Culture medium and culture method for colorectal cancer organoids

Abstract

The present invention provides a culture medium and a culture method for intestinal cancer organoids. The culture medium comprises an MST1 / 2 kinase inhibitor; a B27 additive; an N2 additive; insulin; A8301; human fibroblast growth factor-10; neuregulin-1; amphiregulin; at least one Rho protein kinase inhibitor selected from Y27632, fasudil, and H-1152; fetal bovine serum; basic fibroblast growth factor; insulin-like growth factor-1; forsklin; bovine pituitary extract; and fibroblast growth factor 7. Compared with existing culture methods, in vitro culture using the culture medium of the present invention has higher expansion efficiency. Cultivating intestinal cancer organoids using this culture medium can maintain the morphological structure and pathological characteristics of the original tissue, thereby improving the success rate and survival rate of intestinal cancer organoid culture.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a culture medium and its application, and more specifically to a culture medium for intestinal cancer organoids and a method for culturing intestinal cancer organoids using the culture medium. Background Art

[0002] Colorectal cancer is one of the top ten most common malignant tumors in my country, with an incidence rate of approximately 24 per 100,000 people. It has also been one of the most rapidly increasing cancers in the past two to three decades. Colorectal cancer includes colon and rectal cancer. The incidence rate, from highest to lowest, is rectum, sigmoid colon, cecum, ascending colon, descending colon, and transverse colon, with a recent trend toward the proximal colon (right hemicolon). In the early stages of colorectal cancer, symptoms are asymptomatic or subtle, limited to discomfort, indigestion, and occult blood in the stool. As the cancer progresses, symptoms gradually emerge, manifesting as changes in bowel habits, abdominal pain, blood in the stool, abdominal masses, and intestinal obstruction, with or without systemic symptoms such as anemia, fever, and weight loss. While the incidence and mortality rates of colorectal cancer have declined in Europe and the United States in recent years, they have increased in my country. Currently, colorectal cancer treatment remains primarily surgical, supplemented by radiotherapy and chemotherapy to enhance treatment outcomes. Although significant progress has been made in recent years in comprehensive treatments such as targeted therapies and immunotherapy for colorectal cancer, the overall prognosis is not optimistic.

[0003] The cause of colorectal cancer is not yet fully understood. It is currently believed to be the result of a combination of environmental and genetic factors. The occurrence and development mechanisms of colorectal cancer are complex. Establishing a scientific and rigorous research model will not only benefit basic research on colorectal cancer, but also help in the diagnosis and treatment of colorectal cancer, and help improve the survival rate of colorectal cancer. Organoids are organ-specific cell collections derived from stem cells or precursor cells. Organoids cultured in vitro are highly similar to the corresponding organs in terms of cellular composition and tissue architecture, and have corresponding functional characteristics. Unlike conventional cell culture, which cultivates a single cell population in a two-dimensional environment, organoid culture cultivates multiple cell populations contained in specific tissues and organs in a three-dimensional environment. Its culture system is more similar to the in vivo microenvironment. Therefore, it shows great application prospects in basic research on the physiology and pathology of various organs, precision medicine, drug screening and development, gene therapy, regenerative medicine, etc.

[0004] Although various tumor tissues can be successfully cultured into organoids in vitro using different methods and culture conditions, there are currently few studies and reports on the culture methods of colorectal cancer organoids, especially the specific experimental processes, operating steps, culture conditions, and culture medium formulas.

[0005] Therefore, there is a need in the art for an organoid culture medium formula and culture method that has a high success rate for in vitro culture of intestinal cancer organoids and can maintain the original tissue pathological characteristics. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a culture medium and an in vitro culture method for intestinal cancer organoids.

[0007] One aspect of the present invention is to provide a culture medium for intestinal cancer organoids, the culture medium comprising an MST1 / 2 kinase inhibitor; B27 additive; N2 additive; insulin; A8301; human fibroblast growth factor-10; neuregulin-1; amphiregulin; at least one Rho protein kinase inhibitor selected from Y27632, fasudil, and H-1152; fetal bovine serum; basic fibroblast growth factor; insulin-like growth factor-1; forsklin; bovine pituitary extract; and fibroblast growth factor 7. The MST1 / 2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.

