Culture medium for organoid manufacturing and uses thereof

Abstract

The present application relates to a medium for organoid production containing a prostaglandin, a hepatocyte growth factor (HGF), an interleukin (IL)-6 family protein, and an epidermal growth factor (EGF) family protein, a kit for organoid culture, a method for producing an organoid, and an organoid.

Description

Technical Field

[0001] This invention relates to culture media for organoid manufacturing and their uses. More specifically, this invention relates to culture media for organoid manufacturing, reagent kits for organoid culture, methods for manufacturing organoids, and organoids themselves. This application claims priority based on Japanese Patent Application No. 2024-059184, filed on April 1, 2024, the entire contents of which are incorporated herein by reference. Background Technology

[0002] Organoid culture technology has been developed and holds promise for applications in drug discovery and regenerative medicine. The inventors have pioneered an organoid technology for the permanent three-dimensional culture of tissue stem cells (see, for example, Non-Patent Literature 1, 2).

[0003] However, culturing organoids requires Matrigel. ® Extracellular matrix (e.g., see Patent Document 1, Non-Patent Document 3, etc.). Due to Matrigel ® It is an extract derived from tumors transplanted into mice, and therefore belongs to animal-derived products. Furthermore, due to the possibility of unknown viral contamination and high cost, Matrigel is used in organoid culture. ® This has become an obstacle to its medical application. Furthermore, from an animal welfare perspective, the use of Matrigel also poses a problem. Since the advent of organoid technology, researchers have been actively working to develop Matrigel. ® Alternatives (e.g., see Non-Patent Literature 4).

[0004] [Existing Technical Documents]

[0005] [Patent Documents]

[0006] Patent Document 1: Japanese Patent Application Publication No. 2016-198033

[0007] [Non-patent literature]

[0008] Non-patent document 1: Sato T, et al., Single Lgr5 stem cells build crypt-villusstructures in vitro without a mesenchymal niche, Nature, 459, 262-266, 2009.

[0009] Non-patent document 2: Sato T, et al., Long-term Expansion of EpithelialOrganoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett'sEpithelium, Gastroenterology, 141, 1762-1772, 2011.

[0010] Non-patent literature 3: S Rezakhani, et al., Extracellular matrix requirements for gastrointestinal organoid cultures, Biomaterials, 276, 121020, 2021.

[0011] Non-patent literature 4: Jeong Hyun Heo, et al., Engineering the Extracellular Matrix for Organoid Culture, Int J Stem Cells, 15(1), 60-69, 2022. Summary of the Invention

[0012] [The problem the invention aims to solve]

[0013] The purpose of this invention is to provide a novel organoid culture and manufacturing technology.

[0014] [Methods for solving the problem]

[0015] The present invention includes the following aspects.

[0016] [1] A culture medium for organoid manufacturing, which contains prostaglandins, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins and epidermal growth factor (EGF) family proteins.

[0017] [2] The culture medium for organoid manufacturing according to [1] further contains interferon.

[0018] [3] The culture medium for organoid manufacturing according to [2], wherein the interferon is interferon (IFN)-γ.

[0019] [4] The culture medium for organoid manufacturing according to any one of [1] to [3] further comprises an inhibitor of the Hippo signaling pathway.

[0020] [5] The culture medium for organoid manufacturing according to any one of [1] to [4] further comprises a Wnt agonist, a bone morphogenetic protein (BMP) signaling pathway inhibitor and a transforming growth factor (TGF)-β inhibitor.

[0021] [6] The culture medium for organoid manufacturing according to any one of [1] to [4] is substantially free of Wnt agonists, BMP signaling pathway inhibitors and TGF-β inhibitors.

[0022] [7] Organoid culture kit containing prostaglandins, HGF, IL-6 family proteins and EGF family proteins.

[0023] [8] A method for manufacturing an organoid, comprising culturing cells in a culture medium containing prostaglandins, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins and epidermal growth factor (EGF) family proteins, thereby forming an organoid from said cells, wherein said cells are selected from the following: epithelial cells, epithelial stem cells, mesenchymal cells, mesenchymal stem cells, cancer cells and cancer stem cells.

[0024] [9] According to the manufacturing method described in [8], the culture medium further comprises IFN-γ.

[0025]

[10] The manufacturing method according to [8] or [9], wherein the culture medium is substantially free of ROCK inhibitors.

[0026]

[11] The manufacturing method according to any one of [8] to

[10] , wherein the culture medium further comprises a Wnt agonist, a bone morphogenetic protein (BMP) signaling pathway inhibitor and a TGF-β inhibitor.

[0027]

[12] The manufacturing method according to any one of [8] to

[10] , wherein the culture medium is substantially free of Wnt agonists, bone morphogenetic protein (BMP) signaling pathway inhibitors and transforming growth factor (TGF)-β inhibitors.

[0028]

[13] The manufacturing method according to

[12] , wherein the culture medium is substantially free of ROCK inhibitors.

[0029]

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

[13] , wherein no extracellular matrix is ​​used in the culture of said cells.

[0030]

[15] The manufacturing method according to any one of [8] to

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

[0031]

[16] The manufacturing method according to any one of [8] to

[15] , wherein the cell is a cell that has been dissociated into a single cell.

[0032]

[17] Organoids, which are manufactured by the manufacturing method as described in any one of claims [8] to

[16] .

[0033] Alternatively, the present invention can be said to include the following aspects.

[0034] [P1] Organoid manufacturing culture medium containing prostaglandins, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins and epidermal growth factor (EGF) family proteins.

[0035] [P2] The culture medium for organoid manufacturing according to [P1] also contains interferon.

[0036] [P3] The culture medium for organoid manufacturing according to [P2], wherein the interferon is interferon (IFN)-γ.

[0037] [P4] The culture medium for organoid manufacturing according to any one of [P1] to [P3] further comprises an inhibitor of the Hippo signaling pathway.

[0038] [P5] The culture medium for organoid manufacturing according to any one of [P1] to [P4] further comprises a Wnt agonist, a bone morphogenetic protein (BMP) signaling pathway inhibitor, and a transforming growth factor (TGF)-β inhibitor.

[0039] [P6] The culture medium for organoid manufacturing according to any one of [P1] to [P4] is substantially free of Wnt agonists, bone morphogenetic protein (BMP) signaling pathway inhibitors and transforming growth factor (TGF)-β inhibitors.

[0040] [P7] Organoid culture kit containing prostaglandins, HGF, IL-6 family proteins and EGF family proteins.

[0041] [P8] A method for manufacturing organoids, comprising culturing cells in a culture medium containing prostaglandins, HGF, IL-6 family proteins and EGF family proteins, thereby forming organoids from said cells, wherein said cells are selected from the following: epithelial cells, epithelial stem cells, mesenchymal cells, cancer cells and cancer stem cells.

[0042] [P9] According to the manufacturing method described in [P8], the culture medium further comprises IFN-γ.

[0043] [P10] According to the manufacturing method described in [P8] or [P9], the culture medium further comprises a Wnt agonist, a bone morphogenetic protein (BMP) signaling pathway inhibitor, and a transforming growth factor (TGF)-β inhibitor.

