Method for producing organoid and culture medium for producing organoid

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

[0001] The present invention relates to a method for producing organoids and a culture medium for producing organoids. This application claims priority based on Japanese Patent Application No. 2024-059202, filed on April 1, 2024, the contents of which are incorporated herein by reference.

[0002] Organoids are cultured cells formed by the accumulation of cells, and have a structure and function similar to that of organs in vivo. In recent years, active research has been conducted into the production of 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.

[0003] 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 produced.

[0004] The application of organoids to regenerative medicine is also being considered, which necessitates the establishment of a technology for efficiently expanding and culturing organoids.

[0005] Incidentally, interferon-γ (IFN-γ) is a type of inflammatory cytokine. For example, Non-Patent Document 1 describes that 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 that intestinal organoids were exposed to IFN-γ to reproduce an inflammatory state in order to create an evaluation system for therapeutic drugs for Crohn's disease.

[0006] Nava P, et al., Interferon-gamma regulates intestinal epithelial homeostasis through converging beta-catenin signaling pathways, Immunity, 32 (3), 392-402, 2010.Osaki LH, et al., Interferon-gamma directly induces gastric epithelial cell death and is required for progression to metaplasia, J Pathol., 247 (4), 513-523, 2019.

[0007] The present invention aims to provide a technique for efficiently expanding and culturing organoids.

[0008] 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 interferon-γ receptor. [2] The method for producing organoids according to [1], wherein the medium further contains an agonist of hepatocyte growth factor (HGF) receptor. [3] The method for producing organoids according to [1] or [2], wherein the medium further contains an agonist of vitamin D receptor. [4] The method for producing organoids according to any of [1] to [3], wherein the medium further contains a Hippo signaling pathway inhibitor. [5] The method for producing organoids according to [4], wherein the Hippo signaling pathway inhibitor is a LATS1 / 2 inhibitor. [6] The method for producing organoids according to [5], wherein the medium contains 0.1 μM to 50 μM of the LATS1 / 2 inhibitor. [7] The method for producing organoids according to any of [1] to [6], wherein the medium contains 0.1 pM to 500 pM of the agonist of interferon-γ receptor. [8] The method for producing organoids 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 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.

[10] The method for producing organoids according to any one of [1] to [9], wherein the somatic stem cells are intestinal epithelial stem cells.

[11] The culture medium for producing organoids according to

[11] , further comprising an agonist of the interferon-γ receptor.

[12] The culture medium for producing organoids according to

[11] , further comprising 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.

[0009] According to the present invention, a technique for efficiently expanding and culturing organoids can be provided.

[0010] FIG. 1 is a graph showing the results of evaluating cell proliferation in Experimental Example 1. FIG. 2 is a bright-field observation image and a fluorescence microscope image 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 is a bright-field observation image and a fluorescence microscope image 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 is a bright-field observation image and a fluorescence microscope image 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 is a bright-field observation image and a fluorescence microscope image of small intestinal organoids in Experimental Example 8. 13 is a bright-field observation image of intestinal-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 liver organoids 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 results of evaluating cell yield in Experimental Example 13. FIG. 23 is a graph showing the results of evaluating cell proliferation in Experimental Example 14. Figure 24 shows bright-field observation images and fluorescence microscope images of small intestinal organoids in Experimental Example 14. Figure 25 is a graph showing the results of evaluating cell proliferation in Experimental Example 15. Figure 26 shows bright-field observation images of small intestinal organoids in Experimental Example 15.

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

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

[0013] As used herein, "a medium containing substance X" 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 endogenously express, secrete, or produce substance X, endogenous substance X is distinguished from exogenous substance X, and a medium that does not contain exogenous substance X does not fall under the category of "a medium containing substance X," even if it contains endogenous substance X.

[0014] In this specification, human genes and human proteins are represented by capital letters. Mouse genes are represented by an initial capital letter followed by lowercase letters. Mouse proteins are represented by capital letters. However, in some cases, human genes, mouse genes, human proteins, and mouse proteins may be referred to without strict distinction.

[0015] [Method for producing organoids] In one embodiment, the present invention provides a method for producing organoids, comprising the step of culturing somatic stem cells in a medium containing an agonist of interferon-γ (IFN-γ) receptor.

[0016] As described below in the Examples, the manufacturing method of this embodiment allows efficient expansion and culture of organoids. The inventors discovered that culturing intestinal epithelial stem cells by adding the inflammatory cytokine IFN-γ to the medium increases cell proliferation activity compared to conventional culture methods. They speculate that this is due to the fact that IFN-γ recreates the microenvironment established during tissue regeneration, promoting cell proliferation.

[0017] In the production method of this embodiment, the 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. The 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), and the like.

[0018] The somatic stem cells are preferably derived from mammals, including 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.

[0019] In the production method of this embodiment, it is preferable to suspend somatic stem cells in an extracellular matrix, polymerize the extracellular matrix, and then layer a medium on the polymerized extracellular matrix for culture.

