Culture medium for producing organoid and use of same

Abstract

Provided are: a culture medium which is for producing an organoid and which comprises a prostaglandin, hepatocyte growth factor (HGF), an interleukin (IL)-6 family protein, and an epidermal growth factor (EGF) family protein; a kit for cultivating an organoid; a method for producing an organoid; and an organoid.

Description

Culture medium for producing organoids and its use

[0001] The present invention relates to a culture medium for producing organoids and its use. More specifically, the present invention relates to a culture medium for producing organoids, an organoid culture kit, a method for producing organoids, and organoids. This application claims priority based on Japanese Patent Application No. 2024-059184, filed April 1, 2024, the contents of which are incorporated herein by reference.

[0002] Organoid culture technology has been developed and is expected to be applied to drug discovery and regenerative medicine. The inventors have pioneered the development of organoid technology for permanently culturing tissue stem cells in three dimensions (see, for example, Non-Patent Documents 1 and 2).

[0003] Incidentally, culturing organoids requires an extracellular matrix such as Matrigel (registered trademark) (see, for example, Patent Document 1, Non-Patent Document 3, etc.). Matrigel (registered trademark) is an animal-derived product, as it is an extract from tumors transplanted into mice. Furthermore, the possibility of contamination with unknown viruses and its high cost make the use of Matrigel (registered trademark) in organoid culture a barrier to its medical application. Furthermore, the use of Matrigel is also problematic from the perspective of animal welfare. Since the advent of organoid technology, vigorous efforts have been made to develop alternatives to Matrigel (registered trademark) (see, for example, Non-Patent Document 4).

[0004] Japanese Patent Application Laid-Open No. 2016-198033

[0005] Sato T, et al., Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche, Nature, 459, 262-266, 2009.Sato T, et al., Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium, Gastroenterology, 141, 1762-1772, 2011.S Rezakhani, et al., Extracellular matrix requirements for gastrointestinal organoid cultures, Biomaterials, 276, 121020, 2021.Jeong Hyun Heo, et al., Engineering the Extracellular Matrix for Organoid Culture, Int J Stem Cells, 15 (1), 60-69, 2022.

[0006] The present invention aims to provide a new technique for culturing and producing organoids.

[0007] The present invention includes the following aspects. [1] A culture medium for producing organoids, comprising a prostaglandin, hepatocyte growth factor (HGF), an interleukin (IL)-6 family protein, and an epidermal growth factor (EGF) family protein. [2] The culture medium for producing organoids according to [1], further comprising an interferon. [3] The culture medium for producing organoids according to [2], wherein the interferon is interferon (IFN)-γ. [4] The culture medium for producing organoids according to any of [1] to [3], further comprising an inhibitor of the Hippo signaling pathway. [5] The culture medium for producing organoids according to any of [1] to [4], further comprising a Wnt agonist, a bone morphogenetic protein (BMP) signaling pathway inhibitor, and a transforming growth factor (TGF)-β inhibitor. [6] The culture medium for producing organoids according to any of [1] to [4], which is substantially free of a Wnt agonist, a BMP signaling pathway inhibitor, and a TGF-β inhibitor. [7] A kit for organoid culture comprising a prostaglandin, HGF, an IL-6 family protein, and an EGF family protein. [8] A method for producing organoids, comprising the step of culturing cells in a medium containing a prostaglandin, HGF, an IL-6 family protein, and an EGF family protein, thereby forming organoids from the cells, wherein the cells are selected from the group consisting of epithelial cells, epithelial stem cells, stromal cells, mesenchymal stem cells, cancer cells, and cancer stem cells. [9] The method for producing organoids described in [8], wherein the medium further contains IFN-γ.

[10] The method for producing organoids described in [8] or [9], wherein the medium is substantially free of a ROCK inhibitor.

[11] The method for producing organoids described in any of [8] to

[10] , wherein the medium further contains a Wnt agonist, a BMP signaling pathway inhibitor, and a TGF-β inhibitor.

[12] The method for producing organoids described in any of [8] to

[10] , wherein the medium is substantially free of a Wnt agonist, a BMP signaling pathway inhibitor, and a TGF-β inhibitor.

[13] The method according to

[12] , wherein the medium is substantially free of a ROCK inhibitor.

[14] The method according to any one of [8] to

[13] , wherein an extracellular matrix is ​​not used in culturing the cells.

[15] The method for producing an organoid according to any one of [8] to

[14] , wherein the medium is a serum-free medium.

[16] The method for producing an organoid according to any one of [8] to

[15] , wherein the cells are dissociated into single cells.

[17] An organoid produced by the method for producing an organoid according to any one of [8] to

[16] .

[0008] The present invention can also be said to include the following aspects. [P1] A culture medium for producing organoids, comprising a prostaglandin, hepatocyte growth factor (HGF), an interleukin (IL)-6 family protein, and an epidermal growth factor (EGF) family protein. [P2] The culture medium for producing organoids according to [P1], further comprising an interferon. [P3] The culture medium for producing organoids according to [P2], wherein the interferon is interferon (IFN)-γ. [P4] The culture medium for producing organoids according to any of [P1] to [P3], further comprising an inhibitor of the Hippo signaling pathway. [P5] The culture medium for producing organoids according to any of [P1] to [P4], further comprising a Wnt agonist, a bone morphogenetic protein (BMP) signaling pathway inhibitor, and a transforming growth factor (TGF)-β inhibitor. [P6] The culture medium for producing organoids according to any of [P1] to [P4], which is substantially free of a Wnt agonist, a BMP signaling pathway inhibitor, and a TGF-β inhibitor. [P7] A kit for organoid culture comprising a prostaglandin, HGF, an IL-6 family protein, and an EGF family protein. [P8] A method for producing organoids, comprising the step of culturing cells in a medium containing a prostaglandin, HGF, an IL-6 family protein, and an EGF family protein, thereby forming organoids from the cells, wherein the cells are selected from the group consisting of epithelial cells, epithelial stem cells, stromal cells, cancer cells, and cancer stem cells. [P9] The method for producing organoids described in [P8], wherein the medium further contains IFN-γ. [P10] The method for producing organoids described in [P8] or [P9], wherein the medium further contains a Wnt agonist, a BMP signaling pathway inhibitor, and a TGF-β inhibitor. [P11] The method for producing organoids described in any of [P8] to [P9], wherein the medium is substantially free of a Wnt agonist, a BMP signaling pathway inhibitor, and a TGF-β inhibitor. [P12] The method for producing organoids described in any of [P8] to [P11], wherein an extracellular matrix is ​​not used to culture the cells. [P13] The method of any one of [P8] to [P12], wherein the medium is a serum-free medium. [P14] The method of any one of [P8] to [P13], wherein the cells are dissociated into single cells.[P15] An organoid produced by the production method described in any one of [P8] to "P14".

[0009] According to the present invention, a new organoid culture technique can be provided.