[0044] [P11] The manufacturing method according to [P8] or [P9], wherein the culture medium is substantially free of Wnt agonists, bone morphogenetic protein (BMP) signaling pathway inhibitors and transforming growth factor (TGF)-β inhibitors.

[0045] [P12] The manufacturing method according to any one of [P8] to [P11], wherein no extracellular matrix is ​​used in the culture of said cells.

[0046] [P13] The manufacturing method according to any one of [P8] to [P12], wherein the culture medium is a serum-free culture medium.

[0047] [P14] The manufacturing method according to any one of [P8] to [P13], wherein the cell is a cell that has been dissociated into a single cell.

[0048] [P15] Organoids, which are manufactured by any of the manufacturing methods described in any of [P8] to [P14].

[0049] [The effects of the invention]

[0050] According to the present invention, a new organoid culture technology can be provided.

[0051] [Brief Description of the Attached Image]

[0052]

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[0065] [Organoid Manufacturing Culture Medium]

[0066] In one embodiment, the present invention provides a culture medium for organoid manufacturing, which comprises prostaglandins, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins, and epidermal growth factor (EGF) family proteins.

[0067] The organoid manufacturing culture medium of this embodiment can be used for the culture or establishment of organoids. The organoid manufacturing culture medium of this embodiment can be a serum-free culture medium. As described in the examples described later, the inventors clarify that, compared to culturing organoids using conventional culture media, culturing organoids using the culture medium of this embodiment can promote cell proliferation.

[0068] The inventors further explained that by using the culture medium of this embodiment, in addition to establishing organoids such as those of the digestive tract, lungs, pancreas (islets), liver, salivary glands, bile ducts, respiratory tract, hair follicles, and skin that could be established previously, organoids that could not be established previously could also be established. Examples of organoids that could not be established previously include organoids derived from mesenchymal cells and organoids derived from skin epithelial cells.

[0069] Furthermore, as described in the embodiments described later, organoids can be cultured even in the absence of microenvironmental factors (such as Wnt agonists, bone morphogenetic protein (BMP) signaling pathway inhibitors, and transforming growth factor (TGF)-β inhibitors) that are traditionally considered essential for organoid culture.

[0070] The organoid manufacturing culture medium in this embodiment is a medium obtained by adding prostaglandins, HGF, IL-6 family proteins, and EGF family proteins to a basal culture medium. As the basal culture medium, any serum-free cell culture basal medium can be used. Examples include specific synthetic media buffered to pH 7.2–7.6 with carbonate buffer. More specifically, examples include advanced Dalberco modified Eagle medium / HAM F-12 mixed medium (DMEM / F12), RPMI 1640 medium, and advanced RPMI medium.

[0071] [Prostaglandins]

[0072] Prostaglandins are a group of compounds with a prostaglandic acid skeleton, and they are known to have a variety of physiological activities. In the organoid manufacturing culture medium of this embodiment, the prostaglandin is preferably prostaglandin E2 (CAS No.: 363-24-6). The concentration of prostaglandin E2 contained in the organoid manufacturing culture medium may be, for example, 0.1 to 10 µM, or for example, 1 to 5 µM.

[0073] [HGF]

[0074] HGF is a growth factor that activates the Met receptor, and the activated Met receptor activates the HGF-Met signaling pathway. HGF mimics with the same activity as HGF can also be used as HGF. The concentration of HGF or HGF mimics in the organoid manufacturing culture medium can be, for example, 10 pM to 10 nM, or, for example, 100 pM to 5 nM.

[0075] [Interleukin (IL)-6 family proteins]

[0076] As proteins of the interleukin (IL)-6 family, examples include proteins whose receptors contain gp130, such as IL-6, IL-11, IL-27, IL-35, IL-39, oncostatin M (OSM), leukemia suppressor factor (LIF), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), and their mimics. These can be used individually or in combination. Oncostatin M is particularly suitable. The concentration of oncostatin M in the culture medium for organoid production can be, for example, 1 ng / mL to 10 µg / mL, 5 ng / mL to 1 µg / mL, or 10 to 100 ng / mL.

[0077] [Epidermal growth factor (EGF) family proteins]

[0078] As proteins in the epidermal growth factor (EGF) family, examples include EGF, TGF-α, amphiregulin, heparin-binding EGF-like growth factor (HB-EGF), epithelial regulatory protein, neuromodulatory 1, neuromodulatory 2, neuromodulatory 3, neuromodulatory 4, and mimics of these substances. These substances can be used alone or in combination. Epithelial regulatory protein and neuromodulatory 1 are particularly suitable. The concentration of epithelial regulatory protein in the organoid culture medium is, for example, 1 ng / mL to 10 µg / mL, 5 ng / mL to 1 µg / mL, or 10 ng / mL to 1 µg / mL. The concentration of neuromodulatory 1 in the organoid culture medium is, for example, 1 pM to 1 µM, 100 pM to 100 nM, or 1 nM to 10 nM. In the organoid manufacturing culture medium of this embodiment, the epidermal growth factor (EGF) family proteins may also be factors other than EGF. That is, the organoid manufacturing culture medium of this embodiment may also be substantially free of EGF.

[0079] Interferon

[0080] The organoid manufacturing culture medium of this embodiment may further contain interferon. Interferon (IFN)-γ is preferred as the interferon. Although IFN-γ is not essential, its addition to the culture medium often further promotes organoid proliferation. The concentration of IFN-γ in the organoid manufacturing culture medium may, for example, be 1 pg / mL to 10 ng / mL, 5 pg / mL to 1 ng / mL, or 10 pg / mL to 100 pg / mL.

[0081] [Hippo signaling pathway inhibitors]

[0082] The organoid manufacturing culture medium of this embodiment may also contain an inhibitor of the Hippo signaling pathway. It has been conventionally believed that Matrigel is required for culturing organoids. ® Extracellular matrix, etc. The inventors have previously explained that if a Hippo signaling pathway inhibitor is added to the culture medium for organoid production, organoids can be cultured even without an extracellular matrix.

[0083] Therefore, by using a culture medium for organoid manufacturing that also contains an inhibitor of the Hippo signaling pathway, organoids can be cultured in the absence of an extracellular matrix. Previously, it was impossible to proliferate organoids in the absence of an extracellular matrix. Using the organoid manufacturing culture medium of this embodiment, it is possible to completely eliminate the need for Matrigel. ® Animal-derived products are used to proliferate organoids. Here, animal-derived products refer to substances of undetermined composition derived from humans or non-human animals. Using animal-derived product-free culture techniques holds promise for applying organoids in regenerative medicine. Furthermore, from the perspectives of animal welfare and cost, this method offers significant advantages over previous organoid culture techniques.

[0084] The Hippo signaling pathway is a known signaling pathway involved in cell proliferation, apoptosis, stem cell self-replication, etc., and it is known to be an evolutionarily conserved pathway.