[0020] An extracellular matrix is ​​a substance that serves as a scaffold for cells in cell culture. Examples of components of the extracellular matrix include components contained in basement membranes and glycoproteins present in intercellular spaces. Examples of components contained in basement membranes include type IV collagen, laminin, heparan sulfate proteoglycan, entactin, etc. Examples of glycoproteins present in intercellular spaces include collagen, laminin, entactin, fibronectin, fibrinogen, heparin sulfate, etc. As components of the extracellular matrix, one of these may be used alone, or two or more may be used in combination. Among these, the extracellular matrix preferably contains laminin, and more preferably contains laminin-111.

[0021] 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).

[0022] Hydrogels made of artificial polymers can also be used as scaffolding materials for cells. Commercially available hydrogels made of artificial polymers include Mebiol Gel (Mebiol), VitroGel (The Well Biosciences), and GrowDex (UPM Biomedicals).

[0023] As used herein, the term "receptor agonist" includes factors that mediate signal transduction to the receptor and its downstream transcription factors. It is preferable to use a receptor agonist derived from the same species as the somatic stem cells to be cultured.

[0024] 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.

[0025] The IFN-γ receptor agonist is not particularly limited as long as it specifically binds to the IFN-γ receptor and activates the JAK-STAT pathway, and includes endogenous agonists (i.e., IFN-γ) and exogenous agonists. For example, IFN-γ isolated and purified from a living organism can be used as the endogenous agonist of the IFN-γ receptor. The exogenous agonist of the IFN-γ receptor may be a small organic compound, an inorganic compound, a peptide, a polypeptide, a peptidomimetic, an oligonucleotide, an aptamer, an agonist antibody against the IFN-γ receptor, or a fragment thereof, or the like.

[0026] Examples of NCBI accession numbers for human IFN-γ cDNA include NM_000619.3, etc. Examples of NCBI accession numbers for mouse IFN-γ cDNA include NM_008337.4, etc.

[0027] 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 will be described later 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.

[0028] In the production method of this embodiment, it is preferable that the medium further contains an agonist of hepatocyte growth factor (HGF) receptor. An "agonist of HGF receptor" is a substance that specifically binds to an HGF receptor and exhibits the physiological action of the HGF receptor.

[0029] The HGF receptor is a single-transmembrane receptor tyrosine kinase called MET (also called c-Met). MET signal transduction is initiated by the activation of MET through dimerization upon binding of HGF, which induces the kinase catalytic activity of MET.

[0030] The agonist of the HGF receptor is not particularly limited as long as it is a substance that specifically binds to the HGF receptor and promotes MET signal transduction, and includes endogenous agonists (ie, HGF) and exogenous agonists.

[0031] As an endogenous agonist of the HGF receptor, for example, HGF isolated and purified from a living body can be used.

[0032] The exogenous agonist of the HGF receptor may be a small organic compound, an inorganic compound, a peptide, a polypeptide, a peptidomimetic, an oligonucleotide, an aptamer, etc. Examples of the above peptides include HGF surrogate peptides (c-Met agonists), which are commercially available.

[0033] NCBI accession numbers for human HGF cDNA include NM_000601.6, NM_001010931.3, NM_001010932.3, NM_001010933.3, NM_001010934.3, etc. NCBI accession numbers for mouse HGF cDNA include NM_001289458.1, NM_001289459.1, NM_001289460.2, NM_001289461.1, NM_010427.5, etc.

[0034] 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, and may be 0.1 nM to 5 nM, or may be 0.1 nM to 1 nM. As will be described later 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.

[0035] In the production method of this embodiment, the medium may contain a vitamin D receptor agonist. As will be described later 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 later, it is preferable that the medium further contains a vitamin D receptor agonist.

[0036] A "vitamin D receptor agonist" is a substance that specifically binds to a vitamin D receptor and exhibits the physiological action of the vitamin D receptor. The vitamin D receptor is a nuclear receptor (vitamin D receptor: VDR) that is activated by binding with calcitriol and controls the transcription of target genes.

[0037] The vitamin D receptor agonist is not particularly limited as long as it is a substance that specifically binds to the vitamin D receptor and regulates the transcription of the target gene, and includes endogenous agonists (i.e., calcitriol, CAS number: 32222-06-3) and exogenous agonists.

[0038] As an endogenous agonist of vitamin D receptor, for example, calcitriol isolated and purified from a living body can be used.

[0039] The exogenous agonist of the vitamin D receptor may be an organic small molecule compound, an inorganic compound, a peptide, a polypeptide, a peptidomimetic, an oligonucleotide, an aptamer, etc. The exogenous agonist of the vitamin D receptor may be a vitamin D ２ (Ergocalciferol, CAS number: 50-14-6), Vitamin D ３ (Cholecalciferol, CAS number: 67-97-0), Vitamin D ３ calcipotriol (CAS number: 112965-21-6), a vitamin D analogue ３ calcipotriol hydrate (CAS number: 147657-22-5), a vitamin D analogue ２ An example of such an analog is doxercalciferol (CAS number: 54573-75-0).

