Method for culturing cancerous organ and method for screening substance to be detected

Abstract

A method for culturing a cancerous organ, which comprises a step for culturing a cancerous organ on the top surface or inside of a cell structure in which two or more cell layers are laminated in the thickness direction, said cell structure containing cells constituting a mesenchymal substance, with a culture medium that does not contain an extracellular matrix. A method for screening a substance to be tested, the method comprising: a step for culturing a cell structure comprising a cancerous organ obtained by the method for culturing a cancerous organ in the presence of a substance to be tested; and a step for evaluating the influence of the substance to be tested on the cancerous organ.

Description

Technical Field

[0001] The present invention relates to a method for culturing cancer organoids and a method for screening a substance to be tested.

[0002] This application claims priority from Japanese Patent Application No. 2022-126547 filed in Japan on August 8, 2022, the contents of which are incorporated herein by reference. Background Art

[0003] In the past, the mainstream in cancer research was the use of cancer cell lines established by subculture under optimized culture conditions. However, cancer cell lines that are maintained and continuously cultured in vitro for a long time may not necessarily reflect the properties of clinical tumors and the response to drugs. Therefore, in order to develop more accurate anticancer drugs and select the best treatment for each patient, cancer organoids (also called PDO) derived from the patient's tumor tissue are considered promising (for example, see Non-Patent Document 1, etc.).

[0004] PDO also maintains most of the properties of the tumor tissue of the patient as the source during culture. On the other hand, in vivo, a variety of cells such as cells constituting the stroma constitute the microenvironment of cancer. In contrast, in order to cultivate cancer organoids, extracellular matrices (also referred to as ECM) such as Matrigel (registered trademark) (manufactured by BD Biosciences) are essential. In the extracellular matrix, the cells constituting the stroma cannot stay, so the cancer organoids cannot be co-cultured with the cells constituting the stroma and the blood vessels constituted by the cells. Therefore, it is impossible to evaluate the interaction between cancer organoids and cells constituting the stroma (for example, refer to non-patent literature 1, etc.).

[0005] In addition, the culture of cancer organoids requires a dedicated special culture medium, but the growth factors and low-molecular-weight inhibitors contained in the culture medium may affect the gene expression and signal transduction pathways of cancer organoids and affect drug sensitivity.

[0006] Prior art literature

[0007] Non-patent literature

[0008] Non-patent document 1: Veninga V et al., "Tumor organoids: Opportunities and challenges to guide precision medicine.", Cancer Cell, Vol. 39, Issue 9, pp. 1190-1201, 2021. Summary of the invention

[0009] Problems to be solved by the invention

[0010] The present invention has been made in view of the above circumstances, and provides a method for culturing cancer organoids that can co-culture with cells constituting a stroma while maintaining the properties of cancer organoids without using an extracellular matrix and can evaluate the infiltration of cancer cells, and a method for screening test substances using cancer organoids obtained by the cancer organoid culturing method.

[0011] Means for solving problems

[0012] That is, the present invention includes the following aspects.

[0013] (1) A method for culturing cancer organoids, comprising the step of culturing cancer organoids on the top surface or inside of a cell structure using a culture medium that does not contain an extracellular matrix,

[0014] The cell structure includes cells constituting interstitial tissue and has two or more cell layers stacked in the thickness direction.

[0015] (2) The method for culturing cancer organoids according to (1), wherein the culturing comprises the step of dispersing the cancer organoids in the form of single cells and then culturing them on the top surface or inside the cell structure.

[0016] (3) The method for culturing cancer organoids according to (1) or (2), wherein the culturing comprises the following steps: after inoculating the cancer organoid on the top surface of a first cell structure having two or more cell layers stacked in the thickness direction, further inoculating a cell suspension containing cells constituting the stroma, constructing a second cell structure on the top of the cancer organoid, and culturing the cancer organoid inside the cell structure.

[0017] (4) The method for culturing cancer organoids according to any one of (1) to (3), wherein the culture medium is a culture medium consisting of any one of D-MEM, ESC, EBM2, EGM2 without hydrocortisone as an additive factor, RPMI-1640, Ham's F-12 and Mccoy's5a, or a combination of two or more thereof.

[0018] (5) The method for culturing a cancer organoid according to any one of (1) to (4), wherein the cancer organoid has a glandular duct structure.

[0019] (6) The method for culturing a cancer organoid according to any one of (1) to (5), wherein the cell structure comprises one or more cells selected from the group consisting of fibroblasts, pericytes, endothelial cells, and immune cells as cells constituting the stroma.

[0020] (7) The method for culturing cancer organoids according to (6), wherein the endothelial cells are one or more cells selected from vascular endothelial cells and lymphatic endothelial cells.

[0021] (8) The method for culturing a cancer organoid according to any one of (1) to (7), wherein the cell structure has a vascular network structure.

[0022] (9) The method for culturing a cancer organoid according to any one of (1) to (8), wherein the cell structure has 10 or more cell layers stacked in a thickness direction.

[0023] (10) A method for screening a substance to be tested, comprising:

[0024] a step of culturing a cell structure comprising a cancer organoid obtained by the method for culturing a cancer organoid according to any one of (1) to (9) in the presence of a test substance; and

[0025] The step of evaluating the effect of the test substance on the cancer organoid.

[0026] Effects of the Invention

[0027] According to the cancer organoid culture method of the above aspect, it is possible to co-culture with cells constituting the stroma while maintaining the properties of the cancer organoid without using an extracellular matrix, and to evaluate the infiltration of cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A This is a schematic diagram showing a method for culturing cancer organoids according to one embodiment of the present invention.

[0029] Figure 1B This is a schematic diagram showing a method for culturing cancer organoids according to another embodiment of the present invention.

[0030] Figure 1C This is a schematic diagram showing a method for culturing cancer organoids according to another embodiment of the present invention.

[0031] Figure 1D This is a schematic diagram showing a method for culturing cancer organoids according to another embodiment of the present invention.

[0032] Figure 2A This is a fluorescent immunostaining image using an anti-EpCAM antibody when single-celled patient-derived cancer organoids (PDO) were cultured on the top surface of the cell structure in Example 1.

[0033] Figure 2B This is a graph showing the confluency (%) of PDO when single-celled PDO was cultured on the top surface of the cell structure in Example 1.

[0034] Figure 3A This is a fluorescent immunostaining image using an anti-EpCAM antibody when single-celled PDO was cultured inside the cell structure in Example 1.

[0035] Figure 3B This is a graph showing the fusion (%) of PDO when single-celled PDO was cultured inside the cell structure in Example 1.

[0036] Figure 3C These are immunostaining images of sections obtained when single-celled PDO was cultured inside the cell structure in Example 1 using anti-EpCAM antibodies and anti-CD31 antibodies.

[0037] Figure 3D These are fluorescent immunostaining images of sections obtained when single-celled PDO was cultured inside the cell structure in Example 1 using anti-EpCAM antibodies and anti-CD31 antibodies.

[0038] Figure 3E This is an image of a section obtained by fluorescent immunostaining using an anti-CD31 antibody when single-celled PDO was cultured inside the cell structure in Example 1.

[0039] Figure 4A This is a bright field image observed from the top surface when PDO with its morphology maintained was cultured on the top surface of the cell structure in Example 1.

[0040] Figure 4B This is an immunostaining image using an anti-EpCAM antibody of a section in which PDO with its morphology maintained was cultured on the apical surface of the cell structure in Example 1.

[0041] Figure 5 This is a graph showing the results of a drug efficacy evaluation test of AMG510 alone or AMG510 and cetuximab in combination under the conditions of culturing single-celled PDO on the apical surface of the cell structure in Example 2.

[0042] Figure 6 These are immunostaining images of tissues cultured in various culture media in Example 3 using an anti-CD31 antibody.

[0043] Figure 7 These are immunostaining images of tissues cultured in various culture media in Example 3 using an anti-CD31 antibody.

[0044] Figure 8 These are immunostaining images of tissues cultured in various culture media in Example 3 using an anti-CD31 antibody.

[0045] Fig. 9 These are immunostaining images of tissues cultured in various culture media in Example 3 using an anti-CD31 antibody.

[0046] Fig.10 These are immunostaining images of tissues cultured in various culture media in Example 3 using an anti-CD31 antibody.

[0047] Fig.11 These are immunostaining images of tissues cultured in various culture media in Example 3 using an anti-CD31 antibody.

[0048] Fig.12 These are immunostaining images using an anti-EpCAM antibody of tissue sections of HCT26-1T and HCT26-3LM cultured in each culture medium in Example 4.

[0049] Fig.13 This is a graph showing the time-dependent changes in the fusion (%) of PDO in tissues of HCT26-1T and HCT26-3LM cultured in each culture medium in Example 4.

[0050] Fig.14 This is an immunostaining image of PDO in a tissue section when HCT26-1T cells were cultured on the apical surface of the cell structure in Example 4 using an anti-EpCAM antibody.

[0051] Fig.15 This is an immunostaining image of PDO in a tissue section when HCT26-3LM was cultured on the apical surface of the cell structure in Example 4 using an anti-EpCAM antibody.

[0052] Fig.16 This is an immunostaining image of PDO in a tissue section when HCT26-1T cells were cultured inside the cell structure in Example 4 using an anti-EpCAM antibody.

[0053] Fig.17 This is an immunostaining image of PDO in a tissue section when HCT26-3LM was cultured inside the cell structure in Example 4 using an anti-EpCAM antibody. DETAILED DESCRIPTION

[0054] 《Cultivation Methods of Cancer Organoids》

[0055] A method for culturing a cancer organoid according to one embodiment of the present invention (hereinafter sometimes referred to as “the culturing method according to the present embodiment”) comprises the step of culturing the cancer organoid on the top surface or inside of a cell structure using a culture medium that does not contain an extracellular matrix (hereinafter sometimes referred to as “culturing step”),

[0056] The cell structure includes cells constituting interstitial tissue (hereinafter referred to as "interstitial cells"), and has two or more cell layers stacked in the thickness direction.

[0057] The cell structure used in the culture method of this embodiment includes interstitial cells and is a structure that simulates interstitial tissue. It is not a common culture method in which cancer organoids are adhered to an extracellular matrix such as matrigel for culture, but rather a culture medium that does not contain an extracellular matrix is ​​used as a culture medium for culture on the top surface or inside of the cell structure that simulates the interstitium. Thus, interstitial cells and cancer organoids that could not be co-cultured in the past due to the presence of an extracellular matrix can be co-cultured.