[0085] Figure 1 This is a schematic diagram illustrating the Hippo signal transmission path. For example... Figure 1 As shown, the transcription factor TEAD is known to promote cell proliferation by binding to YAP (Yes-associated protein), an activation cofactor, which activates the transcription of genes involved in cell proliferation. YAP exists in phosphorylated and non-phosphorylated forms. Non-phosphorylated YAP translocates to the nucleus and functions as a transcriptional activation cofactor for TEAD. On the other hand, phosphorylated YAP binds to cytoplasmic protein 14-3-3 and therefore cannot translocate to the nucleus, thus failing to function as an activation cofactor. Therefore, phosphorylation control, which determines YAP nuclear translocation, is a crucial factor in cell proliferation.

[0086] In addition, such as Figure 1 As shown, large tumor suppressor kinases (LATS) are known to negatively regulate YAP’s participation in cell proliferation by phosphorylating YAP, thereby localizing YAP to the cytoplasm.

[0087] As an inhibitor of the Hippo signaling pathway, it can be used without particular restrictions as long as it can inhibit any one stage of the above signaling pathway.

[0088] Hippo signaling pathway inhibitors can be, for example, inhibitors of MST1 or MST2 kinases (MST1 / 2 kinase inhibitors), or inhibitors of LATS1 or LATS2 kinases (LATS1 / 2 kinase inhibitors).

[0089] 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, and NP_066278.3, among others. Similarly, the NCBI accession numbers for the amino acid sequences of human MST2 kinase are NP_001243241.1, NP_001243242.1, and NP_006272.2, among others.

[0090] 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, and NP_004681.1, among others. Additionally, the NCBI accession number for the amino acid sequences of human LATS2 kinase is NP_055387.2, among others.

[0091] More specific examples of Hippo signaling pathway inhibitors include, for example, TRULI (CAS No.: 1424635-83-5), GA-017 (CAS No.: 2351906-74-4), and TDI-011536 (CAS No.: 2687970-96-1), which are inhibitors of LATS1 and LATS2 kinases. These substances can be used alone or in combination of two or more. TRULI and TDI-011536 are preferred. The concentration of TRULI in the organoid manufacturing culture medium can be, for example, 1µM to 10mM, 5µM to 1mM, or 10 to 100µM. The concentration of TDI-011536 in the organoid manufacturing culture medium can be, for example, 1µM to 10mM, 1µM to 1mM, or 1 to 100µM.

[0092] [Fibroblast growth factor (FGF)]

[0093] The culture medium for organoid manufacturing in this embodiment may further contain FGF. Examples of FGF include FGF-2, FGF-4, FGF-7, FGF-10, and their mimics. These substances may be used alone or in combination of two or more. Among them, FGF-7 is preferred.

[0094] The concentration of FGF in the differentiation medium may be, for example, 10–500 ng / mL, 10–300 ng / mL, or 10 ng / mL–100 ng / mL.

[0095] [Forskolin]

[0096] The organoid manufacturing culture medium of this embodiment may further contain trichodin. By adding trichodin to the culture medium, the proliferation of organoids is often further promoted. The concentration of trichodin in the organoid manufacturing culture medium may be, for example, 100 nM to 100 µM, 500 nM to 50 µM, or 1 to 20 µM.

[0097] [Other additives]

[0098] The culture medium for organoid production may also contain a p-kinase (ROCK) inhibitor. Examples of ROCK inhibitors include Y-27632 (CAS No.: 129830-38-2), fasudil (HA1077) (CAS No.: 103745-39-7), and H-1152 (CAS No.: 871543-07-6). When using Y-27632 as a ROCK inhibitor, it is preferable to add it within the first approximately two days of culturing the stem cells after they have been dissociated into single cells. The concentration of Y-27632 in the organoid production culture medium is preferably approximately 10 µM.

[0099] The culture medium for organoid production may also contain at least one amino acid. Examples of amino acids include, for instance, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cysteine, 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 in the organoid culture medium is 0.05 g / L to 1 g / L (typically 0.1 g / L to 0.75 g / L). The concentration of other amino acids in the organoid culture medium is 0.001 g / L to 1 g / L (typically 0.01 g / L to 0.15 g / L). The amino acids may be synthetic.

[0100] The culture medium for organoid production may also contain at least one vitamin. 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), DL-α-tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

[0101] Culture media for organoid production may also contain at least one inorganic salt. Inorganic salts help maintain osmotic balance in cells and help regulate membrane potential. Specific examples of inorganic salts include salts of calcium, copper, iron, magnesium, potassium, sodium, and zinc. Salts are commonly used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. More specific examples include CaCl2, CuSO4·5H2O, Fe(NO3)·9H2O, FeSO4·7H2O, MgCl, MgSO4, KCl, NaHCO3, NaCl, Na2HPO4, Na2HPO4·H2O, ZnSO4·7H2O, etc.

[0102] The culture medium for organoid production may also 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 in the culture medium for organoid production is preferably 1 to 10 g / L.

[0103] The culture medium for organoid culture may also contain at least one trace element. Examples of trace elements include barium, bromine, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum, or their ions.

[0104] The culture medium for organoid culture may also contain at least one additional reagent. Examples of such reagents include nutrients or growth factors reported to improve stem cell culture, such as cholesterol, transferrin, albumin, insulin, progesterone, putrescine, selenite, etc.

[0105] [Microenvironmental factors]

[0106] The culture medium for organoid culture in this embodiment may also contain microenvironmental factors that are conventionally considered essential for organoid culture. Examples of microenvironmental factors include Wnt agonists, inhibitors of the bone morphogenetic protein (BMP) signaling pathway, and inhibitors of transforming growth factor (TGF)-β.

[0107] As described in the embodiments described later, the inventors clarified that, compared with the use of conventional culture media for culturing organoids, when organoids are cultured using a culture medium that further supplements prostaglandins, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins, and epidermal growth factor (EGF) family proteins in a conventional culture medium (containing microenvironmental factors), cell proliferation is further promoted.

[0108] [Wnt agonist]

[0109] Wnt agonists are drugs that can activate T-cell cytokine (TCF) / lymphocyte-enhancing factor (LEF)-mediated transcription within cells. Therefore, Wnt agonists are not limited to Wnt family proteins, but include Wnt agonists that bind to and activate Frizzled receptor family members, inhibitors of intracellular β-catenin degradation, and TCF / LEF activators. Preferably, Wnt agonists are selected from at least one of the following: Wnt protein, R-Spondin, and GSK-3β inhibitors.

[0110] As a Wnt agonist, a complex of Wnt protein and its stabilizing agent Afamin is more preferred, and a complex comprising Wnt protein and Afamin and R-Spondin is even more preferred.

[0111] [Wnt protein]

[0112] There are no particular restrictions on the source of Wnt proteins; Wnt proteins from various organisms can be used. Among these, Wnt proteins derived from mammals are preferred. Mammals will be discussed later. Examples of Wnt proteins derived from mammals include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. Multiple Wnt proteins can also be used in combination in the culture medium for organoid production.