[0040] In the production method of this embodiment, the concentration of the vitamin D receptor agonist in the culture medium is preferably more 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 will be described later in the Examples, when the concentration of the vitamin D receptor agonist in the culture medium is within the above range, the organoid proliferation-promoting effect tends to be high.

[0041] 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.

[0042] In the production method of this embodiment, the medium preferably further contains a Hippo signaling pathway inhibitor.

[0043] The Hippo signaling pathway, also known as the Salvador-Warts-Hippo (SWH) pathway, is a signaling pathway that, although the molecules involved and their mechanisms 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, upon phosphorylation, 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, suggesting that Sav functions as a scaffolding protein.

[0044] Activated Wts phosphorylates and inactivates the transcriptional coactivator Yki (in mammals, the two Yki orthologs are Yes-associated protein (YAP) and transcriptional coactivator with 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 allows 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 nuclear transport. Upon activation, YAP and TAZ can bind to several transcription factors, including p73, Runx2, and several TEADs.

[0045] 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.

[0046] The Hippo signaling pathway inhibitor may be any inhibitor 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.

[0047] The molecule inhibited by the Hippo signaling pathway inhibitor is preferably a molecule constituting the Hippo core kinase cascade, more preferably Wts (LATS1 / 2). Therefore, Hippo signaling pathway inhibitors include LATS1 / 2 inhibitors.

[0048] Examples of 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 the phosphorylation of Yap.

[0049] As described below in the Examples, we have demonstrated that when a 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 combination 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 below 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).

[0050] In the production method of this embodiment, the concentration of the LATS1 / 2 inhibitor in the 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 will be described later in the Examples, when the concentration of the LATS inhibitor in the medium is within the above range, the organoid proliferation promoting effect tends to be high.

[0051] In the production method of this embodiment, the medium may further contain a transforming growth factor β (TGF-β) inhibitor.

[0052] TGF-β signaling contributes to the inhibition of cell proliferation, cell differentiation, induction of apoptosis, etc. TGF-β inhibitors are substances that down-regulate TGF-β signaling, and can also be called TGF-β signaling pathway inhibitors.

[0053] As used herein, the term "TGF-β inhibitor" refers to an inhibitor that inhibits activation of type I or type II serine / threonine kinase receptors, and that is accompanied by inhibition of phosphorylation of Smad2 / 3.

[0054] Examples of TGF-β inhibitors that inhibit the phosphorylation of Smad2 / 3 include A83-01 (CAS number: 909910-43-6), SB-431542 (CAS number: 301836-41-9), SB-505124 (CAS number: 694433-59-5), SB-525334 (CAS number: 356559-20-1), LY364947 (CAS number: 396129-53-6), SD-208 (CAS number: 627536-09-8), and SJN2511 (CAS number: 446859-33-2), of which A83-01 is preferred.

[0055] The concentration of the TGF-β inhibitor contained in the medium may be, for example, 50 nM to 5 μM, for example, 50 nM to 1 μM, or for example, 50 nM to 500 nM.

[0056] 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.

[0057] Furthermore, as described below in the Examples, when the medium contains a combination of IFN-γ, HGF, vitamin D or an analog thereof, and a Hippo-YAP / TAZ signaling pathway inhibitor, a high organoid proliferation promoting effect tends to be obtained even without containing a TGF-β inhibitor.

[0058] In the production method of this embodiment, the medium preferably further contains other factors commonly used in organoid culture. Such factors include, for example, 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, Rho-binding kinase inhibitors, etc. In the production method of this embodiment, the medium preferably further contains one or a combination of two or more of these factors.

[0059] Examples of agonists for epidermal growth factor (EGF) receptors include ligands that have agonistic activity against EGF receptors. Examples of such ligands include EGF and EGF mimetics. Examples of EGF mimetics include agonist antibodies or fragments thereof, peptides, and the like against EGF receptors.

[0060] NCBI accession numbers for human EGF cDNA include NM_001178130.3, NM_001178131.3, NM_001357021.2, NM_001963.6, etc. NCBI accession numbers for mouse EGF cDNA include NM_001310737.1, NM_001329594.1, NM_010113.4, etc.

[0061] 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.

[0062] 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).

[0063] 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.

[0064] Examples of p38 inhibitors include SB202190 (CAS number: 152121-30-7) and Doramapimod (CAS number: 285983-48-4).

[0065] In the production method of this embodiment, the concentration of the p38 inhibitor contained in the medium is preferably 50 nM to 100 μM, more preferably 100 nM to 50 μM, and even more preferably 100 nM to 10 μM.

[0066] Examples of agonists for the insulin-like growth factor 1 (IGF-1) receptor include ligands that have agonistic activity against the IGF-1 receptor. Examples of such ligands include IGF-1 and IGF-1 mimetics. Examples of IGF-1 mimetics include agonist antibodies or fragments thereof, peptides, and the like against the IGF-1 receptor.