[0058] In addition, in the culture method of the present embodiment, on the top surface or inside of the cell structure simulating the interstitial tissue, as a culture medium, in addition to not containing extracellular matrix, cancer organoids can also be cultured without using excessive growth factors or artificial inhibitors. As a result, a cell structure containing cancer organoids is obtained on the top surface or inside of the cell structure. That is, the culture obtained by the present embodiment (i.e., the cell structure containing cancer organoids) is a culture obtained under an environment closer to the organism. Therefore, as shown in the embodiments described later, the cancer organoids in the culture have a glandular duct structure, and in the case of cancer organoids derived from patients, the properties of the tumor tissue of the patient as the source can be maintained. Therefore, the obtained culture is very useful as a sample for evaluating the efficacy of candidate substances of anticancer agents under conditions closer to the body, and for selecting the best anticancer agent for each patient.

[0059] Furthermore, as shown in the Examples described below, in the culture method of the present embodiment, since the infiltration of cancer organoids into the cell structure was observed, the obtained culture can be used to evaluate the infiltration of cancer cells.

[0060] Cancer Organoids

[0061] The cancer organoids used in the culture method of this embodiment can be cancer organoids produced using cultured cell lines, but from the perspective of reflecting the properties of clinical tumors and responsiveness to drugs, cancer organoids are preferably produced using tumor tissues or cancer cells from cancer patients or sick animals, and more preferably organoids derived from cancer patients.

[0062] In the present embodiment and the present specification, cancer cells refer to cells that are derived from somatic cells and have acquired unlimited proliferation ability, and are malignant neoplasms that infiltrate surrounding tissues or cause metastasis.

[0063] Cancer organoids may be composed of multiple types of cells or only of cells of a specific type. In addition, cancer organoids may be composed of only cancer cells or of cancer cells and cells other than cancer cells.

[0064] The tumor tissue or cancer cells from which the cancer organoids are derived can be tissues or cells collected from animals of any biological species. For example, tumor tissue or cancer cells collected from animals such as humans, monkeys, marmosets, dogs, cats, rabbits, pigs, cows, horses, sheep, goats, mice, and rats can be used.

[0065] The tumor tissue from which the cancer organoid is derived can be a solid tissue or a liquid tissue, but solid tissue is preferred. As solid tissue, for example, tissue collected by surgical resection of epithelial tissue, connective tissue, muscle tissue, neural tissue, interstitial tissue or mucosal tissue can be cited. As liquid tissue, for example, body fluids such as blood, lymph, pleural effusion, ascites, cerebrospinal fluid, tears, saliva and urine can be cited. These tissues can be removed by a scalpel or laser, or collected by a syringe or swab, for example, during surgery, endoscopic examination, etc. As human tumor tissue, for example, tissue collected for clinical examination can be used.

[0066] The cancer from which the main tissue or cancer cells originate is preferably a solid cancer. Examples of solid cancers include breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, and inflammatory breast cancer), prostate cancer (e.g., hormone-dependent prostate cancer and hormone-independent prostate cancer), pancreatic cancer (e.g., pancreatic ductal carcinoma), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, and adenosquamous carcinoma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, and malignant mesothelioma), colon cancer (e.g., digestive tract stromal tumor), rectal cancer (e.g., digestive tract stromal tumor), colorectal cancer (e.g., familial colorectal cancer, hereditary non-polyposis colorectal cancer, and digestive tract stromal tumor), small intestinal cancer (e.g., non-Hodgkin's lymphoma and digestive tract stromal tumor), esophageal cancer, duodenal cancer, tongue cancer, pharyngeal cancer (e.g., upper pharyngeal cancer, middle pharyngeal cancer, and lower pharyngeal cancer), head and neck cancer, salivary gland cancer, brain tumors (e.g., pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma), and ulcerative colitis. The invention also includes, but is not limited to, renal cell carcinoma, ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, and ovarian low-grade tumor), bladder cancer, urethral cancer, skin cancer (e.g., intraocular (eye) melanoma and Merkel cell carcinoma), melanoma (malignant melanoma), thyroid cancer (e.g., medullary thyroid cancer), parathyroid cancer, nasal cancer, paranasal cancer, retinal sarcoma, penile cancer, testicular tumor, childhood solid cancer (e.g., Wilms tumor and childhood renal tumor), Kaposi's sarcoma, Kaposi's sarcoma caused by AIDS, maxillary sinus tumor, leiomyosarcoma, and rhabdomyosarcoma.

[0067] Organoids derived from cancer patients can be prepared, for example, by the method shown below. That is, first, the tumor tissue of the cancer patient is surgically removed, immediately cut into small pieces, and washed with PBS. Next, digestion buffers such as collagenase and dispase are added to the small pieces of tumor tissue, and dissociated at a temperature of about 37°C. Then, the tumor tissue is vigorously stirred in PBS by blowing and suction, and the tumor tissue pieces are recovered and centrifuged. The pellets after centrifugation are suspended in an extracellular matrix such as matrigel and transferred to a culture container for culture, thereby obtaining PDO.

[0068] Alternatively, cancer organoids can be made using cancer cells isolated from tumor tissues of cancer patients. As a method for isolating cancer cells from tumor tissues, for example, as a method for isolating cancer cells from epithelial tissues, methods known in the art can be cited. For example, crypts can be separated by placing a chelating agent and tumor tissue at a constant temperature. After washing the tissue, the epithelial cell layer is peeled off from the submucosal layer and chopped with a glass slide. Then, it is placed at a constant temperature in a liquid containing trypsin or preferably at least one of EDTA and EGTA. Then, for example, using at least one of filtration and a centrifuge, undigested tissue fragments and single cells derived from the crypts are separated. Other proteolytic enzymes, such as at least one of collagenase and dispase I, can also be used instead of trypsin. Fragments of the pancreas and stomach are separated using the same method.

[0069] In the case of only sorting cancer cells to make cancer organoids from the tumor tissue derived from cancer patients, it is possible to confirm the amount of cancer cells contained in the tumor tissue before the sorting of cancer cells.Cancer cells can be sorted by the expression of cancer cell-specific proteins and the rise of enzyme activity as cancer markers.Cancer markers are not particularly limited, and for example can be proteins specifically expressed in cancer cells such as EpCAM, CEA, cytokeratin (Cytokeratin) and HER2.In the case of using these proteins as cancer markers, by utilizing immunohistochemistry (also referred to as IHC) staining or immunofluorescence (also referred to as IF) staining for their antibodies, cancer cells can be visualized.In addition, in the case of using the enzyme activity of γ-glutamyl transpeptidase or β-galactosidase rising in cancer cells as cancer markers, fluorescent probes such as ProteoGREEN (registered trademark, Wuling Chemicals) and GlycoGREEN (registered trademark, Wuling Chemicals) can be utilized to measure these enzyme activities.

[0070] The sorting of cancer cells can be carried out by methods such as flow cytometry, magnetic separation, dielectrophoresis, size classification or density gradient classification. The sorting method can be appropriately determined according to the original source organ of the patient's tumor, clinical background or various previous test results. In the case of using flow cytometry, after staining with IF or fluorescent probe, cancer cells can be sorted by separating the cells that are positive for staining. In addition, in flow cytometry, living cells and dead cells can also be determined based on the values ​​of forward scattered light and side scattered light, so living cancer cells can be sorted and recovered more efficiently. In addition, in the case of using magnetic separation, antibodies are used to magnetically mark cells, and any method can be selected from the positive selection method of cancer cells marked by magnetic recovery and the negative selection method of interstitial cells marked by magnetic removal. In addition, in the case of using dielectrophoresis and / or density gradient classification, it is preferred to pre-master the dielectric properties and / or density gradient properties of the cell species used in the construction of interstitial cells.

[0071] Examples of methods for producing cancer organoids include the method described in “Sakahara M et al., “IFN / STAT Signaling Controls Tumorigenesis and the Drug Response in Colorectal Cancer.”, Cancer Sci.,, Vol. 110, Issue 4, pp. 293-1305, 2019. (Reference 1)” and the like.

[0072] The method is a method of allowing cancer cells or tumor tissues containing the cancer cells to adhere to or be embedded in an extracellular matrix for culture.

[0073] [Extracellular Matrix (ECM)]

[0074] Generally, "extracellular matrix" refers to a supramolecular structure present outside cells in organisms. The ECM becomes a scaffold for the proliferation of cancer cells or tumor tissues containing the cancer cells. ECM contains various polysaccharides, water, elastin and glycoproteins. Examples of glycoproteins include collagen, nidogen, fibronectin and laminin.

[0075] ECM can be prepared using connective tissue cells, or commercially available ECM can be used.

[0076] As a method for preparing ECM, for example, after culturing ECM-producing cells (such as fibroblasts), these cells are removed and cancer cells or tumor tissue containing cancer cells are added, whereby the ECM can be used as a scaffold.

[0077] Examples of ECM-producing cells include chondrocytes that mainly produce collagen and proteoglycans, fibroblasts that mainly produce type IV collagen, laminin, procollagen, and fibronectin, and colon myofibroblasts that mainly produce collagen (type I, type III, and type V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin, and cell adhesion molecule-C.

[0078] As commercially available ECMs commonly used in the culture of cancer organoids, for example, extracellular matrix proteins (manufactured by Invitrogen), basement membrane preparations derived from Engelbreth-Holm-Swarm (also referred to as EHS) mouse sarcoma cells (e.g., Matrigel (registered trademark) (manufactured by BD Biosciences)); synthetic ECMs such as ProNectin (Sigma Z378666), etc. In addition, a mixture of natural ECM and synthetic ECM may also be used.

[0079] After the ECM is gelled (or solidified), cancer cells or tumor tissue containing the cancer cells may be placed on it. Alternatively, the ECM and the cancer cells or tumor tissue containing the cancer cells may be mixed and the mixture may be inoculated in a culture container to make it gel (or solidify). Alternatively, when the ECM is gelled (or solidified) at 37°C, a culture medium may be added to diffuse the cancer cells or tumor tissue containing the cancer cells into the ECM before use. The cancer cells or tumor tissue containing the cancer cells in the culture medium may adhere to the ECM by interacting with the surface structure of the ECM (such as integrin, etc.).

[0080] ECM can also be applied to a culture container or the like for use.