[0010] FIG. 1 is a schematic diagram illustrating the Hippo signaling pathway. FIG. 2 is a representative microscopic image of colonic organoids cultured in Experimental Example 1. FIG. 3 is a representative microscopic image of colonic organoids cultured in Experimental Example 1. FIG. 4 is a graph showing the results of measuring the proliferation of colonic organoids in Experimental Example 1. FIG. 5 is a representative microscopic image of small intestinal organoids and liver organoids cultured in Experimental Example 2. FIG. 6 is a representative microscopic image of small intestinal organoids and liver organoids cultured in Experimental Example 2. FIG. 7 is a representative microscopic image of mouse bone marrow stromal cell-derived organoids established in Experimental Example 3. FIG. 8 is a representative microscopic image of human skin epithelial cell-derived organoids established in Experimental Example 4. FIG. 9 is a schematic diagram showing the experimental schedule of Experimental Example 5. FIG. 10 is a microscopic image showing the results of Experimental Example 5. FIG. 11 is an enlarged image of the boxed area in FIG. 10. Fig. 12 shows microscopic images of human small intestinal villi-derived epithelial cell organoids cultured in Experimental Example 6. Fig. 13 shows microscopic images of human small intestinal epithelial cell organoids and human large intestinal epithelial cell organoids cultured in Experimental Example 7.

[0011] [Culture Medium for Organoid Production] In one embodiment, the present invention provides a culture medium for organoid production, comprising a prostaglandin, hepatocyte growth factor (HGF), an interleukin (IL)-6 family protein, and an epidermal growth factor (EGF) family protein.

[0012] The organoid production medium of this embodiment can be used for culturing organoids and establishing organoids. The organoid production medium of this embodiment can be a serum-free medium. As will be described later in the examples, the inventors have found that when organoids are cultured using the medium of this embodiment, cell proliferation is promoted compared to when organoids are cultured using conventional medium.

[0013] The inventors have also demonstrated that the medium of this embodiment can be used to establish organoids that have previously been impossible to establish, such as organoids from the digestive tract, lung, pancreas (pancreatic islets), liver, salivary glands, biliary tract, respiratory tract, hair head, skin, etc. Examples of organoids that have previously been impossible to establish include organoids derived from interstitial cells and organoids derived from skin epithelial cells.

[0014] Furthermore, as will be described later in the Examples, by using the medium of this embodiment, organoids can be cultured even in the absence of niche factors that have traditionally been considered essential for organoid culture, such as Wnt agonists, bone morphogenetic protein (BMP) signaling pathway inhibitors, and transforming growth factor β (TGF)-β inhibitors.

[0015] The organoid production medium of this embodiment is a medium obtained by adding prostaglandin, HGF, IL-6 family proteins, and EGF family proteins to a basal medium. Any serum-free basal cell culture medium can be used as the basal medium. Examples include defined synthetic media buffered to a pH of 7.2 to 7.6 with a carbonate buffer. More specifically, examples include Advanced Dulbecco's Modified Eagle's Medium / Ham's F-12 Mixed Medium (DMEM / F12), RPMI 1640 medium, and Advanced RPMI medium.

[0016] (Prostaglandin) Prostaglandins are a group of compounds having a prostanoic acid skeleton and are known to have various physiological activities. In the organoid production medium of this embodiment, prostaglandin E2 (CAS number: 363-24-6) is preferred as the prostaglandin. The concentration of prostaglandin E2 contained in the organoid production medium may be, for example, 0.1 to 10 μM, for example, 1 to 5 μM.

[0017] (HGF) HGF is a growth factor that activates the Met receptor, and the activated Met receptor activates the HGF-Met signaling pathway. An HGF mimetic having activity similar to that of HGF may be used as HGF. The concentration of HGF or HGF mimetic contained in the organoid production medium may be, for example, 10 pM to 10 nM, or may be, for example, 100 pM to 5 nM.

[0018] (IL-6 Family Proteins) IL-6 family proteins include those containing gp130 as their receptor, such as IL-6, IL-11, IL-27, IL-35, IL-39, oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotropin-1 (CT-1), ciliary neurotrophic factor (CNTF), and mimetics thereof. These may be used alone or in combination of two or more. Of these, oncostatin M is particularly preferred. The concentration of oncostatin M contained in the organoid production medium may be, for example, 1 ng / mL to 10 μg / mL, for example, 5 ng / mL to 1 μg / mL, or for example, 10 to 100 ng / mL.

[0019] (EGF Family Proteins) Examples of EGF family proteins include EGF, TGF-α, amphiregulin, heparin-binding EGF-like growth factor (HB-EGF), epiregulin, neuregulin 1, neuregulin 2, neuregulin 3, neuregulin 4, and mimetics thereof. These may be used alone or in combination of two or more. Of these, epiregulin and neuregulin 1 are particularly suitable. The concentration of epiregulin contained in the organoid production medium may be, for example, 1 ng / mL to 10 μg / mL, for example, 5 ng / mL to 1 μg / mL, for example, 10 ng / mL to 1 μg / mL. The concentration of neuregulin 1 contained in the organoid production medium may be, for example, 1 pM to 1 μM, for example, 100 pM to 100 nM, or for example, 1 nM to 10 nM. In the organoid production medium of this embodiment, the EGF family protein may be a factor other than EGF. In other words, the organoid production medium of this embodiment may be substantially free of EGF.

[0020] (Interferon) The organoid production medium of this embodiment may further contain interferon. Interferon (IFN)-γ is preferred as the interferon. Although IFN-γ is not essential, adding IFN-γ to the medium tends to further promote the growth of organoids. The concentration of IFN-γ contained in the organoid production medium may be, for example, 1 pg / mL to 10 ng / mL, for example, 5 pg / mL to 1 ng / mL, or for example, 10 pg / mL to 100 pg / mL.

[0021] (Hippo signaling pathway inhibitor) The organoid production medium of this embodiment may further contain an inhibitor of the Hippo signaling pathway. It is believed that an extracellular matrix such as Matrigel (registered trademark) is necessary to culture organoids. In contrast, the inventors have previously demonstrated that adding an inhibitor of the Hippo signaling pathway to an organoid production medium allows organoids to be cultured even without an extracellular matrix.

[0022] Therefore, organoids can be cultured in the absence of extracellular matrix using an organoid production medium further containing an inhibitor of the Hippo signaling pathway. Conventionally, it has been impossible to grow organoids in the absence of extracellular matrix. The organoid production medium of this embodiment makes it possible to grow organoids without using any animal-derived products, such as Matrigel (registered trademark). Here, animal-derived products refer to substances of unidentified components derived from humans or non-human animals. It is expected that animal-derived product-free culture techniques will enable the application of organoids to regenerative medicine. Furthermore, from the standpoints of animal welfare and cost, this is more advantageous than conventional organoid culture techniques.

[0023] The Hippo signaling pathway is a signaling pathway known to be involved in cell proliferation, apoptosis, stem cell self-renewal, and the like, and is known to be an evolutionarily conserved pathway.