[0067] 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, NM_001414007.1, etc. Furthermore, NCBI accession numbers for mouse IGF-1 cDNA include NM_001111274.1, NM_001111275.2, NM_001111276.1, NM_001314010.1, NM_010512.5, etc.

[0068] 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.

[0069] Examples of agonists for fibroblast growth factor 2 (FGF2) receptors include 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.

[0070] NCBI accession numbers for human FGF2 cDNA include NM_001361665.2 and NM_002006.6, etc. Furthermore, NCBI accession numbers for mouse FGF2 cDNA include NM_008006.2, etc.

[0071] 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.

[0072] Wnt signaling regulates cell proliferation and differentiation by regulating the protein level of β-catenin, which functions as a transcription promoter. Examples of Wnt signaling pathway activators include inhibitors of the Wnt family, R-spondin family, Norrin, and glycogen synthase (GSK) inhibitors.

[0073] Examples of the Wnt family include 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 these Wnt family members 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.

[0074] Afamin is known to contribute to the stabilization and solubilization of the Wnt family. Therefore, a complex of a Wnt family member with afamin is more preferred as a Wnt signaling pathway activator. Afamin is a glycoprotein that belongs to the albumin family.

[0075] Examples of NCBI accession numbers for human afamin cDNA include NM_001133.2, bovine afamin cDNA include NM_001192175.1, and mouse afamin cDNA include NM_145146.2.

[0076] The Wnt family or a complex of the Wnt family with 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 having a Wnt family concentration within the above range is used as the Wnt family, the content of the conditioned medium in the total volume of the medium is typically 1 (v / v)% to 50 (v / v)%, and may be, for example, 5 (v / v)% to 30 (v / v)%, or may be, for example, 5 (v / v)% to 10 (v / v)%.

[0077] Examples of the R-spondin family include R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4. Among these, R-spondin 1 is preferred. Amino acid sequence information for each protein in the R-spondin family is available from the NCBI database.

[0078] For example, NCBI accession numbers for human R-spondin1 cDNA include NM_001038633.4, NM_001242908.2, NM_001242909.2, NM_001242910.2, NM_173640.1, etc. Furthermore, NCBI accession numbers for mouse R-spondin1 cDNA include NM_138683.2, etc.

[0079] 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.

[0080] 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 in the above range is used as the R-spondin family, the content of the conditioned medium in the culture medium is usually 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)%, based on the total volume of the culture medium.

[0081] GSK inhibitors are inhibitors of glycogen synthase 3β (GSK3β), which phosphorylates β-catenin and promotes its degradation, thereby acting as a Wnt agonist.

[0082] 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).

[0083] As the Wnt signaling pathway activator, it is preferable to use a combination of the Wnt family and the R-spondin family, it is more preferable to use a combination of Wnt3a and R-spondin1, and it is even more preferable to use a combination of a complex of Wnt3a and afamin and R-spondin1.

[0084] 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 number: 850664-21-0), DE-104, H-1152P (CAS number: 872543-07-6), and blebbistatin (CAS number: 856925-71-8).

[0085] In the production method of this embodiment, the concentration of the ROCK inhibitor in the medium is usually 1 μM to 20 μM, and preferably 5 μM to 15 μM.

[0086] In the production method of this embodiment, the medium may contain other components that are typically added to cell culture media, such as amino acids, vitamins, inorganic salts, sugars, trace elements, antibiotics, and other additives.

[0087] Examples of amino acids include 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.

[0088] Examples of vitamins include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), D-calcium 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-α-tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

[0089] Examples of inorganic salts include salts of calcium, copper, iron, magnesium, potassium, sodium, and zinc. The salts are usually used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. More specifically, for example, CaCl ２ , CuSO ４ -5H ２ O, Fe(NO ３ ) -9H ２ O, FeSO ４ -7H ２ O, MgCl, MgSO ４ , KCl, NaHCO ３ , NaCl, Na ２ HPO ４ , Na ２ HPO ４ -H ２ O, ZnSO ４ -7H ２ Examples include O.

[0090] Examples of sugars include glucose, galactose, maltose, and fructose.

[0091] Examples of trace elements include 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 thereof.

[0092] Examples of antibiotics include penicillin, streptomycin, amphotericin B, and nanomycopritin.

[0093] Other additives include B27 supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), gastrin, cholesterol, transferrin, albumin, insulin, progesterone, putrescine, and the like.

[0094] In the production 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 simulating medium, and Fischer's medium, as well as mixtures thereof.

[0095] In one embodiment, the present invention provides the method for producing organoid, comprising the step of culturing somatic stem cell in the medium containing Hippo signaling pathway inhibitor.As described later in Examples, the medium containing Hippo signaling pathway inhibitor tends to have a high effect of promoting the proliferation of organoid.

[0096] In the production method of this embodiment, the somatic stem cells, the Hippo signaling pathway inhibitor, and the like are the same as those described above.

[0097] [Culture medium for organoid production] In one embodiment, the present invention provides a culture medium for organoid production containing 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.