[0081] Cancer organoids can be formed by culturing cancer cells or tumor tissues containing the cancer cells in a state of adhesion to ECM. The culture temperature is preferably above 30°C and below 40°C, more preferably around 37°C. The culture time can be appropriately adjusted according to the cells used. In addition, the CO2 concentration in the culture atmosphere is preferably about 0.03% by volume or more and 10% by volume or less relative to 100% by volume of the culture atmosphere, and most preferably about 5% by volume. In addition, culture can also be carried out in an environment where the O2 concentration is controlled to be lower than that of the atmosphere. In addition, as a culture medium used in the culture of cancer organoids, a culture medium commonly used in the culture of organoids (e.g., IntestiCult Organoid Growth Medium (Human) manufactured by STEMCELL Technologies) and the like can be cited.

[0082] <Cell structure>

[0083] In the present embodiment and the present specification, "cell structure" refers to a three-dimensional cell aggregate containing at least interstitial cells. "The thickness of the cell structure" refers to the length of the structure in the direction of self-weight. The direction of self-weight refers to the direction in which gravity is applied, also referred to as the thickness direction. More specifically, the thickness of the cell structure refers to the thickness of the slice obtained along the line passing through the center of gravity when observed from the upper surface of the cell structure, which is the thickness of the slice of the approximate central part of the cell structure. The shape of the cell structure varies according to the different containers used in the manufacture of the cell structure. For example, in the case of using a cylindrical cell culture plug-in to manufacture the cell structure, it becomes a cylindrical shape. In this case, the shape when the cell structure is observed from the upper surface is a circle, and the center of gravity when observed from the upper surface becomes the center of the circle. The shape of the cell structure is not limited to a cylindrical shape, and can be any shape according to the purpose. Specifically, for example, a triangular prism shape and a quadrangular prism shape and other polygonal prism shapes can be exemplified. "Cell layer" refers to a layer consisting of a group of cells and interstitial substances that exist in a direction orthogonal to the thickness direction and in which the nucleus does not overlap with the thickness direction.

[0084] As described later, the cell structure may include a first cell structure and a second cell structure. In such a cell structure, the cancer organoid is sandwiched between the first and second cell structures. In the case where the cell structure includes the first cell structure and the second cell structure, the thickness of the cell structure is the sum of the thicknesses of the first and second cell structures.

[0085] The cells such as interstitial cells constituting the cell structure are not particularly limited, and may be cells collected from animals, cells obtained by culturing cells collected from animals, cells subjected to various treatments on cells collected from animals, or cultured cell lines. In addition, as cells such as interstitial cells constituting the cell structure, commercially available cells or patient-derived cells may be used. In the case of cells collected from animals, the collection site is not particularly limited, and may be somatic cells derived from bone, muscle, internal organs, nerves, brain, bone, skin or blood, etc., or reproductive cells, or embryonic stem cells (also referred to as ES cells).

[0086] In addition, the biological species from which the cells constituting the cell structure are derived is not particularly limited, and for example, cells derived from animals such as humans, monkeys, marmosets, dogs, cats, rabbits, pigs, cattle, horses, sheep, goats, mice and rats can be used. Cells obtained by culturing cells collected from animals may be primary culture cells or subculture cells. In addition, as cells subjected to various treatments, induced pluripotent stem cells (also referred to as iPS cells) and cells after differentiation induction may be cited.

[0087] The cell structure may be composed only of cells derived from the same biological species, or may be composed of cells derived from multiple biological species.

[0088] As interstitial cells for constructing the cell structure, for example, endothelial cells, fibroblasts, pericytes, immune cells, nerve cells, mast cells, epithelial cells, cardiomyocytes, hepatocytes, islet cells, tissue stem cells and smooth muscle cells can be mentioned. Immune cells are cells involved in immunity. Specifically, lymphocytes, macrophages and dendritic cells can be mentioned. Lymphocytes include T cells, B cells, NK cells and plasma cells. The interstitial cells contained in the cell structure can be one or more. As interstitial cells contained in the cell structure, it is preferably included in one or more selected from fibroblasts, pericytes, endothelial cells and immune cells.

[0089] The number of interstitial cells in the cell structure is not particularly limited. From the perspective of forming a cell structure that better simulates interstitial tissue, the presence ratio (cell number ratio) of interstitial cells to all cells constituting the cell structure is preferably 30% or more, more preferably 50% or more, further preferably 70% or more, and further preferably 80% or more.

[0090] It is believed that the vascular network structure and the lymphatic network structure are important for the cell structure to perform functions similar to those of the interstitial tissue in the organism. Therefore, the cell structure preferably has a cell structure with a vascular network structure. That is, as a cell structure, it is preferred to construct a vascular network structure such as lymphatic vessels and / or blood vessels three-dimensionally inside a laminate of cells that do not form vascular vessels, so as to construct a cell structure that is closer to the tissue in the organism. The vascular network structure can be formed only inside the cell structure, or it can be formed so that at least a part of the vascular network structure is exposed to the surface or bottom surface of the cell structure. In addition, the vascular network structure can be constructed in the entire cell structure, or it can be formed only in a specific cell layer. It should be noted that in this embodiment and this specification, "vascular network structure" refers to a reticular structure such as a vascular network or a lymphatic network in a biological tissue.

[0091] The vascular network structure can be formed by including endothelial cells constituting blood vessels as interstitial cells. The endothelial cells contained in the cell structure can be vascular endothelial cells or lymphatic endothelial cells. In addition, the endothelial cells contained in the cell structure can also include both vascular endothelial cells and lymphatic endothelial cells.

[0092] In the case where the cell structure has a vascular network structure, the cells other than endothelial cells in the cell structure are preferably cells of the peripheral tissues constituting blood vessels in vivo from the perspective that endothelial cells easily form a vascular network that maintains its original function and shape. From the perspective that the environment near the interstitial tissue in the organism and the interstitial tissue in the organism is more similar, the cells other than endothelial cells are more preferably cells that at least contain fibroblasts, and further preferably cells that contain vascular endothelial cells and fibroblasts, cells that contain lymphatic endothelial cells and fibroblasts, or cells that contain vascular endothelial cells, lymphatic endothelial cells and fibroblasts. In addition, the cells other than endothelial cells contained in the cell structure can be cells of the same biological species as the endothelial cells, or cells of different biological species.

[0093] The number of endothelial cells in the cell structure is not particularly limited as long as it is sufficient to form a vascular network structure, and can be appropriately determined in consideration of the size of the cell structure or the cell types of endothelial cells and cells other than endothelial cells. For example, by making the presence ratio (cell number ratio) of endothelial cells to all cells constituting the cell structure 0.1% or more, a cell structure formed with a vascular network structure can be prepared. In the case of using fibroblasts as cells other than endothelial cells, the number of endothelial cells in the cell structure is preferably 0.1% or more of the number of fibroblasts, more preferably 0.1% or more and 5.0% or less. In the case where both vascular endothelial cells and lymphatic endothelial cells are included as endothelial cells, the total number of vascular endothelial cells and lymphatic endothelial cells is preferably 0.1% or more of the number of fibroblasts, more preferably 0.1% or more and 5.0% or less.

[0094] When the culture obtained by the culture method of this embodiment is used for cell-based analysis such as drug sensitivity test, it is preferred that the cell structure used is a structure closer to the stroma in the body. Therefore, as the cell structure, it is preferred that a cell structure with a vascular network structure is formed, more preferably a cell structure with a vascular network structure and containing fibroblasts is formed, and further preferably a cell structure with a vascular network structure and containing fibroblasts and having more than 10 cell layers stacked in the thickness direction.

[0095] Furthermore, in order to meet the need to observe the invasion of cancer cells into blood vessels, it is preferable to have a vascular network structure.

[0096] The size and shape of the cell structure are not particularly limited. From the perspective of being able to form a cell structure that is closer to the state of the interstitial tissue in the body and being able to expect to cultivate cancer organoids in an environment closer to the body, the thickness of the cell structure is preferably 5 μm or more, more preferably 30 μm or more, further preferably 100 μm or more, and further preferably 150 μm or more. The thickness of the cell structure is preferably 500 μm or less, more preferably 400 μm or less, and further preferably 200 μm or less.

[0097] The number of cell layers in the cell structure is preferably at least 2 layers and no more than 60 layers, more preferably at least 3 layers and no more than 60 layers, further preferably at least 5 layers and no more than 60 layers, further preferably at least 5 layers and no more than 20 layers, and particularly preferably at least 10 layers and no more than 20 layers.

[0098] The number of cell layers constituting the cell structure is determined by dividing the total number of cells constituting the three-dimensional structure by the number of cells per layer (the number of cells required to constitute one layer). The number of cells per layer can be studied by culturing the cells in a planar manner in a cell culture container used when constituting the cell structure in advance to achieve fusion (confluent). Specifically, the number of cell layers of the cell structure formed in a cell culture container can be calculated by measuring the total number of cells constituting the cell structure and then dividing it by the number of cells per layer of the cell culture container.

[0099] Usually, the cell structure is constructed in a cell culture container. As the cell culture container, there is no particular limitation as long as it is a container that can construct a cell structure and can culture the constructed cell structure. As the cell culture container, specifically, a culture dish, a cell culture plug-in (such as Transwell (registered trademark) plug-in, Netwell (registered trademark) plug-in, Falcon (registered trademark) cell culture plug-in, Millicell (registered trademark) cell culture plug-in, etc.), a test tube, a flask, a bottle and a plate, etc. can be cited. In the construction of the cell structure, since the constructed cell structure can be directly used for the cultivation of cancer organoids, a culture dish or various cell culture plug-ins are preferred.

[0100] The cell structure can be formed by two or more cell layers including interstitial cells, and the construction method of the cell structure is not particularly limited. For example, it can be a method of constructing layer by layer and then stacking them in sequence, it can be a method of constructing two or more cell layers at a time, or it can be a method of appropriately combining two construction methods to construct a multilayer cell layer.

[0101] In addition, the cell structure may be a multilayer structure in which the cell types constituting each cell layer are different in each layer, or the cell types constituting each cell layer are the same in all layers of the structure. For example, it may be a method of constructing by forming layers for each cell type and stacking the cell layers in sequence, or it may be a method of preparing a cell mixture solution in which a plurality of cells are mixed in advance and constructing a multilayer cell structure at one time from the cell mixture solution.

[0102] As a method of constructing layer by layer and then stacking in sequence, for example, the method described in Japanese Patent Gazette No. 4919464 (Reference 2) can be cited, that is, a method of continuously stacking cell layers by alternately repeating the process of forming a cell layer and the process of contacting the formed cell layer with a solution containing ECM components. For example, when performing the method, a cell mixture obtained by mixing all the cells constituting the cell structure is prepared in advance, and each cell layer is formed using the cell mixture, thereby being able to construct a cell structure in which a vascular network structure is formed in the entire structure. In addition, by forming each cell layer according to each cell type, a cell structure in which a vascular network structure is formed only in the layer formed by endothelial cells can be constructed.

