.

In the manufacturing method of this embodiment, it is preferable that the culture medium further contains a Hippo signaling pathway inhibitor.

The Hippo signaling pathway, also known as the Salvador-Warts-Hippo (SWH) pathway, is a pathway that, although the molecules involved and its mechanism of action remain unclear, can be summarized as follows: Hpo (MST1/2 in mammals), an important signaling factor in Drosophila, is a serine/threonine kinase that, when phosphorylated, activates Wts (LATS1/2 in mammals). Misshapen (Msn, MAP4K4/6/7 in mammals) and Happyhour (Hppy, MAP4K1/2/3/5 in mammals) work in parallel with Hpo to activate Wts. Proteins known to activate Wts are Sav (SAV1 in mammals) and Mob as tumor suppressor (Mats, MOB1 in mammals). Hpo can bind to and phosphorylate Sav, and this Hpo-Sav interaction promotes the phosphorylation of Wts, so Sav is thought to function as a scaffolding protein.

Activated Wts phosphorylates and inactivates the transcriptional coactivator Yki (in mammals, the two Yki orthologs are Yes-associated protein (YAP) and transcriptional coactivator with a PDZ-binding motif (WWTR1, also known as TAZ)). Activated Yki binds to the transcription factor Scalloped (Sd), and the Yki-Sd complex localizes to the nucleus. This enables the expression of several genes that promote organ development, such as cyclin E, which promotes cell cycle progression, and diap1 (Drosophila inhibitor of apoptosis protein-1), which, as its name suggests, prevents apoptosis. By phosphorylating Yki at serine 168, Wts promotes the binding of Yki to 14-3-3 proteins, which anchor Yki in the cytoplasm and prevent its transport to the nucleus. Upon activation, YAP and TAZ can bind to several transcription factors, including p73, Runx2, and several TEADs.

Upstream regulators of the Hpo core kinase cascade include the transmembrane protein Fat and several membrane-associated proteins. The atypical cadherin Fat (FAT1-4 in mammals) may function as a receptor, but its extracellular ligand has not yet been identified. The GPI-anchored cell surface protein glypican-3 (GPC3) is known to interact with Fat1 in human hepatocellular carcinoma. Fat also activates Hpo through the apical protein Expanded (Ex; FRMD6/Willin in mammals). Ex interacts with two other apically localized proteins, Kibra (KIBRA in mammals) and Merlin (Mer; NF2 in mammals), forming the Kibra-Ex-Mer (KEM) complex. The KEM complex physically interacts with the Hpo core kinase cascade, thereby localizing it to the plasma membrane for activation. Fat also regulates Wts independently of Ex/Hpo through inhibition of the atypical myosin Dachs.

The Hippo signaling pathway inhibitor may be one that inhibits any of the Hippo signaling pathways. Molecules inhibited by Hippo signaling pathway inhibitors are molecules contained in or related to the Hippo signaling pathway, and examples thereof include molecules that constitute the Hpo core kinase cascade, such as Hpo (MST1/2), Wts (LATS1/2), Sav (SAV1), Mob as tumor suppressor (MOB1), Misshapen (MAP4K4/6/7), Happyhour (MAP4K1/2/3/5), and Yki (YAP/TAZ); Yki-binding proteins, such as Scalloped and 14-3-3 protein; and upstream regulators of the Hpo core kinase cascade, such as Fat (FAT1-4), glypican-3, Expanded (FRMD6), Kibra (KIBRA), Merlin (NF2), and Dachs. Note that the names in parentheses are mammalian names.

The molecules inhibited by Hippo signaling pathway inhibitors are preferably molecules that constitute the Hippo core kinase cascade, and more preferably Wts (LATS1/2). Therefore, Hippo signaling pathway inhibitors include LATS1/2 inhibitors.

LATS1/2 inhibitors include TRULI (CAS number: 1424635-83-5, also known as "Lats-IN-1"), GA-017 (CAS number: 2351906-74-4), and TDI-011536 (CAS number: 2687970-96-1), which are substances that inhibit LATS1 and LATS2 and suppress Yap phosphorylation.

As described later in the Examples, it was revealed that when the medium contains a combination of an IFN-γ receptor agonist, an HGF receptor agonist, a vitamin D receptor agonist, and a Hippo signaling pathway inhibitor, YAP activation is promoted in conjunction with inflammatory cytokine stimulation, further enhancing cell proliferation activity. More specifically, compared to conventional medium, cell yields can be obtained that are approximately 100-fold higher in one month and approximately 10,000-fold higher in two months. Furthermore, as described later in the Examples, stimulation with these factors can achieve sufficient cell proliferation effects even in a short period of time (the first two days after seeding).

In the production method of this embodiment, the concentration of the LATS1/2 inhibitor in the culture medium is preferably 0.1 μM to 50 μM, more preferably 1 μM to 40 μM, and even more preferably 1 μM to 30 μM. As described below in the Examples, when the concentration of the LATS inhibitor in the culture medium is within the above range, the organoid proliferation-promoting effect tends to be high.

In the manufacturing method of this embodiment, the medium may further contain a transforming growth factor β (TGF-β) inhibitor.

TGF-β signaling contributes to the inhibition of cell proliferation, cell differentiation, and induction of apoptosis. TGF-β inhibitors are substances that downregulate TGF-β signaling and can also be called TGF-β signaling pathway inhibitors.

As used herein, a TGF-β inhibitor refers to an inhibitor that inhibits the activation of type I or type II serine/threonine kinase receptors, and that inhibits the phosphorylation of Smad2/3.

TGF-β inhibitors that inhibit Smad2/3 phosphorylation include, for example, A83-01 (CAS No.: 909910-43-6), SB-431542 (CAS No.: 301836-41-9), SB-505124 (CAS No.: 694433-59-5), SB-525334 (CAS No.: 356559-20-1), LY364947 (CAS No.: 396129-53-6), SD-208 (CAS No.: 627536-09-8), and SJN2511 (CAS No.: 446859-33-2), with A83-01 being preferred.

The concentration of the TGF-β inhibitor contained in the culture medium may be, for example, 50 nM to 5 μM, or may be, for example, 50 nM to 1 μM, or may be, for example, 50 nM to 500 nM.