[0008]

[0009] in,

[0010] R1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl (e.g., phenyl and naphthyl, etc.) optionally substituted by 1-2 independently R6, aryl C1-C6 alkyl (e.g., benzyl, etc.) and heteroaryl (e.g., thienyl, etc.);

[0011] R2 and R3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

[0012] R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, and C3-C6 heterocyclylC1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyranyl, etc.);

[0013] R6 is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoromethyl).

[0014] In a preferred embodiment, the MST1 / 2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,

[0015]

[0016] in,

[0017] R1 is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R6, thienyl optionally substituted by 1-2 independently R6, and benzyl optionally substituted by 1-2 independently R6, more preferably phenyl optionally substituted by 1-2 independently R6;

[0018] R5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R5 is more preferably hydrogen;

[0019] R6 are each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R6 is more preferably fluorine, methyl or trifluoromethyl.

[0020] Preferably, the MST1 / 2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.

[0021]

[0022]

[0023]

[0024]

[0025]

[0026] Most preferably, the MST1 / 2 kinase inhibitor of the present invention is Compound 1.

[0027] In an embodiment of the present invention, the content of each component in the culture medium of the present invention satisfies any one, multiple or all of the following:

[0028] (1) The concentration range of MST1 / 2 kinase inhibitors is generally 1.25 to 10 μM, preferably 2.5 to 10 μM;

[0029] (2) The volume ratio of B27 additive to culture medium is generally in the range of 1:12.5 to 1:200, more preferably 1:50 to 1:100;

[0030] (3) The volume ratio of the N2 additive to the culture medium is generally in the range of 1:25 to 1:400, more preferably 1:100 to 1:400;

[0031] (4) The concentration range of insulin is usually 0.25 to 1 μg / mL, more preferably 0.25 to 0.5 μg / mL;

[0032] (5) The concentration range of A8301 is usually 1 to 9 nM, more preferably 1 to 3 nM;

[0033] (6) The concentration range of human fibroblast growth factor-10 is generally 2.5 to 40 ng / mL, more preferably 5 to 20 ng / mL;

[0034] (7) The concentration of neuregulin-1 is generally in the range of 2.5 to 40 ng / mL, more preferably 2.5 to 10 ng / mL;

[0035] (8) The concentration of amphiregulin is generally in the range of 1 to 27 ng / mL, more preferably 1 to 3 ng / mL;

[0036] (9) The concentration range of the Rho protein kinase inhibitor is generally 1.25 to 20 μM, more preferably 2.5 to 20 μM. The Rho protein kinase inhibitor is preferably Y27632;

[0037] (10) The volume concentration of fetal bovine serum is generally in the range of 1.25% (v / v) to 20% (v / v), more preferably 1.25% (v / v) to 2.5% (v / v);

[0038] (11) The concentration range of basic fibroblast growth factor is usually 2.5 to 40 ng / mL, more preferably 10 to 20 ng / mL;

[0039] (12) The concentration range of insulin-like growth factor-1 is generally 12.5 to 100 ng / mL, more preferably 12.5 to 25 ng / mL;

[0040] (13) The concentration range of forsklin is usually 0.625 to 2.5 μM, more preferably 0.625 to 1.25 μM;

[0041] (14) The volume ratio of bovine pituitary extract to culture medium is usually 1:2000–1:500;

[0042] (15) The concentration range of fibroblast growth factor 7 is usually 0.625 to 2.5 ng / mL.

[0043] In an embodiment of the present invention, the culture medium further contains an initial culture medium selected from DMEM / F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin / penicillin, amphotericin B and primocin.

[0044] In a preferred embodiment, when the antibiotic is selected from streptomycin / penicillin, the concentration of streptomycin is in the range of 25 to 400 μg / mL, and the concentration of penicillin is in the range of 25 to 400 U / mL. When the antibiotic is selected from amphotericin B, the concentration range is 0.25 to 4 μg / mL. When the antibiotic is selected from Primocin, the concentration range is 25 to 400 μg / mL.

[0045] According to a second aspect, the present invention further provides an in vitro culture method for intestinal cancer organoids. In the in vitro culture method for intestinal cancer organoids of the present invention, primary intestinal cancer cells are cultured in vitro using the intestinal cancer organoid culture medium of the present invention.