[0103] As a method for constructing more than two layers of cell layers at a time, for example, the method described in Japanese Patent Gazette No. 5850419 (reference 3) can be cited. The method is as follows: the entire surface of the cell is coated with a polymer containing an arginine-glycine-aspartic acid (RGD) sequence bound by integrin and a polymer that interacts with the polymer containing the RGD sequence, and the coated cells coated by the adhesive film are placed in a cell culture container, and the coated cells are aggregated with each other by centrifugation, thereby constructing a cell structure formed by multiple layers of cell layers. For example, when performing the method, a cell mixture obtained by mixing all the cells constituting the cell structure is prepared in advance, and the coated cells are prepared by adding an adhesive component to the cell mixture. Thus, by a single centrifugation, a cell structure in which the cell composition is uniform in the entire structure can be constructed.

[0104] The cell structure can also be constructed by, for example, the method described in Japanese Patent No. 6639634 (Reference 4) and International Publication No. 2019 / 039457 (Reference 5), that is, a method having the following steps (a) to (c).

[0105] A step (a) of mixing cells and extracellular matrix components in a cationic buffer to obtain a mixture;

[0106] A step (b) of inoculating the mixture obtained in the step (a) into a cell culture container; and

[0107] After the step (b), a step (c) is performed to obtain a cell structure in which cells are stacked in multiple layers in the cell culture vessel.

[0108] In step (a), cells are mixed with a buffer (also referred to as a cationic buffer) containing a cationic substance and an extracellular matrix component, and a cell aggregate is formed by the cell mixture, thus being able to obtain a three-dimensional cell organization with few internal large spaces. In addition, the obtained three-dimensional cell organization is relatively stable, and therefore can be cultivated for at least several days, and the organization is also not easy to collapse when the culture medium is replaced. In addition, in step (b), the step of making the cell mixture inoculated in the cell culture container be precipitated into the cell culture container can be included. The sedimentation of the cell mixture can be actively precipitated by centrifugation, etc., or it can be naturally precipitated.

[0109] In step (a), the cells are preferably further mixed with a strong electrolyte polymer. By mixing the cells with a cationic substance, a strong electrolyte polymer and an extracellular matrix component, in step (b), there is no need for a process such as centrifugation to actively aggregate the cells, and even when the cells are naturally sedimented, a three-dimensional cell tissue with few spaces and thickness can be obtained.

[0110] As the cationic buffer, for example, Tris-hydrochloric acid buffer, Tris-maleic acid buffer, double-Tris-buffer or HEPES etc. can be cited. The concentration and pH of the cationic substance (such as Tris in Tris-hydrochloric acid buffer) in the cationic buffer are not particularly limited as long as they do not have adverse effects on the growth of cells and the construction of cell structures. For example, the concentration of the cationic substance in the cationic buffer can be more than 10mM and less than 100mM, preferably more than 40mM and less than 70mM, more preferably 50mM. In addition, the pH of the cationic buffer can be more than 6.0 and less than 8.0, preferably more than 6.8 and less than 7.8, more preferably more than 7.2 and less than 7.6.

[0111] As the strong electrolyte polymer, for example, glycosaminoglycans such as heparin, chondroitin sulfate (such as 4-chondroitin sulfate, 6-chondroitin sulfate), heparan sulfate, dermatan sulfate, keratan sulfate and hyaluronic acid can be cited; dextran sulfate, rhamnose sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, and polyacrylic acid or their derivatives, but are not limited to these. Only one strong electrolyte polymer can be mixed in the mixture prepared in step (a), or two or more can be combined and mixed. In the construction of the cell structure, it is preferred that the strong electrolyte polymer is glycosaminoglycan. In addition, it is more preferred to use at least one of heparin, dextran sulfate, chondroitin sulfate and dermatan sulfate sulfuric acid. In addition, the strong electrolyte polymer is further preferably heparin.

[0112] The amount of the strong electrolyte polymer mixed in the cationic buffer is not particularly limited as long as it does not have an adverse effect on the growth of cells and the construction of the cell structure. For example, the concentration of the strong electrolyte polymer in the cationic buffer can exceed 0 mg / mL (higher than 0 mg / mL) and be lower than 1.0 mg / mL, preferably more than 0.025 mg / mL and less than 0.1 mg / mL, more preferably more than 0.05 mg / mL and less than 0.1 mg / mL. In addition, the mixture can also be adjusted without mixing the strong electrolyte polymer to construct the cell structure.

[0113] As the extracellular matrix component, for example, collagen, laminin, fibronectin, vitronectin, elastin, cell adhesion factor, nidogen, fibrillin, proteoglycan or their modified or mutants etc. can be enumerated. Proteoglycan includes chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan and dermatan sulfate proteoglycan etc. In the mixture prepared in step (a), only one extracellular matrix component can be mixed, and more than two kinds of mixing can also be combined. In the construction of cell structure, collagen, laminin or fibronectin are preferably used, and collagen is more preferably used. As long as the growth of cells and the formation of cell structure are not adversely affected, modified and mutants of extracellular matrix components described above can also be used.

[0114] The amount of the extracellular matrix components mixed in the cationic buffer is not particularly limited as long as it does not adversely affect the growth of cells and the construction of cell structures. For example, the concentration of the extracellular matrix components relative to the volume of the cationic buffer can exceed 0 mg / mL (higher than 0 mg / mL) and be less than 1.0 mg / mL, preferably 0.025 mg / mL or more and 0.1 mg / mL or less, and more preferably 0.05 mg / mL or more and 0.1 mg / mL or less.

[0115] The ratio of the strong electrolyte polymer to the extracellular matrix component mixed in the cationic buffer is 1:2 to 2:1. In the construction of the cell structure, the ratio of the strong electrolyte polymer to the extracellular matrix component is preferably 1:1.5 to 1.5:1, more preferably 1:1.

[0116] By repeating steps (a) to (c), specifically, repeating step (b) on the cell structure obtained in step (c), inoculating the mixture prepared in step (a), and then performing step (c), a cell structure of sufficient thickness can be constructed. The cell composition of the mixture newly inoculated on the cell structure obtained in step (c) may be the same as or different from the cell composition constituting the constructed cell structure.

[0117] When steps (a) to (c) are repeated, the obtained cell structure can be cultured after step (c) and before step (b). The culture conditions such as the composition of the culture medium used in the culture, the culture temperature, the culture time, and the atmospheric composition during the culture are carried out under conditions suitable for culturing the cells constituting the cell structure. Examples of the culture medium include Dulbecco's modified Eagle's minimal essential medium (D-MEM), Eagle's minimal essential medium (E-MEM), minimal essential medium α (MEMα), RPMI-1640, and Ham's F-12.

[0118] After step (a), a step (a'-1) of removing the liquid portion from the obtained mixture to obtain a cell aggregate and a step (a'-2) of suspending the cell aggregate in a solution may be performed, and then step (b) may be performed. By performing the above steps (a) to (c), a desired tissue can be obtained, but by performing steps (a'-1) and (a'-2) after step (a) and then performing step (b), a more uniform tissue can be obtained.

[0119] In addition, after step (a), the following steps (b'-1) and (b'-2) can be performed instead of step (b). By performing steps (b'-1) and (b'-2), a more uniform tissue can also be obtained. In step (b'-2), the step of allowing the cell mixture inoculated in the cell culture container to settle in the cell culture container can be included in the same way as step (b). The sedimentation of the cell mixture can be actively precipitated by centrifugation, etc., or it can be naturally precipitated. In this embodiment and this specification, "cell viscous body" refers to the gel-like cell aggregate described in "Nishiguchi et al., Macromol Bioscience, 2015, vol.15 (3), p.312-317. (Reference 6)".

[0120] The step (b'-1) is a step of inoculating the mixture obtained in the step (a) into a cell culture vessel and then removing the liquid component from the mixture to obtain a cell slurry.

[0121] The step (b'-2) is a step of suspending the cell slurry in a solvent in a cell culture container.

[0122] As the solvent for preparing the cell suspension, as long as it is non-toxic to the cell, the solvent that does not damage the proliferative property and function is not particularly limited, and water, buffer and cell culture medium etc. can be used. As the buffer, phosphate physiological saline (PBS), HEPES and Hanks buffer etc. can be mentioned. As culture medium, D-MEM, E-MEM, MEMα, RPMI-1640 and Ham's F-12 etc. can be mentioned. In the case of using the culture medium of the cell as the solvent for preparing the cell suspension, it is possible to culture cells without removing the liquid component in the step (c) described later.

[0123] Instead of the step (c), a step (c') of forming a cell layer on the substrate may be performed.

[0124] In the step (c) and the step (c'), the liquid component may be removed from the inoculated mixture.

[0125] The method for removing the liquid component in step (c) and step (c') is not particularly limited as long as it does not adversely affect the growth of cells and the construction of cell structures. As a method for removing the liquid component from a suspension of liquid components and solid components, it can be appropriately carried out by methods known to those skilled in the art. As the method, for example, attraction, centrifugal separation, magnetic separation or filtration can be cited. For example, when a cell culture plug is used as a cell culture container, the cell culture plug inoculated with the mixture is subjected to a centrifugal separation treatment at 10°C, 400×g, and 1 minute, and the cell mixture is precipitated, so that the liquid component can be removed by attraction.

[0126] Next, the steps in the culture method of this embodiment will be described in detail below.

[0127] <Cultivation process>

[0128] In the culture step, cancer organoids are cultured on the top surface or inside the cell structure using a culture medium that does not contain an extracellular matrix.

[0129] Figure 1A This is a schematic diagram showing a method for culturing cancer organoids according to one embodiment of the present invention (a case where cancer organoids are cultured on the top surface of a cell structure).

[0130] That is, when culturing cancer organoid 1 on the top surface of cell structure 2, cancer organoid 1 is added to the culture solution (culture medium not shown) of the cell structure constructed in culture container 3. As a result, cancer organoid 1 adheres to the top surface of cell structure 2, and culture is performed in a state where cancer organoid 1 adheres to the top surface of cell structure 2.

[0131] or, Figure 1B This is a schematic diagram showing a method for culturing cancer organoids according to another embodiment of the present invention (a case of culturing cancer organoids inside a cell structure).

