Method for producing organoid

Abstract

The present invention relates to a method for producing an organoid, the method comprising the steps of: dissociating a biological tissue; and subjecting the dissociated biological tissue to suspension culture in a state in which the dissociated biological tissue is dispersed in a medium containing substantially no p38 inhibitor and containing the following components (i)-(iv): (i) 0.1-10 vol% of an extracellular matrix; (ii) insulin-like growth factor 1 (IGF-1); (iii) fibroblast growth factor 2 (FGF-2); and (iv) at least one substance selected from the group consisting of a Wnt agonist, a bone morphogenetic factor (BMP) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, and an epidermal growth factor (EGF).

Description

Technical Field

[0001] The present invention relates to a method for producing an organoid. More specifically, the present invention relates to a method for producing an organoid and a method for screening an anticancer agent. This application claims priority based on US63 / 317552, a provisional application filed in the United States on March 8, 2022, and its contents are incorporated herein by reference. Background Art

[0002] Drug screening, which searches for the effects of a wide range of candidate therapeutic drugs, is a fundamental technology in drug discovery. Conventional cancer drug discovery has used methods that use cancer cell lines constructed from cancer tissues from patients for preliminary screening and animal models to confirm safety and side effects.

[0003] However, cancer cell lines sometimes lack the properties of actual clinical tumors and many sensitivities to anticancer agents. In addition, even if cancer occurs in the same organ, cancer will show different genetic and biological characteristics for each patient. In cancer cell lines, it is difficult to encompass this diversity. As a result, most of the candidate anticancer agents screened using cancer cell lines have failed to reach clinical application, and the "valley of death" in drug discovery research has become a major medical and social problem.

[0004] The present inventors were the first in the world to develop organoid technology for permanent three-dimensional culture of tissue stem cells (for example, see non-patent documents 1 and 2). The present inventors also constructed cancer organoid biobanks such as colon cancer, pancreatic cancer, and gastric cancer by applying organoid technology to various cancer tissues from patients (for example, see non-patent documents 3 to 5).

[0005] Cancer organoids from patients retain many characteristics of the patients' cancer tissues. As the next generation of cancer research resources, cancer organoid biobanks will be used in basic cancer research, cancer diagnostic tools, drug discovery, personalized treatment, etc.

[0006] Recent overseas clinical trials have shown that the drug sensitivity of cancer organoids from patients is correlated with the response of clinical tumors to anticancer drugs, and there is also hope for the implementation of organoid medicine that uses organoids to predict the therapeutic effects of chemotherapy.

[0007] Prior art literature

[0008] Non-patent literature

[0009] 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.

[0010] Non-patent document 2: Sato T, et al., Long-term Expansion of Epithelial OrganoidsFrom Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium, Gastroenterology, 141, 1762-1772, 2011.

[0011] Non-patent document 3: Fujii M, et al., A Colorectal Tumor Organoid Library Demonstrates Progressive Loss of Niche Factor Requirements during Tumorigenesis, Cell Stem Cell, 18, 827-838, 2016.

[0012] Non-patent document 4: Seino T, et al., Human Pancreatic Tumor Organoids RevealLoss of Stem Cell Niche Factor Dependence during Disease Progression, CellStem Cell, 22, 454-467, 2018.

[0013] Non-patent literature 5: Nanki K, et al., Divergent Routes toward Wnt and R-spondin Niche Independency during Human Gastric Carcinogenesis, Cell, 174, 856-869, 2018. Summary of the invention

[0014] Technical issues to be solved

[0015] However, in previous drug screening, it is difficult to evaluate the side effects and safety of normal tissues. Normal tissues can be cultured by using organoid technology. However, in previous culture technologies, it is difficult to effectively culture organoids from normal tissues in large quantities. Therefore, it is impossible to introduce organoids from normal tissues into drug screening.

[0016] Therefore, an object of the present invention is to provide a technology for efficiently culturing organoids in large quantities, regardless of whether they are derived from normal tissue or tumor tissue.

[0017] Means used to solve problems

[0018] The present invention includes the following aspects.

[0019] [1] A method for producing an organoid, the method comprising the steps of: dissociating a living tissue; and suspending and culturing the dissociated living tissue in a dispersed state in a culture medium, thereby forming an organoid, wherein the culture medium is substantially free of a p38 inhibitor and comprises the following components (i) to (iv): (i) 0.1 to 10% by volume of an extracellular matrix; (ii) insulin-like growth factor 1 (IGF-1); (iii) fibroblast growth factor 2 (FGF-2); and (iv) at least one selected from the group consisting of a Wnt agonist, a bone morphogenetic protein (BMP) inhibitor, a transforming growth factor-β (TGF-β) inhibitor, and an epidermal growth factor (EGF).

[0020] [2] The production method according to [1], wherein the suspension culture is performed while stirring the culture medium.

[0021] [3] The production method according to [1] or [2], wherein the living tissue is normal tissue.

[0022] [4] The production method according to any one of [1] to [3], wherein the Wnt agonist is at least one selected from the group consisting of a Wnt protein, an R-sponge protein, and a GSK-3β inhibitor.

[0023] [5] The production method according to any one of [1] to [4], wherein the Wnt agonist is a Wnt protein and R-sponge protein.

[0024] [6] The production method according to [5], wherein the Wnt protein is a complex with afamin.

[0025] [7] A method for screening anticancer agents, the screening method comprising the following steps: culturing organoids from normal tissues and organoids from tumor tissues in suspension in the presence of a test substance in a dispersed state in a culture medium; and evaluating the effect of the test substance on the organoids from normal tissues and organoids from tumor tissues, wherein a test substance that has little side effects on the organoids from normal tissues and inhibits the proliferation of organoids from tumor tissues is an anticancer agent that is effective for the tumor tissues, the culture medium being substantially free of a p38 inhibitor and comprising the following components (i) to (iv): (i) 0.1 to 10% by volume of an extracellular matrix; (ii) IGF-1; (iii) FGF-2; and (iv) at least one selected from the group consisting of a Wnt agonist, a BMP inhibitor, a TGF-β inhibitor, and EGF.

[0026] [8] The screening method according to [7], wherein the organoid derived from normal tissue is an organoid produced by the production method described in [3].