[0024] Figure 1 is a schematic diagram illustrating the Hippo signaling pathway. As shown in Figure 1, the transcription factor TEAD activates the transcription of genes involved in cell proliferation by binding to the coactivator YAP (Yes-associated protein), thereby promoting cell proliferation. YAP exists in phosphorylated and unphosphorylated forms, and unphosphorylated YAP translocates to the nucleus and acts as a transcriptional coactivator for TEAD. On the other hand, phosphorylated YAP binds to the cytoplasmic protein 14-3-3 and cannot translocate to the nucleus, so it cannot function as a coactivator. Therefore, phosphorylation regulation, which determines the nuclear translocation of YAP, is a very important event in cell proliferation.

[0025] Furthermore, as shown in Figure 1, large tumor suppressor kinase (LATS) is known to phosphorylate YAP, localizing it in the cytoplasm and thereby negatively regulating the involvement of YAP in cell proliferation.

[0026] The inhibitor of the Hippo signaling pathway can be any inhibitor that inhibits any step in the above-mentioned signaling pathway, without any particular limitation.

[0027] The inhibitor of the Hippo signaling pathway may be, for example, an inhibitor of MST1 kinase or MST2 kinase (MST1 / 2 kinase inhibitor), or an inhibitor of LATS1 kinase or LATS2 kinase (LATS1 / 2 kinase inhibitor).

[0028] The NCBI accession numbers for the amino acid sequences of human MST1 kinase are NP_001380510.1, NP_001380511.1, NP_001380512.1, NP_001380513.1, NP_001380514.1, NP_066278.3, etc. The NCBI accession numbers for the amino acid sequences of human MST2 kinase are NP_001243241.1, NP_001243242.1, NP_006272.2, etc.

[0029] The NCBI accession numbers for the amino acid sequences of human LATS1 kinase are NP_001257448.1, NP_001337268.1, NP_001337269.1, NP_001337321.1, NP_004681.1, etc. The NCBI accession numbers for the amino acid sequences of human LATS2 kinase are NP_055387.2, etc.

[0030] More specific examples of inhibitors of the Hippo signaling pathway include, for example, LATS1 kinase and LATS2 kinase inhibitors, such as TRUL1 (CAS number: 1424635-83-5), GA-017 (CAS number: 2351906-74-4), and TDI-011536 (CAS number: 2687970-96-1). These may be used alone or in combination of two or more. Among these, TRUL1 and TDI-011536 are particularly suitable. The concentration of TRUL1 contained in the organoid production medium may be, for example, 1 μM to 10 mM, for example, 5 μM to 1 mM, or for example, 10 to 100 μM. The concentration of TDI-011536 contained in the organoid production medium may be, for example, 1 μM to 10 mM, for example, 1 μM to 1 mM, or for example, 1 to 100 μM.

[0031] (Fibroblast Growth Factor (FGF)) The organoid production medium of this embodiment may further contain FGF. Examples of FGF include FGF-2, FGF-4, FGF-7, FGF-10, and mimetics thereof. These may be used alone or in combination of two or more. Of these, FGF-7 is particularly preferred.

[0032] The concentration of FGF contained in the differentiation medium may be, for example, 10 to 500 ng / mL, for example, 10 to 300 ng / mL, or for example, 10 ng / mL to 100 ng / mL.

[0033] (Forskolin) The organoid production medium of this embodiment may further contain forskolin. Adding forskolin to the medium tends to further promote the growth of organoids. The concentration of forskolin contained in the organoid production medium may be, for example, 100 nM to 100 μM, for example, 500 nM to 50 μM, or for example, 1 to 20 μM.

[0034] (Other Additives) The organoid production medium may further contain a Rho-kinase (ROCK) inhibitor. Examples of ROCK inhibitors include Y-27632 (CAS number: 129830-38-2), fasudil (HA1077) (CAS number: 103745-39-7), and H-1152 (CAS number: 871543-07-6). When using Y-27632 as a ROCK inhibitor, it is preferably added during the first two days of culturing the stem cells dispersed into single cells. The concentration of Y-27632 contained in the organoid production medium is preferably about 10 μM.

[0035] The organoid culture medium may further contain at least one amino acid. 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 combinations thereof. The concentration of L-glutamine contained in the organoid culture medium is 0.05 g / L to 1 g / L (usually 0.1 g / L to 0.75 g / L). The concentration of other amino acids contained in the organoid culture medium is 0.001 g / L to 1 g / L (usually 0.01 g / L to 0.15 g / L). The amino acids may be synthetic.

[0036] The organoid production medium may further contain at least one vitamin, such as 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), DL-α-tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

[0037] The culture medium for organoid preparation may further contain at least one inorganic salt.The inorganic salt is for helping to maintain the osmotic balance of cells and for helping to regulate membrane potential.Specific examples of inorganic salts include calcium, copper, iron, magnesium, potassium, sodium, and zinc salts.Salts are usually used in the form of chloride, phosphate, sulfate, nitrate, and bicarbonate.More specific salts include 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.

[0038] The organoid production medium may further contain at least one sugar that can serve as a carbon energy source. Examples of sugars include glucose, galactose, maltose, and fructose. Among these, glucose is preferred, and D-glucose (dextrose) is particularly preferred. The concentration of sugar contained in the organoid production medium is preferably 1 to 10 g / L.

[0039] The organoid culture medium may further contain at least one trace element, such as barium, bromium, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum, or ions thereof.

[0040] The organoid culture medium may further comprise at least one additional agent, such as a nutrient or growth factor that has been reported to improve stem cell culture, such as cholesterol, transferrin, albumin, insulin, progesterone, putrescine, selenite, etc.

[0041] (Niche Factors) The organoid culture medium of this embodiment may further contain niche factors that have traditionally been considered essential for organoid culture, such as Wnt agonists, bone morphogenetic protein (BMP) signaling pathway inhibitors, and transforming growth factor (TGF)-β inhibitors.

[0042] As described later in the Examples, the inventors have demonstrated that when organoids are cultured in a medium containing a conventional medium containing niche factors and further supplemented with prostaglandins, HGF, IL-6 family proteins, and EGF family proteins, cell proliferation is further promoted compared to when organoids are cultured in a conventional medium.

[0043] (Wnt Agonist) A Wnt agonist refers to a drug that activates T-cell factor (hereinafter also referred to as TCF) / lymphoid enhancer factor (hereinafter also referred to as LEF)-mediated transcription in cells. Therefore, Wnt agonists are not limited to Wnt family proteins, but also include Wnt agonists that bind to and activate Frizzled receptor family members, inhibitors of intracellular β-catenin degradation, and TCF / LEF activators. The Wnt agonist is preferably at least one selected from the group consisting of Wnt proteins, R-spondin, and GSK-3β inhibitors.

[0044] The Wnt agonist is more preferably a complex of a Wnt protein and its stabilizing substance, afamin, and even more preferably includes a complex of a Wnt protein and afamin, and R-spondin.

[0045] <<Wnt Protein>> The origin of the Wnt protein is not particularly limited, and Wnt proteins derived from various organisms can be used. Among them, mammal-derived Wnt proteins are preferred. Mammals will be described later. Examples of mammal-derived Wnt proteins include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. In the culture medium for producing organoids, multiple types of Wnt proteins may be used in combination.