[0098] The medium of this embodiment preferably further contains an agonist of the HGF receptor, which is the same as that described above.

[0099] The medium of this embodiment preferably further contains a vitamin D receptor agonist, which is the same as that described above.

[0100] The medium of this embodiment preferably further contains a Hippo signaling pathway inhibitor, which is the same as that described above.

[0101] The medium of this embodiment preferably further contains a TGF-β inhibitor, which is the same as that described above.

[0102] 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.

[0103] The 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 are the same as those described above.

[0104] As will be described later in the Examples, when a 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-fold higher in one month and approximately 10,000-fold 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).

[0105] The medium of this embodiment may further contain other components that are usually added to cell culture media.

[0106] [Additive Kit] In one embodiment, the present invention provides an additive kit for a culture medium for producing organoids by culturing somatic stem cells, the additive kit comprising a combination of two or more selected from the group consisting of the following components (a) to (d): component (a): an interferon-γ (IFN-γ) receptor ligand, component (b): a Hippo signaling pathway inhibitor, component (c): a hepatocyte growth factor (HGF) receptor agonist, and component (d): a vitamin D receptor agonist.

[0107] 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.

[0108] The present invention will be described in more detail below based on experimental examples. However, the present invention is not limited to 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.

[0109] [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 to 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'', respectively. Advanced DMEM / F12 (Thermo Fisher Scientific) was used as the basal medium.

[0110]

[0111]

[0112]

[0113]

[0114] 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 and used. "CM" means conditioned medium.

[0115] 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.

[0116] 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.

[0117] Noggin CM was prepared according to the procedure described in Vonk AM, et al., Protocol for Application, Standardization and Validation of the Forskolin-Induced Swelling Assay in Cystic Fibrosis Human Colon Organoids, STAR Protoc. 1 (1), 100019, 2020. The concentration of Noggin in Noggin CM was 12 μg / mL.

[0118] (Subculture maintenance of human small intestinal epithelial cells) Human small intestinal epithelial cells derived from human small intestinal tissue fragments were used in the experiment. 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. In addition, IRES-tdTomato was used as the reporter construct. When these cells express LGR5, they express the fluorescent protein tdTomato.

[0119] 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 a concentration of 100 cells / μL.

[0120] Subsequently, 20 μL of the 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 incubated at 37°C and 5% CO ２ The cells were cultured in the presence of HCl. The medium used was the above-mentioned Medium A'. To prevent apoptosis, 10 μM Y-27632 (CAS number: 331752-47-7, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium until the first medium change. The medium was changed every 2 or 3 days.

[0121] As the culture days continued, the cells proliferated within the Matrigel in the form of cell clumps, and after about 7 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 microscopic observation, the lumen became dark. Because cell death occurs after the lumen of the organoids becomes dense due to cell proliferation, around 10 to 11 days after the start of culture, before the lumen becomes excessively dark, the cells were dispersed into single cells using TrypLE Express (Thermo Fisher Scientific) and passaged as described above to maintain human small intestinal epithelial cells.

[0122] (Morphological Observation of Organoids) The morphology of organoids was observed in a bright field using a fluorescence microscope (manufactured by KEYENCE, device name "BZ-X800").

[0123] (Evaluation of organoid proliferation) Organoid proliferation was evaluated using a commercially available kit (CellTiter-Glo （Ｒ） The luminescence signal was detected and read using a 3D Cell Viability Assay (Promega, hereinafter sometimes referred to as "CTG assay") according to the kit's instructions. （Ｒ） This was performed using a Discover Microplate Reader (Promega).

[0124] (Evaluation of Cell Stem Cell Potential) 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, the expression of tdTomato was detected by fluorescence microscopy as an indicator of stem cell potency.

[0125] Experimental Example 1: Study 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.

[0126] Subsequently, 18 μL of the 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 incubated at 37° C. and 5% CO 2 by volume. ２ The cells were cultured in the presence of ATP for 9 days to obtain small intestinal organoids. The above-mentioned media A to P were used. 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.

[0127] FIG. 1 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 and its components. In FIG. 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 alternative 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 the relative value when the control (small intestinal organoids cultured in medium A) is set to 100%.

[0128] As a result, it was observed that cell proliferation was promoted when media B, C, E, F, G, H, K, L, N, O, and P were used, compared to when media A was used. That is, it was observed that cell proliferation was promoted when IFN-γ, HGF, TRULI, vitamin D, or a combination thereof was added to the medium.

[0129] [Experimental Example 2: Study of culture media for organoid production 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.

[0130] Figure 2 shows bright-field images of each small intestinal organoid and fluorescence microscope images in which tdTomato fluorescence was detected. In Figure 2, the upper row is a bright-field image, and the lower row is a fluorescence microscope image. The medium used for culture is indicated at the top of each image. All images were taken at 20x magnification.

