WO2024071212A1 - 細胞外基質の非存在下でオルガノイドを培養するための薬剤 - Google Patents

細胞外基質の非存在下でオルガノイドを培養するための薬剤 Download PDF

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WO2024071212A1
WO2024071212A1 PCT/JP2023/035181 JP2023035181W WO2024071212A1 WO 2024071212 A1 WO2024071212 A1 WO 2024071212A1 JP 2023035181 W JP2023035181 W JP 2023035181W WO 2024071212 A1 WO2024071212 A1 WO 2024071212A1
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organoids
absence
organoid
extracellular matrix
matrigel
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俊朗 佐藤
智樹 矢野
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Keio University
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Keio University
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Priority to CN202380068690.1A priority Critical patent/CN119968460A/zh
Priority to EP23872434.8A priority patent/EP4596677A1/en
Priority to JP2024550395A priority patent/JPWO2024071212A1/ja
Priority to US19/114,635 priority patent/US20250283049A1/en
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    • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase

Definitions

  • the present invention relates to a drug for culturing organoids in the absence of extracellular matrix. More specifically, the present invention relates to a drug for forming and growing organoids in the absence of extracellular matrix, a method for growing organoids, organoids grown by the growth method, a drug for establishing organoids in the absence of extracellular matrix, a method for producing organoids, organoids produced by the production method, a regenerative medicine preparation, and a method for screening a drug that enables the culture of organoids in the absence of extracellular matrix.
  • This application claims priority based on Japanese Patent Application No. 2022-153698 filed in Japan on September 27, 2022, and Japanese Patent Application No. 2023-090460 filed in Japan on May 31, 2023, the contents of which are incorporated herein by reference.
  • Matrigel registered trademark
  • Patent Document 1 Non-Patent Document 1
  • Matrigel registered trademark
  • Matrigel registered trademark
  • the use of Matrigel (registered trademark) in organoid culture is a barrier to medical applications due to the possibility of contamination with unknown viruses and its high cost.
  • the use of Matrigel is also an issue from the perspective of animal welfare. Since the emergence of organoid technology, there has been vigorous development of alternative substances to Matrigel (registered trademark) (see, for example, Non-Patent Document 2).
  • the present invention aims to provide a technique for producing and culturing organoids in the absence of extracellular matrix.
  • the present invention includes the following aspects.
  • a drug for forming and growing organoids in the absence of extracellular matrix the drug comprising an inhibitor of the Hippo signaling pathway as an active ingredient.
  • [4] A drug for forming and growing organoids in the absence of an extracellular matrix according to any one of [1] to [3], wherein the organoid is an epithelial organoid.
  • [5] A method for growing organoids, comprising culturing organoids in the presence of an inhibitor of the Hippo signaling pathway and in the absence of extracellular matrix.
  • [6] The method for proliferating organoids described in [5], wherein the organoids are cultured under serum-free conditions.
  • [8] A drug for establishing organoids in the absence of extracellular matrix, the drug comprising an inhibitor of the Hippo signaling pathway as an active ingredient.
  • the organoid is an epithelial organoid, [8] to [10], wherein the organoid is a drug for establishing an organoid in the absence of an extracellular matrix.
  • [12] A method for producing an organoid, comprising the step of establishing an organoid in the presence of an inhibitor of the Hippo signaling pathway and in the absence of an extracellular matrix. [13] The method for producing an organoid described in [12], wherein the establishment of the organoid is carried out in the presence of a cytokine family that binds to gp130. [14] The method for producing an organoid described in [12] or [13], wherein the establishment of the organoid is performed under serum-free conditions. [15] An organoid produced by the production method according to any one of [12] to [14]. [16] A regenerative medical preparation comprising the organoid according to [15] as an active ingredient.
  • a method for screening for a drug that enables the culture of an organoid in the absence of an extracellular matrix comprising the steps of culturing an organoid in the presence of a test substance and in the absence of an extracellular matrix, and evaluating the proliferation of the organoid, wherein the proliferation of the organoid is higher than that in the absence of the test substance, indicating that the test substance is a drug that enables the culture of an organoid in the absence of an extracellular matrix.
  • the present invention provides a technique for culturing organoids in the absence of extracellular matrix.
  • FIG. 1 is a schematic diagram illustrating the Hippo signaling pathway.
  • FIG. 2 shows representative photographs and graphs illustrating the results of Experimental Example 1.
  • FIG. 3 shows representative photographs and graphs illustrating the results of Experimental Example 2.
  • FIG. 4 is a representative photograph showing the results of Western blotting in Experimental Example 3.
  • FIG. 5 is a representative photograph showing the results of immunostaining of human small intestine-derived organoids in Experimental Example 4.
  • FIG. 6 is a representative photograph showing the results of immunostaining of human colon-derived organoids in Experimental Example 4.
  • FIG. 7 is a graph summarizing the results of FIGS.
  • FIG. 8 is a graph showing the results of Experimental Example 5.
  • FIG. 9 is a representative photograph showing the results of Experimental Example 6.
  • FIG. 10 is a graph showing the results of Experimental Example 7.
  • FIG. 10 is a graph showing the results of Experimental Example 7.
  • FIG. 11 is a representative photograph showing the results of Experimental Example 8.
  • FIG. 12 is a representative photograph showing the results of immunostaining of organoids in Experimental Example 9.
  • FIG. 13 is a representative photograph showing the results of immunostaining of organoids in Experimental Example 10.
  • FIG. 14 is a graph showing the results of Experimental Example 11.
  • FIG. 15 is a graph showing the results of Experimental Example 11.
  • FIG. 16 is a graph showing the results of quantitative real-time PCR in Experimental Example 12.
  • FIG. 17 is a graph showing the results of Western blotting in Experimental Example 12.
  • FIG. 18 is a representative photograph showing the results of immunostaining of organoids in Experimental Example 13.
  • FIG. 19 shows representative microscopic photographs of organoids in Experimental Example 13.
  • FIG. 20 is a representative photograph showing the results of immunostaining of organoids in Experimental Example 14.
  • FIG. 20 is a representative photograph showing the results of immunostaining of organoids in Experimental Example 14.
  • FIG. 21 shows representative microscopic photographs of organoids in Experimental Example 14.
  • FIG. 22 is a graph showing the results of Experimental Example 15.
  • FIG. 23 shows representative microscopic photographs of organoids in Experimental Example 16.
  • FIG. 24 is a graph showing the results of Experimental Example 16.
  • FIG. 25 is a representative photograph showing the results of immunostaining of organoids in Experimental Example 17.
  • FIG. 26 is a graph showing the results of Experimental Example 18.
  • FIG. 27 is a graph showing the results of Experimental Example 19.
  • FIG. 28 is a graph showing the results of Experimental Example 20.
  • FIG. 28 shows micrographs and a graph illustrating the results of Experimental Example 21.
  • FIG. 30 is a schematic diagram illustrating the outline of Experimental Example 22 and a graph showing the results of Experimental Example 22.
  • FIG. 31 is a schematic diagram illustrating an outline of Experimental Example 23.
  • FIG. 32 is a photograph showing the results of Experimental Example 23.
  • the present invention provides a drug for forming and growing organoids in the absence of extracellular matrix, the drug comprising an inhibitor of the Hippo signaling pathway as an active ingredient.
  • the agent of this embodiment may be an agent for growing organoids in the absence of extracellular matrix, containing an inhibitor of the Hippo signaling pathway as an active ingredient.
  • organoids in the absence of an extracellular matrix.
  • the drug of this embodiment makes it possible to grow organoids without using any animal-derived products, such as Matrigel (registered trademark), which is an extracellular matrix.
  • animal-derived products refer to substances derived from humans or non-human animals whose components have not been identified. It is expected that organoids can be applied to regenerative medicine using animal-derived product-free culture technology. In terms of animal welfare and cost, it is also more advantageous than conventional organoid culture technology.