[0046] Methods for preparing Wnt proteins include, for example, methods using Wnt protein-expressing cells. The origin of the Wnt protein-expressing cells (e.g., biological species, culture form) is not particularly limited, as long as they stably express Wnt proteins, and they may also be cells that transiently express Wnt proteins. Examples of Wnt protein-expressing cells include L cells (ATCC CRL-2647) that stably express mouse Wnt3a and L cells (ATCC CRL-2814) that stably express mouse Wnt5a. Furthermore, Wnt protein-expressing cells can be produced using known genetic recombination techniques. Specifically, Wnt protein-expressing cells can be produced by inserting DNA encoding a desired Wnt protein into a known expression vector and then introducing the resulting expression vector into an appropriate host cell. The nucleotide sequence of the gene encoding the desired Wnt protein can be obtained from known databases, such as GenBank.

[0047] The Wnt protein expressed by the Wnt protein-expressing cells may be a fragment of the Wnt protein or may contain an amino acid sequence other than that of the Wnt protein, as long as it has Wnt activity. The amino acid sequence other than that of the Wnt protein is not particularly limited, and examples thereof include the amino acid sequence of an affinity tag. Furthermore, the amino acid sequence of the Wnt protein does not need to be completely identical to an amino acid sequence obtainable from a publicly known database such as GenBank; as long as it has Wnt activity, it may be substantially the same as an amino acid sequence obtainable from a publicly known database.

[0048] Examples of amino acid sequences that are substantially identical to the amino acid sequences of Wnt proteins that can be obtained from publicly known databases such as GenBank include amino acid sequences in which one to several amino acids have been deleted, substituted, or added to the amino acid sequences that can be obtained from publicly known databases.

[0049] An amino acid sequence in which one to several amino acids have been deleted, substituted or added means that the number of amino acids that can be deleted, substituted or added (preferably 10 or less, more preferably 7 or less, and even more preferably 6 or less) has been deleted, substituted or added by, for example, a known method for producing mutant peptides such as site-directed mutagenesis.

[0050] Furthermore, examples of substantially identical amino acid sequences include amino acid sequences that have an identity of at least 80% or more, preferably at least 85% or more, more preferably at least 90% or more, even more preferably at least 92% or more, particularly preferably at least 95% or more, and most preferably at least 99% or more with an amino acid sequence that can be obtained from a publicly known database.

[0051] The concentration of the Wnt protein may be, for example, 50 ng / mL or more, for example, 100 ng / mL to 10 μg / mL, for example, 200 ng / mL to 1 μg / mL, or for example, 300 ng / mL to 1 μg / mL.

[0052] R-spondin Examples of R-spondin include R-spondin family proteins including R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4. R-spondin is a secreted protein known to be involved in the activation and regulation of the Wnt signaling pathway. Multiple types of R-spondin may be used in combination in the organoid production medium. Furthermore, as long as it has R-spondin activity, it may be a fragment of R-spondin or may contain an amino acid sequence other than the amino acid sequence of R-spondin.

[0053] <<GSK-3β Inhibitors>> Examples of GSK-3β inhibitors include CHIR-99021 (CAS No.: 252917-06-9), CHIR-98014 (CAS No.: 252935-94-7), lithium, Kenpaullone (CAS No.: 142273-20-9), 6-bromoindirubin-30-acetoxime, SB216763 (CAS No.: 280744-09-4), SB415286 (CAS No.: 264218-23-7), FRAT family members that inhibit the interaction between GSK-3 and axin, and FRAT-derived peptides.

[0054] Afamin refers to a glycoprotein belonging to the albumin family, and is known to be present in body fluids such as blood. Serum, which is typically added to culture media, contains afamin derived from the animal from which the serum was collected. Because serum contains impurities other than afamin, it is preferable to use afamin alone without serum.

[0055] The origin of afamin contained in the organoid production medium is not particularly limited, and afamin derived from various organisms can be used. Among these, afamin derived from mammals is preferable. Mammals are described below. The amino acid sequences of major mammalian afamins and the nucleotide sequences of the genes encoding them can be obtained from publicly known databases such as GenBank. For example, in GenBank, the amino acid sequence of human afamin is registered as AAA21612, and the nucleotide sequence of the gene encoding it is registered as L32140, while the amino acid sequence of bovine afamin is registered as DAA28569, and the nucleotide sequence of the gene encoding it is registered as GJ060968.

[0056] The afamin contained in the organoid-producing medium may be natural afamin contained in serum or the like, purified by a known method, or it may be recombinant afamin, which can be produced by appropriately using known genetic recombination techniques.

[0057] Recombinant afamin can be produced, for example, by inserting DNA encoding afamin into a known expression vector, introducing the resulting expression vector into an appropriate host cell to express the recombinant afamin, and purifying it using a known purification method. The recombinant afamin may be afamin to which an affinity tag has been added. The affinity tag to be added is not particularly limited, and can be appropriately selected from known affinity tags. The affinity tag is preferably an affinity tag that can be recognized by a specific antibody, and examples include a FLAG tag, a MYC tag, an HA tag, and a V5 tag.

[0058] The Wnt proteins described above are highly hydrophobic because specific serine residues are modified with fatty acids (palmitoleic acid), and therefore Wnt proteins are prone to aggregation or denaturation in aqueous solutions, making them very difficult to purify and store.

[0059] Meanwhile, it has been reported that modification of this specific serine residue with a fatty acid is essential for the physiological activity of Wnt proteins and is involved in binding to members of the Frizzled receptor family.

[0060] It is also known that in aqueous solution, Wnt proteins bind one-to-one with afamin to form a complex, which is solubilized while maintaining high physiological activity. Wnt protein-afamin complexes can be produced by culturing cells that express both Wnt proteins and afamin, or by co-culturing Wnt protein-expressing cells and afamin-expressing cells.

[0061] The concentration of afamin contained in the organoid culture medium is not particularly limited, but may be, for example, 50 ng / mL to 10 μg / mL, or may be, for example, 100 ng / mL to 1 μg / mL or less, or may be, for example, 300 ng / mL to 1 μg / mL.

[0062] (BMP Inhibitors) BMP binds as a dimeric ligand to a receptor complex consisting of two different receptor serine / threonine kinases, type I and type II receptors. The type II receptor phosphorylates the type I receptor, resulting in activation of the receptor kinase. The type I receptor then phosphorylates specific receptor substrates (SMADs), resulting in transcriptional activity via a signal transduction pathway. In general, BMP inhibitors are, for example, drugs that bind to BMP molecules to form complexes that neutralize BMP activity, such as drugs that block or inhibit the binding of BMP molecules to BMP receptors. BMP inhibitors are also, for example, drugs that bind to BMP receptors and block or inhibit the binding of BMP molecules to the receptor, acting as antagonists or inverse agonists.

[0063] The BMP inhibitor preferably has an inhibitory activity of 50% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more, compared to the BMP activity level in the absence of the inhibitor.

[0064] The BMP inhibitor is preferably a natural BMP binding protein, and examples thereof include noggin, gremlin, chordin, chordin-like proteins having a chordin domain; follistatin, follistatin-related proteins having a follistatin domain; DAN, DAN-like proteins having a DAN cysteine-knot domain; sclerostin (SOST), decorin, α-2 macroglobulin, and mimetics thereof.