As described below in the Examples, when the medium contains a combination of HGF and a Hippo-YAP/TAZ signaling pathway inhibitor, or when the medium contains a combination of IFN-γ, HGF, and a Hippo-YAP/TAZ signaling pathway inhibitor, the organoid proliferation-promoting effect tends to be further enhanced by further adding vitamin D or an analog thereof to the medium.

Furthermore, as described below in the Examples, when the medium contains a combination of IFN-γ, HGF, vitamin D or an analogue thereof, and a Hippo-YAP/TAZ signaling pathway inhibitor, a high organoid proliferation-promoting effect tends to be obtained even without the addition of a TGF-β inhibitor.

In the production method of this embodiment, it is preferable that the culture medium further contains other factors that are commonly used in organoid culture. Examples of such factors include epidermal growth factor (EGF) receptor agonists, bone morphogenetic protein (BMP) signaling pathway inhibitors, p38 inhibitors, insulin-like growth factor 1 (IGF1) receptor agonists, fibroblast growth factor 2 (bFGF, FGF2) receptor agonists, Wnt signaling pathway activators, and Rho-associated kinase inhibitors. In the production method of this embodiment, it is preferable that the culture medium further contains one or a combination of two or more of these factors.

Examples of agonists for epidermal growth factor (EGF) receptors include ligands that have agonistic activity against EGF receptors. Such ligands include EGF and EGF mimetics. EGF mimetics include agonist antibodies or fragments thereof, peptides, etc., against EGF receptors.

NCBI accession numbers for human EGF cDNA include NM_001178130.3, NM_001178131.3, NM_001357021.2, and NM_001963.6. NCBI accession numbers for mouse EGF cDNA include NM_001310737.1, NM_001329594.1, and NM_010113.4.

In the production method of this embodiment, the concentration of the EGF receptor agonist in the culture medium is preferably 0.1 ng/mL to 1,000 ng/mL, and may be, for example, 0.1 ng/mL to 500 ng/mL, or may be, for example, 0.1 ng/mL to 200 ng/mL.

Examples of bone morphogenetic protein (BMP) signaling pathway inhibitors include Noggin, Chordin, follistatin, dorsomorphin (CAS number: 866405-64-3), DMH1 (CAS number: 1206711-16-1), and LDN193189 (CAS number: 1062368-24-4).

In the production method of this embodiment, the concentration of the BMP signaling pathway inhibitor contained in the culture medium is preferably, for example, 10 ng/mL to 1,000 ng/mL, and may be, for example, 10 ng/mL to 500 ng/mL, or may be, for example, 10 ng/mL to 300 ng/mL.

Examples of p38 inhibitors include SB202190 (CAS number: 152121-30-7) and doramapimod (CAS number: 285983-48-4).

In the manufacturing method of this embodiment, the concentration of the p38 inhibitor contained in the culture medium is preferably 50 nM to 100 μM, more preferably 100 nM to 50 μM, and even more preferably 100 nM to 10 μM.

Agonists for the insulin-like growth factor 1 (IGF-1) receptor include, for example, ligands that have agonistic activity against the IGF-1 receptor. Such ligands include IGF-1 and IGF-1 mimetics. IGF-1 mimetics include agonist antibodies or fragments thereof, peptides, etc., against the IGF-1 receptor.

NCBI accession numbers for human IGF-1 cDNA include NM_000618.5, NM_001111283.3, NM_001111284.2, NM_001111285.3, NM_001414005.1, NM_001414006.1, and NM_001414007.1. NCBI accession numbers for mouse IGF-1 cDNA include NM_001111274.1, NM_001111275.2, NM_001111276.1, NM_001314010.1, and NM_010512.5.

In the production method of this embodiment, the concentration of the IGF-1 receptor agonist in the culture medium is preferably 10 ng/mL to 1,000 ng/mL, and may be, for example, 10 ng/mL to 500 ng/mL, or may be, for example, 10 ng/mL to 200 ng/mL.

Agonists for the fibroblast growth factor 2 (FGF2) receptor include, for example, ligands that have agonistic activity against the FGF2 receptor. Examples of such ligands include FGF2 and FGF2 mimetics. Examples of FGF2 mimetics include agonist antibodies or fragments thereof, peptides, etc., against the FGF2 receptor.

NCBI accession numbers for human FGF2 cDNA include NM_001361665.2 and NM_002006.6. NCBI accession numbers for mouse FGF2 cDNA include NM_008006.2.

In the production method of this embodiment, the concentration of the FGF2 receptor agonist in the culture medium is preferably 0.1 ng/mL to 1,000 ng/mL, and may be, for example, 0.1 ng/mL to 500 ng/mL, or may be, for example, 0.1 ng/mL to 200 ng/mL.

Wnt signaling controls cell proliferation and differentiation by regulating the protein levels of β-catenin, which functions as a transcription factor. Examples of activators of the Wnt signaling pathway include inhibitors of the Wnt family, R-spondin family, Norrin, and glycogen synthase (GSK) inhibitors.

The Wnt family includes, for example, Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16, with Wnt3a being preferred. Amino acid sequence information for each protein in the Wnt family can be obtained from the NCBI database. For example, NCBI accession numbers for human WNT3A cDNA include NM_033131.4, and for mouse Wnt3a cDNA include NM_009522.3.

Afamin is known to contribute to the stabilization and solubilization of the Wnt family. For this reason, a complex of the Wnt family with afamin is more preferred as a Wnt signaling pathway activator. Afamin is a glycoprotein that belongs to the albumin family.

NCBI accession numbers for human afamin cDNA include NM_001133.2, etc. NCBI accession numbers for bovine afamin cDNA include NM_001192175.1, etc. NCBI accession numbers for mouse afamin cDNA include NM_145146.2, etc.

The Wnt family, or a complex of the Wnt family and afamin, can be used as a conditioned medium containing them. The concentration of the Wnt family in the conditioned medium is preferably 1 μg/mL to 5 μg/mL. When a conditioned medium in which the concentration of the Wnt family is in the above range is used as the Wnt family, the content of the conditioned medium in the culture medium is typically 1 (v/v)% to 50 (v/v)%, for example, 5 (v/v)% to 30 (v/v)%, or for example, 5 (v/v)% to 10 (v/v)%, of the total volume of the culture medium.