[0132] That is, when culturing cancer organoid 1 inside a cell structure, after inoculating cancer organoid 1 on the top surface of a first cell structure 2A having two or more cell layers stacked in the thickness direction, a cell suspension containing stromal cells is further inoculated to construct a second cell structure 2B on the top of the cancer organoid 1, and the cancer organoid 1 is cultured inside the cell structure. In addition, regarding the method for preparing the first cell structure 2A and the second cell structure 2B, the method described in the above "cell structure" can be applied.

[0133] exist Figure 1A and Figure 1B According to the described method, cancer organoids can be cultured while maintaining their morphology.

[0134] On the other hand, cancer organoids can also be cultured on the top surface or inside of the cell structure after being dispersed in the form of single cells. By dispersing cancer organoids in the form of single cells, the number of cells inoculated in the cell structure can be grasped. Therefore, in the analysis of the efficacy evaluation of candidate substances of anticancer agents using the obtained culture, the cell survival rate can be calculated, and quantitative evaluation can be performed.

[0135] Hereinafter, dispersing cancer organoids in the form of single cells will be referred to as “single-cellization”, and cancer organoids dispersed in the form of single cells may be referred to as “single-cellized cancer organoids”.

[0136] Figure 1C This is a schematic diagram showing a method for culturing cancer organoids according to another embodiment of the present invention (a case where single-celled cancer organoids are cultured on the top surface of a cell structure).

[0137] Figure 1C In addition to using single-cellized cancer organoid 1A, Figure 1A The culture method is the same as described in.

[0138] That is, when culturing the single-celled cancer organoid 1A on the top surface of the cell structure 2, the single-celled cancer organoid 1A is added to the culture solution (culture medium not shown) of the cell structure constructed in the culture container 3. As a result, the single-celled cancer organoid 1A adheres to the top surface of the cell structure 2, and the single-celled cancer organoid 1A is cultured in a state of being adhered to the top surface of the cell structure 2.

[0139] or, Figure 1D This is a schematic diagram showing a method for culturing cancer organoids according to another embodiment of the present invention (a case of culturing single-celled cancer organoids within a cell structure).

[0140] That is, when culturing the single-celled cancer organoid 1A inside the cell structure, after the single-celled cancer organoid 1A is inoculated on the top surface of the first cell structure 2A having two or more cell layers stacked in the thickness direction, a cell suspension containing stromal cells is further inoculated, and the second cell structure 2B is constructed on the top of the single-celled cancer organoid 1A, and the single-celled cancer organoid 1A is cultured inside the cell structure. The method for preparing the first cell structure 2A and the second cell structure 2B can be applied to the method described in the above "cell structure".

[0141] exist Figure 1C and Figure 1DAmong the methods described in , examples of methods for converting cancer organoids into single cells include methods of performing enzyme treatment. The enzyme used in the enzyme treatment may be one type or two or more types.

[0142] The enzyme used is not particularly limited as long as it has the target enzyme activity, and may be an enzyme derived from any biological species, or an artificial enzyme obtained by modifying a naturally occurring enzyme. In addition, it may be an enzyme extracted and purified from various cells, or a chemically synthesized enzyme.

[0143] Specifically, the enzyme used for single cell formation is preferably one or more enzymes selected from trypsin, collagenase, dispase, elastase, papain, hyaluronidase, and their substitutes, more preferably trypsin or its substitute, and still more preferably a substitute for trypsin. As a substitute for trypsin, for example, TrypLE TM Expressed enzyme (1×), phenol red-free (registered trademark, Thermo Fisher Scientific), etc.

[0144] The treatment temperature of the enzyme treatment can be any condition as long as the enzyme used can exert its enzymatic activity. From the perspective of inhibiting the influence of biological tissue on cells in the small piece, the treatment temperature of the enzyme treatment is preferably 30°C or more and 40°C or less, and more preferably 37°C. In addition, the treatment time of the enzyme treatment is not particularly limited, and for example, it can be 5 minutes or more and 90 minutes or less, and preferably 10 minutes or more and 60 minutes or less.

[0145] The number of cells of the unicellularized cancer organoid is preferably measured in advance before being inoculated on the top surface or inside of the cell structure, and the number of viable cells is particularly preferably measured in advance. The measurement of the number of cells and the measurement of the number of viable cells can be performed by conventional methods. For example, the measurement of the number of viable cells can include a staining method using trypan blue, etc.

[0146] Cancer organoids can be washed with a buffer or culture medium before enzyme treatment. As a buffer for washing, phosphate buffer, acetate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer and PBS can be used. In addition, antibiotics can be added to the buffer or culture medium for washing. The number of washings can be appropriately determined, preferably more than 1 time and less than 8 times. In addition, washing with a buffer or culture medium can be performed only before enzyme treatment or before and after enzyme treatment.

[0147] When only specific cells in cancer organoids are cultured on the top surface or inside the cell structure, only cancer cells of the target cell type are sorted from the enzyme-treated cancer organoids, and the sorted cancer cells are added to the culture medium of the cell structure. Figure 1D The method described is inoculated into the interior of the cell structure. The method for sorting specific cells from the enzymatically treated product of cancer organoids is not particularly limited, and can be appropriately selected and used from various methods commonly used for cell sorting. For example, specific cells can be sorted by one or more methods selected from flow cytometry, magnetic separation, dielectrophoresis, size classification and density gradient classification.

[0148] In addition, before culturing cancer organoids on the top surface or inside of the cell structure, the cells constituting the cancer organoids can be pre-labeled with fluorescent substances or the like. All cells in the cancer organoids can be labeled, or only target specific cells in the cancer organoids can be labeled. The cell labeling method is not particularly limited, and can be appropriately selected from various labeling methods known in the art. For example, the labeling of cancer cells in cancer organoids can be appropriately utilized by fluorescent labeling performed by CellTracker (registered trademark, Thermo Fisher Scientific), PKH Cell Linker Kit (Sigma-Aldrich Company), etc. In addition, in the case of singulating the cancer organoids and then sorting specific cancer cells, the cancer cells after the sorting process can also be labeled.

[0149] [Cultivation medium]

[0150] The culture medium may be a medium containing growth factors, artificial inhibitors, etc. as long as it does not contain an extracellular matrix, and is preferably a common culture medium commonly used in culturing cell lines. The extracellular matrix is ​​as exemplified in the above "cancer organoid".

[0151] Examples of common culture media commonly used in culturing cell lines include D-MEM, E-MEM, ESC, EBM2, EGM2, MEMα, RPMI-1640, Ham's F-12, and Mccoy's 5A, as well as culture media prepared by adding serum such as CS (bovine serum), FBS (fetal bovine serum), or HBS (fetal horse serum) (to a concentration of about 1% by volume to about 20% by volume (preferably about 10% by volume)), and solutions of antibiotics such as penicillin-streptomycin (to a concentration of about 0.1% by volume to about 5% by volume (preferably about 1% by volume)).

[0152] Among them, as a culture medium, D-MEM, ESC, EBM2, EGM2 without hydrocortisone as an additive factor, RPMI-1640, Ham's F-12 and Mccoy's 5a, and a culture medium to which the above-mentioned serum (reaching 1 volume % or more and 20 volume % or less (preferably about 10 volume %)) and a solution of antibiotics such as penicillin-streptomycin (reaching 0.1 volume % or more and 5 volume % or less (preferably about 1 volume %)) are preferably added. Among them, hydrocortisone inhibits the formation of a vascular network structure in a cell structure, so it is preferably not included in the culture medium.

[0153] [Cultivation conditions]

[0154] The culture conditions can be appropriately set in the same manner as the culture conditions of common animal cells. For example, the culture temperature is preferably about 30° C. or more and 40° C. or less, and most preferably 37° C. In addition, the CO2 concentration is preferably about 1% by volume or more and 10% by volume or less, and most preferably about 5% by volume. In addition, culture can also be performed under an environment in which the O2 concentration is controlled to be lower than that of the atmosphere.

[0155] The cancer organoids seeded on the top surface or inside the cell structure preferably have a ductal structure. When culturing using cancer organoids that have maintained their morphology, cancer organoids that are closer to the in vivo state can be co-cultured with the cell structure containing stromal cells.

[0156] In addition, as shown in the examples described below, the cancer organoids in the culture obtained by the culture method of this embodiment form a duct structure when culturing using single-celled cancer organoids, or maintain the duct structure when culturing using cancer organoids that have a duct structure and maintain their morphology.

[0157] The culture containing cancer organoids having such a ductal structure obtained by the culture method of this embodiment is very useful as a sample for evaluating the efficacy of candidate anticancer agents under conditions closer to in vivo conditions and for selecting the optimal anticancer agent for each patient.

[0158] 《Screening methods for test substances》

[0159] A method for screening a test substance according to one embodiment of the present invention (hereinafter sometimes referred to as “the screening method according to the present embodiment”) comprises: a step of culturing a cell structure comprising a cancer organoid obtained by the above-mentioned cancer organoid culturing method in the presence of a test substance (hereinafter sometimes referred to as “a culturing step in the presence of a test substance”); and

[0160] A step of evaluating the effect of the test substance on the cancer organoid (hereinafter sometimes referred to as "evaluation step").

[0161] The screening method of this embodiment can evaluate the efficacy of candidate anticancer agents under conditions closer to in vivo by using a cell structure containing cancer organoids obtained by the above-mentioned cancer organoid culture method. In addition, when using cancer organoids derived from cancer patients, the screening method of this embodiment can be effectively used to select the most suitable anticancer agent for the cancer patient.

[0162] Next, the steps in the screening method according to the present embodiment will be described in detail below.

[0163] <Culturing step in the presence of a test substance>

[0164] In the culturing step in the presence of a test substance, a cell structure comprising a cancer organoid obtained by the above-mentioned cancer organoid culturing method is cultured in the presence of a test substance. For example, the test substance is brought into contact with the cancer organoid by a method of mixing and adding the test substance to a culture medium.

[0165] As the substance to be tested, for example, a natural compound library, a synthetic compound library, an existing drug library, and a metabolite library can be cited. Existing drugs include well-known anticancer agents such as AMG510 and cetuximab. In addition, new drugs can also be used as the substance to be tested. The substance to be tested can be one, or two or more can be used in combination. The amount of the substance to be tested given can be appropriately selected according to the type and amount of cancer cells constituting the cancer organoid.

[0166] <Evaluation Process>

[0167] In the evaluation step, the effect of the test substance on the cancer organoid is evaluated.

[0168] In the evaluation step, the effect of the test substance on cancer organoids can be examined or evaluated by immunoblotting, ELISA, immunostaining, or the like.