[0046] The in vitro culture method of intestinal cancer organoids of the present invention comprises the following steps:

[0047] 1. Isolation of Primary Colorectal Cancer Cells

[0048] (1) Isolate colorectal cancer tissue samples, add basal culture medium and tissue digestion solution at a volume ratio of 1:3 (Note: the amount of tissue digestion solution added is about 5-10 mL of tissue digestion solution for 1 g of tumor tissue), and place in a constant temperature shaker for digestion. The digestion temperature range is 4-37°C and the digestion speed range is 200 rpm-350 rpm;

[0049] (2) Digestion can be terminated when no obvious tissue blocks are observed. The digestion time is 3 to 6 hours.

[0050] (3) After centrifugation, the supernatant is discarded. The centrifugal speed range is 1200-1600 rpm, and the centrifugation time is 2-6 minutes. The intestinal cancer organoid culture medium of the present invention is added and resuspended for later use.

[0051] The basal culture medium includes an initial culture medium selected from DMEM / F12, DMEM, F12, or RPMI-1640; and one or more antibiotics selected from streptomycin / penicillin, amphotericin B, and primocin. The tissue digestion solution includes 1640 culture medium, collagenase II (1-2 mg / mL), collagenase IV (1-2 mg / mL), DNase (50-100 U / mL), hyaluronidase (0.5-1 mg / mL), calcium chloride (1-5 mM), and bovine serum albumin (BSA) (5-10 mg / mL).

[0052] 2. Cultivation using the intestinal cancer organoid culture medium of the present invention

[0053] The primary intestinal cancer cells obtained in step 1 above were resuspended in basal culture medium and counted, and mixed evenly with matrigel on ice at a volume ratio of 1:1. The final cell density was 1-8×10 5 / mL, take the matrix gel and cell suspension to form solidified droplets in the culture plate, let the culture plate stand at 4-37°C for 10-60 minutes to wait for the matrix gel to completely solidify, add the intestinal cancer organoid culture medium of the present invention, and culture in an incubator.

[0054] The technical solution of the present invention can achieve the following technical effects:

[0055] (1) Improve the success rate of colorectal cancer organoid culture, and be able to culture tumor tissue from multiple sources such as colon and rectum, with a culture success rate of over 80%;

[0056] (2) Intestinal cancer organoids cultured in vitro can maintain the patient's pathological characteristics;

[0057] (3) The cultured intestinal cancer organoids are not interfered with by stromal cells such as fibroblasts and adipocytes;

[0058] (4) High amplification efficiency, colorectal cancer organoids can be successfully cultured within about a week, and the amplified colorectal cancer organoids can be continuously passaged;

[0059] (5) The culture cost is controllable, and the culture medium does not need to be added with expensive Wnt agonists, R-spondin family proteins and other factors;

[0060] (6) The colorectal cancer organoids obtained by the above technology are large in number and highly homogenized, which are suitable for high-throughput screening of new candidate compounds and providing high-throughput in vitro drug sensitivity functional testing for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 A graph showing the effects of different combinations of added factors in colorectal cancer organoid culture medium on the growth of colorectal cancer organoids.

[0062] Figures 2A-2O A graph showing the effects of different concentrations of added factors to colorectal cancer organoid culture medium on the growth of colorectal cancer organoids.

[0063] Figures 3A-3F These are photographs of intestinal cancer organoids cultured using the intestinal cancer organoid culture medium of the present invention observed under a microscope.

[0064] Figures 4A-4B The photographs are of intestinal cancer organoids cultured to different passages using the intestinal cancer organoid culture medium of the present invention observed under a microscope.

[0065] Figures 5A-5D These are the immunohistochemical results of primitive intestinal cancer tissue cells.

[0066] Figures 6A-6D These are the immunohistochemical results of intestinal cancer organoids obtained by culturing primary intestinal cancer tissue cells to the sixth passage using the intestinal cancer organoid culture medium of the present invention.

[0067] Figure 7 These are microscopic photographs of intestinal cancer organoids obtained by culturing them using the intestinal cancer organoid culture medium of the present invention, the culture medium from literature, and the commercial culture medium. DETAILED DESCRIPTION

[0068] For a better understanding of the present invention, the present invention is further described below in conjunction with embodiments and drawings. The following embodiments are merely illustrative of the present invention and are not intended to limit the present invention.