[0113] Methods for preparing Wnt protein include, for example, using Wnt protein expression cells. There are no particular limitations on the source (biological species, culture method, etc.) of Wnt protein expression cells, as long as they are cells that stably express Wnt protein, or cells that transiently express Wnt protein. Examples of Wnt protein expression cells include L cells stably expressing mouse Wnt3a (ATCC CRL-2647) and L cells stably expressing mouse Wnt5a (ATCC CRL-2814). Furthermore, Wnt protein expression cells can be prepared using known gene recombination techniques. That is, DNA encoding the desired Wnt protein is inserted into a known expression vector, and the resulting expression vector is introduced into a suitable host cell to prepare Wnt protein expression cells. The base sequence of the gene encoding the desired Wnt protein can be obtained, for example, from known databases such as GenBank.

[0114] The Wnt protein expressed by cells expressing Wnt protein can be a fragment of the Wnt protein, or it can contain amino acid sequences other than the Wnt protein sequence, as long as it possesses Wnt activity. There are no particular restrictions on the amino acid sequences other than the Wnt protein sequence; for example, amino acid sequences of affinity tags can be listed. Furthermore, the amino acid sequence of the Wnt protein does not need to be completely identical to the amino acid sequence obtained from known databases such as GenBank; as long as it possesses Wnt activity, it can also be a substantially identical amino acid sequence to the amino acid sequence obtained from known databases.

[0115] An amino acid sequence that is substantially the same as the amino acid sequence of the Wnt protein obtained from known databases such as GenBank can be, for example, an amino acid sequence in which one or more amino acids are missing, replaced, or added, as can be listed in the amino acid sequences obtained from known databases.

[0116] "An amino acid sequence with one or more amino acids missing, replaced or added" refers to an amino acid sequence with a number of missing, replaced or added amino acids (preferably 10 or less, more preferably 7 or less, and even more preferably 6 or less) by a known mutant peptide preparation method, such as site-directed mutagenesis.

[0117] Furthermore, as substantially identical amino acid sequences, examples include amino acid sequences that have a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 92%, particularly preferably at least 95%, and most preferably at least 99%.

[0118] The concentration of Wnt protein can be, for example, above 50 ng / mL, for example, 100 ng / mL to 10 µg / mL, for example, 200 ng / mL to 1 µg / mL, for example, 300 ng / mL to 1 µg / mL.

[0119] [R-Spondin]

[0120] As R-Spondins, the R-Spondin family of proteins, including R-Spondin1, R-Spondin2, R-Spondin3, and R-Spondin4, can be listed. R-Spondins are secreted proteins and are known to participate in the activation and regulation of the Wnt signaling pathway. Multiple R-Spondins can also be used in combination in organoid culture media. Furthermore, any protein possessing R-Spondin activity can be a fragment of R-Spondin or contain amino acid sequences other than the R-Spondin amino acid sequence.

[0121] [GSK-3β inhibitors]

[0122] 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-bromoindorubin-30-acetone oxime, SB216763 (CAS No.: 280744-09-4), SB415286 (CAS No.: 264218-23-7), FRAT family members that prevent the interaction between GSK-3 and axin, and peptides derived from FRAT.

[0123] [Afamin]

[0124] Afamin is a glycoprotein belonging to the albumin family, known to be present in bodily fluids such as blood. Serum added to culture media typically 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 using serum.

[0125] The source of Afamin contained in the culture medium for organoid production is not particularly limited, and Afamin from various organisms can be used. Preferably, Afamin is derived from mammals. Mammals will be described later. The amino acid sequences of Afamin from major mammals and the nucleotide sequences of the genes encoding them can be obtained, for example, from known databases such as GenBank. For instance, in GenBank, the amino acid sequence of human Afamin is registered with accession number AAA21612, and the nucleotide sequence of the gene encoding it is registered with accession number L32140; the amino acid sequence of bovine Afamin is registered with accession number DAA28569, and the nucleotide sequence of the gene encoding it is registered with accession number GJ060968.

[0126] The Afamin contained in the culture medium for organoid manufacturing can be either natural Afamin purified from serum or other sources using known methods, or recombinant Afamin. Recombinant Afamin can be prepared by appropriately applying known gene recombination techniques.

[0127] As a method for preparing recombinant Afamin, for example, DNA encoding Afamin can be inserted into a known expression vector, the resulting expression vector can be introduced into a suitable host cell to express recombinant Afamin, and then purified using a known purification method. Recombinant Afamin can also be Afamin with an added affinity tag. The added affinity tag is not particularly limited and can be appropriately selected from known affinity tags. Preferably, the affinity tag is one that can be recognized by a specific antibody; for example, FLAG tags, MYC tags, HA tags, V5 tags, etc., are examples.

[0128] The Wnt protein described above exhibits strong hydrophobicity due to the modification of specific serine residues with fatty acids (palmitoic acid). Therefore, Wnt proteins readily aggregate or denature in aqueous solutions, making their purification and preservation extremely difficult, a fact widely known in the industry.

[0129] On the other hand, it has been reported that fatty acid modification of this specific serine residue is essential for the physiological activity of the Wnt protein and is involved in its binding to members of the Frizzled receptor family.

[0130] Furthermore, it is known that in aqueous solution, Wnt protein and Afamin bind in a 1:1 ratio to form a complex, which can achieve solubilization while maintaining high physiological activity. The Wnt protein-Afamin complex can be prepared by culturing cells that simultaneously express both Wnt protein and Afamin, or by co-culturing cells expressing both Wnt protein and Afamin.

[0131] The concentration of Afamin in the culture medium for organoid culture is not particularly limited. For example, it can be 50 ng / mL to 10 µg / mL, or less than 100 ng / mL to 1 µg / mL, or 300 ng / mL to 1 µg / mL.

[0132] [BMP inhibitors]

[0133] BMP, as a dimer ligand, binds to a receptor complex composed of two different receptor serine / threonine kinases: type I and type II receptors. The type II receptor phosphorylates the type I receptor, resulting in the activation of that receptor kinase. The type I receptor then phosphorylates a specific receptor substrate (SMAD), leading to transcriptional activity via a signaling pathway. Generally, BMP inhibitors are substances that prevent or inhibit the binding of BMP molecules to BMP receptors; that is, agents that bind to BMP molecules to form a complex that neutralizes BMP activity. Alternatively, BMP inhibitors are substances that bind to BMP receptors, preventing or inhibiting the binding of BMP molecules to receptors; that is, agents that act as antagonists or inverse agonists.

[0134] Compared to the BMP activity level in the absence of the inhibitor, the BMP inhibitor preferably has an inhibitory activity of 50% or more, more preferably 70% or more, further preferably 80% or more, and particularly preferably 90% or more.

[0135] BMP inhibitors are preferably natural BMP-binding proteins, such as Noggin, Gremlin, Chordin, Chordin-like proteins with a Chordin domain; follistatin, follistatin-related proteins with a follistatin domain; DAN, DAN-like proteins with a DAN cysteine ​​domain; Sclerosing protein (SOST), core proteoglycans, α-2 macroglobulin, and other similar proteins.

[0136] Chordin-like or DAN-like proteins are preferred as BMP inhibitors contained in the culture medium for organoid culture, with chordin-like proteins being more preferred. Noggin is preferred as a chordin-like protein. Chordin-like and DAN-like proteins are diffusible proteins that can bind to BMP molecules with different affinities, thereby inhibiting the approach of BMP molecules to signal transduction receptors.