Examples of the R-spondin family include R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4. Of these, R-spondin 1 is preferred. Amino acid sequence information for each protein in the R-spondin family is available from the NCBI database.

For example, NCBI accession numbers for human R-spondin1 cDNA include NM_001038633.4, NM_001242908.2, NM_001242909.2, NM_001242910.2, and NM_173640.1. NCBI accession numbers for mouse R-spondin1 cDNA include NM_138683.2.

When the R-spondin family binds to Lgr5 in the cell membrane, it is removed from the cell membrane by autoubiquitination, resulting in Frezzled, which induces activation of the Wnt signaling pathway, and activates the β-catenin pathway in the cell membrane.

The R-spondin family can be used as a conditioned medium containing the same. The concentration of the R-spondin family in the conditioned medium is preferably 1 μg/mL to 20 μg/mL. When a conditioned medium having an R-spondin family concentration within the above range is used as the R-spondin family, the content of the conditioned medium in the culture medium is typically 1 (v/v)% to 50 (v/v)%, for example, 1 (v/v)% to 30 (v/v)%, or for example, 1 (v/v)% to 10 (v/v)%, of the total volume of the culture medium.

GSK inhibitors are inhibitors of glycogen synthase 3β (GSK3β). GSK3β phosphorylates β-catenin and promotes its degradation, thereby acting as a Wnt agonist.

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).

As a Wnt signaling pathway activator, it is preferable to use a combination of the Wnt family and the R-spondin family, more preferably a combination of Wnt3a and R-spondin1, and even more preferably a combination of a complex of Wnt3a and afamin and R-spondin1.

When cells are dissociated into single cells, apoptosis can be suppressed by adding a Rho-associated kinase (ROCK) inhibitor to the culture medium. Examples of ROCK inhibitors include Y-27632 (CAS number: 331752-47-7), Fasudil (CAS number: 105628-07-7), Y-39983 (CAS number: 203911-26-6), Wf-536 (CAS number: 539857-64-2), and SLx-2119 (CAS number: 911417-8). 7-3), azabenzimidazole-aminofurazans (CAS No.: 850664-21-0), DE-104, H-1152P (CAS No.: 872543-07-6), and blebbistatin (CAS No.: 856925-71-8).

In the production method of this embodiment, the concentration of the ROCK inhibitor in the culture medium is typically 1 μM to 20 μM, and preferably 5 μM to 15 μM.

In the manufacturing method of this embodiment, the medium may contain other components that are typically added to cell culture media. Such components include, for example, amino acids, vitamins, inorganic salts, sugars, trace elements, antibiotics, and other additives.

Amino acids include, for example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and N-acetylcysteine.

Examples of vitamins include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), calcium D-pantothenate (vitamin B5), pyridoxal/pyridoxamine/pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), calcitriol (vitamin D3), DL-alpha tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

Examples of inorganic salts include calcium, copper, iron, magnesium, potassium, sodium, and zinc salts. The salts are usually used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. More specific examples include CaCl ₂ , CuSO ₄ -5H ₂ O, Fe(NO ₃ )-9H ₂ O, FeSO ₄ -7H ₂ O, MgCl 2 , MgSO ₄ , KCl, NaHCO ₃ , NaCl, Na _{2 HPO 4 , Na 2 HPO 4 -H 2} O, and ZnSO ₄ -7H ₂ O.

Examples of sugars include glucose, galactose, maltose, fructose, etc.

Trace elements include, for example, barium, bromium, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum, and ions of these elements.

Examples of antibiotics include penicillin, streptomycin, amphotericin B, and nanomycopritin.

Other additives include B27 supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), gastrin, cholesterol, transferrin, albumin, insulin, progesterone, putrescine, etc.

In the manufacturing method of this embodiment, a medium obtained by adding the above-mentioned components to a basal medium can be used. Examples of basal media that can be used include DMEM medium, F-12 medium, DMEM/F12 medium, Advanced DMEM/F12 medium, 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, Ham's medium, RPMI 1640 medium, serum imitation medium, and Fischer's medium, as well as mixtures of these.

In one embodiment, the present invention provides a method for producing organoids, comprising the step of culturing somatic stem cells in a medium containing a Hippo signaling pathway inhibitor. As described below in the Examples, a medium containing a Hippo signaling pathway inhibitor tends to have a strong effect in promoting organoid proliferation.

In the manufacturing method of this embodiment, the somatic stem cells, Hippo signaling pathway inhibitor, etc. are the same as those described above.

[Organoid production medium]
In one embodiment, the present invention provides a culture medium for producing organoids, which comprises an IFN-γ receptor agonist. As described below in the Examples, the culture medium of this embodiment enables efficient expansion of organoids. The IFN-γ receptor agonist is the same as that described above.

The medium of this embodiment preferably further contains an HGF receptor agonist. The HGF receptor agonist is the same as that described above.

The medium of this embodiment preferably further contains a vitamin D receptor agonist. The vitamin D receptor agonist is the same as that described above.

The culture medium of this embodiment preferably further contains a Hippo signaling pathway inhibitor. The Hippo signaling pathway inhibitor is the same as that described above.

The medium of this embodiment preferably further contains a TGF-β inhibitor. The TGF-β inhibitor is the same as that described above.

The medium of this embodiment preferably further contains other factors commonly used in organoid culture. Examples of such factors include EGF receptor agonists, BMP signaling pathway inhibitors, p38 inhibitors, IGF-1 receptor agonists, FGF2 receptor agonists, Wnt signaling pathway activators, and Rho-associated kinase inhibitors. The medium of this embodiment preferably further contains one or a combination of two or more of these factors.

The same applies to EGF receptor agonists, BMP signaling pathway inhibitors, p38 inhibitors, IGF-1 receptor agonists, FGF2 receptor agonists, Wnt signaling pathway activators, and Rho-associated kinase inhibitors as described above.

As will be described later in the Examples, when the medium contains a combination of an IFN-γ receptor agonist, an HGF receptor agonist, a vitamin D receptor agonist, and a Hippo signaling pathway inhibitor, it is possible to obtain a cell yield that is approximately 100 times higher in one month and approximately 10,000 times higher in two months compared to conventional medium. Furthermore, as will be described later in the Examples, stimulation with these factors can achieve a sufficient cell proliferation effect even in a short period of time (the first two days after seeding).