[0069] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0070] As used herein, an MST1 / 2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1 / 2 signaling. Generally, an MST1 / 2 kinase inhibitor, for example, binds to and reduces the activity of MST1 / 2 kinases. Due to the structural similarities between MST1 and MST2, an MST1 / 2 kinase inhibitor may also be a compound that binds to and reduces the activity of either MST1 or MST2.

[0071] 1. Preparation of MST1 / 2 kinase inhibitor compound 1

[0072] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide 1

[0073]

[0074] Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): Add 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) to a round-bottom flask, followed by methanol (30 mL). Thionyl chloride (1.2 mL) was then added dropwise under an ice bath. The reaction was allowed to react at 85°C overnight. After completion of the reaction, the solvent was evaporated under reduced pressure to yield a white solid, which was used directly in the next step.

[0075] Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (A3): To a round-bottom flask, add methyl 2-amino-2-(2,6-difluorophenyl)acetate (2 g), followed by acetone (30 ml) and potassium carbonate (2.2 g). The mixture was then cooled to -10°C in an ice-salt bath. An acetone solution of 2,4-dichloro-5-nitropyrimidine (3.1 g) was then slowly added. The reaction mixture was stirred at room temperature overnight. After completion of the reaction, the mixture was filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by pressurized silica gel column chromatography to yield compound A3. LC / MS: M+H 359.0.

[0076] 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): To a round-bottom flask, add methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g), followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction system was stirred at 60°C for two hours. After completion of the reaction, the solvent was evaporated under reduced pressure, and the resulting product was neutralized with saturated sodium bicarbonate until alkaline. Extraction was performed with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain compound A4. LC / MS: M+H 297.0.

[0077] 2-Chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (A5): 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (2 g) and N,N-dimethylacetamide (10 mL) were added to a round-bottom flask. The mixture was cooled to -35°C, and iodomethane (0.9 mL) was added, followed by sodium hydride (615 mg). The reaction system was stirred for two hours. After completion of the reaction, the mixture was quenched with water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, then dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain compound A5. LC / MS: M+H 325.0.

[0078] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzenesulfonamide (1): 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (100 mg), sulfonamide (53 mg), p-toluenesulfonic acid (53 mg), and sec-butanol (5 mL) were added to a round-bottom flask. The reaction system was stirred at 120°C overnight. After completion of the reaction, the mixture was filtered and washed with methanol and diethyl ether to obtain compound 1. LC / MS: M+H 461.1.

[0079] 2. Preparation of other MST1 / 2 inhibitor compounds of the present invention

[0080] Other MST1 / 2 inhibitor compounds of the present invention were synthesized in a similar manner to compound 1, and their structures and mass spectrometry data are shown in the following table.

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Example 1 Effects of various added factors in the intestinal cancer organoid culture medium on the growth of intestinal cancer organoids

[0087] (1) Preparation of intestinal cancer organoid culture medium

[0088] First, a basal culture medium containing an initial culture medium is prepared. The initial culture medium can be selected from DMEM / F12, DMEM, F12, or RPMI-1640 commonly used in the art. In this embodiment, the formula of the basal culture medium is: DMEM / F12 culture medium (purchased from Corning) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available product concentration 50 mg / ml). Different types of additives (see Table 1) are added to the basal culture medium to prepare a colorectal cancer organoid culture medium containing different additives.

[0089] (2) Isolation and processing of primary colorectal cancer cells

[0090] 1. Sample selection

[0091] Intestinal cancer solid tumor tissue samples (intraoperative / endoscopic) were obtained from patients by professional medical staff of professional medical institutions, and all patients signed informed consent. 3 , endoscopic sample 0.025cm 3 Commercial tissue preservation solution (manufacturer: Miltenyi Biotec) was used for storage and transportation.

[0092] 2. Material Preparation

[0093] After surface disinfection, sterile 15mL centrifuge tubes, pipettes, 10mL pipettes, and sterile pipette tips should be placed in a clean bench and exposed to UV light for 30 minutes. Remove the basal culture medium from the 4°C refrigerator 30 minutes in advance, and remove the tissue digestion solution (recipe below) from the -20°C refrigerator 30 minutes in advance.