[0027] Effects of the Invention

[0028] According to the present invention, it is possible to provide a technology for efficiently culturing a large number of organoids regardless of whether they are derived from normal tissues or tumor tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a graph showing the results of measuring the growth of organoids in Experimental Example 1.

[0030] Figure 2 This is a graph showing the results of calculating the doubling time of organoids in Experimental Example 1.

[0031] Figure 3 This is a graph showing the results of measuring the growth of organoids in Experimental Example 2.

[0032] Figure 4 This is a graph showing the results of calculating the coefficient of variation of the area of ​​the fluorescent dye in Experimental Example 2.

[0033] Figure 5 This is a graph showing the results of calculating the Z'-factor in each organ in Experimental Example 2.

[0034] Figure 6 : is a graph showing the results of all drug-organoid combinations in the high-throughput screening (HTS) performed in Experimental Example 2.

[0035] Figure 7 This is a graph showing the distribution of Z'-factors in 237 screening plates in Experimental Example 3.

[0036] Figure 8 This is a graph showing the root mean square error (rmse) of the sigmoid response curve fitted to the dose-response curve in Experimental Example 3.

[0037] Fig. 9 It shows that the log2 (IC 50 ) results.

[0038] Fig.10 It means that in Experiment 3, based on IC 50 Graph of the results of Spearman's correlation coefficients calculated for all pairs of organoid lines.

[0039] Fig.11 It is a volcano plot showing the results of multivariate analysis of variance (MANOVA) in Experimental Example 4.

[0040] Fig.12 is the IC of (+)-JQ1 measured in Experimental Example 4 for normal colon organoids (n=6) and CRC organoids (n=15). 50 of charts.

[0041] Fig.13 These are representative micrographs showing the responses of normal colon organoids and CRC organoids obtained in Experimental Example 4 to (+)-JQ1. The scale bar is 500 μm. DETAILED DESCRIPTION

[0042] [Method for producing organoids]

[0043] In one embodiment, the present invention provides a method for producing an organoid, the method comprising the steps of: dissociating a living tissue; and suspending and culturing the dissociated living tissue in a state dispersed in a culture medium, thereby forming an organoid, wherein the culture medium does not substantially contain a p38 inhibitor and comprises the following components (i) to (iv):

[0044] (i) 0.1 to 10% by volume of extracellular matrix;

[0045] (ii) IGF-1;

[0046] (iii) FGF-2; and

[0047] (iv) at least one selected from the group consisting of a Wnt agonist, a BMP inhibitor, a TGF-β inhibitor, and EGF.

[0048] As described later in the experimental examples, the manufacturing method of this embodiment can stably culture and mass-produce organoids from normal tissues. The manufacturing method of this embodiment can also be referred to as a method for culturing organoids. In this embodiment, mass culturing refers to, for example, proliferating 100% of tissue from a living body. 6 ~10 7 Cell organoids.

[0049] In the manufacturing method of this embodiment, the living tissue can be living tissue from a patient or a constructed organoid. In the case where the living tissue is an organoid, the manufacturing method of this embodiment can also be called an organoid passaging method.

[0050] In the manufacturing method of the present embodiment, the living tissue is preferably epithelial tissue. Intestinal epithelial tissue can be cited as the epithelial tissue. The intestine is the organ with the largest contact area with the outside world in the human body, and has functions such as digestion and absorption that are indispensable for maintaining life. Most of the intestinal functions are undertaken by the intestinal epithelium covering its inner layer. The intestinal epithelium is composed of two divided areas: villi composed of differentiated cells of three systems (mucus producing cells, absorptive epithelial cells, and endocrine cells) and crypts mainly composed of undifferentiated proliferating cells. The epithelial tissue is preferably a tissue containing epithelial stem cells or epithelial cells. Epithelial cells include differentiated epithelial cells and epithelial stem cells. "Epithelial stem cells" refer to cells with long-term self-renewal function and the ability to differentiate into epithelial differentiated cells, and refer to stem cells derived from epithelial tissue. Examples of epithelial tissues include cornea, oral mucosa, skin, conjunctiva, bladder, renal tubules, kidneys, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), large intestine (including colon), liver, pancreas, mammary gland, salivary gland, lacrimal gland, prostate, hair roots, trachea, lungs, and the like.

[0051] The living tissue is preferably a tissue from a human or non-human mammal. As non-human mammals, there are no particular limitations, and mice, rats, cattle, pigs, rabbits, monkeys, etc. can be cited. In the manufacturing method of this embodiment, the living tissue can be a tumor tissue or a cancer tissue, or it can be a normal tissue. Among them, tumor tissue refers to a tumorized tissue, and cancer tissue refers to a cancerous tissue.

[0052] Dissociating living tissue means fragmenting living tissue and dividing it into cell masses of one to a plurality of cells. The plurality may be about 2 to 100 cells. The dissociation of living tissue may be performed by mechanical shearing or by enzyme treatment such as collagenase, dispase I, liberase, trypsin, TrypLE Express (Thermo Fisher Scientific).

[0053] It is known that organoids sometimes recover poorly after being dissociated into single cells. In contrast, the present inventors have discovered that the p38 inhibitor, which has been considered necessary for the culture of human intestinal organoids in recent years, inhibits the growth of organoids derived from single cells. The present inventors have also discovered that the growth of organoids derived from single cells can be promoted by replacing the p38 inhibitor with a combination of IGF-1 and FGF-2.

[0054] In addition, conventional organoid culture schemes in the past include embedding the dissociated organoids in extracellular matrices such as Matrigel (registered trademark). However, the organoids embedded in the extracellular matrix may be difficult to expand the culture scale. In addition, the organoids embedded in the extracellular matrix may deviate and become larger when growing.

[0055] In contrast, in the manufacturing method of the present embodiment, the dissociated living tissue is suspended and cultured in a state dispersed in a culture medium. That is, in the manufacturing method of the present embodiment, the dissociated living tissue is not embedded in an extracellular matrix, but is suspended and cultured in a culture medium containing an extracellular matrix in a state where cells are dispersed.

[0056] As described later in the experimental examples, the manufacturing method of this embodiment can stably culture not only cancer organoids but also organoids from normal tissues in large quantities. In addition, when seeding cells, by accurately aligning the number of cells, the deviation of the number of cells between wells can be suppressed. In order to align the number of cells, for example, a cell sorter can be used for sorting.