The medium of this embodiment may further contain other components that are typically added to cell culture media.

[Additive Kit]
In one embodiment, the present invention provides an additive kit for a culture medium for culturing somatic stem cells to produce organoids, the additive kit comprising a combination of two or more components selected from the group consisting of the following components (a) to (d):
Component (a): a ligand for the interferon-gamma (IFN-gamma) receptor,
Component (b): Hippo signaling pathway inhibitor,
Component (c): a hepatocyte growth factor (HGF) receptor agonist, and component (d): a vitamin D receptor agonist.

In the additive kit of this embodiment, the somatic stem cells, culture medium, IFN-γ receptor ligand, Hippo signaling pathway inhibitor, HGF receptor agonist, vitamin D receptor agonist, etc. are the same as those described above.

The present invention will be explained in more detail below based on experimental examples. However, the present invention is in no way limited by these experimental examples. Sterilized instruments were used in all experiments in the following experimental examples. Furthermore, all experiments were conducted in accordance with ethical research plans approved by the Keio University School of Medicine Ethics Committee.

Materials and Methods
(Preparation of culture medium for organoid production)
Each component shown in Tables 1 to 4 below was added to the basal medium at the concentrations shown in Tables 1 to 4 below to prepare media A to P and media A', B', E', K', L', P', K'', and G''. Advanced DMEM/F12 (Thermo Fisher Scientific) was used as the basal medium.

In Tables 1 to 4 above, HEPES, Penicillin/Streptmycin, GlutaMAX-I, B-27 supplement (50x), and human EGF were obtained from Thermo Fisher Scientific. Leu15-Gastrin I was obtained from ANASPEC. N-Acetyl-L-cysteine and calcitriol (vitamin D3) were obtained from Fujifilm Wako Pure Chemical Industries. Human IGF-1 was obtained from Biolegend. Human FGF2 and IFN-γ were obtained from Peprotech. A83-01 (CAS number: 909910-43-6) was obtained from Tocris. HGF substitute peptide (c-Met agonist) was obtained from PeptiGrowth. Recombinant human HGF (insect-derived) was obtained from Peprotech. TRULI (CAS number: 1424635-83-5, also known as "Lats-IN-1") was obtained from Medchemexpress. R-spondin 1 CM, Afamin/Wnt3a CM, and Noggin CM were prepared in-house. "CM" stands for conditioned medium.

The Wnt agonist Wnt3a was used in the form of a conditioned medium containing a complex of Wnt3a and afamin. Afamin/Wnt3a CM was prepared according to the procedure described in Mihara E., et al., Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/alpha-albumin, elife, 5, e11621, 2016. The concentration of Wnt3a in Afamin/Wnt3a CM was 3 μg/mL.

R-spondin 1 CM was prepared according to the procedure described in Ootani A, et al., Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche, Nat Med, 15 (6), 701-706, 2009. The concentration of R-spondin 1 in R-spondin 1 CM was 13 μg/mL.

Noggin CM was prepared according to the procedure described in Vonk A. M., et al., Protocol for Application, Standardization and Validation of the Forskolin-Induced Swelling Assay in Cystic Fibrosis Human Colon Organoids, STAR Protocol 1 (1), 100019, 2020. The concentration of Noggin in Noggin CM was 12 μg/mL.

(Maintenance of human small intestinal epithelial cells through multiple cultures)
Human small intestinal epithelial cells derived from human small intestinal tissue fragments were used in the experiments. These cells had a reporter construct introduced into exon 18 of the LGR5 gene locus by genome editing. The LGR5 gene is a stem cell marker. Furthermore, IRES-tdTomato was used as the reporter construct. When these cells express LGR5, they express the fluorescent protein tdTomato.

A micropipette (Thermo Fisher Scientific) was used to measure and dispense various reagents. Human small intestinal epithelial cells were dispersed into single cells using TrypLE Express (Thermo Fisher Scientific) and suspended in Matrigel (registered trademark, BD Biosciences) at 100 cells/μL.

Next, 20 μL of the above suspension was seeded into each well of a 48-well tissue culture plate (Greiner Bio-One) and incubated at 37°C for 10 minutes to polymerize the Matrigel. After Matrigel polymerization, 300 μL of medium was added per well and the plates were cultured at 37°C in the presence of 5% CO2 ₍ volume). The medium used was Medium A' described above. To prevent apoptosis, 10 μM Y-27632 (CAS number: 331752-47-7, Fujifilm Wako Pure Chemical Industries) was added to the medium until the first medium change. The medium was changed every 2 or 3 days.

As the culture days continued, the cells proliferated within the Matrigel in the form of cell clusters, and after about seven days, they formed cyst-like small intestinal organoids with bud-like protrusions (buds). As the culture continued, the differentiated cells folded toward the lumen, and under a microscope, the lumen appeared dark. Because cell death occurs after the lumen of the organoids becomes dense due to cell proliferation, the cells were dispersed into single cells using TrypLE Express (Thermo Fisher Scientific) around 10-11 days after the start of culture, before the lumen became excessively dark, and then passaged using the procedure described above to maintain human small intestinal epithelial cells.

(Morphological observation of organoids)
The morphology of the organoids was observed in bright field using a fluorescence microscope (manufactured by KEYENCE, device name "BZ-X800").

(Evaluation of organoid proliferation)
Organoid proliferation was assessed using a commercially available kit (CellTiter- ^Glo® 3D Cell Viability Assay, Promega, hereinafter sometimes referred to as the "CTG assay") according to the kit's instructions. Luminescence signals were detected and read using a ^GloMax® Discover Microplate Reader (Promega).

(Evaluation of stem cell properties of cells)
Stem cells contained in organoids transfected with the Lgr5-tdTomato reporter construct express the fluorescent protein tdTomato along with the stem cell marker LGR5 protein. Therefore, tdTomato expression was detected by fluorescence microscopy as an indicator of stemness.

[Experimental Example 1: Examination of culture medium for organoid production 1]
Human small intestinal epithelial cells were dispersed into single cells using TrypLE Express (Thermo Fisher Scientific) and suspended in Matrigel (registered trademark, BD Biosciences) at 100 cells/μL.