  • the Hippo signaling pathway is a signaling pathway that is known to be involved in cell proliferation, apoptosis, stem cell self-renewal, etc., and is known to be an evolutionarily conserved pathway.
  • FIG. 1 is a schematic diagram explaining the Hippo signaling pathway.
  • the transcription factor TEAD is known to activate the transcription of genes involved in cell proliferation by binding with the coactivator YAP (Yes-associated protein), thereby promoting cell proliferation.
  • YAP coactivator YAP
  • phosphorylated YAP binds to the cytoplasmic protein 14-3-3 and cannot translocate to the nucleus, so it cannot act as a coactivator. For this reason, phosphorylation control, which determines the nuclear translocation of YAP, is a very important event in cell proliferation.
  • LATS large tumor suppressor kinase
  • any inhibitor that inhibits any stage of the above signaling pathway can be used without any particular limitations.
  • MST1 kinase or MST2 kinase MST1/2 kinase inhibitor
  • LATS1/2 kinase inhibitor an inhibitor of LATS1 kinase or LATS2 kinase
  • NCBI accession numbers for the amino acid sequence 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 sequence of human MST2 kinase are NP_001243241.1, NP_001243242.1, NP_006272.2, etc.
  • NCBI accession numbers for the amino acid sequence 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 sequence of human LATS2 kinase are NP_055387.2, etc.
  • inhibitors of the Hippo signaling pathway include, for example, Lats-IN-1 (CAS number: 1424635-83-5, hereinafter sometimes referred to as "Lats-IN”; also called “TRULI”), GA-017 (CAS number: 2351906-74-4), and TDI-011536 (CAS number: 2687970-96-1), which are inhibitors of LATS1 kinase and LATS2 kinase.
  • Extracellular matrices include Matrigel (registered trademark), collagen, fibronectin, proteoglycan, laminin, etc. Previously, it was impossible to grow organoids in the absence of an extracellular matrix.
  • a trace amount of extracellular matrix produced by the organoids themselves may be mixed into the culture medium, or for a trace amount of extracellular matrix to be mixed into the culture medium unintentionally.
  • a trace amount may be at the detection limit.
  • the organoids include epithelial organoids, such as small intestine-derived organoids, large intestine-derived organoids, stomach-derived organoids, biliary tract-derived organoids, pancreatic organoids, mammary gland-derived organoids, hepatocyte organoids, lung organoids, airway organoids, esophageal organoids, salivary gland organoids, etc.
  • epithelial organoids such as small intestine-derived organoids, large intestine-derived organoids, stomach-derived organoids, biliary tract-derived organoids, pancreatic organoids, mammary gland-derived organoids, hepatocyte organoids, lung organoids, airway organoids, esophageal organoids, salivary gland organoids, etc.
  • the organoid may be derived from a human or a non-human animal.
  • Non-human animals include mammals, such as rodents, such as mice, rats, hamsters, and guinea pigs; ungulates, such as pigs, cows, goats, horses, and sheep; carnivores, such as dogs and cats; and primates, such as rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees.
  • the drug of this embodiment is added to an organoid culture medium for use.
  • concentration of the drug of this embodiment in the organoid culture medium is preferably about 10 ⁇ M or more and 60 ⁇ M or less, and more preferably about 10 ⁇ M or more and 30 ⁇ M or less.
  • the organoid culture medium may be a conventional organoid culture medium from which the extracellular matrix has been removed.
  • the organoid culture medium is preferably a medium in which at least one of the following components i) to v) has been added to a basal medium.
  • the organoid culture medium may contain serum, but is preferably serum-free from the viewpoint of being free of animal-derived products.
  • Wnt agonist ii) at least one selected from the group consisting of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2), EGF (epidermal growth factor) and epiregulin (EREG), fibroblast growth factor 10 (FGF10), and gastrin-1 iii) bone morphogenetic protein (BMP) inhibitor iv) transforming growth factor- ⁇ (TGF- ⁇ ) inhibitor v) gamma secretase inhibitors
  • the organoid culture medium preferably contains at least one selected from the group consisting of IGF1, FGF2, EGF, and epiregulin.
  • the IGF1, FGF2, EGF, and epiregulin to be contained can be selected as appropriate.
  • the organoid culture medium preferably contains IGF1 and epiregulin, FGF2 and epiregulin, or IGF1 and FGF2, and more preferably contains IGF1 and FGF2.
  • any serum-free cell culture medium can be used as the basic medium.
  • a prescribed synthetic medium buffered to pH 7.2 or higher and pH 7.6 or lower with a carbonate buffer can be used.
  • an Advanced Dulbecco's Modified Eagle Medium/Ham's F-12 Mixed Medium DMEM/F12
  • DMEM/F12 an Advanced Dulbecco's Modified Eagle Medium/Ham's F-12 Mixed Medium
  • DMEM/F12 Advanced Dulbecco's Modified Eagle Medium/Ham's F-12 Mixed Medium
  • RPMI 1640 medium, Advanced RPMI medium, etc. can be used instead of DMEM/F12 medium.
  • the 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, the Wnt agonist is not limited to Wnt family proteins, but includes Wnt agonists that bind to and activate Frizzled receptor family members, inhibitors of intracellular ⁇ -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-3 ⁇ inhibitors.
  • the organoid culture medium preferably contains a Wnt agonist. More preferably, the Wnt agonist contains a complex of a Wnt protein and its stabilizing substance, afamin, and even more preferably, a complex of a Wnt protein and afamin, and R-spondin.
  • Wnt proteins derived from various organisms can be used. Among them, Wnt proteins derived from mammals are preferable. Examples of mammals include those similar to those mentioned above. Examples of mammalian 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 organoid culture medium, a combination of multiple Wnt proteins may be used.
  • the method of preparing Wnt protein includes, for example, a method of preparing Wnt protein using Wnt protein-expressing cells.
  • the origin of the Wnt protein-expressing cells is not particularly limited as long as the cells stably express Wnt protein, and may be cells that transiently express Wnt protein.
  • 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.
  • Wnt protein-expressing cells can be produced using known gene recombination techniques.
  • Wnt protein-expressing cells can be produced by inserting DNA encoding a desired Wnt protein into a known expression vector and introducing the resulting expression vector into an appropriate host cell.
  • the base sequence of the gene encoding the desired Wnt protein can be obtained from a known database such as GenBank.
  • 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 the amino acid sequence of the Wnt protein, so long as it has Wnt activity.
  • amino acid sequence other than the amino acid sequence of the Wnt protein examples include the amino acid sequence of an affinity tag.
  • the amino acid sequence of the Wnt protein does not need to be completely identical to an amino acid sequence that can be obtained from a publicly known database such as GenBank, and may be substantially the same as an amino acid sequence that can be obtained from a publicly known database, so long as it has Wnt activity.
  • amino acid sequence that is substantially identical to an amino acid sequence of a Wnt protein that can be obtained from a publicly known database such as GenBank is an amino acid sequence obtained from a publicly known database in which one or several amino acids have been deleted, substituted, or added.
  • 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 known methods for producing mutant peptides, such as site-directed mutagenesis.
  • 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 amino acid sequences that can be obtained from publicly known databases.
  • the concentration of Wnt protein is preferably 50 ng/mL or more, more preferably 100 ng/mL to 10 ⁇ g/mL or less, even more preferably 200 ng/mL to 1 ⁇ g/mL or less, and particularly preferably 300 ng/mL to 1 ⁇ g/mL or less.
  • R-Spondin examples include the R-spondin family consisting of R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4.
  • the R-spondin family is a secretory protein, and is known to be involved in the activation and control of the Wnt signaling pathway. In the organoid culture medium, a combination of multiple types of R-spondin may be used. In addition, 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.