[0057] Examples of p38 inhibitors used in conventional organoid culture include SB202190 (CAS number: 152121-30-7), SB203580 (CAS number: 152121-47-6), VX-702 (CAS number: 745833-23-2), VX-745 (CAS number: 209410-46-8), PD169316 (CAS number: 152121-53-4), RO4402257 (CAS number: 1449811-01-2), and BIRB796 (CAS number: 285983-48-4).

[0058] In contrast, in the production method of the present embodiment, a culture medium substantially free of p38 inhibitors is used. In this specification, "substantially free of p38 inhibitors" means that p38 inhibitors are not intentionally added to the culture medium, and the inevitable mixing of p38 inhibitors is allowed within the range of achieving the effect of the present embodiment.

[0059] The culture medium used in the manufacturing method of the present embodiment includes an extracellular matrix. The extracellular matrix is ​​a non-cellular component, including various polysaccharides, water, elastin and glycoprotein. As glycoprotein, for example, collagen, nidogen (Nidogen), fibronectin, laminin, etc. can be listed.

[0060] As the extracellular matrix, commercially available substances may also be used, for example, extracellular matrix protein (Thermo Fisher Scientific), basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Matrigel (registered trademark), Corning), ProNectin (Sigma-Aldrich), etc.

[0061] The culture medium used in the production method of the present embodiment preferably contains 0.1 to 10% by volume, for example, 0.1 to 5% by volume, for example, 0.1 to 3% by volume, for example, about 2% by volume of the extracellular matrix.

[0062] The culture medium used in the manufacturing method of this embodiment contains IGF-1. IGF-1 is also called growth factor C, and is a factor that is mainly secreted by stimulation caused by growth hormone (GH) in the liver. The concentration of IGF-1 contained in the culture medium can be, for example, 5 ng / mL to 1 μg / mL, can be, for example, 10 ng / mL to 1 μg / mL, or can be, for example, 50 to 500 ng / mL.

[0063] The culture medium used in the manufacturing method of this embodiment contains FGF-2. FGF-2 is basic fibroblast growth factor, also known as bFGF. The concentration of FGF-2 contained in the culture medium can be, for example, 5 ng / mL to 1 μg / mL, can be, for example, 10 ng / mL to 1 μg / mL, or can be, for example, 50 to 500 ng / mL.

[0064] As described later in the experimental examples, by replacing the p38 inhibitor that was previously considered necessary for the cultivation of organoids with a combination of IGF-1 and FGF-2, the growth of organoids derived from a single cell was promoted. As a result, not only cancer organoids but also organoids from normal tissues could be cultured stably in large quantities.

[0065] The culture medium used in the manufacturing method of the present embodiment includes at least one selected from the group consisting of a Wnt agonist, a BMP inhibitor, a TGF-β inhibitor, and EGF. The culture medium used in the manufacturing method of the present embodiment may include two selected from the group consisting of a Wnt agonist, a BMP inhibitor, a TGF-β inhibitor, and EGF, or three selected from the group consisting of a Wnt agonist, a BMP inhibitor, a TGF-β inhibitor, and EGF, or may contain a Wnt agonist, a BMP inhibitor, a TGF-β inhibitor, and EGF.

[0066] Wnt agonists refer to substances that activate transcription mediated by T cell factor (hereinafter, sometimes referred to as "TCF") / lymphoid enhancer factor (hereinafter, sometimes referred to as "LEF") in cells. Wnt agonists are not limited to Wnt family proteins, and include agonists that bind to and activate Frizzled receptor family members, inhibitors of intracellular β-catenin degradation, and TCF / LEF activating substances.

[0067] The Wnt agonist is preferably at least one selected from the group consisting of Wnt protein, R-sponge protein, and GSK-3β inhibitor.

[0068] A Wnt agonist is a substance that stimulates Wnt activity in a cell by at least 10%, preferably at least 20%, more preferably at least 30%, further preferably at least 50%, particularly preferably at least 90%, and most preferably 100% compared to the level of Wnt activity in the absence of the Wnt agonist. Wnt activity can be evaluated by measuring the transcriptional activity of Wnt, for example, by a TCF reporter gene assay.

[0069] The Wnt agonist is preferably a combination of a Wnt protein and an R-sponge protein. As the Wnt protein, the source is not particularly limited, and Wnt proteins from various organisms can be used. Among them, preferably a Wnt protein from a mammal. The mammal is the same as the above-mentioned animal. As a mammalian Wnt protein, Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, etc. can be listed. One Wnt protein can be used alone, or two or more can be used in combination.

[0070] Wnt protein can be a protein produced using Wnt protein expression cells, or it can be a commercially available protein. As long as the Wnt protein has Wnt activity, it can be a fragment of the Wnt protein, or it can be a protein containing an amino acid sequence other than the amino acid sequence of the Wnt protein. As amino acid sequences other than the amino acid sequence of the Wnt protein, for example, the amino acid sequence of an affinity tag can be cited. In addition, the amino acid sequence of the Wnt protein does not have to be completely consistent with the amino acid sequence that can be obtained from known databases such as GenBank. As long as it has Wnt activity, it can also be an amino acid sequence that is deleted, substituted or added to an amino acid sequence that can be obtained from a known database. Among them, as 1 to multiple, preferably 10 or less, more preferably 7 or less, and further preferably 6 or less.

[0071] The concentration of the Wnt protein contained in the culture medium may be, for example, 50 ng / mL to 10 μg / mL, 100 ng / mL to 10 μg / mL, 200 ng / mL to 1 μg / mL, or 300 ng / mL to 1 μg / mL.

[0072] The Wnt protein is preferably a protein that forms a complex with alfamine. Alfamin is a glycoprotein belonging to the albumin family and is known to be present in body fluids such as blood. The serum added to the cell culture medium contains alfamine from the animal from which the serum was collected. Since the serum contains impurities other than alfamine, it is preferred to use alfamine alone without using serum.

[0073] The source of alfammine is not particularly limited, and alfammine derived from various organisms can be used. Among them, alfammine derived from mammals is preferred. Mammals are the same as the animals described above.