Next, 18 μL of the above suspension was seeded into each well of a 48-well tissue culture plate (Greiner Bio-One) and incubated at 37°C for 10 minutes to polymerize the Matrigel. After the Matrigel polymerized, 300 μL of medium was added per well and cultured at 37°C in the presence of 5% CO2 by _volume for 9 days to obtain small intestinal organoids. The above-mentioned media A to P were used as the medium. To prevent apoptosis, 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries) was added to the medium until the first medium change. The medium was changed every 2 or 3 days.

Figure 1 is a graph showing the results of a CTG assay of the resulting small intestinal organoids. The horizontal axis of the graph indicates the medium used and its components. In Figure 1, "vitD" and "D" indicate culture in a medium containing calcitriol (synonymous with vitamin D). "HGF" and "H" indicate culture in a medium containing an "HGF substitute peptide." "IFNg" and "I" indicate culture in a medium containing "IFN-γ." "TRULI" and "T" indicate culture in a medium containing "TRULI." "HD," "ID," "TD," "HI," "HID," "TI," "TID," "TH," "THD," "THI," and "THID" indicate culture in a medium containing a combination of "D," "H," "I," and "T." The vertical axis of the graph indicates relative values when the control (small intestinal organoids cultured in medium A) is set to 100%.

As a result, it was found that cell proliferation was promoted when medium B, C, E, F, G, H, K, L, N, O, and P were used compared to medium A. In other words, the addition of IFN-γ, HGF, TRULI, vitamin D, or a combination of these to the medium promoted cell proliferation.

[Experimental Example 2: Examination of organoid production medium 2]
Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the culture period was set to 7 days and the above-mentioned media A, B, and G were used, and the stem cell properties of the cells were evaluated.

Figure 2 shows bright-field images of each small intestinal organoid and fluorescence microscope images detecting tdTomato fluorescence. In Figure 2, the upper row is a bright-field image, and the lower row is a fluorescence microscope image. The culture medium used for culture is indicated at the top of each image. All images were taken at 20x magnification.

As a result, it was found that the use of media B and G promoted cell proliferation compared to the use of media A, and also increased the number of stem cells indicated by tdTomato signaling. In other words, it was found that adding IFN-γ, or a combination of IFN-γ, HGF, TRULI, and vitamin D to the media promoted cell proliferation and increased the number of stem cells.

[Experimental Example 3: Examination of organoid production medium 3]
Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the above-mentioned media A, A', B', G', K', L', and P' were used, respectively.

Figure 3 is a graph showing the results of a CTG assay of the resulting small intestinal organoids. The horizontal axis of the graph indicates the medium used and its components. In Figure 3, "D" indicates culture in a medium containing calcitriol (synonymous with vitamin D). "H" indicates culture in a medium containing an "HGF alternative peptide." "IFNg" and "I" indicate culture in a medium containing "IFN-γ." "T" indicates culture in a medium containing "TRULI." "HI," "HID," "HIT," and "THID" indicate culture in a medium containing a combination of "D," "H," "I," and "T." The vertical axis of the graph indicates relative values when the control (small intestinal organoids cultured in medium A) is set to 100%.

As a result, it was found that adding A83-01 to the medium, along with IFN-γ, HGF, TRULI, vitamin D, or a combination of these, promoted cell proliferation.

[Experimental Example 4: Evaluation of the effect of inflammatory cytokines on organoid culture]
Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the above-mentioned medium A' was supplemented with IFN-γ at concentrations of 0, 0.01 nM, 0.03 nM, 0.1 nM, 0.3 nM, and 1 nM.

Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the above-mentioned medium A' was supplemented with IL11 at concentrations of 0, 0.2 nM, 0.6 nM, and 2.1 nM.

Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the above-mentioned medium A' was supplemented with IL22 at concentrations of 0, 0.1 nM, 0.4 nM, and 1.2 nM.

Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the above-mentioned medium A' was supplemented with IL6 at concentrations of 0, 0.05 nM, 0.15 nM, 0.5 nM, 1.5 nM, and 5 nM.

Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the above-mentioned medium A' was supplemented with IFN-γ at concentrations of 0, 0.1 pM, 1 pM, 10 pM, 100 pM, and 1000 pM.

Figure 4 is a graph showing the results of a CTG assay of the resulting small intestinal organoids. The vertical axis of the graph shows the relative value when the control (small intestinal organoids cultured in medium A' without cytokines) is set at 100%.

As a result, it was found that adding the inflammatory cytokines IL11, IL22, IL6, and IFN-γ to the culture medium tended to promote cell proliferation. Of these, the addition of IFN-γ tended to promote cell proliferation the most.

Figure 5 shows bright-field images of small intestinal organoids obtained using the above-mentioned medium A' to which IFN-γ was added at concentrations of 0, 0.01 nM, 0.1 nM, and 1 nM. All images in Figure 5 were taken at a magnification of 100x.

As a result, promotion of cell proliferation was observed at IFN-γ concentrations in the range of greater than 0 nM and less than 1 nM.

[Experimental Example 5: Evaluation of the effect of HGF on organoid culture]
Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the above-mentioned medium A' was supplemented with HGF substitute peptides at concentrations of 0, 0.1 nM, 0.3 nM, and 1 nM.

Figure 6 is a graph showing the results of a CTG assay of the resulting small intestinal organoids. The vertical axis of the graph shows relative values, with the control (small intestinal organoids cultured in medium A' without HGF) set at 100%.

As a result, it was found that the addition of HGF promoted cell proliferation.

[Experimental Example 6: Evaluation of the effect of recombinant HGF on organoid culture]
Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the culture period was 7 days and the above-mentioned media G, K, G", and K" were used. Media G" and K" were prepared by replacing the HGF substitute peptide contained in media G and K with recombinant human HGF (insect-derived, PeproTech). Recombinant human HGF was used at a concentration of 50 ng/mL, with reference to Yu Takahashi, et al., Drug cytotoxicity screening using human intestinal organoids propagated with extensive cost-reduction strategies, Sci Rep., 13 (1), 5407, 2023, and other publications.