[0065] Among the BMP inhibitors contained in the organoid culture medium, chordin-like protein or DAN-like protein is preferred, with chordin-like protein being more preferred. As the chordin-like protein, noggin is preferred. Chordin-like protein and DAN-like protein are diffusible proteins that bind to BMP molecules with varying affinities and can inhibit the BMP molecules from approaching signaling receptors.

[0066] The concentration of the BMP inhibitor contained in the organoid culture medium may be, for example, 10 ng / mL to 100 ng / mL, for example, 20 to 100 ng / mL, or for example, 50 to 100 ng / mL.

[0067] (TGF-β Inhibitors) TGF-β is a type of growth factor that is produced by almost all cells, including those in the kidney, bone marrow, and platelets. There are five subtypes of TGF-β (β1 to β5). TGF-β is known to promote the proliferation of osteoblasts and the synthesis and proliferation of connective tissues such as collagen, while suppressing the proliferation of epithelial cells and osteoclasts. In general, TGF-β inhibitors are, for example, drugs that block or inhibit the binding of TGF-β to TGF-β receptors, and bind to TGF-β to form a complex that neutralizes TGF-β activity. TGF-β inhibitors are, for example, drugs that bind to TGF-β receptors and block or inhibit the binding of TGF-β to the receptor, and act as antagonists or inverse agonists.

[0068] Examples of TGF-β inhibitors include A-83-01 (CAS number: 909910-43-6), ALK5 inhibitor I (3-(pyridin-2-yl)-4-(4-quinonyl)-1H-pyrazole), LDN193189 (CAS No.: 1062368-24-4), SB-431542 (CAS No.: 301836-41-9), SB-505124 (CAS No.: 694433-59-5), SD-208 (CAS No.: 627536-09-8), SB-525334 (CAS No.: 356559-20-1), LY364947 (CAS No.: 396129-53-6), LY2157299 (CAS No.: 700874-72-2), TGF-β RI Kinase Inhibitor II 616452 (CAS No.: 446859-33-2), TGF-β RI Kinase Inhibitor III 616453 (CAS No.: 356559-13-2), TGF-β RI Kinase Inhibitor IX 616463 (4-((4-((2,6-dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide), TGF-β RI Kinase Inhibitor VII 616458 (CAS No.: 666729-57-3), TGF-β RI Kinase Inhibitor VIII 616459 (CAS number: 356559-20-1), AP12009 (TGF-β2 antisense compound "Trabedersen"), Belagenpumatucel-L (TGF-β2 antisense gene-modified allogeneic tumor cell vaccine), CAT-152 (Glaucoma-lerdelimumab (anti-TGF-β-2 monoclonal antibody)), CAT-192 (Metelimumab (human IgG4 monoclonal antibody that neutralizes TGFβ1)), GC-1008 (anti-TGF-β monoclonal antibody), and the like. Of these, A-83-01 is preferred as a TGF-β inhibitor.

[0069] The concentration of the TGF-β inhibitor contained in the organoid culture medium may be, for example, 100 nM to 10 μM, for example, 500 nM to 5 μM, or for example, 500 nM to 2 μM.

[0070] (EGF) The organoid production medium of this embodiment may contain EGF as a niche factor. The concentration of EGF contained in the organoid production medium may be, for example, 5 ng / mL to 1 μg / mL, for example, 10 ng / mL to 1 μg / mL, or for example, 50 to 500 ng / mL.

[0071] (Organoid) The organoids that can be cultured in the organoid producing medium of this embodiment are not particularly limited, and include organoids derived from epithelial cells, epithelial stem cells, interstitial cells, mesenchymal stem cells, cancer cells, and cancer stem cells. More specifically, they include organoids derived from the digestive tract (esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon)), liver, pancreas (pancreatic islets), salivary gland, mammary gland, biliary tract, lung, respiratory tract, hair head, skin, cartilage, and bone marrow interstitial cells.

[0072] Organoid can be the organoid of human origin, or can be the organoid of non-human animal origin.Non-human animal can include mammals, for example, rodents such as mouse, rat, hamster, guinea pig, etc.; ungulates such as pig, cow, goat, horse, sheep, etc.; carnivores such as dog, cat, etc.; primates such as rhesus monkey, cynomolgus monkey, marmoset, orangutan, chimpanzee, etc.

[0073] [Organoid Culture Kit] In one embodiment, the present invention provides an organoid culture kit comprising prostaglandin E2, HGF, an IL-6 family protein, and an EGF family protein.

[0074] The factors contained in the kit of this embodiment can be added to the basal medium to prepare the above-described medium for producing organoids.

[0075] The kit of this embodiment may further include a basal medium, which is the same as that described above.

[0076] The kit of this embodiment may further contain IFN-γ. Although IFN-γ is not essential, adding IFN-γ to the organoid production medium tends to further promote the growth of organoids. The details of IFN-γ are the same as those described above.

[0077] The kit of this embodiment may further contain an inhibitor of the Hippo signaling pathway. Usually, an extracellular matrix such as Matrigel (registered trademark) is required to culture organoids. In contrast, adding an inhibitor of the Hippo signaling pathway to the organoid production medium makes it possible to culture organoids without an extracellular matrix. The inhibitor of the Hippo signaling pathway is the same as that described above.

[0078] The kit of this embodiment may further contain factors such as FGF and forskolin depending on the organoid to be cultured. The FGF and forskolin are the same as those described above.

[0079] The kit of this embodiment may further include a niche factor. Examples of niche factors include a Wnt agonist, a BMP signaling pathway inhibitor, and a TGF-β inhibitor. The Wnt agonist, the BMP signaling pathway inhibitor, and the TGF-β inhibitor are the same as those described above. The kit of this embodiment may also include EGF as a niche factor. The EGF is the same as those described above.

[0080] The kit of this embodiment may further contain other additives as described above. In the kit of this embodiment, the factors or additives may be contained in separate containers, or two or more of them may be mixed and contained in one or more containers.

[0081] [Method for producing organoids] In one embodiment, the present invention provides a method for producing organoids, comprising the step of culturing cells in a medium containing prostaglandin E2, HGF, an IL-6 family protein, and an EGF family protein, thereby forming organoids from the cells, wherein the cells are selected from the group consisting of epithelial cells, epithelial stem cells, stromal cells, mesenchymal stem cells, cancer cells, and cancer stem cells. The prostaglandin E2, HGF, IL-6 family protein, and EGF family protein are the same as those described above.

[0082] The method of the present embodiment allows the establishment of organoids that have not been established in the past, as well as organoids that have not been established in the past, such as chondrocyte-derived organoids, stromal cell-derived organoids, and skin epithelial cell-derived organoids.

[0083] In the production method of this embodiment, the cells are not particularly limited, and examples thereof include epithelial cells, epithelial stem cells, stromal cells, mesenchymal stem cells, cancer cells, cancer stem cells, etc. More specifically, examples thereof include cells derived from the digestive tract (esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon)), liver, pancreas (pancreatic islets), salivary glands, mammary glands, biliary tract, lung, airway, hair head, skin, cartilage, bone marrow stroma, etc. Organoids can be produced from these cells by the production method of this embodiment. Organoids can also be established from single cells by the production method of this embodiment.