[0137] The concentration of the BMP inhibitor contained in the culture medium for organoid culture may be, for example, 10 ng / mL to 100 ng / mL, 20 to 100 ng / mL, or 50 to 100 ng / mL.

[0138] [TGF-β inhibitors]

[0139] TGF-β is a growth factor produced by almost all cells, such as those in the kidneys, bone marrow, and platelets. There are five subtypes of TGF-β (β1–β5). Furthermore, TGF-β is known to promote osteoblast proliferation and the synthesis and proliferation of connective tissues such as collagen, while inhibiting the proliferation of epithelial cells and osteoclasts. Generally, TGF-β inhibitors refer to substances that, for example, prevent or inhibit TGF-β from binding to TGF-β receptors, or agents that bind to TGF-β to form a complex that neutralizes TGF-β activity. In addition, TGF-β inhibitors are agents that, for example, bind to TGF-β receptors, prevent or inhibit TGF-β from binding to receptors, and act as antagonists or inverse agonists.

[0140] Examples of TGF-β inhibitors include A-83-01 (CAS No.: 909910-43-6), ALK5 inhibitor I (3-(pyridin-2-yl)-4-(4-quinolinyl)-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), and 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 No.: 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 neutralizing TGFβ1)), GC-1008 (anti-TGF-β monoclonal antibody), etc. As a TGF-β inhibitor, A-83-01 is preferred.

[0141] The concentration of the TGF-β inhibitor contained in the culture medium for organoid culture can be, for example, 100 nM to 10 µM, 500 nM to 5 µM, or 500 nM to 2 µM.

[0142] [EGF]

[0143] The organoid manufacturing culture medium of this embodiment may contain EGF as a microenvironmental factor. The concentration of EGF contained in the organoid manufacturing culture medium may be, for example, 5 ng / mL to 1 µg / mL, 10 ng / mL to 1 µg / mL, or 50 to 500 ng / mL.

[0144] [Organoids]

[0145] The organoids that can be cultured using the organoid manufacturing culture medium of this embodiment are not particularly limited, and examples include organoids derived from epithelial cells, epithelial stem cells, mesenchymal cells, mesenchymal stem cells, cancer cells, and cancer stem cells. More specifically, examples include organoids derived from the digestive tract (esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon)), liver, pancreas (islets of Langerhans), salivary glands, mammary glands, bile ducts, lungs, respiratory tract, hair follicles, skin, cartilage, and bone marrow mesenchymal cells.

[0146] Organoids can be of human origin or of non-human animal origin. Examples of non-human animals include mammals such as rodents (mice, rats, hamsters, guinea pigs, etc.); ungulates such as pigs, cattle, goats, horses, sheep, etc.; carnivores such as dogs and cats; and primates such as macaques, cynomolgus monkeys, marmosets, orangutans, chimpanzees, etc.

[0147] [Organoid Culture Kit]

[0148] In one embodiment, the present invention provides an organoid culture kit comprising prostaglandin E2, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins, and epidermal growth factor (EGF) family proteins.

[0149] The same applies to prostaglandin E2, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins, and epidermal growth factor (EGF) family proteins. The organoid manufacturing culture medium described above can be prepared by adding the factors included in the kit of this embodiment to the basal culture medium.

[0150] The kit in this embodiment may also include a basal culture medium. The basal culture medium is the same as described above.

[0151] The kit in this embodiment may also contain interferon (IFN)-γ. Although interferon (IFN)-γ is not essential, the addition of interferon (IFN)-γ to the culture medium for organoid production tends to further promote organoid proliferation. The same applies to interferon (IFN)-γ as described above.

[0152] The kit in this embodiment may also contain an inhibitor of the Hippo signaling pathway. Typically, Matrigel is required for culturing organoids. ® Extracellular matrix. In contrast, if a Hippo signaling pathway inhibitor is added to the culture medium for organoid production, organoids can be cultured even without an extracellular matrix. The same applies to Hippo signaling pathway inhibitors.

[0153] The kit in this embodiment may also include fibroblast growth factor (FGF), trichodin, and other factors, depending on the cultured organoids. The same applies to fibroblast growth factor (FGF) and trichodin as described above.

[0154] The kit of this embodiment may also contain microenvironmental factors. Examples of microenvironmental factors include Wnt agonists, inhibitors of the bone morphogenetic protein (BMP) signaling pathway, and transforming growth factor (TGF)-β inhibitors. The same applies to Wnt agonists, BMP signaling pathway inhibitors, and TGF-β inhibitors. The kit of this embodiment may also contain epidermal growth factor (EGF) as a microenvironmental factor. The same applies to EGF.

[0155] The kit of this embodiment may also contain other additives as described above. In the kit of this embodiment, each of the above factors or additives may be contained in a separate container, or two or more may be mixed and contained in one or more containers.

[0156] [Methods for manufacturing organoids]

[0157] In one embodiment, the present invention provides a method for manufacturing organoids, comprising culturing cells in a culture medium containing prostaglandin E2, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins, and epidermal growth factor (EGF) family proteins, thereby forming organoids from said cells, wherein said cells are selected from the following: epithelial cells, epithelial stem cells, mesenchymal cells, mesenchymal stem cells, cancer cells, and cancer stem cells. The same applies to prostaglandin E2, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins, and epidermal growth factor (EGF) family proteins.

[0158] The manufacturing method of this embodiment enables the creation of organoids that were previously possible, as well as those that were previously impossible to create. Examples of organoids that were previously impossible to create include chondrocyte-derived organoids, mesenchymal cell-derived organoids, and skin epithelial cell-derived organoids.

[0159] In the manufacturing method of this embodiment, the cells used are not particularly limited, and examples include epithelial cells, epithelial stem cells, mesenchymal cells, mesenchymal stem cells, cancer cells, and cancer stem cells. More specifically, examples include cells derived from the digestive tract (esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon)), liver, pancreas (islets of Langerhans), salivary glands, mammary glands, bile ducts, lungs, respiratory tract, hair follicles, skin, cartilage, and bone marrow matrix. The manufacturing method of this embodiment enables the creation of organoids from these cells. The manufacturing method of this embodiment also enables the creation of organoids from single cells.

[0160] In the manufacturing method of this embodiment, the culture medium may further contain interferon (IFN)-γ. Although interferon (IFN)-γ is not essential, the addition of interferon (IFN)-γ to the culture medium tends to further promote organoid proliferation. The same applies to interferon (IFN)-γ as described above.

[0161] In the manufacturing method of this embodiment, the culture medium may further contain an inhibitor of the Hippo signaling pathway. Typically, Matrigel is required for culturing organoids. ® Extracellular matrix. In contrast, if a Hippo signaling pathway inhibitor is added to the culture medium, organoids can be cultured even in the absence of an extracellular matrix. The same applies to Hippo signaling pathway inhibitors.

[0162] In the manufacturing method of this embodiment, the culture medium can be a serum-free culture medium. By establishing organoids under conditions where there is no extracellular matrix and no serum, organoids manufactured without animal-derived components from the time of establishment can be obtained.