[0074] Alfamin may be obtained by purifying natural afamin contained in serum or the like by a known method, or may be recombinant afamin. Recombinant afamin may be produced by appropriately using known gene recombination techniques. As a method for producing recombinant afamin, for example, DNA encoding afamin may be inserted into a known expression vector, the obtained expression vector may be introduced into a suitable host cell to express the recombinant afamin, and the recombinant afamin may be produced by purifying the recombinant afamin using a known purification method.

[0075] Recombinant afamin can be afamin to which an affinity tag is added. The additional affinity tag is not particularly limited and can be appropriately selected from known affinity tags for use. As the affinity tag, it is preferably an affinity tag recognized by a specific antibody, for example, a FLAG tag, a MYC tag, a HA tag, a V5 tag, etc. can be cited.

[0076] The Wnt protein has strong hydrophobicity because a specific serine residue is modified by a fatty acid (palmitoleic acid). Therefore, it is well known that the Wnt protein is easily aggregated or denatured in an aqueous solution and is very difficult to purify and preserve. On the other hand, it is reported that the modification of this specific serine residue by a fatty acid is necessary for the physiological activity of the Wnt protein and is involved in binding to members of the Frizzled receptor family.

[0077] In aqueous solution, Wnt protein forms a complex by binding to afamin in a one-to-one ratio, and can be dissolved while maintaining high physiological activity. Therefore, cells expressing both Wnt protein and afamin can be cultured to produce Wnt protein-afamin complex, or Wnt protein-expressing cells and afamin-expressing cells can be co-cultured to produce Wnt protein-afamin complex. Wnt protein-afamin complex can also be added to the culture medium in the form of a conditioning medium.

[0078] The concentration of afamin contained in the culture medium may be, for example, 50 ng / mL to 10 μg / mL, 100 ng / mL to 10 μg / mL, 200 ng / mL to 1 μg / mL, or 300 ng / mL to 1 μg / mL.

[0079] As R-sponge protein, the R-sponge protein family consisting of R-sponge protein 1, R-sponge protein 2, R-sponge protein 3 and R-sponge protein 4 can be enumerated. Known R-sponge protein family is a secretory protein, which is related to the activation and control of the Wnt signal transduction pathway. As R-sponge protein, as long as there is R-sponge protein activity, a fragment of R-sponge protein can be used, or a substance having an amino acid sequence other than the amino acid sequence of R-sponge protein such as an affinity tag can be used. R-sponge protein can be used alone or in combination with more than two kinds.

[0080] The concentration of R-sponge protein contained in the culture medium can be, for example, 50 ng / mL to 10 μg / mL, can be, for example, 100 ng / mL to 10 μg / mL, can be, for example, 200 ng / mL to 1 μg / mL, can also be, for example, 300 ng / mL to 1 μg / mL. R-sponge protein can also be added to the culture medium in the form of a conditioning medium.

[0081] Examples of the GSK-3β inhibitor include CHIR99021 (CAS No.: 252917-06-9), Kenpaullone (CAS No.: 142273-20-9), and 6-Bromoindirubin-3′-oxime (BIO, CAS No.: 667463-62-9).

[0082] Examples of BMP inhibitors include substances having an inhibitory activity of preferably 50% or more, more preferably 70% or more, further preferably 80% or more, and particularly preferably 90% or more compared to the BMP activity level in the absence of the inhibitor. BMP inhibitory activity can be evaluated by measuring the transcriptional activity of BMP.

[0083] BMP inhibitors are preferably natural BMP binding proteins, for example, Noggin, gremlin, chordin, chordin domain and other chordin-like proteins; Follistatin, chorlistatin domain and other chorlistatin-related proteins; Differential screening-selected gene Aberrative in Neuroblastoma (DAN), DAN cysteine ​​knot domain and other DAN-like proteins; SOST; Decorin; α-2 macroglobulin, etc.

[0084] The concentration of the BMP inhibitor contained in the culture medium may be, for example, 10 ng / mL to 1 μg / mL, 20 ng / mL to 1 μg / mL, 50 ng / mL to 1 μg / mL, or about 100 ng / mL.

[0085] TGF-β is a type of growth factor, produced in almost all cells such as the kidney, bone marrow, and platelets. There are 5 subtypes of TGF-β (β1 to β5). In addition, it is known that TGF-β promotes the proliferation of osteoblasts and the synthesis and proliferation of connective tissues such as collagen, and has an inhibitory effect on the proliferation of epithelial cells and osteoclasts. Generally, TGF-β inhibitors are substances that, for example, prevent or inhibit TGF-β from binding to TGF-β receptors, and are drugs that bind to TGF-β to form a complex that neutralizes the activity of TGF-β. In addition, TGF-β inhibitors are, for example, substances that bind to TGF-β receptors and prevent or inhibit TGF-β from binding to receptors, and are drugs used as antagonists or inverse agonists.

[0086] Examples of TGF-β inhibitors include substances having an inhibitory activity of preferably 50% or more, more preferably 70% or more, further preferably 80% or more, particularly preferably 90% or more, compared to the TGF-β activity level in the absence of the inhibitor.

[0087] Examples of TGF-β inhibitors include A83-01 (CAS No.: 909910-43-6), SB431542 (CAS No.: 301836-41-9), SB505124 (CAS No.: 694433-59-5), SB525334 (CAS No.: 356559-20-1), LY364947 (CAS No.: 396129-53-6), SD208 (CAS No.: 627536-09-8), and SJN2511 (CAS No.: 446859-33-2). Among them, A83-01 is preferred.

[0088] The concentration of the TGF-β inhibitor contained in the culture medium may be, for example, 100 nM to 1 μM, 200 nM to 1 μM, 300 nM to 1 μM, or about 500 nM.

[0089] In the manufacturing method of the present embodiment, in addition to the above-mentioned components, the culture medium may also contain mitogenic growth factors. Examples of mitogenic growth factors include epidermal growth factor (EGF), transforming growth factor-α (TGF-α), keratinocyte growth factor (KGF), etc. These mitogenic growth factors may be used alone or in combination of two or more.

[0090] EGF is a potent mitogenic factor for various cultured ectodermal and mesodermal cells and has a significant effect on the specific cell differentiation of a portion of fibroblasts. The EGF precursor exists as a membrane-bound molecule that is proteolytically cleaved to produce a 53-amino acid peptide hormone that stimulates cells.

[0091] Among them, EGF is preferred as the mitogenic growth factor. The concentration of EGF contained in the culture medium may be, for example, 5 to 500 ng / mL, 10 to 400 ng / mL, or about 50 ng / mL.