[0094] Tissue digestion solution: 1640 culture medium (Corning, 10-040-CVR), collagenase II (2 mg / mL), collagenase IV (2 mg / mL), DNase (50 U / mL), hyaluronidase (0.75 mg / mL), calcium chloride (3.3 mM), and BSA (10 mg / mL).

[0095] The above-mentioned collagenase II, collagenase IV, DNA enzyme, and hyaluronidase were purchased from Sigma; calcium chloride was purchased from Shanghai Shenggong Biotechnology Co., Ltd.; and BSA was purchased from Biofroxx.

[0096] 3. Isolation of Primary Colorectal Cancer Cells

[0097] 3.1 Take the tissue sample in the clean bench and place it in a culture dish. Remove the blood-stained tissue and rinse it twice with basal culture medium. Transfer the tissue to another culture dish and perform mechanical separation with a sterile scalpel. Cut the tissue into 1×1×1 mm 3 size;

[0098] 3.2 Aspirate the cut intraoperative or endoscopic tissue into a 15 mL centrifuge tube, add 5 mL of basal culture medium, mix well, and centrifuge at 1500 rpm for 4 minutes;

[0099] 3.3 Discard the supernatant and add basal culture medium and tissue digestion solution in a 1:3 ratio (Note: the amount of tissue digestion solution added is approximately 10 mL for 1 g of tumor tissue). Label the sample name and number, seal with sealing film, and digest at 37°C in a shaker (Zhichu Instrument ZQLY-180N) at 300 rpm. Observe the digestion completion every 30 minutes, judging by the absence of visible particulate matter. The digestion time is approximately 4 hours.

[0100] 3.4 After digestion is complete, filter the undigested tissue clumps through a 100 μm filter. Rinse the tissue clumps on the filter with basal culture medium into a centrifuge tube to reduce cell loss and centrifuge at 1500 rpm for 4 minutes at 25°C.

[0101] 3.5 Discard the supernatant and observe whether there are blood cells. If there are blood cells, add 8 mL of blood cell lysis buffer (purchased from Sigma), mix well, and lyse at 4°C for 20 minutes, inverting once during the process. Centrifuge at 1500 rpm at 25°C for 4 minutes.

[0102] 3.6 Discard the supernatant and add 2 mL of basal culture medium to resuspend the cells for later use.

[0103] 4. Cell Counting and Processing

[0104] 4.1 Observation under microscope: Pipette a small amount of resuspended cells and spread them flatly in a culture dish. Observe the density and morphology of cancer cells under a microscope (CNOPTEC, BDS400).

[0105] 4.2 Viable cell counting: 12 μL of the resuspended cell suspension was added to 12 μL of trypan blue dye (Shanghai Sangon Biotech Co., Ltd.), mixed thoroughly, and 20 μL was added to a cell counting plate (Countstar, specification: 50 plates / box). The percentage of viable large cells (cell size >10 μm) was calculated using a cell counter (Countstar, IC1000) as follows: number of viable cells / total number of cells × 100%.

[0106] (3) Culture of colorectal cancer organoids

[0107] The primary colorectal cancer cells isolated from three colorectal cancer tissues (numbered OE(E)067, OE(E)071, and OE(E)084) according to the above step (2) were resuspended in basal culture medium and counted, and then mixed with matrigel ( 356231) were mixed evenly on ice, and the final cell density was 5×10 5 / mL, take 7μL of matrix gel and cell suspension to form a solidified droplet in the center of each well of a 96-well culture plate, and let the culture plate stand at 37°C for 30 minutes to wait for the matrix gel to completely solidify. The culture medium with different components in Table 1 was added to the 96-well plate at a volume of 100μL / well. Among them, as an experimental control, a basic culture medium without any added components was used. After culturing for 7 to 10 days, 50μL of CellTiter-Glo (purchased from Promega) luminescent reagent was added to each well, allowed to stand for 10 minutes and then mixed, and detected using a multifunctional microplate reader (PerkinElmer Envision). The relative cell viability (%) was calculated according to the formula = chemiluminescence value of the experimental well / chemiluminescence value of the control well × 100%, and the promoting effect of different added components on the growth of intestinal cancer organoids was inferred. The experimental results are shown in Table 1.