[0169] In addition, in the evaluation step, when single-celled cancer organoids are used, the survival rate of the cancer cells can be calculated by measuring the number of viable cancer cells at the time of inoculation and measuring the number of viable cancer cells after contact with the test substance. That is, the evaluation step can be a step of calculating the ratio of the number of viable cancer cells after contact with the test substance to the number of viable cancer cells at the time of inoculation.

[0170] The method for determining the number of viable cells of cancer cells is described in detail. The number of viable cells of cancer cells can be evaluated using signals related to the viable cells of cancer cells or their presence. As long as the number of viable cells of cancer cells at the evaluation time point can be determined, it is not necessarily necessary to measure in a viable state. For example, cancer cells can be marked to distinguish them from other cells, and the signal from the mark is studied as an indicator. For example, after the cancer cells are fluorescently labeled, the life and death of the cells is determined, so that the viable cells in the cell structure can be directly counted. At this time, image analysis technology can also be used. The life and death of cells can be determined by known cell life and death determination methods such as trypan blue staining or PI (Propdium Iodide, propidium iodide) staining. Among them, the fluorescent labeling of cancer cells can be performed by using antibodies against substances specifically expressed on the cell surface of cancer cells as primary antibodies, using immunostaining methods such as fluorescently labeled secondary antibodies that specifically bind to the primary antibodies. The life and death determination of cells and the determination of the number of viable cells can be performed in the state of the cell structure, or in the state where the cell structure is destroyed to the single cell level. For example, after destroying the three-dimensional structure of a cell structure in which cancer cells and dead cells are labeled, only cancer cells alive at the evaluation time point may be directly counted by FACS (fluorescence activated cell sorting) using the label as an index.

[0171] As the labeling material that cancer cells are marked, for example, fluorescent pigments, fluorescent beads, quantum dots, biotin, antibodies, antigens, energy absorbing materials, radioisotopes, chemiluminescent bodies and enzymes, etc. can be enumerated, but are not limited to these. Wherein, fluorescent pigments are preferably used as labeling materials. As fluorescent pigments, more specifically, for example, FAM (carboxyfluorescein), JOE (6-carboxyl-4', 5'-dichloro 2', 7'-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), HEX (5'-hexachloro-fluorescein-CE phosphoramide), Cy3, Cy5, Alexa568, Alexa647, PKH26 and PKH67GL, etc. can be enumerated.

[0172] In addition, in the evaluation step, the survival rate of cancer cells in the cell structure after culturing the cell structure comprising the cancer organoid in the absence of the test substance may be calculated as a control group. That is, the evaluation step may be a step of comparing the survival rate of cancer cells in the cell structure after culturing the cell structure comprising the cancer organoid in the absence of the test substance with the survival rate of cancer cells in the cell structure after culturing the cell structure comprising the cancer organoid in the presence of the test substance.

[0173] In this case, if the survival rate of cancer cells in the group containing the test substance is lower than that in the group not containing the test substance, it can be determined that the cancer organoids are sensitive to the drug of the test substance and the test substance is effective for the cancer organoids.

[0174] On the other hand, when the survival rate of cancer cells in the group with the test substance remains unchanged or increases compared to the survival rate of cancer cells in the group without the test substance, it can be judged that the cancer cells have acquired resistance to the test substance and the test substance has no effect on the cancer organoids.

[0175] Example

[0176] Hereinafter, the above-mentioned embodiments of the present invention will be specifically described by way of examples, but the present invention is not limited to the following examples at all, and a person having ordinary knowledge in the field can make various modifications within the scope of the present invention.

[0177] <Materials>

[0178] 1. Culturing cells and cancer organoids

[0179] The cell lines and cancer organoids used, and the medium used for culturing them are shown in the following Table 1. Patient-derived cancer organoid HT191 is a cancer organoid derived from a patient with colorectal cancer (KRAS G12C mutation-positive), and was constructed based on the method described in Reference 2 (Japanese Patent No. 6639634).

[0180] Table 1

[0181]

[0182] 2. Reagents, etc.

[0183] The reagents, consumables such as culture vessels, and antibodies used are shown in Tables 2 to 4 below.

[0184] Table 2

[0185]

[0186] Table 3

[0187]

[0188] Table 4

[0189] Antibody Manufacturer Name Host clone Dilution ratio Secondary Antibodies CD31 DAK0 Mouse JC70A 1 / 50 647 EpCAM CST Rabbit D4k8R 1 / 100 594

[0190] 3. Measurement device

[0191] The number of cells in the cell suspension was counted using an automatic cell counter Countss (registered trademark) II FL (Thermo Fisher Scientific). Fluorescent immunostaining images were captured and analyzed using Operetta CLS TM High-content imaging system (Perkin Elmer).

[0192] [Example 1]

[0193] Cancer organoids are inoculated directly or in the form of single cells onto the top surface or inside of a cell structure for culture, and the proliferation of the cancer organoids is confirmed.

[0194] It should be noted that D-MEM or IntestiCult organoid growth medium was used in the co-culture of PDO and cell structures described below. D-MEM was D-MEM to which FBS (up to 10 (v / v)%) and penicillin-streptomycin solution (up to 1 (v / v)%) were added. All cells were sterilely operated in a safety cabinet and cultured in an incubator at 37°C and 5 (v / v)% CO2 atmosphere.

[0195] 1. Pre-culture of Patient-Derived Organoids (PDO)

[0196] While maintaining the morphology of PDO during passaging, medium was added and pipetted to physically disperse the Matrigel and PDO.

[0197] When PDO was isolated into single cells, it was washed with PBS and then isolated using TrypLE TM The enzyme was expressed (1×) and treated at 37°C for 15 minutes without phenol red to disperse the cells. Then, the reaction was stopped in a culture medium and the single-celled PDO was recovered.

[0198] Then, while reseeding PDO, cool the test tube of the cell pellet on ice and suspend it with Matrigel. At this time, be careful and quick to avoid bubbles during suspension. Apply 20μL of cell suspension gel to the center of a 24-well plate cooled on ice, and use the tip of the chip to apply the gel to about half the diameter of the well. Be careful not to let the gel touch the edge of the well at this time. Incubate the plate upside down, and after the gel solidifies, add 500μL / well of culture medium (IntestiCult Organoid Growth Medium). In the case of 48 wells, add 250μL / well of culture medium (IntestiCult Organoid Growth Medium).

[0199] 2. Preparation of Cell Structures

[0200] The cell structure using the heparin method was prepared according to the previously reported procedure.

[0201] Specifically, first, 5 mM acetic acid solution containing 0.2 mg / mL collagen and 100 mM Tris-HCl buffer (pH 7.4) containing 0.2 mg / mL heparin were mixed in equal amounts and diluted to a final concentration of 0.1 mg / mL to prepare a cell coating solution.

[0202] In the case of culturing PDO on the top surface of the cell structure, mix the cell suspension so that the NHDF reaches 2 × 10 6 cells / well (20 layers), and HUVEC reached 3×10 4 cells / well (1.5% of NHDF), and after centrifugation, the supernatant was removed. On the other hand, when PDO was cultured inside the cell structure, the cell suspension was mixed so that the NHDF reached 1×10 6 cells / well (10 layers), and HUVEC reached 1.5×10 4 cells / well (1.5% of NHDF), and after centrifugation, the supernatant was removed.

[0203] Next, suspend with cell coating solution, centrifuge, and remove the supernatant. Then, suspend with culture medium and inoculate into a cell culture plug-in for a 24-well plate. The plug-in is pre-coated with a 0.1 mg / mL fibronectin solution for more than 30 minutes and the solution is removed. After dispensing 1 mL of D-MEM culture medium into the wells of the 24-well plate and 300 μL of cell suspension into the plug-in, centrifuge the plate to settle the cells and let them stand in the incubator for 2 hours. Then, add 1 mL of culture medium and incubate at 37°C for 1 day.

[0204] 3. Co-culture of PDO and cell structures

[0205] Next, the PDO or the single-cellized PDO in the same form was dispersed by the method described in "1." above, and the number of cells was measured, and the number of cells was adjusted to a desired number of inoculations (1×10 for single-cellized PDO). 4 The cells / well and the PDO cells maintaining their morphology were seeded in the cell structure at 200±20 cells / well.

[0206] Specifically, when culturing PDO on the top surface of the cell structure, after inoculation of PDO, the cell structure was left to stand in an incubator for more than 2 hours, and then the medium was added. On the other hand, when culturing PDO inside the cell structure, after inoculation of PDO, the cell structure was left to stand in an incubator for more than 2 hours, and then a medium containing 1×10 6 Cells / well (10 layers) NHDF and 1.5×10 4A HUVEC cell suspension of 1.5% cells / well (NHDF) was added to the insert and centrifuged at 400×g for 1 minute. After standing in an incubator for more than 2 hours, 1 mL of culture medium was added and incubated at 37°C.

[0207] The start of the preparation of the cell structure was taken as day 0. After the second day (i.e., the start day of co-culture of PDO and the cell structure), the tissue was washed twice with PBS and fixed with 10 v / v% neutral buffered formalin. The samples obtained by inoculating the single-celled PDO on the top surface of the cell structure and culturing were fixed on the second, sixth, ninth and 14th days. In addition, on the second day, fixation was performed 8 hours after inoculation of PDO. The samples obtained by inoculating the single-celled PDO inside the cell structure and culturing were fixed on the fourth, seventh and ninth days. One hour after the start of fixation, the formalin was removed, and the tissue was washed twice with PBS and then immersed in PBS.

[0208] For samples that were continuously cultured, the medium was replaced with an arbitrary medium once every two days or once every three days.

[0209] 4. Fluorescent Immunostaining (IF)

[0210] The fixed tissue is blocked for 1 to 2 hours with a PBS solution containing 1 (v / v)% BSA and 0.2 (v / v)% Triton. Then, add the primary antibody according to the dilution ratio recorded in the above table and treat overnight (O / N). The next day, remove the primary antibody solution, wash with PBS, and add the secondary antibody according to the dilution ratio recorded in the above table and treat for 2 hours. Remove the secondary antibody solution and wash with PBS. Both the primary and secondary antibodies are diluted with a PBS solution containing 1 (v / v)% BSA and 0.2 (v / v)% Triton. In addition, wash with PBS as needed, dehydrate with 99.5 (v / v)% ethanol, and then transparentize with Visikol reagent.

[0211] 5. Image capture and image analysis

[0212] Image capture and image analysis of stained samples using Operetta CLS TM After alignment was performed using PreScan with a 1.25x objective lens, images were taken in nine fields of view for each well using a 5x objective lens.