Figure 7 is a graph showing the results of a CTG assay of the resulting small intestinal organoids. The vertical axis of the graph shows relative values, with the control (small intestinal organoids cultured in media G and K containing HGF substitute peptides) set at 100%. The horizontal axis of the graph shows the media used and their components. In Figure 7, "D" indicates culture in a medium containing calcitriol (synonymous with vitamin D). "H" indicates culture in a medium containing "recombinant human HGF." "I" indicates culture in a medium containing "IFN-γ." "T" indicates culture in a medium containing "TRULI." "HI" and "THID" indicate culture in a medium containing a combination of "D," "H," "I," and "T."

As a result, it was confirmed that the effect on cell proliferation was the same regardless of whether HGF alternative peptides or recombinant human HGF were added to the culture medium.

Figure 8 shows bright-field images of the resulting small intestinal organoids and fluorescence microscope images showing tdTomato fluorescence. All images in Figure 8 are at 20x magnification. The top row is a bright-field image, and the bottom row is a fluorescence microscope image.

As a result, it was confirmed that the effect on cell proliferation was the same regardless of whether HGF alternative peptides or recombinant human HGF were added to the culture medium.

[Experimental Example 7: Evaluation of the effect of vitamin D on organoid culture]
Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the above-mentioned medium A' was supplemented with vitamin D at concentrations of 0, 3 nM, 10 nM, 30 nM, and 100 nM.

Figure 9 is a graph showing the results of a CTG assay of the resulting small intestinal organoids. The vertical axis of the graph shows the relative value, with the control (small intestinal organoids cultured in medium A' without added vitamin D) set at 100%.

Figure 10 shows bright-field images and fluorescence microscope images of small intestinal organoids obtained using the above-mentioned medium A' to which vitamin D was added at concentrations of 0, 3 nM, 10 nM, 30 nM, and 100 nM, respectively, showing tdTomato fluorescence. In Figure 10, the top two rows are at 20x magnification, and the bottom row is at 100x magnification. The top and bottom rows are bright-field images, and the second row from the top is a fluorescence microscope image.

As a result, cell proliferation was observed to be promoted at vitamin D concentrations in the range of greater than 0 nM and less than 100 nM.

[Experimental Example 8: Evaluation of the effect of TRUL1 on organoid culture]
Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the above-mentioned medium A' was used to which TRUL1 was added so as to give concentrations of 0, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM.

Figure 11 is a graph showing the results of a CTG assay of the resulting small intestinal organoids. The vertical axis of the graph shows relative values, with the control (small intestinal organoids cultured in Medium A' without Truli) set at 100%.

Figure 12 shows bright-field images and fluorescence microscope images of small intestinal organoids obtained using the above-mentioned medium A' to which TRUL1 was added at concentrations of 0, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM, respectively. The top two rows in Figure 12 are at 20x magnification, and the bottom row is at 100x magnification. The top and bottom rows are bright-field images, and the second row from the top is a fluorescence microscope image.

Normally, organoids form monolayered cysts with bud-like processes similar to intestinal crypts found in vivo. In contrast, when cultured in a medium containing TRULI, they took on a spherical shape with a solid lumen. Furthermore, as shown in Figures 11 and 12, TRULI promoted cell proliferation in a concentration-dependent manner up to a concentration of approximately 30 μM, and also enhanced stemness, as indicated by tdTomato signaling. However, at a 100 μM concentration of TRULI, recrystallization of TRULI was observed, and cell death was also observed.

[Experimental Example 9: Evaluation of organoid cultures derived from the duodenum, ileum, and colon]
Intestinal organoids were prepared in the same manner as in Experimental Example 1, using media A' and G', except that human small intestinal (ileal) epithelial cells derived from human small intestinal tissue fragments, duodenal epithelial cells derived from human duodenal tissue fragments, or human large intestinal epithelial cells derived from human large intestinal tissue fragments were used instead of the human small intestinal epithelial cells derived from the above-mentioned human small intestinal tissue fragments.

Figure 13 shows bright-field observation images of each intestinal-derived organoid. In Figure 13, the top row shows organoids derived from duodenal epithelial cells, the middle row shows organoids derived from ileal epithelial cells, and the bottom row shows organoids derived from colonic epithelial cells. The culture medium used for culture is indicated at the top of each image. From left to right, the results of culture using medium A', medium G', medium A', and medium G' are shown. The magnification of the left two columns is 20x, and the magnification of the right two columns is 100x.

Figure 14 is a graph showing the results of a CTG assay of the resulting intestinal organoids. In Figure 14, "Dudenum" indicates the results for organoids derived from duodenal epithelial cells, "Ileum" indicates the results for organoids derived from ileal epithelial cells, and "Colon" indicates the results for organoids derived from colonic epithelial cells. The horizontal axis of the graph indicates the medium used and its components. "D" indicates calcitriol (synonymous with vitamin D), "H" indicates "HGF substitute peptide," "I" indicates "IFN-γ," and "T" indicates "TRULI." "THID" indicates that the medium was cultured in a medium containing a combination of "D," "H," "I," and "T." The vertical axis of the graph indicates the measured ATP concentration value.

As a result, promotion of cell proliferation was observed in each intestinal-derived organoid.

[Experimental Example 10: Evaluation of liver-derived organoid culture]
Instead of the human small intestinal epithelial cells derived from the human small intestinal tissue fragment described above, organoid culture was performed using frozen human primary hepatocytes. The frozen human primary hepatocytes used were those shown in Table 5 below.

Cultures Q to W were prepared by adding the components shown in Table 6 below to the basal medium at the concentrations shown in Table 6. Advanced DMEM/F12 (Thermo Fisher Scientific) was used as the basal medium.

In Table 6 above, HEPES, Penicillin/Streptmycin, GlutaMAX-I, B-27 supplement (50x), and human EGF were obtained from Thermo Fisher Scientific. Leu15-Gastrin I was obtained from Sigma. N-Acetyl-L-cysteine and calcitriol (vitamin D3) were obtained from Fujifilm Wako Pure Chemical Industries. Noggin, human FGF10, Oncostatin M, IFN-γ, and recombinant human HGF were obtained from Peprotech. A83-01 (CAS number: 909910-43-6) was obtained from Tocris. HGF substitute peptide (c-Met agonist) was obtained from PeptiGrowth. TRULI (CAS number: 1424635-83-5) was obtained from Selleck. R-spondin 1 CM and Afamin/Wnt3a CM were prepared in-house. "CM" stands for conditioned medium.