[0108] Table 1 Additives in culture medium and their effects on promoting cell proliferation

[0109]

[0110] Among them, "+" indicates that compared with the basic culture medium, the culture medium with the addition of this supplement has a proliferation-promoting effect on at least two of the primary intestinal cancer cells isolated from intestinal cancer tissue; "-" indicates that the culture medium with the addition of this supplement has an inhibitory effect on at least one of the primary intestinal cancer cells isolated from intestinal cancer tissue; "○" indicates that the culture medium with the addition of this supplement has no obvious effect on the proliferation of the primary intestinal cancer cells isolated from intestinal cancer tissue.

[0111] Based on the above results, it is planned to select vitamin E, SB202190, compound 1, B27, N2, insulin, insulin-transferrin-selenium supplement, A8301, human fibroblast growth factor-10, neuregulin-1, amphiregulin, Y-27632, fetal bovine serum, basic fibroblast growth factor, insulin-like growth factor-1, Forsklin, bovine pituitary extract, fibroblast growth factor 7 and other added ingredients for further culture experiments.

[0112] Example 2 Effects of different combinations of added factors in intestinal cancer organoid culture medium on the growth of intestinal cancer organoids

[0113] According to the ingredients in Table 2, colorectal cancer organoid culture media with different combinations of added factors were prepared to investigate the growth-promoting effects of different combinations of added factors on colorectal cancer organoids.

[0114] Table 2 Preparation of different components of culture medium (concentration is final concentration)

[0115]

[0116] Primary intestinal cancer cells were obtained from intestinal cancer tissues (numbered OE(E)067, OE(E)071, OE(E)084, and OE(E)099) according to the method of step (2) of Example 1, resuspended in basal culture medium and counted, and then mixed with matrigel ( 356231) were mixed evenly on ice, and the final cell density was 5×10 5 / mL, take 20μL of matrix gel and cell suspension to form a solidified droplet in the center of each well of a 48-well culture plate, and let the culture plate stand at 37°C for 30 minutes to wait for the matrix gel to completely solidify. Finally, add 1mL of BM culture medium and culture medium corresponding to No. 1 to No. 19 to the 48-well plate respectively. After culturing for 7 to 10 days, add 20μL of CellTiter-Glo (purchased from Promega) luminescent reagent to each well, let it stand for 10 minutes and mix well, and use a multifunctional microplate reader (PerkinElmer Envision) for detection. According to the formula, the relative viability of cells is calculated (%) = chemiluminescence value of the experimental well / chemiluminescence value of the control well × 100%, and the effect of different component culture media on the growth promotion of colorectal cancer organoids is obtained. The experimental results are shown in Figure 1 .

[0117] according to Figure 1 The results show that compared with the basal culture medium, when using the above-mentioned No. 1 to No. 19 culture media, the proliferation of primary intestinal cancer cells can be promoted to varying degrees. When the added factors vitamin E (No. 2), SB202190 (No. 3), and insulin-transferrin-selenium supplement (No. 8) are omitted, the proliferation-promoting effect of the culture medium formula is more obvious. Therefore, in subsequent embodiments, factors such as B27, N2, insulin, A8301, compound 1, human fibroblast growth factor-10, neuregulin-1, amphiregulin, Y-27632, fetal bovine serum, basic fibroblast growth factor, insulin-like growth factor-1, Forsklin, bovine pituitary extract, and fibroblast growth factor 7 are used as culture medium formulas for culturing intestinal cancer organoids for further study.

[0118] Example 3 Effects of Different Concentrations of Factors Added to Intestinal Cancer Organoid Culture Medium on the Proliferation of Intestinal Cancer Organoids

[0119] Primary intestinal cancer cells were obtained from intestinal cancer tissues (numbered OE(E)071, OE(O)015, and OE(E)113) according to the method of step (2) of Example 1, resuspended in basal culture medium and counted, and then mixed with matrigel ( 356231) were mixed evenly on ice, and the final cell density was 5×10 5 7 μL of matrix gel and cell suspension were taken to form a solidified droplet in the center of each well of a 96-well culture plate. The culture plate was placed at 37°C for 30 minutes to allow the matrix gel to completely solidify for the following culture experiments.