[0092] In the production method of this embodiment, it is preferred to perform suspension culture of the dissociated living tissue while stirring the culture medium. As described later in the experimental examples, when performing suspension stirring culture, organoids can be proliferated more rapidly than in suspension culture.

[0093] [Screening method for anticancer agents]

[0094] In one embodiment, the present invention provides a method for screening an anticancer agent, the screening method comprising the steps of: culturing organoids from normal tissues and organoids from tumor tissues in suspension in the presence of a test substance in a dispersed state in a culture medium; and evaluating the effect of the test substance on the organoids from normal tissues and the organoids from tumor tissues, wherein the test substance has little side effects on the organoids from normal tissues and is effective on the organoids from tumor tissues, indicating that it is an anticancer agent effective for the tumor tissues, wherein the culture medium does not substantially contain a p38 inhibitor and comprises the following components (i) to (iv):

[0095] (i) 0.1 to 10% by volume of extracellular matrix;

[0096] (ii) IGF-1;

[0097] (iii) FGF-2; and

[0098] (iv) at least one selected from the group consisting of a Wnt agonist, a BMP inhibitor, a TGF-β inhibitor, and EGF.

[0099] In the past, it was difficult to culture organoids from normal tissues in large quantities and stably, so it was not possible to include organoids from normal tissues in drug screening. As a result, it was difficult to evaluate the side effects and safety of drugs on normal tissues.

[0100] In contrast, as described later in the experimental examples, the screening method of this embodiment can be used for drug screening together with organoids from tumor tissues and organoids from normal tissues. As a result, the side effects and safety of drugs on normal tissues can be evaluated. In addition, as described later in the experimental examples, it is possible to screen drugs that do not affect the proliferation of organoids from normal tissues and specifically inhibit the growth of organoids from tumor tissues.

[0101] As described later in the experimental examples, when inoculating the organoids, by accurately aligning the number of cells, it is possible to suppress the variation in the number of cells between wells. To align the number of cells, for example, sorting can be performed using a cell sorter.

[0102] In the screening method of this embodiment, 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 screening method of this embodiment enables high-throughput screening (HTS) with high accuracy, robustness, and reproducibility.

[0103] In the screening method of the present embodiment, the organoid from normal tissue is preferably an organoid produced by the above-mentioned production method. In the screening method of the present embodiment, the extracellular matrix, IGF-1, FGF-2, Wnt agonist, BMP inhibitor, TGF-β inhibitor, EGF, organoid, etc. are the same as the above-mentioned substances.

[0104] [Experimental example]

[0105] Next, the present invention will be described in more detail with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[0106] [Materials and Methods]

[0107] (Construction of patient-derived organoids)

[0108] Seven newly constructed organoid strains and organoid strains from patients constructed in previous studies were used. All samples used in organoid construction and biological analysis were obtained from Keio University Hospital or the University of Tokyo Hospital with written informed consent. This study was approved by the ethics committees of both institutions.

[0109] Normal colon tissue and tumorous colon tissue were obtained from either endoscopic biopsy or surgical resection. The samples were stored in ice-cold phosphate-buffered saline (PBS) until processing. Fresh tissue samples were cut into small pieces, washed at least 10 times with ice-cold PBS, and then digested with digestive enzymes (TH-grade, Roche) at 37°C for 60 minutes, with vigorous pipetting every 15 minutes. The remaining fragments were further treated in TrypLE Express (Thermo Fisher Scientific) at 37°C for 20 minutes. Then, the supernatant was recovered and centrifuged at 200×g and 4°C for 3 minutes. Then, the cell pellet was suspended in Matrigel (registered trademark) (growth factor reduced, BD Biosciences) and dispensed into 48-well culture plates (25 μL of Matrigel (registered trademark) per well). The names of the various organ strains used and the properties of the tissues from the various organs are shown in Table 1 below. In the "Histology" column of Table 1, "NL" indicates normal tissue, "TA" indicates tubular adenoma, "SSL" indicates serrated lesion, "well" indicates well-differentiated adenocarcinoma, "mod" indicates moderately differentiated adenocarcinoma, "por" indicates poorly differentiated adenocarcinoma, and "muc" indicates mucinous adenocarcinoma. In addition, in the "Location" column, "A" indicates ascending colon, "T" indicates transverse colon, "D" indicates descending colon, "S" indicates sigmoid colon, "R" indicates rectum, "LM" indicates liver metastasis, and "C" indicates cecum. In addition, in the "Sex" column, "F" indicates female and "M" indicates male. In addition, in the "Culture Conditions" column, "W" indicates Wnt, "E" indicates EGF, "N" indicates Nogin, "R" indicates R-Spondin1, "A" indicates A83-01, "S" indicates SB202190, and "-" indicates that growth factors are not required.

[0110] [Table 1]

[0111] Organoid name Histology Location gender age stage Culture conditions NLSL1 NL A F 68 - WENARFI NLCRC15 NL T？ F 84 - WENARFI NLCRC25 NL T？ M 50 - WENARFI NLCRC26 NL ？ F 66 - WENARFI NLCRC32 NL D M 68 - WENARFI NLCRC33 NL T F 74 - WENARFI NLCRC35 NL R M 50 - WENARFI NLSL3 NL A M 69 - WENARFI Ad2 TA A A 40 - ENAFI SL3 SSL A M 69 - WENRA CRC5 well D M 58 I ENAFI CRC7 mod S M 85 II ENA CRC11 mod C M 75 II - CRC12 mod A M 45 II ENA CRC13 muc T F 70 II WENAR CRC15 por T F 84 II ENA CRC17a por,mod A M 82 III ENA CRC17b por,mod A M 82 III - CRC18a muc C F 70 III WENARAFI CRC18b muc C F 70 III WENARAFI CRC20 mod R F 67 IV E CRC23b mod R M 72 IV EFI CRC24b mod R F 48 IV ENAFI CRC25a mod T M 50 IV ENA CRC25c mod LM M 50 IV ENA CRC28 mod LM M 63 IV EA CRC31 por,mod R M 45 II ENFI CRC32 por,mod D M 68 IV - CRC33 mod T F 74 II WNRA CRC34 mod R F 40 IV ENAFI

[0112] (Organoid Culture)

[0113] Human colorectal cancer (CRC) organoids were cultured in a medium containing 2% by volume of Matrigel (registered trademark) (corning). As a culture medium, various combinations of growth factors were added to the basal medium in the case of organoids from normal tissue or tumor tissue, and in the case of tumor tissue, depending on the origin of the tumor. When stirring the medium, a CO2-resistant orbital shaker MaxQ 2000CO2 plus (Thermo Fisher Scientific) was used.