[0084] In the production method of this embodiment, the medium may further contain IFN-γ. Although IFN-γ is not essential, adding IFN-γ to the medium tends to further promote the growth of organoids. The IFN-γ is the same as that described above.

[0085] In the manufacturing method of this embodiment, the culture medium may further contain an inhibitor of the Hippo signaling pathway. Usually, in order to culture organoids, an extracellular matrix such as Matrigel (registered trademark) is required. In contrast, if an inhibitor of the Hippo signaling pathway is added to the culture medium, it becomes possible to culture organoids without an extracellular matrix. The inhibitor of the Hippo signaling pathway is the same as that described above.

[0086] In the method of the present embodiment, the culture medium may be a serum-free medium. By establishing organoids in the absence of extracellular matrix and serum, organoids can be obtained that are free of animal-derived products from the time of establishment.

[0087] The kit of this embodiment may further contain factors such as FGF and forskolin depending on the organoid to be cultured. The FGF and forskolin are the same as those described above.

[0088] In the production method of this embodiment, the medium may further contain a niche factor. Examples of niche factors include Wnt agonists, BMP signaling pathway inhibitors, and TGF-β inhibitors. The Wnt agonists, BMP signaling pathway inhibitors, and TGF-β inhibitors are the same as those described above. In the production method of this embodiment, the medium may contain EGF as a niche factor. The EGF is the same as that described above.

[0089] In the production method of this embodiment, the medium may further contain other additives as described above.

[0090] (Extracellular matrix) When organoid is produced or cultured in the presence of extracellular matrix, examples of the extracellular matrix include Matrigel (registered trademark), collagen, fibronectin, proteoglycan, laminin, etc. Growing organoid in the absence of extracellular matrix means that no extracellular matrix is ​​added from the outside to the culture medium of organoid, and it is acceptable that a trace amount of extracellular matrix produced by organoid itself is mixed into the culture medium, or that a trace amount of extracellular matrix is ​​mixed into the culture medium unintentionally.Here, a trace amount may be at the detection limit.

[0091] [Organoid] In one embodiment, the present invention provides an organoid produced by the above-described production method.

[0092] The organoid of this embodiment includes not only the organoid that has been established in the past, but also the organoid that has not been established in the past.The organoid that has not been established in the past includes, for example, stromal cell-derived organoid, skin epithelial cell-derived organoid, etc.That is, in one embodiment, the present invention provides stromal cell-derived organoid.In addition, in one embodiment, the present invention provides skin epithelial cell-derived organoid.

[0093] As will be described later in the Examples, organoids obtained by culturing cells in a medium containing prostaglandin E2, HGF, an IL-6 family protein, and an EGF family protein have a different morphology from conventional organoids.

[0094] Specifically, for example, small intestinal organoids established or cultured by conventional methods exhibit a cystic morphology covered with a single cell layer with budding, whereas small intestinal organoids established or cultured in a medium containing prostaglandin E2, HGF, an IL-6 family protein, and an EGF family protein exhibit the morphology of a stratified cell mass.

[0095] For example, liver organoids established or cultured by conventional methods exhibit a cystic morphology covered with a monolayer of cells, whereas liver organoids established or cultured in a medium containing prostaglandin E2, HGF, an IL-6 family protein, and an EGF family protein exhibit the morphology of stratified cell masses.

[0096] As described below in the Examples, the inventors have demonstrated that organoids established or cultured in a medium containing prostaglandin E2, HGF, an IL-6 family protein, and an EGF family protein, and morphologically different from conventional organoids, revert to the morphology of conventional organoids when cultured in a conventional medium in an extracellular matrix.

[0097] The organoid of this embodiment is considered to have differences in gene expression patterns and the like with conventional organoids.However, it is unclear whether it is possible to identify the differences in gene expression patterns and clearly distinguish between conventional organoids and the organoid of this embodiment, and this is not practical.Therefore, it is considered practical to define the organoid of this embodiment by manufacturing method.

[0098] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0099] Experimental Example 1: Culture of human colon-derived organoids. Human colon-derived organoids were cultured under various conditions and their dynamics were observed. Human colon-derived organoids were established from tissues derived from patients with gastrointestinal tumors who provided informed consent, in accordance with an ethical research plan approved by the Keio University School of Medicine Ethics Committee.

[0100] Colon organoids were dissociated into single cells using TrypLE Express (Thermo Fisher Scientific) to obtain a cell suspension. When cultured in extracellular matrix, 1 × 10 ５ Cells were seeded at 1 x 10 cells / well together with 25 μL of Matrigel® onto a 48-well plate. After the Matrigel® gelled, 100 μL of a medium of the desired composition was added to each well and cultured at 37°C. When culturing in the absence of extracellular matrix, 1 x 10 cells / well were added. ５ Cells were suspended at a concentration of 1 / well in 100 μL / well of a medium of the desired composition, seeded onto a 48-well plate, and cultured at 37°C.

[0101] 2 and 3 are representative microscopic images of colon organoids after 7 days of culture. In Figures 2 and 3, "conventional organoid" indicates the results of culturing in an extracellular matrix, and "MfLO" indicates the results of culturing in the absence of an extracellular matrix.

[0102] In Figure 2, "Full" indicates that the medium contained EGF, Noggin, Wnt, R-spondin, and A-83-01 (a TGF-β inhibitor) (hereinafter, sometimes referred to as "niche factor"). A complex of Wnt3a and afamin was used as the Wnt. The EGF concentration in the medium was 50 ng / mL, the Noggin concentration was 4%, the Wnt3a concentration was 10%, the R-spondin concentration was 4%, and the A-83-01 concentration was 500 nM. "Control" indicates the result of culturing in a normal medium containing niche factors, "TRULI" indicates the result of culturing in a medium to which 20 μM TRULI (CAS number: 1424635-83-5) was further added to the normal medium containing niche factors, and "PHOENIX" indicates the result of culturing in a medium to which 2.5 μM prostaglandin E2, 0.5 nM hepatocyte growth factor (HGF), 20 ng / mL oncostatin M, 100 ng / mL epiregulin, 5 nM neuregulin 1, 30 pg / mL interferon (IFN)-γ, and ...0 μM TRULI were further added to the normal medium containing niche factors. The results are shown as being obtained by culturing in a medium supplemented with 20 μM of CI 20489 and 10 μM of TDI-011536 (CAS number: 2687970-96-1) (hereinafter sometimes referred to as "PHOENIX").

[0103] In Figure 3, "-ENWRA" indicates that the medium does not contain the above-mentioned niche factors, "Control" indicates the result of culturing in a medium that does not contain niche factors, "TRULI" indicates the result of culturing in a medium that does not contain niche factors but to which 20 μM TRULI was added, and "PHOENIX" indicates the result of culturing in a medium that does not contain niche factors but to which the above-mentioned PHOENIX was added.