[0120] Next, intestinal cancer organoid culture medium containing basal medium BM, 10 μM compound 1, 1:50 (v / v) B27, 1:100 (v / v) N2, 1 μg / mL insulin, 9 nM A8301, 5 ng / mL human fibroblast growth factor-10, 5 ng / mL neuregulin-1, 9 ng / mL amphiregulin, 2.5 μM Y-27632, 1.25% (v / v) fetal bovine serum, 10 ng / mL basic fibroblast growth factor, 50 ng / mL insulin-like growth factor-1, 2.5 μM Forsklin, 1:500 (v / v) bovine pituitary extract, and 1.25 ng / mL fibroblast growth factor 7 and the following 15 formula culture media were prepared for experiments.

[0121] Recipe 1: The above-mentioned intestinal cancer organoid culture medium components do not contain B27;

[0122] Recipe 2: The above-mentioned intestinal cancer organoid culture medium components do not contain N2;

[0123] Recipe 3: The above-mentioned intestinal cancer organoid culture medium components do not contain insulin;

[0124] Recipe 4: The above-mentioned intestinal cancer organoid culture medium components do not contain A8301;

[0125] Recipe 5: The above-mentioned intestinal cancer organoid culture medium components do not contain human fibroblast growth factor-10;

[0126] Recipe 6: The above-mentioned intestinal cancer organoid culture medium components do not contain neuregulin-1;

[0127] Recipe 7: The above-mentioned intestinal cancer organoid culture medium components do not contain amphiregulin;

[0128] Recipe 8: The above-mentioned intestinal cancer organoid culture medium components do not contain Y-27632;

[0129] Recipe 9: The above-mentioned intestinal cancer organoid culture medium components do not contain fetal bovine serum;

[0130] Recipe 10: The above-mentioned intestinal cancer organoid culture medium components do not contain basic fibroblast growth factor;

[0131] Recipe 11: The above-mentioned intestinal cancer organoid culture medium components do not contain insulin-like growth factor-1;

[0132] Recipe 12: The above-mentioned intestinal cancer organoid culture medium components do not contain Forsklin;

[0133] Formulation 13: The above-mentioned intestinal cancer organoid culture medium components do not contain bovine pituitary extract;

[0134] Recipe 14: The above-mentioned intestinal cancer organoid culture medium components do not contain fibroblast growth factor 7;

[0135] Formulation 15: The above-mentioned intestinal cancer organoid culture medium components do not contain compound 1.

[0136] When using the culture medium of Formula 1, 100 μL of prepared B27 was added to each well of a 96-well plate seeded with primary cells. The volume ratios of B27 to culture medium were 1:200, 1:100, 1:50, 1:25, and 1:12.5, respectively. Control wells (BC) were set up using the culture medium of Formula 1.

[0137] When using the culture medium of Formula 2, 100 μL of prepared N2 was added to each well of a 96-well plate seeded with primary cells. The volume ratios of N2 to the culture medium were 1:400, 1:200, 1:100, 1:50, and 1:25, respectively; and control wells (BC) were set up using the culture medium of Formula 2.

[0138] When using the culture medium of Formula 3, 100 μL of prepared insulin was added to each well of a 96-well plate seeded with primary cells. The final concentrations of insulin were 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 3.

[0139] When using the culture medium of Formula 4, 100 μL of prepared A8301 was added to each well of a 96-well plate seeded with primary cells. The final concentrations of A8301 were 1 nM, 3 nM, 9 nM, 27 nM, and 81 nM, respectively. Control wells (BC) were set using the culture medium of Formula 4.

[0140] When using the culture medium of Formula 5, 100 μL of prepared human fibroblast growth factor-10 was added to each well of a 96-well plate seeded with primary cells. The final concentrations of human fibroblast growth factor-10 were 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively; and control wells (BC) were set using the culture medium of Formula 5.

[0141] When using the culture medium of Formula 6, 100 μL of the prepared neuregulin-1 was added to each well of a 96-well plate seeded with primary cells, with final concentrations of neuregulin-1 of 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively; and control wells (BC) were set using the culture medium of Formula 6.

[0142] When using the medium of Formula 7, 100 μL of the prepared amphiregulin was added to each well of a 96-well plate seeded with primary cells. The final concentrations of amphiregulin were 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, and 81 ng / mL, respectively. Control wells (BC) were set up using the medium of Formula 7.