[0213] The acquired images were analyzed by the image analysis software Harmony. The analysis was performed by tiling the images of 4 fields of view as a global image in the case of 96 wells and tiling the images of 9 fields of view as a global image in the case of 24 wells. The images of each plane with different Z positions were synthesized into one in Maximum projection. The ROI (Region of interest) was set so that it could cover most of the culture surface of the cell culture plug-in. After the area with high fluorescence intensity (EpCAM positive) in the ROI was identified as cancer cells, the area of ​​the cancer cells was calculated. In addition, the number of Ki-67 cells contained in the EpCAM positive area was counted.

[0214] 6. Preparation of sections and immunohistochemical staining (IHC)

[0215] (1) Preparation of paraffin sections

[0216] The embedding was performed using a paraffin embedding device CT-Pro20 (Genostaff) based on the scheme of using a low-toxic solvent G-Nox (Genostaff) as a substitute for xylene to prepare the block. The block was thinly sliced ​​at a thickness of about 6 μm to prepare serial sections.

[0217] (2)IHC

[0218] Tissue sections were deparaffinized with xylene and rehydrated with an ethanol series and TBS (Tris-buffered saline). Antigens were activated by enzyme treatment (proteinase K, 5 μg / mL, 37°C, 10 minutes). After washing with PBS at room temperature (about 25°C) for 5 minutes, three times, endogenous peroxidase was blocked with methanol containing 0.3 v / v% hydrogen peroxide for 30 minutes, washed with TBS at room temperature (about 25°C) for 5 minutes, three times, blocked with G-Block (Genostaff), and incubated with an avidin / biotin blocking kit (avidin / biotin blocking kit, Vector). The sections were incubated overnight at 4°C with anti-EpCAM mouse monoclonal antibody (CST).

[0219] After washing with TBS-T at room temperature (about 25°C) for 5 minutes, twice, and then washing with TBS at room temperature (about 25°C) for 5 minutes, once, add biotin-conjugated goat anti-mouse IgG (Vector) and incubate at room temperature (about 25°C) for 30 minutes. Thereafter, add peroxidase-conjugated streptavidin (Nichirei) for 5 minutes. After washing with TBS-T at room temperature (about 25°C) for 5 minutes, twice, and then washing with TBS at room temperature (about 25°C) for 5 minutes, once, the peroxidase activity was visualized with diaminobenzidine. The sections were contrast stained with Mayer's Hematoxylin (MUTO), dehydrated, and sealed with Malinol (MUTO).

[0220] 7. Results and Investigation

[0221] (1) Co-culture of single-celled PDO on the top surface of the cell structure

[0222] The results of confirming proliferation when PDO dispersed in single cells was seeded on the top surface of the cell structure are shown in FIG. Figure 2A (Fluorescent immunostaining images of PDO) and Figure 2B (Graph showing fusion (%) of PDO). Figure 2A The scale bar in the figure is 1 mm.

[0223] like Figure 2A and Figure 2B As shown, when PDO dispersed in single cells is inoculated on the top surface of the cell structure, it can be seen that it proliferates over time even if either D-MEM or organoid culture medium is used. Focusing on the difference in culture medium, when cultured in D-MEM, PDOs aggregate with each other and cells proliferate, whereas when cultured in organoid culture medium, PDOs proliferate while remaining at the position at the inoculation time point.

[0224] (2) Co-culture of single-celled PDO inside a cell structure

[0225] The results of confirming the proliferation of PDO dispersed in single cells when seeded inside the cell structure are shown in FIG. Figure 3A (Fluorescent immunostaining image of PDO), Figure 3B (Graph showing the fusion of PDO (%)), Figure 3C (Immunostaining images of PDO and vascular network structure in sections), Figure 3D (Fluorescent immunostaining images of PDO and vascular network structure in sections) and Figure 3E (Fluorescent immunostaining images of vascular network structures in sections). Figure 3A The scale bar in the figure is 1 mm. Figure 3CThe arrows in the figure indicate PDO infiltrating into the blood vessel-like vascular network structure. Figure 3D The scale bar in is 200 μm. Figure 3D The arrows in the figure indicate PDO infiltrating into the blood vessel-like vascular network structure.

[0226] like Figure 3A and Figure 3B As shown, it was found that even when PDO dispersed in single cells was inoculated inside the cell structure, PDO proliferated over time.

[0227] In addition, from Figure 3C The immunostaining images of the sections using the anti-EpCAM antibody confirmed that, under the condition that PDO dispersed in single cells was inoculated inside the cell structure, a glandular duct structure was formed after 7 days.

[0228] In addition, from Figure 3C Immunostained images of sections using anti-CD31 antibodies and Figure 3D It was confirmed that PDO infiltrated into a part of the blood vessel-like vascular network structure. In addition, it was observed that the blood vessel-like vascular network structure was enlarged in morphology compared with the normal state, and that the blood vessel-like lumen structure was induced by PDO.

[0229] like Figure 3E As shown, it was clear that endothelial cells in organoid culture medium aggregated with each other and could not form a vascular network structure like blood vessels. It is speculated that organoid culture medium is not suitable as a culture medium for evaluating the interaction between vascular network structure like blood vessels and PDO, but D-MEM is suitable.

[0230] (3) Co-culture of PDO on the top surface of the cell structure while maintaining its morphology

[0231] The results of confirming proliferation when PDO with maintained morphology was seeded on the top surface of the cell structure are shown in Figure 4A (Bright field image) and Figure 4B (Immunostaining images of PDO in sections). Figure 4A The scale bar in the upper row is 1 mm, and the scale bar in the lower row is 500 μm.

[0232] like Figure 4A As shown, it was confirmed that PDO adhered to the top surface of the cell structure, and the three-dimensional structure of PDO was slightly flattened as the culture time passed. Due to the bright field, it is difficult to quantitatively evaluate, but it is known that even if any culture medium of D-MEM and organoid culture medium is used, the cells themselves proliferate over time.

[0233] like Figure 4BAs shown, it was found that when cultured in D-MEM, PDO maintained the morphology of the ductal structure, whereas when the organoid culture medium was used, the ductal structure disappeared and existed in a flattened form on the top surface of the cell structure.

[0234] From the above, we can know the following.

[0235] When the single-celled PDO was cultured on the top surface or inside the cell structure, PDO proliferated even if either D-MEM or organoid culture medium was used. In addition, it was confirmed that when D-MEM was used as the culture medium, PDO formed a glandular duct structure on the top surface or inside the cell structure. In addition, infiltration into a blood vessel-like vascular network structure was also observed.

[0236] When PDO with its morphology maintained was cultured on the top surface of the cell structure, PDO proliferated regardless of whether D-MEM or organoid culture medium was used. In addition, it was found that when D-MEM was used as a culture medium, the duct structure was maintained, whereas when the organoid culture medium was used as a culture medium, the duct structure of PDO could not be maintained.

[0237] Furthermore, it was found that when D-MEM was used as the culture medium, the cell structure was able to form a vascular network structure, whereas when the organoid culture medium was used as the culture medium, the vascular network structure could not be formed.

[0238] [Example 2]

[0239] From the results of Example 1, it is known that under the condition of seeding PDO with the morphology maintained, it is difficult to measure the number of cells and seed any number of cells in the cell structure, but under the condition of seeding PDO after single cellization, the number of cells can be measured and seeded, which is suitable for drug efficacy evaluation. Therefore, the drug efficacy evaluation test was performed under the condition of seeding single cellized PDO on the top surface of the cell structure.

[0240] The PDO used had a G12C mutation in the KRAS gene, so the G12C inhibitor AMG510 was used alone (at a concentration of 0, 0.1, 1, and 10 μM in the culture medium) and the EGFR inhibitor cetuximab (at a concentration of 50 μg / mL in the culture medium) was used as a drug. D-MEM was used as the culture medium.

[0241] First, the same method as in Example 1 was used, with the construction start date of the cell structure as day 0, the medium was replaced on day 1, and single-celled PDO (5×10 3cells / well; 96-well plate) were seeded on the top surface of the cell structure. Then, the above-mentioned agent was added on the 5th day, fixed on the 8th day, and the number of surviving cancer cells was measured. Then, the ratio of the number of surviving cells under each condition to the number of cells under the condition of AMG510 at 0μM and cetuximab at 0μg / mL was calculated as the survival rate (%) of cancer cells. The results are shown in Figure 5 .exist Figure 5 In the graph, the horizontal axis represents the concentration of AMG510 in the culture medium, and the vertical axis represents the survival rate (%) of cancer cells.

[0242] like Figure 5 As shown, in both the case of using AMG510 alone and the case of using AMG510 and cetuximab in combination, a drug concentration-dependent decrease in cancer cell AMG510 was confirmed.

[0243] From the above results, it was confirmed that the efficacy of the drug expected to have the effect presumed based on the PDO gene mutation was actually achieved.

[0244] [Example 3]

[0245] Organoids different from those in Example 1 were seeded on the top surface or inside of the cell structure and cultured to confirm the proliferation of cancer organoids. In addition, the influence of the culture medium on the formation of the vascular network was studied.

[0246] <Materials>

[0247] 1. Culturing cells and cancer organoids

[0248] The cell lines and cancer organoids used are shown in Table 5. Patient-derived PDO HCT26 1T is a PDO of a primary lesion derived from a patient with colorectal cancer. Patient-derived PDO HCT26 3LM is a PDO of a large intestine of a metastatic lesion derived from a patient with colorectal cancer. Patient-derived PDO HCT26 1T and patient-derived PDO HCT26 3LM used PDOs established based on the method described in Reference 1.

[0249] Table 5

[0250]

[0251] The HUVEC and NHDF used in the construction of the cell structure were the same as those in Example 1.

[0252] 2. Reagents, etc. and measuring devices

[0253] The reagents used are shown in Table 6. Consumables such as culture containers, antibodies, and measurement devices were the same as those in Example 1.

[0254] Table 6

[0255]

[0256] Table 7 shows the composition of the culture medium used and the factors added to the culture medium.

[0257] Table 7

[0258]

[0259] <Cultivation and Image Analysis>

[0260] The pre-culture of patient-derived PDO and the preparation of the cell structure were performed by the same method as in Example 1. For the co-culture of PDO and the cell structure, a method of using single-celled PDO to culture on the top surface of the cell structure was adopted. In addition, for the samples to be cultured continuously, the co-culture of PDO and the cell structure was performed by the same method as in Example 1, except that the culture medium was replaced with each culture medium once every 3 or 4 days.

[0261] Fluorescent immunostaining, image photography and image analysis were performed in the same manner as in Example 1.