[0163] The kit of this embodiment may also include fibroblast growth factor (FGF), trichodin, and other factors, depending on the cultured organoids. The same applies to fibroblast growth factor (FGF) and trichodin as described above.

[0164] In the manufacturing method of this embodiment, the culture medium may further include microenvironmental factors. Examples of microenvironmental factors include Wnt agonists, inhibitors of the bone morphogenetic protein (BMP) signaling pathway, and inhibitors of transforming growth factor (TGF)-β. The same applies to Wnt agonists, BMP signaling pathway inhibitors, and TGF-β inhibitors. In the manufacturing method of this embodiment, the culture medium may include epidermal growth factor (EGF) as a microenvironmental factor. The same applies to epidermal growth factor (EGF).

[0165] In the manufacturing method of this embodiment, the culture medium may also contain the other additives mentioned above.

[0166] [Extracellular matrix]

[0167] When creating or culturing organoids in the presence of an extracellular matrix, examples of extracellular matrices include Matrigel. ® The components include collagen, fibronectin, proteoglycans, and laminin. Proliferating organoids in the absence of extracellular matrix means not adding extracellular matrix to the organoid culture medium from the outside, allowing trace amounts of extracellular matrix produced by the organoid itself to mix into the culture medium, and also allowing unintentional trace amounts of extracellular matrix to mix into the culture medium. Here, "trace" can refer to the detection limit.

[0168] [Organoids]

[0169] In one embodiment, the present invention provides an organoid manufactured by the above-described manufacturing method.

[0170] The organoids in this embodiment include not only conventionally available organoids but also those that were previously impossible to create. Examples of conventionally impossible organoids include, for instance, mesenchymal cell-derived organoids and skin epithelial cell-derived organoids. Specifically, in one embodiment, the present invention provides mesenchymal cell-derived organoids. In another embodiment, the present invention provides skin epithelial cell-derived organoids.

[0171] As described in the examples below, organoids obtained by culturing cells in a culture medium containing prostaglandin E2, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins and epidermal growth factor (EGF) family proteins have morphologies different from those of conventional organoids.

[0172] Specifically, for example, small intestinal organoids established or cultured using conventional methods exhibit a vesicular morphology with budding and covered by a single cell layer. In contrast, small intestinal organoids established or cultured in a medium containing prostaglandin E2, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins, and epidermal growth factor (EGF) family proteins exhibit a multilayered cell mass morphology.

[0173] Furthermore, liver organoids established or cultured using conventional methods exhibit a vesicular morphology covered by a single cell layer. In contrast, liver organoids established or cultured in a medium containing prostaglandin E2, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins, and epidermal growth factor (EGF) family proteins exhibit a multilayered cell mass morphology.

[0174] As described in the embodiments below, the inventors have demonstrated that for organoids established or cultured in a culture medium containing prostaglandin E2, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins and epidermal growth factor (EGF) family proteins, and whose morphology differs from that of previous organoids, when cultured in an extracellular matrix with the previous culture medium, their morphology will revert to that of the previous organoids.

[0175] The organoids of this embodiment are considered to differ from conventional organoids in terms of gene expression patterns, etc. However, it is unclear and impractical to definitively distinguish between conventional and conventional organoids based solely on differences in specific gene expression patterns. Therefore, it is considered appropriate to define the organoids of this embodiment through their manufacturing method.

[0176]

Example

[0177] The present invention will be further described in detail below through embodiments, but the present invention is not limited to the following embodiments.

[0178] [Experimental Example 1: Culture of Human Large Intestine-Derived Organoids]

[0179] Human colony-derived organoids were cultured under various conditions, and their growth dynamics were observed. The human colony-derived organoids used were established from tissue samples of patients with gastrointestinal tumors who had obtained informed consent, based on an ethical research plan approved by the Ethics Committee of the Faculty of Medicine, Keio University.

[0180] Large intestine organoids were dissociated into single cells using TrypLE Express (Thermo Fisher Scientific) to obtain a cell suspension. During culture in the extracellular matrix, 1×10⁶ cells were added. 5 Cells per well with 25 µL Matrigel ® They were inoculated together into 48-well plates. Then, Matrigel... ® After gelation, 100 µL of the target composition culture medium was added to each well and incubated at 37°C. When culturing in the absence of extracellular matrix, 1×10⁻⁶ cells / well was added. 5 Cells per well were suspended in 100 µL of the target composition medium per well, seeded into 48-well plates, and cultured at 37°C.

[0181] Figure 2 and Figure 3 Representative microscopic images of the large intestine organoids after 7 days of culture. Figure 2 and Figure 3In this context, "conventional organoid" refers to the result of culture in an extracellular matrix, while "MfLO" refers to the result of culture in the absence of an extracellular matrix.

[0182] exist Figure 2 In this context, "Full" indicates that the culture medium contains EGF, Noggin, Wnt, R-Spondin, and A-83-01 (a transforming growth factor (TGF)-β inhibitor) (hereinafter sometimes referred to as a "niche factor"). As Wnt, a complex of Wnt3a and Afamin was used. The concentrations of EGF in the culture medium were 50 ng / mL, Noggin 4%, Wnt3a 10%, R-Spondin 4%, and A-83-01 500 nM. In addition, "Control" indicates the results of culture in a standard medium containing microenvironmental factors; "TRULI" indicates the results of culture in a standard medium containing microenvironmental factors with the addition of 20 µM TRULI (CAS No.: 1424635-83-5); "PHOENIX" indicates the results of culture in a standard medium containing microenvironmental factors with the addition of prostaglandin E2 2.5 µM, hepatocyte growth factor (HGF) 0.5 nM, oncostatin M 20 ng / mL, epithelial regulatory protein 100 ng / mL, neuromodulatory factor 15 nM, interferon (IFN)-γ 30 pg / mL, TRULI 20 µM, and TDI-011536 (CAS No.: 2687970-96-1) 10 µM (hereinafter sometimes referred to as "PHOENIX").

[0183] exist Figure 3 In the text, "-ENWRA" indicates that the culture medium does not contain the above-mentioned microenvironmental factors; "Control" indicates the results of culture in a culture medium without microenvironmental factors; "TRULI" indicates the results of culture in a culture medium without microenvironmental factors with 20 µM TRULI added; and "PHOENIX" indicates the results of culture in a culture medium without microenvironmental factors with the above-mentioned PHOENIX added.

[0184] The results are as follows Figure 2 As shown, in Matrigel ®During culture, the addition of the LATS inhibitor TRULI to the culture medium clearly promoted organoid proliferation compared to the control group. Furthermore, a further proliferation-promoting effect was observed in the group that received PHOENIX to standard culture medium containing microenvironmental factors. Additionally, when cultured without extracellular matrix, large intestine organoids could not survive in standard culture medium containing microenvironmental factors; however, the addition of the LATS inhibitor TRULI to the culture medium clearly promoted organoid proliferation compared to the control group. Again, a further proliferation-promoting effect was observed in the group that received PHOENIX to standard culture medium containing microenvironmental factors.