[0131] As a result, it was found that the use of media B and G promoted cell proliferation and increased stem cells as indicated by tdTomato signaling compared to the use of media A. In other words, it was found that the addition of IFN-γ or a combination of IFN-γ, HGF, TRULI, and vitamin D to the medium promoted cell proliferation and increased stem cells.

[0132] [Experimental Example 3: Study of culture media for organoid production 3] Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that the above-mentioned culture media A, A', B', G', K', L', and P' were used, respectively.

[0133] Figure 3 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 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%.

[0134] As a result, it was found that the addition of A83-01 to the medium, together with IFN-γ, HGF, TRULI, vitamin D or a combination thereof, promoted cell proliferation.

[0135] [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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] In addition, 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.

[0140] 4 is a graph showing the results of a CTG assay of the obtained 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 to 100%.

[0141] As a result, it was found that the addition of inflammatory cytokines IL11, IL22, IL6, and IFN-γ to the culture medium tended to promote cell proliferation, with the addition of IFN-γ being the most effective.

[0142] Figure 5 shows bright-field images of small intestinal organoids obtained using 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.

[0143] As a result, promotion of cell proliferation was observed when the concentration of IFN-γ was in the range of more than 0 nM and less than 1 nM.

[0144] [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 peptide at concentrations of 0, 0.1 nM, 0.3 nM, and 1 nM.

[0145] 6 is a graph showing the results of a CTG assay of the obtained small intestinal organoids. The vertical axis of the graph shows relative values ​​when the control (small intestinal organoids cultured in medium A′ without HGF) is set to 100%.

[0146] As a result, it was found that the addition of HGF promoted cell proliferation.

[0147] 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 the like.

[0148] Figure 7 is a graph showing the results of a CTG assay of the obtained small intestinal organoids. The vertical axis of the graph shows the relative value when the control (small intestinal organoids cultured in media G and K using HGF substitute peptides) is set to 100%. The horizontal axis of the graph shows the medium used and its 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".

[0149] As a result, it was confirmed that the effect on cell proliferation was the same regardless of whether HGF alternative peptide or recombinant human HGF was added to the culture medium.

[0150] Figure 8 shows bright-field images of the obtained small intestinal organoids and fluorescence microscope images in which tdTomato fluorescence was detected. All images in Figure 8 are magnified 20 times. The upper row shows bright-field images, and the lower row shows fluorescence microscope images.

[0151] As a result, it was confirmed that the effect on cell proliferation was the same regardless of whether HGF alternative peptide or recombinant human HGF was added to the culture medium.

[0152] [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.

[0153] 9 is a graph showing the results of a CTG assay of the obtained small intestinal organoids. The vertical axis of the graph shows the relative value when the control (small intestinal organoids cultured in medium A' without vitamin D) is set to 100%.

[0154] 10 shows bright-field images of small intestinal organoids obtained using a medium to which vitamin D was added to the above-mentioned medium A' at concentrations of 0, 3 nM, 10 nM, 30 nM, and 100 nM, as well as fluorescent microscope images of tdTomato fluorescence. In FIG. 10, the magnification of the top two rows is 20x, and the magnification of the bottom row is 100x. The top and bottom rows are bright-field images, and the second row from the top is a fluorescent microscope image.

[0155] As a result, promotion of cell proliferation was observed when the vitamin D concentration was in the range of more than 0 nM and less than 100 nM.

[0156] [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 supplemented with TRUL1 at concentrations of 0, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM.

[0157] 11 is a graph showing the results of a CTG assay of the obtained small intestinal organoids. The vertical axis of the graph shows relative values ​​when the control (small intestinal organoids cultured in Medium A' without TRUL1) is set to 100%.

[0158] 12 shows bright-field images of small intestinal organoids obtained using a medium to which TRUL1 was added at concentrations of 0, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM to the above-mentioned medium A', and fluorescent microscope images in which tdTomato fluorescence was detected. In FIG. 12, the magnification of the top two rows is 20x, and the magnification of the bottom row is 100x. The top and bottom rows are bright-field images, and the second row from the top is a fluorescent microscope image.

[0159] Typically, organoids form monolayered cysts with bud-like processes similar to the intestinal crypts found in vivo. In contrast, by culturing in a medium containing TRUL1, the organoids took on a spherical form with a solid lumen. Furthermore, as shown in Figures 11 and 12, TRUL1 promoted cell proliferation in a concentration-dependent manner up to a concentration of about 30 μM, and also enhanced stemness as indicated by tdTomato signaling. However, at a concentration of 100 μM, TRUL1 recrystallized, and cell death was observed.

[0160] Experimental Example 9: Evaluation of organoid culture derived from duodenum, ileum, and large intestine Using media A' and G', intestinal organoids were prepared in the same manner as in Experimental Example 1, except that human small intestinal (ileal) epithelial cells derived from a human small intestinal tissue fragment, duodenal epithelial cells derived from a human duodenal tissue fragment, or human large intestinal epithelial cells derived from a human large intestinal tissue fragment were used instead of the human small intestinal epithelial cells derived from the human small intestinal tissue fragment described above.