  • GSK-3 ⁇ inhibitors examples include CHIR-99021 (CAS number: 252917-06-9), CHIR-98014 (CAS number: 252935-94-7), lithium, Kenpaullone (CAS number: 142273-20-9), 6-bromoindirubin-30-acetoxime, SB216763 (CAS number: 280744-09-4), SB415286 (CAS number: 264218-23-7), FRAT family members that block the interaction between GSK-3 and axin, and FRAT-derived peptides.
  • Afamin Afamin refers to a glycoprotein belonging to the albumin family, and is known to be present in blood or body fluids.
  • Serum which is usually added to a culture medium, contains afamin derived from the animal from which the serum was collected. Since serum contains impurities other than afamin, it is preferable to use afamin alone without using serum.
  • the afamin contained in the organoid culture medium is not particularly limited in origin, and afamin derived from various organisms can be used. Among these, afamin derived from mammals is preferable. Examples of mammals include those similar to those mentioned above.
  • the amino acid sequences of afamin from major mammals and the nucleotide sequences of the genes encoding them can be obtained from publicly known databases such as GenBank.
  • the amino acid sequence of human afamin is registered under the accession number AAA21612, and the nucleotide sequence of the gene encoding it is registered under the accession number L32140, while the amino acid sequence of bovine afamin is registered under the accession number DAA28569, and the nucleotide sequence of the gene encoding it is registered under the accession number GJ060968.
  • the afamin contained in the organoid culture medium may be natural afamin contained in serum, etc., purified by a known method, or it may be recombinant afamin. Recombinant afamin can be produced by appropriately using known gene recombination techniques.
  • 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 recombinant afamin, and purifying it using a known purification method.
  • the recombinant afamin may be afamin to which an affinity tag has been added.
  • affinity tag There is no particular limitation on the affinity tag to be added, and any known affinity tag can be appropriately selected for use.
  • the affinity tag is preferably an affinity tag that is recognized by a specific antibody, and examples of such tags include the FLAG tag, MYC tag, HA tag, and V5 tag.
  • Wnt proteins have strong hydrophobicity because certain serine residues are modified with fatty acids (palmitoleic acid). For this reason, it is widely known that Wnt proteins are very difficult to purify and store because they are prone to aggregation or denaturation in aqueous solutions.
  • Wnt proteins bind one-to-one with afamin to form a complex, which is soluble while maintaining high physiological activity.
  • Wnt protein-afamin complexes may be produced by culturing cells that express both Wnt proteins and afamin, or by co-culturing Wnt protein-expressing cells and afamin-expressing cells.
  • the concentration of afamin contained in the organoid culture medium is not particularly limited, but is preferably from 50 ng/mL to 10 ⁇ g/mL, more preferably from 100 ng/mL to 1 ⁇ g/mL, and even more preferably from 300 ⁇ g/mL to 1 ⁇ g/mL.
  • IGF1 also known as somatomedin C, is a factor secreted mainly by the liver in response to stimulation by growth hormone (GH). It is known that most cells in the human body (particularly those of muscles, bones, liver, kidneys, nerves, skin, lungs, etc.) are affected by IGF1. In addition to insulin-like effects, IGF1 has the function of regulating cell growth (particularly nerve cells), development, and cellular DNA synthesis.
  • the concentration of IGF1 contained in the organoid culture medium is not particularly limited, but is preferably 5 ng/mL or more and 1 ⁇ g/mL or less, more preferably 10 ng/mL or more and 1 ⁇ g/mL or less, and even more preferably 50 ng/mL or more and 500 ng/mL or less.
  • FGF2 is a basic fibroblast growth factor that binds to fibroblast growth factor receptors (FGFR) and promotes the proliferation and organization of vascular endothelial cells into tubular structures, i.e., angiogenesis.
  • Human FGF2 is known to have two isoforms, low molecular weight (LWL) and high molecular weight (HWL).
  • LWL is mainly present in the cytoplasm and acts in an autocrine manner, while HWL is present in the nucleus and is active via an intracellular intracrine mechanism.
  • the concentration of FGF2 contained in the organoid culture medium is not particularly limited, but is preferably 5 ng/mL or more and 500 ⁇ g/mL or less, more preferably 10 ng/mL or more and 300 ⁇ g/mL or less, and even more preferably 50 ng/mL or more and 100 ⁇ g/mL or less.
  • FGF10 is a molecule that is mainly secreted from mesenchymal tissues and acts on epithelia via FGFR2b, and is responsible for important epithelial-mesenchymal interactions in the formation and repair of various tissues.
  • the concentration of FGF10 contained in the organoid culture medium is not particularly limited, but is preferably 5 ng/mL or more and 1 ⁇ g/mL or less, more preferably 10 ng/mL or more and 1 ⁇ g/mL or less, and even more preferably 30 ng/mL or more and 500 ng/mL or less.
  • EGF EGF is a potent mitogen for a variety of cultured ectodermal and mesodermal cells and has profound effects on the differentiation of certain fibroblasts and specific cells.
  • the EGF precursor exists as a membrane-bound molecule that is proteolytically cleaved to generate a 53-amino acid peptide hormone that stimulates cells.
  • the concentration of EGF contained in the organoid culture medium is preferably 5 ng/mL or more and 500 ng/mL or less, more preferably 10 ng/mL or more and 400 ng/mL or less, and even more preferably 50 ng/mL or more and 200 ng/mL or less.
  • EREG EREG is an EGF-like growth factor that specifically binds to ErbB1 and ErbB4 of the tyrosine kinase (ErbB) family receptors (ErbB1-4). It is known to stimulate the proliferation of keratinocytes, hepatocytes, fibroblasts, and vascular endothelial cells. EREG is also expressed mainly in malignant tumors of the bladder, lung, kidney, colon, etc., as well as in the placenta and peripheral blood leukocytes.
  • ErbB tyrosine kinase
  • the concentration of EREG contained in the organoid culture medium is not particularly limited, but is preferably 5 ng/mL or more and 1 ⁇ g/mL or less, more preferably 10 ng/mL or more and 1 ⁇ g/mL or less, and even more preferably 50 ng/mL or more and 500 ng/mL or less.
  • 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. Type II receptor phosphorylates type I receptor, which activates the receptor kinase. Type I receptor then phosphorylates specific receptor substrates (SMADs), which lead to transcriptional activity by signal transduction pathways.
  • SADs specific receptor substrates
  • BMP inhibitors are agents that bind to BMP molecules to form complexes that neutralize BMP activity, e.g., agents that bind to BMP receptors and block or inhibit the binding of BMP molecules to the receptor, acting as antagonists or inverse agonists.
  • 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.
  • BMP inhibitors are preferably natural BMP binding proteins, examples of which include chordin-like proteins such as Noggin, Gremlin, Chordin, and Chordin domain; follistatin-related proteins such as Follistatin and Follistatin domain; DAN-like proteins such as DAN and DAN cysteine-knot domain; sclerostin/SOST, decorin, ⁇ -2 macroglobulin, etc.
  • chordin-like protein or DAN-like protein is preferred, with chordin-like protein being more preferred.
  • noggin is preferred.
  • Chordin-like protein and DAN-like protein are diffusible proteins that bind to BMP molecules with various affinities and can inhibit the BMP molecules from approaching signaling receptors. When culturing epithelial stem cells, the loss of stem cells can be prevented by adding these BMP inhibitors to the organoid culture medium.
  • the concentration of the BMP inhibitor contained in the organoid culture medium is preferably 10 ng/mL or more and 100 ng/mL or less, more preferably 20 ng/mL or more and 100 ng/mL or less, and even more preferably 50 ng/mL or more and 100 ng/mL or less.
  • TGF- ⁇ inhibitor TGF- ⁇ is a type of growth factor, and is produced by almost all cells, including those in the kidney, bone marrow, and platelets. There are five subtypes of TGF- ⁇ ( ⁇ 1 to ⁇ 5). It is also known that TGF- ⁇ promotes the proliferation of osteoblasts, as well as the synthesis and proliferation of connective tissues such as collagen, and acts inhibiting the proliferation of epithelial cells and osteoclasts.