(Maintenance of human primary hepatocytes through subculture)
A micropipette (Thermo Fisher Scientific) was used to measure and dispense various reagents. Frozen human primary hepatocytes shown in Table 5 were thawed and dispersed into single cells using TrypLE Express (Thermo Fisher Scientific), and then suspended in Matrigel (registered trademark, BD Biosciences) at a concentration of 100 cells/μL.

Next, 20 μL of the above suspension was seeded into each well of a 48-well tissue culture plate (Greiner Bio-One) and incubated at 37°C for 10 minutes to polymerize the Matrigel. After Matrigel polymerization, 300 μL of medium was added per well and the plates were cultured at 37°C in the presence of 5% CO2 ₍ volume). The medium used was Medium S, as described above. The medium was changed every two or three days. Approximately 10 to 11 days after the start of culture, the cells were dispersed into single cells using TrypLE Express (Thermo Fisher Scientific) and passaged as described above. Human primary hepatocytes were maintained for 119 days.

Subsequently, the human primary hepatocytes after subculture were cultured for 13 days using media Q to W to produce hepatic organoids. Figure 15 shows bright-field images of hepatic organoids. The media used for culture is indicated at the top of each image. All images are at 20x magnification.

Figure 16 is a graph showing the results of a CTG assay of the obtained hepatic organoids. The horizontal axis of the graph indicates the medium used and its components. In Figure 16, "IFNg" and "I" indicate culture in a medium containing "IFN-γ." "HGF" and "H" indicate culture in a medium containing "HGF alternative peptide." "HGF-P" and "HP" indicate culture in a medium containing "recombinant human HGF." "D" indicates calcitriol (synonymous with vitamin D), "T" indicates "TRULI," and "HI," "HPI," and "THPID" indicate culture in a medium containing a combination of "D," "H," "HGF-P," "I," and "T." The vertical axis of the graph indicates the measured ATP concentration.

As a result, it was found that adding IFN-γ and/or HGF, or a combination of IFN-γ, HGF, TRULI, and vitamin D to the culture medium promoted cell proliferation of hepatic organoids.

[Experimental Example 11: Evaluation of the effect of EGF on organoid culture]
Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the medium used was one to which human EGF, one of the additive factors in the above-mentioned media A, G, A', and G', was added at concentrations of 0, 5 ng/mL, 50 ng/mL, and 500 ng/mL.

Figure 17 is a graph showing the results of a CTG assay of the resulting small intestinal organoids. In Figure 17, "w/o A83-01" indicates culture in a medium without A83-01, and "w/ A83-01" indicates culture in a medium containing A83-01. The horizontal axis of the graph indicates the components of the medium used and the concentration of human EGF. "D" indicates calcitriol (synonymous with vitamin D), "H" indicates "HGF substitute peptide," "I" indicates "IFN-γ," and "T" indicates "TRULI." "THID" indicates culture in a medium containing a combination of "D," "H," "I," and "T." "(-)" indicates culture in a medium without "D," "H," "I," or "T." The vertical axis of the graph indicates the measured ATP concentration.

Figures 18 and 19 are bright-field images of small intestinal organoids grown using culture media containing human EGF at concentrations of 0 ng/mL, 5 ng/mL, 50 ng/mL, and 500 ng/mL. In Figures 18 and 19, the four left columns show the results of culturing in a medium that did not contain A83-01, while the four right columns show the results of culturing in a medium that contained A83-01. The top columns show the results of culturing in media A and A', which contained human EGF at concentrations of 0 ng/mL, 5 ng/mL, 50 ng/mL, and 500 ng/mL, respectively. The bottom columns show the results of culturing in media G and G', which contained human EGF at concentrations of 0 ng/mL, 5 ng/mL, 50 ng/mL, and 500 ng/mL. The magnification of the image in Figure 18 was 20x. The magnification of the image in Figure 19 was 100x.

As a result, when medium G or G' was used, even though the medium did not contain human EGF, promotion of cell proliferation was observed compared to when medium A or A' was used. Furthermore, promotion of cell proliferation was observed when the concentration of human EGF was in the range of more than 0 ng/mL and up to 500 ng/mL.

[Experimental Example 12: Evaluation of the effects of IGF-1 and FGF2 on organoid culture]
Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that a medium was used that did not contain human IGF-1 and/or human FGF2 among the supplemental factors in the above-mentioned media A' and G'.

Figure 20 is a graph showing the results of a CTG assay of the resulting small intestinal organoids. In Figure 20, "IGF1/FGF2" indicates culture in a medium supplemented with IGF-1 and FGF2, "(-)" indicates culture in a medium without IGF-1 or FGF2, "IGF1" indicates culture in a medium supplemented with IGF-1 but without FGF2, and "FGF2" indicates culture in a medium supplemented with FGF2 but without IGF-1. The vertical axis of the graph shows the relative value when the control (small intestinal organoids cultured in medium A' supplemented with IGF-1 and human FGF2) is set at 100%.

Figure 21 shows bright-field images of the resulting small intestinal organoids. In Figure 21, (from left) the results of culturing in medium A' or medium G', culturing in medium A' or G' without human IGF-1 or human FGF2, culturing in medium A' or G' without human FGF2, and culturing in medium A' or G' without human IGF-1. The first and third rows from the top show the results of culturing in medium A' or medium A' without human IGF-1 and/or human FGF2, while the second and fourth rows from the top show the results of culturing in medium G' or medium G' without human IGF-1 and/or human FGF2. The magnification of the images in the first and second rows from the top in Figure 21 was 20x. The magnification of the images in the third and fourth rows from the top was 100x.

As a result, when medium G' was used, even though the medium did not contain human IGF-1 and/or human FGF2, promotion of cell proliferation was observed compared to when medium A' was used.

[Experimental Example 13: Evaluation of organoid cell yield]
Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the above-mentioned media A', E', G', and L' were used and cultured at 37°C in the presence of 5% _CO2 by volume for 61 to 68 days. In the case of cystic organoids with bud-like protrusions, as the number of culture days increased, the proliferated or differentiated cells folded into the lumen, and subsequently underwent cell death. Therefore, subculture was performed when the lumen became dark during bright field observation, before cell death. Spherical organoids were also subcultured when the center became dark during bright field observation.