[0114] As the basal medium, a medium prepared by adding penicillin / streptomycin, 10 mM HEPES, 2 mM GlutaMAX, 1× B27 (Thermo Fisher Scientific), 10 nM gastrin I (Sigma-Aldrich), and 1 mM N-acetylcysteine ​​(Fujifilm Wako Pure Chemical Industries, Ltd.) to advanced DMEM / F12 was used.

[0115] Organoids from normal tissues are maintained in either modified medium or regular medium. Modified medium is a medium that has the following seven growth factors added to the basal medium.

[0116] 20% Afamin-Wnt3A serum-free conditioned medium

[0117] 10% R-Spongin-1 Conditioning Medium

[0118] 50ng / mL mouse recombinant EGF (Thermo Fisher Scientific)

[0119] 500nM A83-01(Tocris)

[0120] 100ng / mL mouse recombinant Nogin (Peptec)

[0121] 100ng / mL recombinant human IGF-1 (Biolegend)

[0122] 50ng / mL recombinant human FGF-basic (FGF-2) (Peptech)

[0123] The regular medium was the same as the modified medium except that 10 μM SB202190 (Sigma-Aldrich) was added to replace IGF-1 and FGF-2. In tumor organoids, some growth factors are not required due to genetic mutations.

[0124] Passaging was performed by collecting and washing organoids and fragmenting them by mechanical shearing or TrypLE Express (Thermo Fisher Scientific)-based digestion. Two days after passaging, organoid fragments were cultured in fresh medium containing 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) and inhibiting cell death.

[0125] (Drug Screening Test)

[0126] The drug library of the Standard Inhibitor Kit (SCADS Inhibitor Kit) was provided by the Anticancer Drug Screening Committee of the Cancer Research Support Project for New Academic Research under the Ministry of Education, Culture, Sports, Science and Technology.

[0127] In this study, a total of 23 organoid systems were screened. Before screening, organoids were labeled with green fluorescent protein (GFP) using PB-CMV-MCS-EF1α-GFP-Puro PiggyBac (PB513B-1, System Biosciences) and SuperPiggyBac transposase (PB210-20, System Biosciences) vectors.

[0128] Organoids were dissociated using TrypLE Express (Thermo Fisher Scientific) and filtered using a cell strainer (Sysmex) with a pore size of 20 μm. 800 to 1500 cells were sorted into each well of a 384-well plate using a cell sorter (SH800, Sony).

[0129] Organoids were treated with test compounds 24 or 96 hours after seeding. Organoids were grown for 6-8 days and colony area was measured based on GFP fluorescence using the IN Cell Analyzer 6000 confocal imaging system (GE Healthcare Lifesciences). In addition, bright field imaging and luminescence-based ATP measurement were performed using Cell3iMager duos (SCREEN) and CellTiter-Glo cell viability assay (Promega).

[0130] Brightfield images were analyzed using software embedded in Cell3iMager. Default parameters were used for preprocessing and object detection, and supervised life and death judgment was applied to machine learning. For evaluation of (+)-JQ1, CellTiter-Glo Cell Viability Assay (Promega) was used to determine cell viability.

[0131] (Dose response curve fitting)

[0132] Dose response curves were fitted to the drug response data using a Bayesian sigmoid model. Drug response data were acquired in 3-fold fashion with more than 6 controls without drug and 7-point drug responses at 2-fold concentrations. In all screening panels, the test proteasome inhibitor MG-132 was tested at a maximum concentration of 1 μM, accurately estimating 100% cell death and background signal.

[0133] A sigmoid response curve of the four parameters was fitted as follows. First, the viability of the organoids was normalized by dividing by the mean of the drug-free control. lc It is estimated to be the average value of the following formula.

[0134] X Ic =I min +(I max -I min ) / (1+e (Ic-βpos) / βshape) )…(1)

[0135] In formula (1), I max represents the survival rate of the drug-free control, I min represents the average value of the background signal, βpos represents the position of the S-shaped curve, βshape represents the steepness, and lc represents the logarithm of the concentration (log concentration).

[0136] Assume that the survival rate of the organoid is X lc There is a variance Var(X lc )=BE(X lc ) is a γ distribution. Where B represents the noise parameter. Assume that the survival rate of the drug-free control is X max There is also a variance Var(X max )=BI max The gamma distribution of .

[0137] Markov chain Monte Carlo simulations were performed using rstan (v.2.18.0) to estimate the parameters. Initial values ​​were randomly generated from past distributions. After 1000 burn-in iterations, the posterior distribution was obtained by sampling 1000 iterations from 4 independent chains. The median of the posterior distribution was used as the parameter estimate. βpos had a normal prior distribution with a 99% probability mass covering the range from one hundredth of the minimum concentration tested to 100 times the maximum tested concentration. I max With a uniform prior distribution of 0 to 5. min As I min =αI max (where α has a uniform prior distribution from 0 to 1) was reparameterized. B and βshape have low-information prior probabilities (half-Cauchy distribution with position = 0 and scale = 10). In the case where the R convergence metric is greater than 1.1 or the mean absolute error is greater than 0.3, the curve fit is determined to be insufficient and discarded, and no downstream analysis is performed.

[0138] The AUC values ​​were estimated using the trapezoidal approximation. The AUC values ​​were calculated as the concentration range covered and vertically divided by I max and I min The area enclosed is determined by a portion of the rectangle.

[0139] [Experimental Example 1]

[0140] (Suspension Culture of Organoids)

[0141] Human colorectal cancer (CRC) organoids were suspended in a culture medium supplemented with 2% by volume of Matrigel (registered trademark) and their growth was studied. For comparison, a culture embedded in Matrigel (registered trademark) droplets as a previous culture method was also performed. In addition, a suspension agitation culture in which the organoids were suspended and the culture medium was agitated was also performed.