[0185] In addition, such as Figure 3 As shown, in a culture medium free of microenvironmental factors, whether in Matrigel... ® Whether cultured in vitro or in the absence of extracellular matrix, large intestine organoids cannot survive. Similarly, in media containing the LATS inhibitor TRULI (which does not contain microenvironmental factors), whether in Matrigel or other mediums, large intestine organoids cannot survive. ® Whether cultured in vitro or in the absence of extracellular matrix, large intestine organoids could not survive. On the other hand, in media containing PHOENIX (which does not contain microenvironmental factors), whether in Matrigel or other media, organoids could not survive. ® Whether cultured in vitro or in the absence of extracellular matrix, large intestine organoids can survive and proliferate.

[0186] Figure 4 This indicates that the results were obtained using a commercially available kit (product name "Celltiter-Glo", Promega). Figure 2 and Figure 3 The chart shows the proliferation results of large intestine organoids cultured under the same culture conditions. Figure 4 In this context, "conventional" indicates the result of culture in an extracellular matrix, while "Matrigel-free" indicates the result of culture in the absence of an extracellular matrix. Furthermore, "full" indicates that the culture medium contains the aforementioned microenvironmental factors, "-ENWRA" indicates that the culture medium does not contain these microenvironmental factors, "TRULI" indicates the result of culture using a medium supplemented with 20 µM TRULI, "PHOENIX" indicates the result of culture using a medium supplemented with the aforementioned PHOENIX, and "control" indicates the result of culture using a control medium that does not contain TRULI or PHOENIX.

[0187] The results showed that, regardless of Matrigel ®Whether cultured internally or in the absence of extracellular matrix, colonic organoids cultured in a medium containing PHOENIX but without microenvironmental factors exhibited the same proliferation as previous colonic organoid culture conditions (cultured in an extracellular matrix in a medium containing the aforementioned microenvironmental factors).

[0188] [Experimental Example 2: Study of Organoid Morphology]

[0189] Organoids were cultured under conventional organoid culture conditions (i.e., cultured in a medium containing the aforementioned microenvironmental factors in an extracellular matrix) and under conditions where organoids were cultured in a medium containing PHOENIX but without an extracellular matrix and without the aforementioned microenvironmental factors. Their morphology was then compared.

[0190] As organoids, human small intestine organoids and human liver organoids were cultured. These organoids were developed from patient-derived tissues with informed consent, based on an ethical research program approved by the Ethics Committee of Keio University School of Medicine.

[0191] Figure 5 These are representative microscopic images showing the morphology of various organs after 7 days of culture following dissociation into single cells. The scale bar is 100µm. Figure 5 The top section shows an image of a small intestinal organoid, and the bottom section shows an image of a liver organoid. Furthermore, "standard culture" indicates the result of culturing organoids under conventional culture conditions (i.e., cultured in an extracellular matrix in a medium containing the aforementioned microenvironmental factors). "No Matrigel, No ENWRA, PHOENIX" indicates the result of culturing organoids under conditions lacking an extracellular matrix and without the aforementioned microenvironmental factors but containing PHOENIX.

[0192] The results showed that conventionally cultured small intestinal organoids exhibited a budding, sac-like morphology covered by a single cell layer. In contrast, small intestinal organoids cultured in a medium containing PHOENIX but without the aforementioned microenvironmental factors, in the absence of extracellular matrix, exhibited a multilayered cell cluster morphology.

[0193] Furthermore, conventionally cultured liver organoids exhibit a sac-like morphology covered by a single cell layer. In contrast, liver organoids cultured in a medium containing PHOENIX but without the aforementioned microenvironmental factors, in the absence of extracellular matrix, exhibit a multilayered cell cluster morphology.

[0194] Subsequently, the small intestinal organoids and liver organoids, which had been cultured in a medium containing PHOENIX but without the extracellular matrix and without the aforementioned microenvironmental factors, were cultured again under conventional culture conditions for 7 days, and their morphology was observed.

[0195] Figure 6 These are representative microscope images showing the morphology of various organs. The scale bar is 100µm. Figure 6 The top section shows an image of a small intestinal organoid, and the bottom section shows an image of a liver organoid. Furthermore, "Matrigel-free, ENWRA-free, PHOENIX-free" indicates the results of culturing organoids under conditions where the extracellular matrix is ​​absent and the aforementioned microenvironmental factors are absent, but PHOENIX is present. "Routine culture" indicates the results of culturing organoids under conventional culture conditions (i.e., culturing in an extracellular matrix within a medium containing the aforementioned microenvironmental factors).

[0196] The results showed that, for both small intestinal and liver organoids, when the culture conditions were restored to normal, the morphologically changed organoids could be restored to their original form.

[0197] [Experimental Example 3: Establishment of organoids derived from mouse bone marrow mesenchymal cells]

[0198] An attempt was made to create organoids derived from mouse bone marrow mesenchymal cells. The creation of organoids derived from mouse bone marrow mesenchymal cells has not been reported before.

[0199] Mouse bone marrow mesenchymal cells were cultured in the extracellular matrix using a medium containing the aforementioned microenvironmental factors and PHOENIX. As a result, organoids derived from mouse bone marrow mesenchymal cells were successfully established.

[0200] Figure 7 These are representative microscopic images of established organoids derived from mouse bone marrow mesenchymal cells. Figure 7 The scale bar in the image above is 500µm. Figure 7 The scale bar in the image below is 50µm.

[0201] [Experiment Example 4: Establishing an organoid derived from human skin epithelial cells]

[0202] An attempt was made to create organoids derived from skin epithelial cells from human hair roots. The creation of organoids derived from skin epithelial cells has not been reported before.

[0203] Human hair root cells were cultured in an extracellular matrix using a medium containing the aforementioned microenvironmental factors, as well as prostaglandin E2 2.5 µM, hepatocyte growth factor (HGF) 0.5 nM, oncostatin M 20 ng / mL, epithelial regulatory protein 100 ng / mL, neuromodulatory 1 5 nM, interferon (IFN)-γ 30 pg / mL, fibroblast growth factor (FGF)-7 50 ng / mL, and trichodin 10 µM. Hereinafter, prostaglandin E2, hepatocyte growth factor (HGF), oncostatin M, epithelial regulatory protein, neuromodulatory 1, and interferon (IFN)-γ are sometimes referred to as "PHOENI". As a result, organoids derived from human skin epithelial cells were successfully established.

[0204] Figure 8 It shows the process of organoid formation and representative microscope images of the established organoids. Figure 8 The scale bar in the lower right image is 100µm. Figure 8 In the text, "D0.5", "D2", "D3", "D7", "D13", and "D15" represent images taken 0.5 days, 2 days, 3 days, 7 days, 13 days, and 15 days after the start of cell culture from human hair roots, respectively. Additionally, "Passage 2 (D3)" represents an image taken 3 days after the cells from human hair roots were dissociated into single cells and cultured again under the same conditions 15 days after the start of the culture. Figure 8 The image in the lower right corner is... Figure 8 A magnified portion of the image in the lower left corner.

[0205] [Experimental Example 5: Differentiation Induction of Organoids Derived from Human Skin Epithelial Cells]

[0206] An attempt was made to induce differentiation of the organoids derived from human skin epithelial cells established in Experiment 4. Figure 9 This is a schematic diagram illustrating the differentiation induction experimental procedure.