[0161] Figure 13 shows bright-field observation images of each intestinal-derived organoid. In Figure 13, the upper row shows organoids derived from duodenal epithelial cells, the middle row shows organoids derived from ileal epithelial cells, and the lower row shows organoids derived from colonic epithelial cells. The media used for culture are 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.

[0162] Figure 14 is a graph showing the results of a CTG assay of the obtained intestinal organoids. In Figure 14, "duodenum" indicates the results of organoids derived from duodenal epithelial cells, "ileum" indicates the results of organoids derived from ileal epithelial cells, and "colon" indicates the results of 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.

[0163] As a result, promotion of cell proliferation was observed in each intestinal-derived organoid.

[0164] Experimental Example 10: Evaluation of liver-derived organoid culture Instead of the human small intestinal epithelial cells derived from the above-mentioned human small intestinal tissue fragment, organoid culture was performed using frozen human primary hepatocytes. The frozen human primary hepatocytes used were those shown in Table 5 below.

[0165]

[0166] Media Q to W were prepared by adding each component 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.

[0167]

[0168] 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 Peprotec. 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" means conditioned medium.

[0169] (Subculture and Maintenance of Human Primary Hepatocytes) 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.

[0170] Subsequently, 20 μL of the 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 incubated at 37°C and 5% CO ２ The cells were cultured in the presence of HCl. The medium used was the above-mentioned Medium S. 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, maintaining the human primary hepatocytes for 119 days.

[0171] Subsequently, the human primary hepatocytes after passage maintenance 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 are shown at the top of each image. All images are at 20x magnification.

[0172] Figure 16 is a graph showing the results of a CTG assay of the obtained liver organoids. The horizontal axis of the graph indicates the medium used and its components. In Figure 16, "IFNg" and "I" indicate that the cells were cultured in a medium containing "IFN-γ". "HGF" and "H" indicate that the cells were cultured in a medium containing an "HGF alternative peptide". "HGF-P" and "HP" indicate that the cells were cultured in a medium containing "recombinant human HGF". "D" indicates calcitriol (synonymous with vitamin D), "T" indicates "TRULI", and "HI", "HPI", and "THPID" indicate that the cells were cultured 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 value.

[0173] 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.

[0174] 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 among the additive factors in the above-mentioned media A, G, A', and G', media containing human EGF added at concentrations of 0, 5 ng / mL, 50 ng / mL, and 500 ng / mL were used.

[0175] Figure 17 is a graph showing the results of a CTG assay of the obtained small intestinal organoids. In Figure 17, "w / o A83-01" indicates that the organoids were cultured in a medium that did not contain A83-01, and "w / A83-01" indicates that the organoids were cultured in a medium that contained 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," "T" indicates "TRULI," and "THID" indicates that the organoids were cultured in a medium containing a combination of "D," "H," "I," and "T." "(-)" indicates that the organoids were cultured in a medium that did not contain "D," "H," "I," or "T." The vertical axis of the graph indicates the measured ATP concentration.

[0176] 18 and 19 are bright-field observation images of small intestinal organoids obtained using medium to which human EGF was added at concentrations of 0 ng / mL, 5 ng / mL, 50 ng / mL, and 500 ng / mL. In FIGS. 18 and 19, the left four columns show the results of culturing in medium not containing A83-01, and the right four columns show the results of culturing in medium containing A83-01. The upper row shows the results of using medium A and A' to which human EGF was added at concentrations of 0 ng / mL, 5 ng / mL, 50 ng / mL, and 500 ng / mL, and the lower row shows the results of using medium G and G' to which human EGF was added at concentrations of 0 ng / mL, 5 ng / mL, 50 ng / mL, and 500 ng / mL. The magnification of the image in FIG. 18 was 20x. The magnification of the image in FIG. 19 was 100x.

[0177] As a result, when media G and G' were used, even when the media did not contain human EGF, promotion of cell proliferation was observed compared to when media A and A' were 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 not more than 500 ng / mL.

[0178] 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 medium A' and G' described above were used without human IGF-1 and / or human FGF2, which were added as additives.

[0179] Figure 20 is a graph showing the results of the CTG assay of the obtained small intestinal organoids. In Figure 20, "IGF1 / FGF2" indicates that the organoids were cultured in a medium supplemented with IGF-1 and FGF2, "(-)" indicates that the organoids were cultured in a medium without the addition of IGF-1 and FGF2, "IGF1" indicates that the organoids were cultured in a medium supplemented with IGF-1 and without FGF2, and "FGF2" indicates that the organoids were cultured in a medium supplemented with FGF2 but not with IGF-1. The vertical axis of the graph indicates the relative value when the control (small intestinal organoids cultured in medium A' supplemented with IGF-1 and human FGF2) is taken as 100%.