[0142] Figure 1 This is a graph showing the results of measuring the growth of organoids under various culture conditions (n=3). Figure 1 In the table, "CRC17b", "CRC25a", "CRC32", and "NLCRC32" are the names of organoid strains. The properties of each organoid strain are shown in Table 1 above. CRC17b, CRC25a, and CRC32 are CRC organoids, and NLCRC32 is an organoid derived from normal tissue. The results show that in any organoid, when suspended culture is performed, compared with culture embedded in Matrigel (registered trademark) droplets, the growth rate is faster than that of the normal tissue. 6 ~10 7 In addition, organoids proliferate more rapidly when cultured in suspension.

[0143] Figure 2 The graph shows the results of calculating the doubling time of organoids under various culture conditions. The results show that the doubling time tends to be shorter in suspension culture than in culture embedded in Matrigel (registered trademark) droplets, and the doubling time tends to be even shorter in suspension agitation culture.

[0144] [Experimental Example 2]

[0145] (A robust high-throughput screening (HTS) system for patient-derived colorectal organoids)

[0146] When organoids are dissociated unevenly, particularly by mechanical shearing, selective organoid proliferation from large organoid fragments occurs and organoid proliferation is biased between wells.

[0147] Therefore, organoids were labeled with GFP and sorted into each well of a 384-well plate by flow cytometry-based cell sorting, so that each well was accurately distributed with the same number of live single cells. By fluorescently labeling organoids, it is also possible to use a high-content cell imaging analyzer for automated imaging of organoids and measurement of organoid proliferation.

[0148] Using a drug library containing 56 drugs, the drug sensitivity of each drug to CRC organoids and normal colon organoids was analyzed. The drug library contains 9 clinically used anticancer agents and 47 small molecule inhibitors targeting cancer-related signaling pathways. Among the organoid strains shown in Table 1 above, 6 normal colon organoid strains and 20 CRC organoid strains were provided for HTS. Using 237 screening plates, HTS was performed with a total of 1438 drug-organoid combinations.

[0149] Drugs from the drug library were added 4 days after cell seeding, and the proliferation of organoids was calculated based on the area of ​​fluorescent dye. To confirm that this method is equivalent to the results based on other methods, the survival rate of organoids was also analyzed by ATP-based luminescence detection and bright field imaging analysis.

[0150] Fluorescence measurement and luminescence detection based on ATP show the same results in many drugs. In addition, the same results as those based on luminescence detection based on ATP were obtained by bright field imaging analysis. However, due to the different morphologies between CRC organoids, the life and death determination of cells by bright field imaging analysis requires subsequent parameter calibration for each organoid strain. Therefore, the life and death determination of cells by bright field imaging analysis has the tendency to damage the simplicity of screening and reduce throughput.

[0151] HTS using normal human colon organoids has been difficult to implement in the past because of the poor recovery of normal human colon organoids after dissociation into single cells. In contrast, the present inventors have discovered that the p38 inhibitor, which has been considered necessary for the culture of human intestinal organoids in recent years, inhibits the growth of organoids derived from single cells. The present inventors have also discovered that the growth of organoids derived from single cells is promoted by replacing the p38 inhibitor with a combination of IGF-1 and FGF-2.

[0152] Organoids were cultured in regular medium containing a p38 inhibitor (SB202190) and in a modified medium containing IGF-1 and FGF-2 instead of the p38 inhibitor, and their growth was measured based on the area of ​​the fluorescent dye. Each of 800 cells was cultured in a 384-well plate.

[0153] Figure 3 This is a graph showing the results of measuring the growth of organoids. Figure 3 The left side shows the results of normal colon organoids. Figure 3 The right side shows the results of CRC organoids. Figure 3 In the graphs, each point represents the measurement result in one well. The bars in the graphs represent the mean ± standard deviation. Figure 3 In the table, “*” indicates that when P = 9.51 × 10 -13 (NLCRC32 strain, n = 22 wells), P = 1.12 × 10-21 (CRC24b strain, n = 44 wells), P = 4.52 × 10 -21 (CRC31 strain, n=36 wells) There was a significant difference.

[0154] Figure 4 This is a graph showing the results of calculating the coefficient of variation of the area of ​​the fluorescent dye. Figure 4 The left side shows the results of normal colon organoids. Figure 4 The right side shows the results of CRC organoids. Figure 4 In the table, “*” indicates that when P = 7.28 × 10 -8 (NLCRC32 strain, n = 22 wells), P = 5.69 × 10 -5 (CRC24b strain, n = 44 wells), P = 3.95 × 10 -2 (CRC31 strain, n=36 wells) There was a significant difference.

[0155] Figure 5 This is a graph showing the results of calculating the Z'-factor for each organ. The results show that the use of the improved culture medium not only improves the efficiency of organoid culture, but also enables uniform culture of normal colon organoids classified as single cells. In addition, since the Z'-factor is higher than 0.5, it shows that this screening system is a high-quality HTS system.

[0156] The above results indicate that the HTS system of this experimental example can be used to perform drug screening on various organoids including organoids derived from normal tissues.

[0157] [Experimental Example 3]

[0158] (Summary of drug responses in colorectal organoids)

[0159] Figure 6 is a graph showing the results of all drug-organoid combinations in HTS performed in Experimental Example 2. Figure 6 In the above example, log2(IC 50 ) indicates drug response. For some drugs, molecular targets are shown. In HTS, Z'-factor was greater than 0.5 in 82% of the screening panels.

[0160] Figure 7 Graph showing the distribution of Z'-factors in 237 screening plates. 58 screening plates were seeded with normal colon organoids, and 139 screening plates were seeded with CRC organoids. Figure 7 As shown, no significant difference in Z'-factor was observed between normal colon organoids and CRC organoids.

[0161] The response of each drug was expressed as 50% inhibitory concentration (IC 50 ), the steepness of the dose-response curve, and the area under the dose-response curve. Figure 8 is a graph showing the root mean square error (rmse) of the sigmoidal response curve fitted to the dose response curve. The average value of the rmse was 0.119, indicating the robustness of the curve fit.

[0162] Fig. 9 The log2 (IC 50 ) The results were shown in a graph. As a result, it was confirmed that the three independent test results showed a high correlation.

[0163] The above results show that the HTS system of Experimental Example 2 has high accuracy, robustness and reproducibility.