  • a TGF- ⁇ inhibitor is, for example, a drug that blocks or inhibits the binding of TGF- ⁇ to a TGF- ⁇ receptor, and binds to TGF- ⁇ to form a complex that neutralizes TGF- ⁇ activity.
  • a TGF- ⁇ inhibitor is, for example, a drug that binds to a TGF- ⁇ receptor and blocks or inhibits the binding of TGF- ⁇ to the receptor, and acts as an antagonist or inverse agonist.
  • TGF- ⁇ inhibitors include, for example, A83-01 (CAS number: 909910-43-6), ALK5 Inhibitor I (3-(pyridin-2-yl)-4-(4-quinonyl)-1H-pyrazole), LDN193189 (CAS number: 1062368-24-4), SB-431542 (CAS number: 301836-41-9), SB-505124 (CAS number: 694433-59-5), SD-208 (CAS number: 627536-09-8), SB-5 25334 (CAS number: 356559-20-1), LY364947 (CAS number: 396129-53-6), LY2157299 (CAS number: 700874-72-2), TGF- ⁇ RI Kinase Inhibitor II 616452 (CAS number: 446859-33-2), TGF- ⁇ RI Kinase Inhibitor III 616453 (CAS number: 356559-13-2), TGF- ⁇ RI Kinase Inhibitor or IX 616463 (4-((4-((2,6-
  • the concentration of the TGF- ⁇ inhibitor contained in the organoid culture medium is preferably 100 nM or more and 10 ⁇ M or less, more preferably 500 nM or more and 5 ⁇ M or less, and even more preferably 500 nM or more and 2 ⁇ M or less.
  • gamma-secretase inhibitor examples include BMS299897 (CAS number: 290315-45-6), DAPT (CAS number: 208255-80-5), DBZ (CAS number: 209984-56-5), JLK6 (CAS number: 62252-26-0), L-685458 (CAS number: 292632-98-5), LY411575 (CAS number: 209984-57-6), etc. Among them, LY411575 is preferable.
  • the concentration of the gamma-secretase inhibitor contained in the organoid culture medium is preferably 1 nM or more and 10 ⁇ M or less, and more preferably 100 nM or more and 1 ⁇ M or less.
  • the organoid culture medium may further contain a Rho-kinase (Rock) inhibitor.
  • 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).
  • Y-27632 As a Rock inhibitor, it is preferable to add it during the first about 2 days of culturing the stem cells dispersed into single cells.
  • the Y-27632 contained in the organoid culture medium is preferably about 10 ⁇ M.
  • the organoid culture medium further contains a cytokine of the cytokine family that binds to gp130, such as oncostatin M.
  • the concentration of oncostatin M contained in the organoid culture medium may be, for example, 1 ng/mL to 10 ⁇ g/mL, for example, 1 ng/mL to 1 ⁇ g/mL, or for example, 5 ng/mL to 100 ng/mL.
  • the organoid culture medium may further contain gastrin (or a suitable substitute such as Leu15-gastrin).
  • the concentration of gastrin or a suitable substitute contained in the organoid culture medium may be, for example, 1 ng/mL to 10 ⁇ g/mL, for example, 1 ng/mL to 1 ⁇ g/mL, or for example, 5 ng/mL to 100 ng/mL.
  • the organoid culture medium may further contain at least one amino acid.
  • 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 or more and 1 g/L or less (usually 0.1 g/L or more and 0.75 g/L or less).
  • the concentration of other amino acids contained in the organoid culture medium is 0.001 g/L or more and 1 g/L (usually 0.01 g/L or more and 0.15 g/L or less).
  • the amino acids may be synthetic.
  • the organoid culture medium may further contain at least one vitamin.
  • vitamins include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), calcium D-pantothenate (vitamin B5), pyridoxal/pyridoxamine/pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL- ⁇ -tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).
  • vitamins include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), calcium D-pantothenate (vitamin B5), pyridoxal/pyridoxamine/pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascor
  • the organoid culture medium may further contain at least one inorganic salt.
  • the inorganic salt is for helping to maintain the osmotic balance of the cells and for helping to regulate the 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 chlorides, phosphates, sulfates, nitrates, and bicarbonates.
  • 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 O, and the like.
  • the organoid culture medium may further contain at least one sugar that can be a carbon energy source.
  • sugars include glucose, galactose, maltose, and fructose. Among them, glucose is preferred, and D-glucose (dextrose) is particularly preferred.
  • concentration of sugar contained in the organoid culture medium is preferably 1 g/L or more and 10 g/L or less.
  • the organoid culture medium may further contain at least one trace element.
  • trace elements include barium, bromium, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum, or ions thereof.
  • the organoid culture medium may further contain at least one additional drug.
  • drugs include nutrients or growth factors that have been reported to improve stem cell culture, such as cholesterol, transferrin, albumin, insulin, progesterone, putrescine, selenite, etc.
  • the present invention provides a method for the proliferation of organoid, comprising culturing organoid in the presence of the inhibitor of Hippo signaling pathway and in the absence of extracellular matrix.
  • the method for the proliferation of organoid in this embodiment can also be said to be the method for the production of organoid.
  • the method of this embodiment makes it possible to grow organoids in the absence of extracellular matrix.
  • growing means increasing the number of cells that make up the organoid, and can also be called “culturing.”
  • the inhibitor of the Hippo signaling pathway, the extracellular matrix, the organoid, and the like are the same as those described above. That is, in the method of this embodiment, the inhibitor of the Hippo signaling pathway is preferably an MST1/2 kinase inhibitor or a LATS1/2 kinase inhibitor. Also, in the method of this embodiment, it is preferable that the organoid is an epithelial organoid.
  • the organoids can also be cultured under serum-free conditions. By growing the organoids in the absence of extracellular matrix and serum, it is possible to obtain organoids produced without using any animal-derived products.
  • the present invention provides organoids grown by the above-described growth method.
  • the organoids of this embodiment are produced free of animal-derived products. This makes them easy to apply to regenerative medicine.
  • the organoid of this embodiment may be a gastrointestinal organoid.
  • Conventional gastrointestinal organoids cultured in the presence of Matrigel are spherical in shape, with a monolayer of epithelial cells that maintains apical polarity and the inside facing the apical side.
  • the organoids of this embodiment have a spherical morphology in which the cells are stratified and the apical polarity of the epithelial cells is not maintained, which differs from the morphology of conventional gastrointestinal organoids cultured in the presence of Matrigel (registered trademark).
  • the organoids of this embodiment are thought to differ from conventional organoids in terms of gene expression patterns, etc. However, it is unclear and unrealistic whether it is possible to identify the differences in gene expression patterns and clearly distinguish between conventional organoids and organoids of this embodiment. For this reason, it is thought to be practical to define the organoids of this embodiment by the manufacturing method.
  • the present invention provides an agent for establishing an organoid in the absence of an extracellular matrix, the agent comprising an inhibitor of the Hippo signaling pathway as an active ingredient.
  • organoids can be grown in the absence of extracellular matrix by adding an inhibitor of the Hippo signaling pathway to the culture medium.
  • organoids can be established from tissues derived from living organisms by adding an inhibitor of the Hippo signaling pathway to the culture medium.
  • organoids can be established from tissues derived from living organisms in the absence of extracellular matrix, and that organoids can also be established from single cells without the use of extracellular matrix.
  • the inhibitor of the Hippo signaling pathway, the extracellular matrix, the organoid, etc. are the same as those described above. That is, in the method of this embodiment, the inhibitor of the Hippo signaling pathway is preferably an MST1/2 kinase inhibitor or a LATS1/2 kinase inhibitor. Also, in the drug of this embodiment, it is preferable that the organoid is an epithelial organoid.