[0180] Figure 21 is a bright-field observation image of the obtained small intestinal organoids. In Figure 21, from the left, the results of culturing in medium A' or medium G', the results of culturing in medium A', G' without the addition of human IGF-1 and human FGF2, the results of culturing in medium A', G' without the addition of human FGF2, and the results of culturing in medium A', G' without the addition of human IGF-1 are shown. The first and third rows from the top show the results of culturing in medium A' or medium A' without the addition of human IGF-1 and / or human FGF2, and the second and fourth rows from the top show the results of culturing in medium G' or medium G' without the addition of human IGF-1 and / or human FGF2. In Figure 21, the magnification of the images in the first and second rows from the top was 20 times. The magnification of the images in the third and fourth rows from the top was 100 times.

[0181] 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.

[0182] Experimental Example 13: Evaluation of cell yield of organoids Using the above-mentioned media A', E', G', and L', the cells were cultured at 37°C and 5% by volume of CO ２Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that they were cultured for 61 to 68 days in the presence of α-glucan. In the case of cystic organoids with bud-like protrusions, the proliferated or differentiated cells folded into the lumen as the culture days progressed, and subsequently underwent cell death. Therefore, subculture was performed when the lumen became dark under bright-field observation, before cell death. Spherical organoids were also subcultured when the center became dark under bright-field observation.

[0183] The cell yield at the first passage was calculated by multiplying the volume of the dispersed cell suspension by the counted cell density. For calculation of the cell yield at the second and subsequent passages, the cells discarded during passage were assumed to proliferate in the same manner as the cells that were continuously 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, and the cell yield at that time point was calculated.

[0184] Figure 22 is a graph showing the evaluation results of cell yield. 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 a medium containing IFN-γ or a medium containing TRULI 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 at one month and approximately 10,000-fold higher at two months.

[0185] Experimental Example 14: Evaluation of the effect of short-term treatment with IFN-γ, HGF, and vitamin D on organoid culture. The above-described media A′ and L′ were used, and the culture medium was incubated at 37° C. and 5% by volume CO ２ Small intestinal organoids were formed in the same manner as in Experimental Example 1, except that they were cultured for 9 days in the presence of medium L'. In addition, a sample was also prepared in which medium L' was used until the second day of culture and medium A' was used from the second day of culture onwards.

[0186] 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 of culture 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%.

[0187] 24 shows bright-field images of the obtained small intestinal organoids and fluorescence microscope images in which tdTomato fluorescence was detected. In FIG. 24, the magnification of the top two rows is 20x, and the magnification of the bottom row is 100x. The top and bottom rows are bright-field images, and the second row from the top is a fluorescence microscope image.

[0188] As a result, it was revealed that stimulation of cells with IFN-γ, HGF alternative peptides, and vitamin D was effective in promoting cell proliferation even for a short period of time, such as two days after seeding.

[0189] 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.

[0190] 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 containing "HGF substitute peptide," "IFN-γ," and "calcitriol (synonymous with vitamin D)." "TRULI" indicates that the cells were cultured in a medium containing "TRULI." "TDI-011536" indicates that the cells were cultured in a medium containing "TDI-011536."

[0191] As a result, it was confirmed that adding TDI-011536 to the medium instead of TRUL1 was also effective in promoting cell proliferation.

[0192] Figure 26 shows bright-field images of the resulting small intestinal organoids. All images in Figure 26 are at 100x magnification.

[0193] As a result, it was confirmed that adding TDI-011536 to the medium instead of TRUL1 was also effective in promoting cell proliferation.

[0194] According to the present invention, a technique for efficiently expanding and culturing organoids can be provided.

Claims

1. A method for producing organoids, comprising a step of culturing somatic stem cells in a medium containing an agonist of the interferon-γ receptor.

2. The method for producing organoids described in claim 1, wherein the culture medium further contains an agonist of the hepatocyte growth factor (HGF) receptor.

3. A method for producing organoids described in claim 1 or 2, wherein the culture medium further contains a vitamin D receptor agonist.

4. A method for producing organoids described in claim 1 or 2, wherein the culture medium further contains a Hippo signaling pathway inhibitor.

5. The method for producing organoids described in claim 4, wherein the Hippo signaling pathway inhibitor is a LATS1 / 2 inhibitor.

6. The method for producing organoids described in claim 1, wherein the culture medium contains 0.1 pM to 500 pM of an agonist of the interferon-gamma receptor.

7. 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.

8. A method for producing organoids described in claim 1 or 2, wherein the somatic stem cells are cultured in the culture medium for more than 24 hours from the start of culture.

9. A method for producing an organoid 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.

10. A method for producing organoids described in claim 1 or 2, wherein the somatic stem cells are intestinal epithelial stem cells.

11. Organoid production medium containing an interferon-gamma receptor agonist.

12. The organoid production medium of claim 11, further comprising an agonist of the hepatocyte growth factor (HGF) receptor.

13. A culture medium for producing organoids described in claim 11 or 12, further comprising a vitamin D receptor agonist.

14. A culture medium for producing organoids as described in claim 11 or 12, further comprising a Hippo signaling pathway inhibitor.

15. A culture medium for producing organoids as described in claim 11 or 12, further comprising 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.

Images