The cell yield at the first passage was calculated by multiplying the volume of the dispersed cell suspension by the counted cell density. When calculating the cell yield at the second and subsequent passages, the cells discarded during passage were assumed to proliferate in the same way as the cells that continued to be cultured, and the cell yield based on the discarded cells was used as the cumulative cell yield. The cell yield calculated by multiplying the volume of the dispersed cell suspension by the counted cell density was then added to the cumulative cell yield described above to calculate the cell yield at that point.

Figure 22 is a graph showing the results of cell yield evaluation. In Figure 22, "Medium A'," "Medium E'," "Medium G'," and "Medium L'" indicate the media used for culture. As shown in Figure 22, organoids cultured in media containing IFN-γ or TRUL1 had a high cell yield. Compared to the cell yield of organoids cultured in medium A', the cell yield of organoids cultured in medium G' was approximately 100-fold higher after one month and approximately 10,000-fold higher after two months.

[Experimental Example 14: Evaluation of the effects of short-term treatment with IFN-γ, HGF, and vitamin D on organoid culture]
Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the above-mentioned media A' and L' were used and cultured at 37°C in the presence of 5% by volume _CO2 for 9 days. In addition, samples were prepared using medium L' up to the second day of culture and medium A' from the second day onwards.

Figure 23 is a graph showing the results of a CTG assay of the obtained small intestinal organoids. The horizontal axis of the graph indicates the medium used. "2 days Medium L' → Medium A'" indicates that Medium L' was used until the second day of culture, and Medium A' was used from the second day onwards. The vertical axis of the graph indicates the relative value when the control (small intestinal organoids cultured in Medium A') is set to 100%.

Figure 24 shows bright-field images of the resulting small intestinal organoids and fluorescence microscope images in which tdTomato fluorescence was detected. In Figure 24, the top two rows are at 20x magnification, and the bottom row is at 100x magnification. The top and bottom rows are bright-field images, and the second row from the top is a fluorescence microscope image.

As a result, it was revealed that stimulating cells with IFN-γ, HGF substitute peptides, and vitamin D was effective in promoting cell proliferation even within the short period of two days after seeding.

[Experimental Example 15: Evaluation of the effect of LATS1/2 inhibitors on organoid culture]
Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the culture period was 6 days and the above-mentioned media A', L', E', G', E'', and G''' were used. Media E'' and G'' were prepared by replacing the TRULl contained in media E' and G' with TDI-011536. TDI-011536 (CAS number: 2687970-96-1, also known as "Lats-IN-1") was obtained from Selleck Biotechnology. TDI-011536 was added to the culture medium to a concentration of 3 μM.

Figure 25 is a graph showing the results of a CTG assay of the obtained small intestinal organoids. The vertical axis of the graph shows the measured ATP concentration value. The horizontal axis of the graph shows the medium used and its components. In Figure 25, "(-) HID" indicates that the cells were cultured in a medium that did not contain "HGF substitute peptide," "IFN-γ," or "calcitriol (synonymous with vitamin D)." "(+) HID" indicates that the cells were cultured in a medium that contained "HGF substitute peptide," "IFN-γ," and "calcitriol (synonymous with vitamin D)." "TRULI" indicates that the cells were cultured in a medium that contained "TRULI." "TDI-011536" indicates that the cells were cultured in a medium that contained "TDI-011536."

As a result, it was confirmed that adding TDI-011536 to the culture medium instead of TRUL1 was effective in promoting cell proliferation.

Figure 26 shows bright-field images of the resulting small intestinal organoids. All images in Figure 26 are at 100x magnification.

As a result, it was confirmed that adding TDI-011536 to the culture medium instead of TRUL1 was effective in promoting cell proliferation.

The present invention provides a technology for efficiently expanding and culturing organoids.

Claims (15)

A method for producing organoids, comprising the step of culturing somatic stem cells in a medium containing an agonist for the interferon-gamma receptor. The method for producing organoids described in claim 1, wherein the culture medium further contains an agonist of the hepatocyte growth factor (HGF) receptor. The method for producing organoids described in claim 1 or 2, wherein the culture medium further contains a vitamin D receptor agonist. The method for producing organoids described in claim 1 or 2, wherein the culture medium further contains a Hippo signaling pathway inhibitor. The method for producing organoids described in claim 4, wherein the Hippo signaling pathway inhibitor is a LATS1/2 inhibitor. The method for producing organoids described in claim 1, wherein the culture medium contains 0.1 pM to 500 pM of the interferon-γ receptor agonist. The method for producing organoids described in claim 5, wherein the culture medium contains the LATS1/2 inhibitor at a concentration of 0.1 μM to 50 μM. The method for producing organoids described in claim 1 or 2, wherein the somatic stem cells are cultured in the medium for 24 hours or more from the start of culture. The method for producing organoids described in claim 1 or 2, wherein the culture medium further contains one or a combination of two or more selected from the group consisting of an epidermal growth factor (EGF) receptor agonist, a bone morphogenetic protein (BMP) signaling pathway inhibitor, a p38 inhibitor, an insulin-like growth factor 1 (IGF1) receptor agonist, a fibroblast growth factor 2 (bFGF, FGF2) receptor agonist, a Wnt signaling pathway activator, and a Rho-associated kinase inhibitor. The method for producing organoids described in claim 1 or 2, wherein the somatic stem cells are intestinal epithelial stem cells. Culture medium for organoid production containing an interferon-gamma receptor agonist. The organoid production medium of claim 11, further comprising an agonist of the hepatocyte growth factor (HGF) receptor. The organoid production medium according to claim 11 or 12, further comprising a vitamin D receptor agonist. The organoid production medium according to claim 11 or 12, further comprising a Hippo signaling pathway inhibitor. The organoid production medium according to claim 11 or 12, further comprising one or a combination of two or more selected from the group consisting of epidermal growth factor (EGF) receptor agonists, bone morphogenetic protein (BMP) signaling pathway inhibitors, p38 inhibitors, insulin-like growth factor 1 (IGF1) receptor agonists, fibroblast growth factor 2 (bFGF, FGF2) receptor agonists, Wnt signaling pathway activators, and Rho-associated kinase inhibitors.