  • the present invention provides the method for producing organoid, comprising the step of establishing organoid in the presence of the inhibitor of Hippo signaling pathway and in the absence of extracellular matrix.
  • the method for producing organoid of this embodiment can also be said to be the method for establishing organoid.
  • the method of this embodiment allows organoids to be established from biological tissue in the absence of extracellular matrix, and also allows organoids to be established from single cells without using extracellular matrix. Furthermore, the method of this embodiment allows organoids to be established under serum-free conditions. By establishing organoids in the absence of extracellular matrix and serum, it is possible to obtain organoids that are free of animal-derived products from the time of establishment.
  • the inhibitor of the Hippo signaling pathway, the extracellular matrix, the organoid, and the like are the same as those described above. That is, in the production method of this embodiment, the inhibitor of the Hippo signaling pathway is preferably an MST1/2 kinase inhibitor or a LATS1/2 kinase inhibitor. Also, in the production method of this embodiment, it is preferable that the organoid is an epithelial organoid.
  • the present invention provides an organoid produced by the above-mentioned production method.
  • the inventors have demonstrated that the above-mentioned production method can establish organoids from tissues derived from a living body in the absence of extracellular matrix, and can also establish organoids from single cells without using extracellular matrix.
  • the organoids of this embodiment particularly when established under serum-free conditions, are free of animal-derived products from the time of establishment. This makes them particularly easy to transplant into a living body and apply to regenerative medicine. Therefore, in one embodiment, the present invention provides a regenerative medicine preparation containing, as an active ingredient, organoids produced by the above-mentioned production method.
  • the organoids of this embodiment are thought to differ from conventional organoids in terms of gene expression patterns, etc. However, it is unclear and unrealistic whether it is possible to identify the differences in gene expression patterns and clearly distinguish between conventional organoids and organoids of this embodiment. For this reason, it is thought to be practical to define the organoids of this embodiment by the manufacturing method.
  • the present invention provides a method for screening for a drug that enables the culture of an organoid in the absence of an extracellular matrix, comprising the steps of culturing an organoid in the presence of a test substance and in the absence of an extracellular matrix, and evaluating the proliferation of the organoid, wherein a higher proliferation of the organoid compared to that in the absence of the test substance indicates that the test substance is a drug that enables the culture of an organoid in the absence of an extracellular matrix.
  • the screening method of this embodiment makes it possible to screen for drugs that enable the cultivation of organoids in the absence of extracellular matrix.
  • the extracellular matrix, organoids, etc. are the same as those described above.
  • the test substance is not particularly limited, and for example, a natural compound library, a synthetic compound library, an existing drug library, etc. can be used.
  • the proliferation of organoids can be evaluated by any suitable method, such as measuring the number of cells based on microscopic observation, measuring the number of cells by measuring ATP, or measuring the number of cells using a tetrazolium compound such as MTT. If the proliferation of organoids is higher than in the absence of the test substance, it can be determined that the test substance is a drug that enables the culture of organoids in the absence of extracellular matrix.
  • Human small intestine-derived organoids and human large intestine-derived organoids were established from tissues of patients with gastrointestinal tumors who had given informed consent, in accordance with an ethical research plan approved by the Keio University School of Medicine Ethics Committee.
  • Each organoid was dissociated into single cells using TrypLE Express (Thermo Fisher Scientific) to obtain a cell suspension. Then, 1 x 10 5 cells/well were seeded on a 48-well plate together with 25 ⁇ L of Matrigel (registered trademark). Then, after the Matrigel (registered trademark) gelled, 100 ⁇ L/well of medium having the composition shown in Table 1 below was added and cultured at 37 ° C. 10 mM Lats-IN was added to the medium of the test group. Then, the medium was replaced every 2 days.
  • Matrigel registered trademark
  • Figure 2 shows representative photographs of each organoid after 7 days of culture and a graph showing the cell count.
  • the top panel of Figure 2 shows the results for organoids derived from human small intestine, and the bottom panel of Figure 2 shows the results for organoids derived from human large intestine.
  • Cell proliferation was measured by measuring adenosine triphosphate (ATP) using a commercially available kit (product name: CellTiter-Glo 3D, Promega).
  • ATP adenosine triphosphate
  • the top left of Figure 2 is a photograph of a control (Ctrl) human small intestine-derived organoid cultured in the absence of Lats-IN
  • the top center of Figure 2 is a photograph of a human small intestine-derived organoid cultured in the presence of Lats-IN.
  • the scale bar is 200 ⁇ m.
  • the top right of Figure 2 is a graph showing the results of measuring the number of cells per well of human small intestine-derived organoids cultured in the presence of Lats-IN and as a control (Ctrl).
  • the vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present.
  • the bottom left of Figure 2 is a photograph of a control (Ctrl) human colon-derived organoid cultured in the absence of Lats-IN
  • the bottom center of Figure 2 is a photograph of a human colon-derived organoid cultured in the presence of Lats-IN.
  • the scale bar is 200 ⁇ m.
  • the bottom right of Figure 2 is a graph showing the results of measuring the number of cells per well of human colon-derived organoids cultured in the presence of Lats-IN and as a control (Ctrl).
  • the vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present.
  • Figure 3 shows representative photographs of each organoid after 7 days of culture and a graph showing the cell count.
  • the top panel of Figure 3 shows the results for human small intestine-derived organoids, and the bottom panel of Figure 3 shows the results for human large intestine-derived organoids.
  • the top left of Figure 3 is a photograph of a control (Ctrl) human small intestine-derived organoid cultured in the absence of Lats-IN
  • the top center of Figure 3 is a photograph of a human small intestine-derived organoid cultured in the presence of Lats-IN.
  • the scale bar is 100 ⁇ m.
  • the top right of Figure 3 is a graph showing the results of measuring the cell count of human small intestine-derived organoids cultured in the presence of Lats-IN and as a control (Ctrl) by measuring ATP using a commercially available kit (product name: CellTiter-Glo 3D, Promega).
  • the vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present.
  • the bottom left of Figure 3 is a photograph of a control (Ctrl) human colon-derived organoid cultured in the absence of Lats-IN
  • the bottom center of Figure 3 is a photograph of a human colon-derived organoid cultured in the presence of Lats-IN.
  • the scale bar is 100 ⁇ m.
  • the bottom right of Figure 3 is a graph showing the results of measuring the cell count of human colon-derived organoids cultured in the presence of Lats-IN and as a control (Ctrl).
  • the vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present.
  • Figure 4 shows representative photographs showing the results of Western blotting.
  • pYAP indicates phosphorylated YAP
  • YAP indicates total YAP (total of phosphorylated YAP and non-phosphorylated YAP).
  • Phospho-YAP Ser127
  • YAP indicates total YAP (total of phosphorylated YAP and non-phosphorylated YAP).
  • Phospho-YAP Ser127
  • YAP Total-YAP (#14074, Cell Signaling Technology) was used as the antibody against total YAP.
  • the leftmost column of Figure 5 is a photograph showing the results of immunostaining of human small intestine-derived organoids cultured in the presence of Matrigel (registered trademark) but in the absence of Lats-IN (Ctrl).
  • the top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc)
  • the second row from the top is an image showing the detection results of Ki67
  • the third row from the top is an image showing the detection results of YAP
  • the fourth row from the top is an image showing the detection results of nuclei (Nuc).
  • the fields of view in the second, third, and fourth rows from the top correspond to the area enclosed in a square in the top row.
  • the second column from the left in Figure 5 is a photograph showing the results of immunostaining of human small intestine-derived organoids cultured in the presence of Matrigel (registered trademark) and Lats-IN.
  • the top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc)
  • the second row from the top is an image showing the detection results of Ki67
  • the third row from the top is an image showing the detection results of YAP
  • the fourth row from the top is an image showing the detection results of nuclei (Nuc).
  • the fields of view in the second, third, and fourth rows from the top correspond to the area enclosed in a square in the top row.