[0164] Fig.10 Is based on IC 50 Figure 3. Graph of the results of Spearman's correlation coefficients calculated for all pairs of organoid lines. Fig.10 Each point represents a pair of organoid lines. The bars in the graph represent the mean ± standard deviation. In addition, “*” indicates that P = 1.34 × 10 -6 There is a significant difference, "**" indicates that P = 3.61 × 10 -7 In addition, "Normal-Normal" represents the results of normal colon organoid strain pairs (n=15), "Normal-CRC" represents the results of normal colon organoid strain pairs and CRC organoid strain pairs (n=120), and "CRC-CRC" represents the results of CRC organoid strain pairs (n=190).

[0165] As a result, the six normal colon organoid strains showed the same drug response. In contrast, the drug response differences between the CRC organoid strains were large. Although the drug response differences between the CRC organoid strains were large, Figure 6 As shown, CRC organoids showed similar responses to drugs with the same target, such as EGFR inhibitors, mTOR inhibitors, and topoisomerase inhibitors.

[0166] Based on these results, we believe that the heterogeneity in drug response of CRC organoids is not based on technical issues but rather due to the genetic or epigenetic diversity of CRC organoids.

[0167] [Experimental Example 4]

[0168] (Bromodomain and extra-terminal motif (BET) bromodomain protein inhibitors inhibit CRC growth)

[0169] Based on the results of HTS performed in Experimental Example 2, multivariate analysis of variance (MANOVA) was used to compare drug responses of normal colon organoids and CRC organoids.

[0170] Fig.11 is a volcano plot showing the results of MANOVA. Fig.11 In the figure, each dot represents one drug. The dot with a slash indicates a drug for which a statistically significant difference was observed. More than 60% of the drugs showed higher proliferation inhibitory activity in normal colon organoids than in (36 / 56, 64.3%) CRC organoids. This is believed to be due to the rapid proliferation of normal colon organoids. Despite this bias, (+)-JQ1 (CAS No.: 1268524-70-4), a BET bromodomain protein inhibitor, still showed cancer-specific inhibition of organoid growth.

[0171] Fig.12 is the IC of (+)-JQ1 relative to normal colon organoids (n=6) and CRC organoids (n=15). 50 . Fig.12 Each point represents one organoid strain. The bars in the graph represent the mean ± standard deviation. “*” indicates that the -3 There are significant differences.

[0172] Fig.13 Representative micrographs showing the response of normal colon organoids and CRC organoids to (+)-JQ1. Scale bar is 500 μm. Fig.13 In the figure, “Normal” indicates the result of normal colon organoids, and “CRC7” and “CRC17a” indicate the names of CRC organoid strains.

[0173] These results suggest that it is useful to use both normal and cancer organoids for the discovery of potential therapeutic approaches.

[0174] [Experimental Example 5]

[0175] All animal experiments were performed with the approval of the Laboratory Animal Center of Keio University School of Medicine. CRC organoids were xenotransplanted into nude mice to verify whether the HTS results using in vitro cultured organoids were the same as those in vivo.

[0176] CRC organoids were cultured in the same manner as in Experimental Example 1. The CRC organoids obtained by suspension culture were centrifuged and the precipitate was suspended in cold Matrigel. 0.2 mL (1×106 Cells / injection) CRC organoid suspension was injected into four left and right subcutaneous sites of nude mice and transplanted.

[0177] Tumor volume was measured over time by electron carriers. Tumor volume reached 100-150 mm 3 After that, cetuximab (40 mg / kg, Merck) was injected intraperitoneally 7 times every 3 days. In addition, nab-paclitaxel (20 mg / kg, Taiho Pharmaceutical Co., Ltd.) was injected intravenously 6 times twice a week. The results of continuing to measure tumor volume confirmed that the results of drug effects in vivo were very consistent with the results of HTS obtained in vitro.

[0178] Industrial Applicability

[0179] According to the present invention, a technology for efficiently culturing a large number of organoids, whether derived from normal tissue or tumor tissue, can be provided. According to the present invention, HTS screening of drugs that have little side effects on normal tissue and exhibit specific effects on tumor tissue can be performed.

Claims

1. A method for producing an organoid, the method comprising the following steps: Dissociating living tissue; and The dissociated living tissue is suspended and cultured in a dispersed state in a culture medium, thereby forming an organoid. The culture medium is substantially free of p38 inhibitors and comprises the following components (i) to (iv): (i) 0.1 to 10% by volume of extracellular matrix; (ii) Insulin-like growth factor 1 (IGF-1); (iii) Fibroblast growth factor 2 (FGF-2); and (iv) at least one selected from the group consisting of a Wnt agonist, a bone morphogenetic protein (BMP) inhibitor, a transforming growth factor-β (TGF-β) inhibitor, and an epidermal growth factor (EGF).

2. The manufacturing method according to claim 1, wherein: The suspension culture is performed while stirring the culture medium.

3. The manufacturing method according to claim 1 or 2, wherein: The living tissue is normal tissue.

4. The manufacturing method according to any one of claims 1 to 3, wherein: The Wnt agonist is at least one selected from the group consisting of Wnt protein, R-sponge protein and GSK-3β inhibitor.

5. The manufacturing method according to any one of claims 1 to 4, wherein: The Wnt agonists are Wnt protein and R-sponge protein.

6. The manufacturing method according to claim 5, wherein: The Wnt protein is in a complex with afamin.

7. A method for screening an anticancer agent, comprising the following steps: In the presence of a test substance, culturing the normal tissue-derived organoid and the tumor tissue-derived organoid in suspension in a dispersed state in a culture medium; and evaluating the effect of the test substance on the organoids derived from normal tissue and the organoids derived from tumor tissue, A test substance that has little side effects on the organoids derived from normal tissue and inhibits the proliferation of the organoids derived from tumor tissue indicates that it is an anticancer agent effective for the tumor tissue. The culture medium is substantially free of p38 inhibitors and comprises the following components (i) to (iv): (i) 0.1 to 10% by volume of extracellular matrix; (ii) IGF-1; (iii) FGF-2; and (iv) at least one selected from the group consisting of a Wnt agonist, a BMP inhibitor, a TGF-β inhibitor, and EGF.

8. The screening method according to claim 7, wherein The normal tissue-derived organoid is an organoid produced by the production method according to claim 3.

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