[0104] As a result, as shown in Figure 2, in Matrigel (registered trademark) culture, it was revealed that the addition of LATS inhibitor TRULl to the medium enhanced organoid proliferation compared to the control group (Control). Furthermore, a further proliferation-promoting effect was observed in the group in which PHOENIX was further added to a normal medium containing niche factors. Furthermore, when cultured in the absence of extracellular matrix, colon organoids could not survive in a normal medium containing niche factors, but by adding LATS inhibitor TRULl to the medium, it was revealed that organoid proliferation was enhanced compared to the control group (Control). Furthermore, a further proliferation-promoting effect was observed in the group in which PHOENIX was further added to a normal medium containing niche factors.

[0105] 3, in a medium containing no niche factors, when cultured in Matrigel (registered trademark), in the absence of extracellular matrix, colonic organoids could not survive. Also, in a medium containing no niche factors and the LATS inhibitor TRUL1, when cultured in Matrigel (registered trademark), in the absence of extracellular matrix, colonic organoids could not survive. On the other hand, in a medium containing no niche factors and the above-mentioned PHOENIX, when cultured in Matrigel (registered trademark), in the absence of extracellular matrix, colonic organoids could survive and proliferate.

[0106] FIG. 4 is a graph showing the results of measuring the growth of colonic organoids cultured under the same culture conditions as in FIGS. 2 and 3 using a commercially available kit (product name "Celltiter-Glo", Promega). In FIG. 4, "conventional" indicates the results of culturing in an extracellular matrix, and "Matrigel free" indicates the results of culturing in the absence of an extracellular matrix. In addition, "Full" indicates that the medium contains the above-mentioned niche factors, "-ENWRA" indicates that the medium does not contain the above-mentioned niche factors, "TRULI" indicates the results of culturing in a medium supplemented with 20 μM TRULI, "PHOENIX" indicates the results of culturing in a medium supplemented with the above-mentioned PHOENIX, and "Control" indicates the results of culturing in a control medium containing neither TRULI nor PHOENIX.

[0107] As a result, it was revealed that, whether cultured in Matrigel (registered trademark) or in the absence of extracellular matrix, culturing colonic organoids in a medium containing no niche factors and to which PHOENIX was added resulted in growth equivalent to that observed under conventional colonic organoid culture conditions, in which the organoids were cultured in a medium containing niche factors in an extracellular matrix.

[0108] [Experimental Example 2] (Study of organoid morphology) Organoids were cultured under conventional organoid culture conditions, in which they were cultured in a medium containing the above-mentioned niche factors in an extracellular matrix, and under culture conditions in which organoids were cultured in a medium containing the above-mentioned PHOENIX in the absence of an extracellular matrix, and the morphologies were compared.

[0109] Human small intestinal and liver organoids were cultured. These organoids were established from tissues derived from patients who provided informed consent, in accordance with an ethical research plan approved by the Keio University School of Medicine Ethics Committee.

[0110] Figure 5 shows representative microscopic images of the morphology of each organoid dissociated into single cells after 7 days of culture. The scale bar is 100 μm. In Figure 5, the upper row shows images of small intestinal organoids, and the lower row shows images of liver organoids. "Normal culture" indicates the results of culturing organoids under conventional culture conditions in an extracellular matrix medium containing the above-mentioned niche factors. "Matrigel-free ENWRA-free PHOENIX" indicates the results of culturing organoids under culture conditions in the absence of an extracellular matrix, in a medium containing the above-mentioned PHOENIX, and without the above-mentioned niche factors.

[0111] As a result, the small intestinal organoids cultured under normal conditions showed a cystic morphology covered with a single cell layer with budding.In contrast, the small intestinal organoids cultured in a medium containing the above-mentioned PHOENIX and not containing the above-mentioned niche factors in the absence of extracellular matrix showed the morphology of stratified cell masses.

[0112] In addition, the liver organoids cultured under normal conditions showed a cystic morphology covered with a single cell layer.In contrast, in the absence of extracellular matrix, the liver organoids cultured in the medium containing the above-mentioned PHOENIX and not containing the above-mentioned niche factors showed the morphology of stratified cell masses.

[0113] Next, small intestinal organoids and liver organoids cultured in a medium containing the above-mentioned PHOENIX, without the above-mentioned niche factors, in the absence of extracellular matrix, were cultured again under normal culture conditions for 7 days, and their morphology was observed.

[0114] Figure 6 is a representative microscopic image showing the morphology of each organoid. The scale bar is 100 μm. In Figure 6, the upper row is an image of a small intestinal organoid, and the lower row is an image of a liver organoid. In addition, "Matrigel-free ENWRA-free PHOENIX" indicates the result of culturing organoids under culture conditions in the absence of extracellular matrix, without the above-mentioned niche factors, and in a medium containing the above-mentioned PHOENIX. "Normal culture" indicates the result of culturing organoids under conventional culture conditions, in which they are cultured in an extracellular matrix and in a medium containing the above-mentioned niche factors.

[0115] As a result, it was revealed that for both small intestinal and liver organoids, the altered morphology of the organoids returned to its original form when the culture conditions were returned to normal.

[0116] [Experimental Example 3] (Establishment of organoids derived from mouse bone marrow stromal cells) An attempt was made to establish organoids derived from mouse bone marrow stromal cells. The establishment of organoids derived from mouse bone marrow stromal cells has not been reported previously.

[0117] Mouse bone marrow stromal cells were cultured in a medium containing the above-mentioned niche factors and PHOENIX in an extracellular matrix, and as a result, organoids derived from mouse bone marrow stromal cells were established.

[0118] Figure 7 shows representative microscopic images of established mouse bone marrow stromal cell-derived organoids. The scale bar in the upper image is 500 μm, and the scale bar in the lower image is 50 μm.

[0119] [Experimental Example 4] (Establishment of organoids derived from human skin epithelial cells) An attempt was made to establish organoids derived from skin epithelial cells from human hair roots. The establishment of organoids derived from skin epithelial cells has not been reported previously.

[0120] In addition to the niche factors mentioned above, human hair root-derived cells were cultured in a medium containing the following: extracellular matrix, prostaglandin E2 2.5 μM, hepatocyte growth factor (HGF) 0.5 nM, oncostatin M 20 ng / mL, epiregulin 100 ng / mL, neuregulin 1 5 nM, interferon (IFN)-γ 30 pg / mL, fibroblast growth factor (FGF)-7 50 ng / mL, and forskolin 10 μM. Hereinafter, prostaglandin E2, hepatocyte growth factor (HGF), oncostatin M, epiregulin, neuregulin 1, and interferon (IFN)-γ may be referred to as "PHOENI." As a result, human skin epithelial cell-derived organoids were established.

[0121] Figure 8 shows representative microscopic images of the process of organoid establishment and the established organoids. The scale bar in the image at the bottom right of Figure 8 is 100 μm. In Figure 8, "D0.5", "D2", "D3", "D7", "D13" and "D15" indicate images taken 0.5 days, 2 days, 3 days, 7 days, 13 days and 15 days after the start of culture of human hair root-derived cells, respectively. In addition, "Passage 2 D3" indicates an image taken 3 days after dissociating cells into single cells 15 days after the start of culture of human hair root-derived cells and starting culture again under the same conditions. The image at the bottom right of Figure 8 is an enlarged image of a portion of the image at the bottom left of Figure 8.