  • the scale bar indicates 50 ⁇ m.
  • the third column from the left in Figure 5 is a photograph showing the results of immunostaining of human small intestine-derived organoids cultured in the absence of Matrigel (registered trademark) and in the absence of Lats-IN (Ctrl).
  • the top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc)
  • the second row from the top is an image showing the detection results of Ki67
  • the third row from the top is an image showing the detection results of YAP
  • the fourth row from the top is an image showing the detection results of nuclei (Nuc).
  • the fields of view in the second, third, and fourth rows from the top correspond to the area enclosed in a square in the top row.
  • the rightmost column of Figure 5 is a photograph showing the results of immunostaining of human small intestine-derived organoids cultured in the presence of Lats-IN in the absence of Matrigel (registered trademark).
  • the top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc)
  • the second row from the top is an image showing the detection results of Ki67
  • the third row from the top is an image showing the detection results of YAP
  • the fourth row from the top is an image showing the detection results of nuclei (Nuc).
  • the fields of view in the second, third, and fourth rows from the top correspond to the area enclosed in a square in the top row.
  • the leftmost column of Figure 6 is a photograph showing the results of immunostaining of human colon-derived organoids cultured in the presence of Matrigel (registered trademark) but in the absence of Lats-IN (Ctrl).
  • the top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc)
  • the second row from the top is an image showing the detection results of Ki67
  • the third row from the top is an image showing the detection results of YAP
  • the fourth row from the top is an image showing the detection results of nuclei (Nuc).
  • the fields of view in the second, third, and fourth rows from the top correspond to the area enclosed in a square in the top row.
  • the second column from the left in Figure 6 is a photograph showing the results of immunostaining of human colon-derived organoids cultured in the presence of Matrigel (registered trademark) and Lats-IN.
  • the top row is an image merging the detection results of Ki67, YAP, and nucleus (Nuc)
  • the second row from the top is an image showing the detection results of Ki67
  • the third row from the top is an image showing the detection results of YAP
  • the fourth row from the top is an image showing the detection results of nucleus (Nuc).
  • the fields of view in the second, third, and fourth rows from the top correspond to the area enclosed in a square in the top row.
  • the third column from the left in Figure 6 is a photograph showing the results of immunostaining of human colon-derived organoids cultured in the absence of Matrigel (registered trademark) and in the absence of Lats-IN (Ctrl).
  • the top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc)
  • the second row from the top is an image showing the detection results of Ki67
  • the third row from the top is an image showing the detection results of YAP
  • the fourth row from the top is an image showing the detection results of nuclei (Nuc).
  • the fields of view in the second, third, and fourth rows from the top correspond to the area enclosed in a square in the top row.
  • the rightmost column of Figure 6 is a photograph showing the results of immunostaining of human colon-derived organoids cultured in the presence of Lats-IN in the absence of Matrigel (registered trademark).
  • the top row is an image merging the detection results of Ki67, YAP, and nuclei (Nuc)
  • the second row from the top is an image showing the detection results of Ki67
  • the third row from the top is an image showing the detection results of YAP
  • the fourth row from the top is an image showing the detection results of nuclei (Nuc).
  • the fields of view in the second, third, and fourth rows from the top correspond to the area enclosed in a square in the top row.
  • Figure 7 is a graph summarizing the results of Figures 5 and 6.
  • Matrigel registered trademark
  • YAP was localized in the cytoplasm, and when the number of Ki67-positive cells, which indicates cell proliferation, was counted, the percentage of Ki67-positive cells was approximately 10% of the total cells.
  • Figure 8 is a graph showing the results of measuring the proliferation of human small intestine-derived organoids and human large intestine-derived organoids in the absence of Lats-IN (Ctrl), in the presence of Lats-IN (Lats-IN), in the presence of Lats-IN and MYF01-37 (Lats-IN+MYF), and in the presence of MYF01-37 (MYF).
  • Cell proliferation was measured by measuring ATP using a commercially available kit (product name: CellTiter-Glo 3D, Promega).
  • the vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present.
  • Figure 9 shows representative photographs showing the results of organoid formation from single cells in the presence or absence of Matrigel (registered trademark) and in the absence or presence of Lats-IN (final concentration 10 ⁇ M).
  • the arrowheads indicate cells or organoids.
  • D1, D4, and D7 indicate the first, fourth, and seventh days after the start of culture, respectively.
  • the denominator indicates the number of single cells seeded
  • the numerator indicates the number of organoids formed. Also shown is the percentage of cells that formed organoids among the single cells seeded.
  • Crypts were isolated from human small intestine and large intestine tissues.
  • the isolated crypts were cultured in the absence of Matrigel (registered trademark) and in the absence or presence of Lats-IN (final concentration 10 ⁇ M).
  • Figure 10 is a graph showing the results of measuring the proliferation of crypts isolated from human colon-derived tissue over time.
  • Cell proliferation was measured by measuring ATP using a commercially available kit (product name: CellTiter-Glo 3D, Promega).
  • the vertical axis of the graph shows luminescence intensity (relative value), which indicates the amount of ATP present.
  • the isolated crypts increased in number enough to be passaged once, but then gradually underwent apoptosis, and completely died after the second passage.
  • Figure 11 shows representative photographs of the results of culturing gastric, biliary, and mammary organoids in the absence of Matrigel (registered trademark) and in the absence of Lats-IN (Ctrl), or in the absence of Matrigel (registered trademark) and in the presence of Lats-IN (Lats-IN).
  • Scale bar is 100 ⁇ m.
  • FIG. 12 shows a representative photograph of immunostained organoids derived from human small intestine.
  • the bottom of Figure 12 shows a representative photograph of immunostained organoids derived from human large intestine.
  • the scale bar in Figure 12 indicates 50 ⁇ m.
  • Immunostaining detected the basement membrane marker integrin ⁇ 4 (Int ⁇ 4), the apical marker Ezrin, the cell-cell adhesion marker ⁇ -catenin ( ⁇ Cat), the Paneth cell marker lysozyme, the germ cell marker Mucin2 (MUC2), the enteroendocrine cell marker Chromogranin A (CHGA), and the nucleus (Nuc).
  • expression of the stem cell marker Lgr5 was detected by the fluorescence of TdTomato, a fluorescent protein introduced into the Lgr5 locus of the cells.
  • Matrigel registered trademark
  • spherical with a single layer of epithelial cell sheets that maintain apical polarity, with the apical side facing inwards, and each organoid contains differentiated cells such as stem cells, embryonic cells, and enteroendocrine cells.
  • FIG. 13 The top of Figure 13 is a representative photograph of immunostained human small intestine-derived organoids.
  • the bottom of Figure 13 is a representative photograph of immunostained human large intestine-derived organoids.
  • the scale bar in Figure 13 indicates 50 ⁇ m.
  • Immunostaining detected the basement membrane marker integrin ⁇ 4 (Int ⁇ 4), the apical marker Ezrin, the cell-cell adhesion marker ⁇ -catenin ( ⁇ Cat), the Paneth cell marker lysozyme, the germ cell marker Mucin2 (MUC2), the enteroendocrine cell marker Chromogranin A (CHGA), and the nucleus (Nuc).
  • expression of the stem cell marker Lgr5 was detected by the fluorescence of TdTomato, a fluorescent protein introduced into the Lgr5 locus of the cells.
  • Figure 14 is a graph showing the results of measuring the proliferation of human small intestine-derived organoids and human large intestine-derived organoids cultured under conditions in which all niche factors of WRENAIF were added and under conditions in which some of these niche factors were removed.
  • the medium to which all niche factors of WRENAIF were added the medium with the composition shown in Table 1 above was used, and as the medium from which some of these niche factors were removed, the medium with the composition shown in Table 1 above from which the corresponding niche factors were removed was used.
  • Cell proliferation was measured by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D", Promega).