[0262] <Results and Investigation>

[0263] PDO and cell structures were co-cultured using the culture media D-MEM, IntestiCult Organoid Growth Medium, RPMI-1640, McCoy5A, and Ham's F12 shown in Table 6 and the culture media AIM(+), EGM2, and ESC shown in Table 7 to evaluate vascular network formation. Figure 6 These are images of immunostaining using anti-CD31 antibodies using tissues cultured in each culture medium. Figure 6 The scale bar in the figure represents 500 μm. Figure 6 In the table, DMEM denotes D-MEM, OM denotes IntestiCult Organoid Growth Medium, RPMI denotes RPMI-1640, McCoy denotes McCoy5A, F12 denotes Ham's F12, and ESC-Y-bFGF denotes ESC.

[0264] The results showed that vascular networks were formed when D-MEM, RPMI-1640, and McCoy 5A were used. When Ham's F12 and ESC were used, vascular networks were formed, although they were suppressed compared to D-MEM. In addition, when EGM2, IntestiCult Organoid Growth Medium, and AIM(+) were used, endothelial cells aggregated and vascular network formation itself was suppressed.

[0265] EGM2 and AIM(+) were supplemented with basal medium and the supplementary factors listed in Table 7, respectively. Therefore, we distinguished whether the inhibitory factor of vascular network formation was basal medium or supplementary factors. It should be noted that IntestiCult organoid growth medium already contained supplementary factors, so it was not possible to distinguish and was therefore discarded.

[0266] As culture medium, D-MEM, AIM(+), and D-MEM and AIM with added factors of AIM(+) were used respectively to perform co-culture of PDO and cell structures. The results are shown in Figure 7 . Figure 7 The scale bar in the figure represents 500 μm. Figure 7 In the table, DMEM+AIM supplement means that D-MEM to which the supplement factor AIM(+) was added was used.

[0267] When D-MEM to which AIM(+) was added as a factor was used, a vascular network was formed, whereas in the AIM medium to which no additional factor was added, the formation of a vascular network was inhibited. Therefore, it was suggested that the basal medium of AIM is a vascular network inhibitory factor.

[0268] PDO and cell structures were co-cultured using D-MEM supplemented with D-MEM, EBM2, and EGM2 as culture medium. The results are shown in Figure 8 . Figure 8 The scale bar in the figure represents 500 μm. Figure 8 In the above, DMEM+EGM2 supplement means that D-MEM to which the supplement factor of EGM2 is added is used. In EBM2, which is the basal medium of EBM2 medium, a vascular network is formed, whereas in D-MEM to which the supplement factor of EGM2 is added, the formation of a vascular network is inhibited.

[0269] Next, in order to identify the inhibitory factors among the added factors, mediums were prepared in which each of the added factors of EGM2 listed in Table 7 was added to D-MEM and EBM2, and mediums in which each of the added factors listed in Table 7 was excluded from EBM2, and vascular network evaluation was performed. Fig. 9 and Fig.10 . Fig. 9 and Fig.10 The scale bar in the figure represents 500 μm. Fig. 9 In the text, DMEM+EGM2 supplement means that D-MEM to which all the supplementary factors of EGM2 were added was used. Fig.10In the table, + indicates that an added factor is present, and - indicates that an added factor is excluded. As a result, when only hydrocortisone was added to DMEM and EBM2, the formation of the vascular network was inhibited, while when only hydrocortisone was excluded from the EGM2 medium, the vascular network was formed, indicating that hydrocortisone is involved in the inhibition of the formation of the vascular network.

[0270] IntestiCult Organoid Growth Medium contains several additional factors in the basal medium listed in Table 7. Therefore, the basal medium was used as the culture medium and the co-culture of PDO and cell structures was performed in the same manner as in Example 3. The results are shown in Fig.11 The “foundation” of. Fig.11 Also shown are the results of co-culture using IntestiCult Organoid Growth Medium. Fig.11 The scale bar in the figure represents 500 μm. Fig.11 As shown in the figure, it can be seen that a vascular network is formed when a basal medium is used. The medium in which various supplementary factors (B27-vitamin A, N-acetylcysteine, gastrin, EGF, R-spondin 1, Noggin, nicotinamide, A-83-01, and SB202190) are added to the basal medium is the IntestiCult Organoid Growth Medium, so it is believed that these supplementary factors have factors that inhibit the formation of a vascular network.

[0271] In addition, in order to study the effect of FBS on vascular network formation, D-MEM without FBS (hereinafter referred to as DMEM(-)) and D-MEM containing FBS (hereinafter referred to as DMEM(+)) were used to co-culture PDO and cell structures in the same manner as in Example 3. The results are shown in Fig.11 When D-MEM was used, the density and amount of vascular network formation differed depending on the presence or absence of FBS, but vascular network formation was observed in all cases.

[0272] Based on the above results, it is suggested that the basal medium in AIM medium may be the main factor for the inhibition of vascular network. It is suggested that the factor hydrocortisone added to EGM2 medium is the factor for the inhibition of vascular network.

[0273] [Example 4]

[0274] Cancer proliferation and morphology evaluation were performed using HCT26-1T and HCT26-3LM, which are different types of PDO2 from Example 1. DMEM(-), DMEM(+), basal medium, and IntestiCult organoid growth medium were used as culture media and culture was performed in the same manner as in Example 3. In the method of culturing PDO inside a cell structure using single-celled PDO, for samples that were continuously cultured, co-culture of PDO and a cell structure was performed in the same manner as in Example 1, except that the culture medium was replaced with each culture medium once every 3 or 4 days.

[0275] The results are shown in Fig.12 . Fig.12 The scale bar in the figure represents 500 μm. Fig.12 In the figure, “on 3D” means culturing PDO on the top surface of the cell structure, and “in 3D” means culturing PDO inside the cell structure. Fig.12 The images shown show the calculation of the confluence (%) of PDOs on days 4, 8, and 11 of culture. Fig.13 It is a graph showing the time-dependent change in the fusion (%) of PDO in tissues obtained by culturing HCT26-1T and HCT26-3LM in each culture medium. It can be seen that HCT26-1T and HCT26-3LM proliferated significantly in culture media other than IntestiCult Organoid Growth Medium, whether PDO was cultured on the top surface of the cell structure or inside the cell structure.

[0276] Next, in order to confirm the morphology of PDO, sections of these tissues were prepared and evaluated. Fig.14 This is an image showing immunostaining of PDO in tissue sections when HCT26-1T cells were cultured on the apical surface of the cell structure. Fig.15 This is an image showing immunostaining of PDO in tissue sections when HCT26-3LM was cultured on the apical surface of a cell structure. Fig.16 This figure shows an immunostaining image of PDO in a tissue section when HCT26-1T cells were cultured inside a cell structure. Fig.17 This figure shows an image of PDO immunostaining in a tissue section when HCT26-3LM was cultured inside a cell structure.

[0277] Proliferation was poor under the conditions of using IntestiCult organoid growth medium. On the other hand, even when HCT26-1T and HCT26-3LM were cultured in either the top surface or the interior of the cell structure, PDO with ductal structure was confirmed under the conditions of the culture medium other than IntestiCult organoid growth medium. In addition, it is known that it is very interesting that even if single-celled HCT26-1T and HCT26-3LM are inoculated on the top surface of the cell structure, in most cases, the ductal structure is formed by proliferation inside the cell structure. Although there are also colonies of PDO that are locally present in the cell structure, PDO is at a high density in most cases. When PDO is at a high density, a ductal structure is not formed but a cylindrical epithelial-like structure is formed.

[0278] The above results confirmed that in HCT26 1T and HCT26 3LM, PDOs established from clinical samples, vascular network formation and PDO proliferation occurred in D-MEM and basal medium, but vascular network formation and PDO proliferation were inhibited in IntestiCult organoid growth medium containing additives. In addition, it was confirmed that even if PDO was inoculated on the top surface of the cell structure, it proliferated in the cell structure and formed a glandular duct structure.

[0279] Industrial availability

[0280] According to the cancer organoid culture method of the present embodiment, it is possible to co-culture with cells constituting the stroma while maintaining the properties of the cancer organoid without using an extracellular matrix, and to evaluate the infiltration of cancer cells.

[0281] Description of Reference Numerals

[0282] 1 Cancer Organoids

[0283] 1A Unicellularized cancer organoids (cancer organoids dispersed into single cells)

[0284] 2 Cell structure

[0285] 2A First cell structure

[0286] 2B Second cell structure

[0287] 3 Culture container

Claims

1. A method for culturing cancer organoids, comprising the step of culturing cancer organoids on the top surface or inside of a cell structure using a culture medium that does not contain an extracellular matrix, in, The cell structure includes cells constituting interstitial tissue and has two or more cell layers stacked in a thickness direction.

2. The method for culturing cancer organoids according to claim 1, wherein: The culturing comprises the step of dispersing the cancer organoid in the form of single cells and then culturing the cells on the top surface or inside the cell structure.

3. The method for culturing cancer organoids according to claim 1 or 2, wherein: The culture comprises the following steps: after inoculating the cancer organoid on the top surface of a first cell structure having two or more cell layers stacked in the thickness direction, further inoculating a cell suspension containing cells constituting the stroma, constructing a second cell structure on the top of the cancer organoid, and culturing the cancer organoid inside the cell structure.

4. The method for culturing cancer organoids according to claim 1 or 2, wherein: The culture medium is any one of D-MEM, ESC, EBM2, EGM2 without hydrocortisone as an added factor, RPMI-1640, Ham's F-12 and Mccoy's 5a, or a combination of two or more thereof.

5. The method for culturing cancer organoids according to claim 1 or 2, wherein: The cancer organoids have a glandular duct structure.

6. The method for culturing cancer organoids according to claim 1 or 2, wherein: The cell structure includes, as cells constituting the stroma, one or more cells selected from the group consisting of fibroblasts, pericytes, endothelial cells, and immune cells.

7. The method for culturing cancer organoids according to claim 6, wherein: The endothelial cells are one or more cells selected from vascular endothelial cells and lymphatic endothelial cells.

8. The method for culturing cancer organoids according to claim 1 or 2, wherein: The cell structure has a vascular network structure.

9. The method for culturing cancer organoids according to claim 1 or 2, wherein: The cell structure has 10 or more cell layers stacked in the thickness direction.

10. A method for screening a substance to be tested, comprising: a step of culturing a cell structure comprising a cancer organoid obtained by the method for culturing a cancer organoid according to claim 1 or 2 in the presence of a test substance; and The step of evaluating the effect of the test substance on the cancer organoid.

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