[0207] First, the organoids derived from human skin epithelial cells established in Experiment 4 were seeded into cell culture chambers and cultured for 6 days in a medium containing, in addition to the microenvironmental factors mentioned above, prostaglandin E2 2.5µM, hepatocyte growth factor (HGF) 0.5nM, tumor suppressor M 20ng / mL, epithelial regulatory protein 100ng / mL, neuromodulatory factor 15nM, interferon (IFN)-γ 30pg / mL, fibroblast growth factor (FGF)-7 50ng / mL, and pilocarpine 10µM.

[0208] Subsequently, gas-liquid interface culture was performed until day 18 to induce differentiation of organoids derived from human skin epithelial cells. The culture media used for gas-liquid interface culture were: (1) "all + PHOENI + FGF7 + trichodin" medium, (2) "all + FGF7 + trichodin" medium with PHOENI removed from the medium in (1), (3) "-ENWRA + PHOENI" medium with microenvironmental factors, FGF7 and trichodin removed from the medium in (1), and (4) "-ENWRA" medium with PHOENI removed from the medium in (3). Subsequently, the organoids were fixed and thin sections were prepared, stained with hematoxylin-eosin and observed under a microscope.

[0209] Figure 10 These are microscope images of samples that underwent differentiation induction in various culture media. Figure 11 It is Figure 10 The image shows a magnified view of the rectangular area within the image. The results showed that when cultured in a medium free of microenvironmental factors, the cells differentiated and exhibited a morphology remarkably similar to skin.

[0210] [Experimental Example 6: Establishment of organoids derived from human small intestinal villi epithelial cells]

[0211] An attempt was made to create organoids from epithelial cells derived from human small intestinal villi. Since small intestinal villi do not contain intestinal epithelial stem cells, the creation of organoids derived from small intestinal villi epithelial cells is challenging and has not been previously reported.

[0212] In the extracellular matrix, human villus-derived intestinal epithelial cells lacking intestinal epithelial stem cells were cultured using a medium containing the aforementioned microenvironmental factors and PHOENIX. As a result, human small intestinal villus-derived epithelial cell organoids were successfully established. These human small intestinal villus-derived epithelial cells were isolated and established from tissues derived from patients with gastrointestinal tumors who provided informed consent, based on an ethical research program approved by the Ethics Committee of the Faculty of Medicine, Keio University.

[0213] Figure 12 The image on the left is a microscope image of human small intestinal villi-derived epithelial cell organoids 10 days after the start of culturing. Figure 12 The right side is Figure 12 A magnified image of one organoid in the image on the left.

[0214] [Example 7: Culturing organoids in a culture medium without ROCK inhibitors]

[0215] ROCK inhibitors are crucial for the survival of organoids after cell dispersion and must be added when passaged human small intestinal epithelial cell organoids and human large intestinal epithelial cell organoids. It is known that organoid formation efficiency is significantly reduced when organoids are dispersed and cultured in media without ROCK inhibitors. In this experimental example, human small intestinal epithelial cell organoids and human large intestinal epithelial cell organoids were dispersed in the extracellular matrix and cultured in PHOENIX medium without ROCK inhibitors.

[0216] Figure 13 Microscopic images showing the culture results. The results indicate that human small intestinal epithelial cell organoids and human large intestinal epithelial cell organoids cultured in the extracellular matrix only formed organoids in a culture medium containing ROCK inhibitors (comparative). Figure 13 Left side and Figure 13 Central). In culture media without ROCK inhibitors, organoid formation was significantly inhibited ( Figure 13 (Central). In contrast, when PHOENIX was added to a culture medium without ROCK inhibitors, human small intestinal epithelial cell organoids and human large intestinal epithelial cell organoids were formed even without ROCK inhibitors. Figure 13 (Right side).

[0217] [Industrial Applicability]

[0218] According to the present invention, a novel organoid culture and manufacturing technology is provided. Organoids manufactured using the method of the present invention exhibit shapes and properties different from conventional organoids, and upon returning to conventional culture conditions, they transform into the shapes of conventional 3D organoids, thus being considered to reflect a certain morphology of the organoid formation process. To date, although the characteristics of the regenerative state after tissue injury have been elucidated through single-cell RNA analysis, there is no culture method capable of simulating this state, resulting in a lag in drug development focused on in vivo regeneration and tissue repair. Therefore, the culture method and the cultured organoids of the present invention can serve as organoids simulating a certain stage of the tissue regeneration process, and can be used as novel research tools such as human epithelial cell culture technology to simulate tissue regeneration after injury or inflammatory diseases, or for assisting tissue regeneration, demonstrating new industrial application possibilities.

Claims

1. Culture medium for organoid production, comprising prostaglandins, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins, and epidermal growth factor (EGF) family proteins.

2. The culture medium for organoid manufacturing according to claim 1, further comprising interferon.

3. The culture medium for organoid manufacturing according to claim 2, wherein the interferon is interferon (IFN)-γ.

4. The culture medium for organoid manufacturing according to claim 1 or 2, further comprising an inhibitor of the Hippo signaling pathway.

5. The culture medium for organoid manufacturing according to 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. The culture medium for organoid manufacturing according to claim 1 or 2, which substantially does not contain Wnt agonists, BMP signaling pathway inhibitors, or TGF-β inhibitors.

7. Organoid culture kit containing prostaglandins, HGF, IL-6 family proteins and EGF family proteins.

8. A method for manufacturing organoids, comprising the step of culturing cells in a culture medium containing prostaglandins, hepatocyte growth factor (HGF), interleukin (IL)-6 family proteins, and epidermal growth factor (EGF) family proteins, thereby forming organoids from said cells. The cells mentioned therein are selected from the following: epithelial cells, epithelial stem cells, mesenchymal cells, mesenchymal stem cells, cancer cells, and cancer stem cells.

9. The manufacturing method according to claim 8, wherein the culture medium further comprises interferon (IFN)-γ.

10. The manufacturing method according to claim 8 or 9, wherein the culture medium is substantially free of ROCK inhibitors.

11. The manufacturing method according to claim 8 or 9, wherein the culture medium further comprises a Wnt agonist, a bone morphogenetic protein (BMP) signaling pathway inhibitor, and a transforming growth factor (TGF)-β inhibitor.

12. The manufacturing method according to claim 8 or 9, wherein the culture medium is substantially free of Wnt agonists, bone morphogenetic protein (BMP) signaling pathway inhibitors, and transforming growth factor (TGF)-β inhibitors.

13. The manufacturing method according to claim 12, wherein the culture medium is substantially free of ROCK inhibitors.

14. The manufacturing method according to claim 8 or 9, wherein an extracellular matrix is ​​not used when culturing the cells.

15. The manufacturing method according to claim 8 or 9, wherein the culture medium is a serum-free culture medium.

16. The manufacturing method according to claim 8 or 9, wherein the cell is a cell dissociated into a single cell.

17. An organoid, which is obtained by the manufacturing method according to claim 8 or 9.

Images