  • the vertical axis of the graph shows the luminescence intensity (relative value) indicating the amount of ATP present.
  • Figure 15 is a graph showing the results of measuring the proliferation of human small intestine-derived organoids cultured in the absence of Matrigel (registered trademark), in the absence or presence of Lats-IN (final concentration 10 ⁇ M), under conditions in which all niche factors of WRENAIF were added, and under conditions in which some of these niche factors were omitted.
  • P0 to P16 represent passage numbers 0 to 16.
  • darker colors in the graph indicate that passage was successful, and lighter colors indicate that passage was not successful.
  • Human small intestine-derived organoids and human large intestine-derived organoids were cultured in the absence or presence of Matrigel (registered trademark) and in the absence or presence of Lats-IN (final concentration 10 ⁇ M), and the expression level of the AXIN2 gene, whose expression is regulated by Wnt, was examined by quantitative real-time PCR.
  • Figure 16 is a graph showing the results of quantitative real-time PCR. The results showed that when organoids were cultured in the presence of Matrigel (registered trademark), expression of AXIN2 was observed, whereas when organoids were cultured in the absence of Matrigel (registered trademark), the expression level of AXIN2 was significantly reduced. Furthermore, when organoids were cultured in the absence of Matrigel (registered trademark) but in the presence of Lats-IN, the expression level of AXIN2 was increased to the same level as in the presence of Matrigel (registered trademark).
  • human small intestine-derived organoids were cultured in the absence or presence of Matrigel (registered trademark) and in the absence or presence of Lats-IN (final concentration 10 ⁇ M), and ERK, which is phosphorylated by EGF signaling, was detected by Western blotting.
  • Figure 17 is a graph showing the results of Western blotting.
  • pERK indicates phosphorylated ERK
  • total ERK indicates total ERK (the sum of phosphorylated ERK and non-phosphorylated ERK).
  • pYAP indicates phosphorylated YAP
  • total YAP indicates total YAP (the sum of phosphorylated YAP and non-phosphorylated YAP).
  • Phospho-ERK Thr202/Tyr204
  • Total-ERK #4695, Cell Signaling Technology
  • Phospho-YAP Ser127
  • Ser127 was used as the antibody against phosphorylated YAP.
  • the antibody used for total YAP was total-YAP (#14074, Cell Signaling Technology).
  • ERK phosphorylation was observed when organoids were cultured in the presence of Matrigel (registered trademark), whereas ERK phosphorylation was significantly reduced when organoids were cultured in the absence of Matrigel (registered trademark). Furthermore, it was shown that ERK phosphorylation increased to a level equivalent to that in the presence of Matrigel (registered trademark) when organoids were cultured in the absence of Matrigel (registered trademark) but in the presence of Lats-IN.
  • Figure 18 shows a representative photograph of immunostained human colon-derived organoids on the 7th day of culture after 18 passages following establishment.
  • the scale bar in Figure 18 indicates 50 ⁇ m.
  • Immunostaining detected the apical marker Ezrin, the basement membrane marker integrin ⁇ 4 ( ⁇ 4-Integrin), the cell-cell adhesion marker ⁇ -catenin ( ⁇ -Catenin), the germ cell marker Mucin 2 (MUC2), the enteroendocrine cell marker Chromogranin A (CHGA), and nuclei (Nuc).
  • Matrigel registered trademark
  • spherical with a monolayer of epithelial cells that maintains apical polarity, with the apical side facing inward, and each organoid contains differentiated cells such as stem cells, embryonic cells, and enteroendocrine cells.
  • Figure 19 shows micrographs of human colon-derived organoids on the 7th day of culture, which had been established in the absence of Matrigel (registered trademark) and in the presence of Lats-IN and had been passaged 18 times.
  • the scale bar in the left photograph of Figure 19 is 500 ⁇ m, and the scale bar in the right photograph of Figure 19 is 100 ⁇ m.
  • Figure 20 shows representative photographs of immunostained human colon-derived organoids that were established in the absence of Matrigel (registered trademark) and the presence of Lats-IN, and then cultured for 7 days in the presence of Matrigel (registered trademark) for the first time after 18 passages.
  • the scale bar in Figure 20 indicates 50 ⁇ m.
  • Immunostaining detected the apical marker Ezrin, the basement membrane marker integrin ⁇ 4 ( ⁇ 4-Integrin), the cell-cell adhesion marker ⁇ -catenin ( ⁇ -Catenin), the germ cell marker Mucin 2 (MUC2), the enteroendocrine cell marker Chromogranin A (CHGA), and nuclei (Nuc).
  • Figure 21 shows a micrograph of a human colon-derived organoid on the 7th day after it was established in the absence of Matrigel (registered trademark) and the presence of Lats-IN, and cultured in the presence of Matrigel (registered trademark) for the first time after 18 passages.
  • the scale bar in the left photograph of Figure 21 is 500 ⁇ m, and the scale bar in the right photograph of Figure 21 is 100 ⁇ m.
  • the cells were harvested and suspended in growth medium supplemented with 2, 5, 10, 20, and 50 ⁇ M TDI-011536 (CAS number: 2687970-96-1), and seeded on an Ultra Low attachment 96-well plate at 2,000 to 4,000 cells/well, preferably 2,000 cells/well. Cells in the growth medium without TDI-011536 were also prepared as a control.
  • the growth medium was a basal medium (Advanced DMEM/F-12 medium, 10 mM HEPES, 2 mM GlutaMAX, 100 U penicillin, 100 ⁇ g/mL streptomycin) supplemented with 10% Afamin-Wnt-3A conditioned medium, 2% R-spondin 1 conditioned medium, 2% Noggin conditioned medium, and 20 ng/mL Oncostatin M.
  • Basal medium Advanced DMEM/F-12 medium, 10 mM HEPES, 2 mM GlutaMAX, 100 U penicillin, 100 ⁇ g/mL streptomycin
  • Figure 22 is a graph showing the results of measuring the number of viable cells.
  • the number of viable cells was measured to be 16 and 17 times higher than in the control group.
  • An increase in the number of viable cells of approximately 3 times was also observed with other LATS-INs, such as TRULI, and it was revealed that an increase in the number of viable cells of human hepatocyte organoids was observed in the presence of LATS-IN in the absence of Matrigel.
  • each thawed primary human hepatocyte was stained with APC-labeled mouse anti-human EpCAM antibody, and EpCAM-negative cells were collected using a cell sorter.
  • the collected cells were then suspended in growth medium supplemented with 20 ⁇ M TDI-011536, and seeded onto an Ultra Low attachment 96-well plate at 3,000-4,000 cells/well. The cells were then cultured to obtain hepatocyte organoids. For comparison, cells were also prepared using growth medium without TDI-011536 and growth medium without Oncostatin M.
  • Hepatocyte organoids were passaged in the absence of Matrigel (registered trademark) as follows. First, calcium ions and magnesium ions were chelated using 1 mM EDTA/phosphate buffered saline (PBS). Next, TrypLE Express (Thermo Fisher Scientific) was added and incubated at 37°C for 5 minutes to dissociate into single cells.
  • Matrigel registered trademark
  • the collected cells were then suspended in growth medium supplemented with 20 ⁇ M TDI-011536 and seeded onto an Ultra Low attachment 96-well plate at 2,000 cells/well.
  • Figure 23 shows micrographs of each cell taken 1 day and 9 days after the start of culture.
  • the scale bar is 500 ⁇ m.
  • Figure 24 is a graph showing the results of measuring changes in cell proliferation over time.
  • the vertical axis of the graph shows cell proliferation (fold increase), and the horizontal axis shows the number of days of culture.
  • OSM stands for oncostatin M
  • Lats IN stands for Lats kinase inhibitor (TDI-011536).