[0122] [Experimental Example 5] (Induction of Differentiation of Human Skin Epithelial Cell-Derived Organoids) An attempt was made to induce differentiation of the human skin epithelial cell-derived organoids established in Experimental Example 4. Figure 9 is a schematic diagram showing the experimental schedule for differentiation induction.

[0123] First, the human skin epithelial cell-derived organoids established in Experimental Example 4 were seeded into cell culture inserts and cultured for 6 days in a medium containing, in addition to the above-mentioned niche factors, prostaglandin E2 2.5 μM, hepatocyte growth factor (HGF) 0.5 nM, oncostatin M 20 ng / mL, epiregulin 100 ng / mL, neuregulin 1 5 nM, interferon (IFN)-γ 30 pg / mL, fibroblast growth factor (FGF)-7 50 ng / mL, and forskolin 10 μM (hereinafter, sometimes referred to as "Full + PHOENI + FGF7 + Forskolin" medium).

[0124] Subsequently, air-liquid interface culture was performed until day 18, and human skin epithelial cell-derived organoids were induced to differentiate. As the culture medium for air-liquid interface culture, (1) "Full + PHOENI + FGF7 + Forskolin" medium, (2) "Full + FGF7 + Forskolin" medium obtained by removing PHOENI from the medium (1), (3) "-ENWRA + PHOENI" medium obtained by removing niche factors, FGF7, and Forskolin from the medium (1), and (4) "-ENWRA" medium obtained by removing PHOENI from the medium (3) were used. Subsequently, the organoids were fixed and thin sections were prepared, stained with hematocrit and eosin, and observed under a microscope.

[0125] Figure 10 shows microscopic images of samples induced to differentiate in each medium. Figure 11 shows an enlarged image of the boxed area in Figure 10. The results showed that cells differentiated when cultured in a medium that did not contain niche factors, and exhibited a morphology very similar to that of skin.

[0126] [Experimental Example 6] (Establishment of human small intestinal villi-derived epithelial cell organoids) We attempted to establish organoids from human small intestinal villi-derived epithelial cells. Because small intestinal villi do not contain intestinal epithelial stem cells, the establishment of small intestinal villi-derived epithelial cell organoids is difficult and has not been reported previously.

[0127] Human villi-derived intestinal epithelial cells without intestinal epithelial stem cells were cultured in a medium containing the above-mentioned niche factors and PHOENIX in an extracellular matrix. As a result, human small intestinal villi-derived epithelial cell organoids were established. Human small intestinal villi-derived epithelial cells were established from tissues derived from patients with gastrointestinal tumors who provided informed consent, in accordance with an ethical research plan approved by the Keio University School of Medicine Ethics Committee.

[0128] The left image of Figure 12 is a microscopic image of human small intestinal villi-derived epithelial cell organoids 10 days after the start of culture, and the right image is a magnified image of one organoid in the left image of Figure 12.

[0129] [Example 7] (Culturing organoids in a medium without a ROCK inhibitor) A ROCK inhibitor is essential for the survival of organoids after cell dispersion, and it is essential to add it when subculturing human small intestinal epithelial cell organoids and human large intestinal epithelial cell organoids. It is known that when organoids are dispersed and cultured in a medium without a ROCK inhibitor, the efficiency of organoid formation is significantly reduced. In this experimental example, human small intestinal epithelial cell organoids and human large intestinal epithelial cell organoids were dispersed in an extracellular matrix and cultured in PHOENIX medium without a ROCK inhibitor.

[0130] Figure 13 shows microscopic images of the culture results. As a result, human small intestinal epithelial cell organoids and human colonic epithelial cell organoids cultured in extracellular matrix formed organoids only in media containing a ROCK inhibitor (compare Figure 13, left and center). Organoid formation was significantly inhibited in media without a ROCK inhibitor (Figure 13, center). In contrast, when PHOENIX was added to media without a ROCK inhibitor, human small intestinal epithelial cell organoids and human colonic epithelial cell organoids were formed, even without a ROCK inhibitor (Figure 13, right).

[0131] The present invention provides a novel organoid culture and production technique. Organoids produced using the production method of the present invention exhibit different shapes and properties from conventional organoids. When returned to normal culture conditions, they regain the shape of conventional 3D organoids, which is believed to represent one aspect of the organoid formation process. While single-cell RNA analysis has revealed the characteristics of the regenerative state after tissue injury, no culture method has been able to mimic this, hindering drug development focused on in vivo regeneration and tissue repair. Therefore, the culture method and cultured organoids of the present invention, which mimic part of the tissue regeneration process, can be used as new research tools, such as a culture technique for human epithelial cells that mimic tissue regeneration from injury or inflammatory diseases, and can also be used to assist regeneration, demonstrating new industrial potential.

Claims

1. A medium for producing organoids, comprising prostaglandins, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins, and epidermal growth factor (EGF) family proteins.

2. The organoid production medium of claim 1, further comprising interferon.

3. The organoid production medium of claim 2, wherein the interferon is interferon (IFN)-γ.

4. A culture medium for producing organoids as described in claim 1 or 2, further comprising an inhibitor of the Hippo signaling pathway.

5. A culture medium for producing organoids described in claim 1 or 2, further comprising a Wnt agonist, a bone morphogenetic protein (BMP) signaling pathway inhibitor, and a transforming growth factor (TGF)-β inhibitor.

6. A culture medium for producing organoids described in claim 1 or 2, which is substantially free of Wnt agonists, BMP signaling pathway inhibitors, and TGF-β inhibitors.

7. A kit for organoid culture comprising prostaglandin, HGF, IL-6 family protein, and EGF family protein.

8. A method for producing organoids, comprising the step of culturing cells in a medium containing prostaglandin, HGF, an IL-6 family protein, and an EGF family protein, thereby forming organoids from the cells, wherein the cells are cells selected from the group consisting of epithelial cells, epithelial stem cells, stromal cells, mesenchymal stem cells, cancer cells, and cancer stem cells.

9. The method of claim 8, wherein the medium further contains IFN-γ.

10. The production method according to claim 8 or 9, wherein the medium is substantially free of a ROCK inhibitor.

11. The production method according to claim 8 or 9, wherein the medium further contains a Wnt agonist, a BMP signaling pathway inhibitor, and a TGF-β inhibitor.

12. The production method according to claim 8 or 9, wherein the medium is substantially free of Wnt agonists, BMP signaling pathway inhibitors, and TGF-β inhibitors.

13. The method of claim 12, wherein the medium is substantially free of a ROCK inhibitor.

14. The method of claim 8 or 9, wherein no extracellular matrix is ​​used to culture the cells.

15. The method of claim 8 or 9, wherein the medium is a serum-free medium.

16. The method of claim 8 or 9, wherein the cells are dissociated into single cells.

17. An organoid produced by the production method described in claim 8 or 9.

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