  • human hepatocyte organoids were fixed using 1% formaldehyde/HEPES-buffered saline (HBS). Then, the fixed human hepatocyte organoids were perforated using 1% Triton-X100/PBS. Then, the human hepatocyte organoids were blocked using 1% BSA/PBS. Next, HNF4 ⁇ , a hepatocyte marker, was immunostained using mouse anti-HNF4 ⁇ antibody (Thermo Fisher Scientific).
  • Figure 25 is a micrograph showing the results of fluorescent immunostaining.
  • the scale bar is 50 ⁇ m.
  • “Matrigel (+)” indicates the results for hepatocyte organoids cultured in the presence of Matrigel (registered trademark)
  • “Matrigel (-)” indicates the results for hepatocyte organoids cultured in the absence of Matrigel (registered trademark).
  • “Nuc” indicates the results of staining the nucleus.
  • LATS kinase inhibitors used were GA-017 (CAS number: 2351906-74-4), TDI-011536 (CAS number: 2687970-96-1), and TRULI (CAS number: 1424635-83-5, also known as "Lats-IN” or "Lats-IN-1").
  • Figure 26 is a graph showing the results of measuring the cell count of each organoid after six days of culture using a commercially available kit (product name: CellTiter-Glo 3D, Promega) to measure ATP.
  • the vertical axis of the graph shows the luminescence intensity (relative value) indicating the amount of ATP present.
  • the left side of Figure 26 shows the results for organoids derived from human large intestine, and the right side of Figure 26 shows the results for organoids derived from human small intestine.
  • Figure 27 is a graph showing the results of organoid formation from single cells in the presence (+) or absence (-) of Matrigel (registered trademark), and in the absence (-) or presence (+, final concentration 10 ⁇ M) of TRULIS.
  • the denominator shows the number of single cells seeded
  • the numerator shows the number of organoids formed.
  • the vertical axis of the graph shows the percentage of cells that formed organoids out of the single cells seeded.
  • FIG. 28 is a graph showing the results of forming organoids from single cells in the presence (+) or absence (-) of Matrigel (registered trademark), in the absence (-) or presence (+, final concentration 10 ⁇ M) of TRUL I, and measuring the colony size.
  • the colony size was measured using software (ImageJ).
  • the vertical axis indicates colony size (mm 2 ).
  • LATS kinase inhibitors used were TDI-011536 (CAS number: 2687970-96-1) and TRULI (CAS number: 1424635-83-5, also known as "Lats-IN” or “Lats-IN-1”).
  • concentration of the LATS kinase inhibitor varied depending on the organoid, and was used at 5 to 20 ⁇ M.
  • Figure 29 shows bright-field micrographs of each organoid after six days of culture, and a graph showing the results of measuring the cell count of each organoid by measuring ATP using a commercially available kit (product name "CellTiter-Glo 3D", Promega).
  • the vertical axis of the graph shows luminescence intensity (relative value) indicating the amount of ATP present.
  • the top left shows the results for human liver-derived organoids
  • the top center shows the results for human salivary gland-derived organoids
  • the top right shows the results for human alveolar-derived organoids
  • the bottom left shows the results for human airway-derived organoids
  • the bottom second from the left shows the results for human pancreatic-derived organoids
  • the bottom third from the left shows the results for human stomach-derived organoids
  • the bottom right shows the results for human biliary tract-derived organoids.
  • Control indicates the results of the control
  • TDI indicates the results for the group to which TDI-011536 was added.
  • the top panel of Figure 30 is a schematic diagram outlining this experimental example.
  • the bottom panel of Figure 30 is a graph showing the results of measuring organoid proliferation.
  • TRUL I cell proliferation was observed to be comparable to that in the presence of Matrigel (registered trademark), even in the absence of Matrigel (registered trademark).
  • FIG 31 is a schematic diagram outlining this experimental example.
  • Human colon-derived organoids established in the absence of Matrigel (registered trademark) and in the presence of TRULIS were electroporated with a green fluorescent protein (GFP) expression vector to obtain GFP-labeled human colon-derived organoids.
  • GFP green fluorescent protein
  • GFP-labeled human colon-derived organoids were dissociated into single cells and precultured for 4 days in a medium supplemented with 2% Matrigel (registered trademark) or 2% collagen gel (50% type IA, 50% type IC).
  • the medium was supplemented with 2.5 ⁇ M 16,16-dimethyl prostaglandin E2 (dmPGE2, Cayman Chemical), 50 ng/mL human recombinant hepatocyte growth factor (HGF, Peprotech), 20 ng/mL human recombinant oncostatin M (OSM, Peprotech), and 5 nM human recombinant heregulin ⁇ 1 (NRG1, Peprotech).
  • the colonic epithelium and mucosa of NOG mice were then removed, and GFP-labeled human colon-derived organoids were orthotopically xenografted.
  • NOG mice fed a normal diet were anesthetized by inhalation of 2-3% isoflurane.
  • the mouse colon was then washed with PBS to remove the luminal contents.
  • a catheter with a small balloon attached was then inserted into the anus, and the balloon was inflated with air to hold the catheter in place.
  • Hot EDTA 250 mM, 50-55°C was then injected into the rectum to remove the colonic mucosa and chelate Ca 2+ and Mg 2+ , which are necessary for cell-cell adhesion and cell-extracellular matrix adhesion.
  • the colonic epithelium and mucosa were then scraped using a vibrating electric toothbrush (EW-DL22, EW0945, Panasonic). Successful removal of the epithelium and mucosa was confirmed by releasing the isolated colonic epithelium from the vibrating electric toothbrush into a water-filled Petri dish.
  • EW-DL22, EW0945, Panasonic a vibrating electric toothbrush
  • the total cell number of the precultured GFP-labeled human colon-derived organoids was counted, and the cells were suspended in Advanced DMEM/F12 medium supplemented with 10% (vol/vol) Matrigel® or type IA collagen, and 70 ⁇ L of the organoid suspension containing 1 ⁇ 10 7 cells was injected into the colon of a NOG mouse (recipient mouse) from which the colonic epithelium and mucosa had been removed, using a 200 ⁇ L pipette.
  • the anus of the mouse was temporarily blocked with soft paper using adhesive. To examine the development of intestinal obstruction, the feces of the recipient mouse were carefully observed for one week.
  • the leftmost photograph in the top row of Figure 32 is a bright-field photograph of the colon of a NOG mouse into which a GFP-labeled human colon-derived organoid had been transplanted.
  • the second photograph from the left in the top row of Figure 32 is a photograph of GFP fluorescence in the same field of view as the leftmost photograph in the top row of Figure 32.
  • the third photograph from the left in the top row of Figure 32 is a photograph of a mouse intestine with stained damaged areas.
  • the rightmost photograph in the top row of Figure 32 is a micrograph showing the results of immunostaining with an anti-human cytokeratin antibody of a section of the colon of a NOG mouse into which a GFP-labeled human colon-derived organoid had been transplanted.
  • the scale bar is 200 ⁇ m.
  • the leftmost photograph in the bottom row of Figure 32 is a fluorescence micrograph showing the results of staining the area enclosed by the dotted line in the top right of Figure 32 by in situ hybridization with mRNA of the human LGR5 gene.
  • the second photograph from the left in the bottom row of Figure 32 is an enlarged photograph of the area enclosed by the dotted line in the top right of Figure 32.
  • the scale bar is 100 ⁇ m.
  • the third photograph from the left in the bottom row of Figure 32 is a fluorescence micrograph showing the results of staining the area enclosed by the dotted line in the top right of Figure 32 with anti-human chromogranin A (CHGA) antibody and anti-human villin antibody.
  • the scale bar is 100 ⁇ m.
  • the rightmost photograph in the bottom row of Figure 32 is a fluorescence micrograph showing the results of staining the area enclosed by the dotted line in the top right of Figure 32 with anti-human mucin 2 (MUC2) antibody and anti-human villin antibody.
  • the scale bar is 100 ⁇ m.
  • the present invention provides a technique for culturing organoids in the absence of extracellular matrix.

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