[0143] When using the culture medium of Formula 8, 100 μL of the prepared Y-27632 was added to each well of a 96-well plate seeded with primary cells. The final concentrations of Y-27632 were 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, respectively. Control wells (BC) were set using the culture medium of Formula 8.

[0144] When using the culture medium of Formula 9, 100 μL of prepared fetal bovine serum was added to each well of a 96-well plate seeded with primary cells. The volume ratios of fetal bovine serum to culture medium were 1.25%, 2.5%, 5%, 10%, and 20%, respectively. Control wells (BC) were set up using the culture medium of Formula 9.

[0145] When using the culture medium of Formula 10, 100 μL of the prepared basic fibroblast growth factor was added to each well of a 96-well plate seeded with primary cells. The final concentrations of basic fibroblast growth factor were 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively. Control wells (BC) were set using the culture medium of Formula 10.

[0146] When using the culture medium of Formula 11, 100 μL of the prepared insulin-like growth factor-1 was added to each well of a 96-well plate seeded with primary cells, and the final concentrations of insulin-like growth factor-1 were 12.5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL, respectively; and control wells (BC) were set using the culture medium of Formula 11.

[0147] When using the culture medium of Formula 12, 100 μL of the prepared Forsklin was added to each well of a 96-well plate seeded with primary cells. The final concentrations of Forsklin were 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM, respectively. Control wells (BC) were set using the culture medium of Formula 12.

[0148] When using the culture medium of Formula 13, 100 μL of the prepared bovine pituitary extract was added to each well of a 96-well plate seeded with primary cells. The volume ratios of the bovine pituitary extract to the culture medium were 1:2000, 1:1000, 1:500, 1:250, and 1:125, respectively; and control wells (BC) were set using the culture medium of Formula 13.

[0149] When using the culture medium of Formula 14, 100 μL of the prepared fibroblast growth factor 7 was added to each well of a 96-well plate seeded with primary cells. The final concentrations of fibroblast growth factor 7 were 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 5 ng / mL, and 10 ng / mL, respectively. Control wells (BC) were set using the culture medium of Formula 14.

[0150] When using the culture medium of Formula 15, 100 μL of the prepared compound 1 was added to each well of a 96-well plate seeded with primary cells, and the final concentrations of compound 1 were 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, respectively; and control wells (BC) were set using the culture medium of Formula 15.

[0151] After 7-10 days of organoid culture, the relative viability was calculated by comparing the cell numbers in the control wells (BC) and the results were shown in Figures 2A to 2O . Figures 2A to 2O In the data, relative viability is the ratio of the viability of colorectal cancer organoids cultured in each medium for 7–10 days to the viability of colorectal cancer organoids cultured in the corresponding control wells for 7–10 days. A ratio greater than 1 indicates that the culture medium containing the factor or small molecule at the different concentrations is more effective in promoting proliferation than the culture medium in the control wells. A ratio less than 1 indicates that the culture medium containing the factor or small molecule at the different concentrations is less effective in promoting proliferation than the culture medium in the control wells.

[0152] according to Figures 2A to 2O The results show that the volume ratio of B27 additive to culture medium is preferably in the range of 1:12.5 to 1:200, more preferably 1:50 to 1:100, and most preferably 1:50; the volume ratio of N2 additive to culture medium is preferably in the range of 1:25 to 1:400, more preferably 1:100 to 1:400, and most preferably 1:200; the concentration range of insulin is preferably 0.25 to 1 μg / mL, more preferably 0.25 to 0.5 μg / mL, and most preferably 0.5 μg / mL; the concentration range of A8301 is preferably 1 to 9 nM, more preferably 1 to 3 nM. The concentration range of human fibroblast growth factor-10 is preferably 2.5 to 40 ng / mL, more preferably 5 to 20 ng / mL, and most preferably 10 ng / mL; the concentration range of neuregulin-1 is preferably 2.5 to 40 ng / mL, more preferably 2.5 to 10 ng / mL, and most preferably 5 ng / mL; the concentration range of amphiregulin is preferably 1 to 27 ng / mL, more preferably 1 to 3 ng / mL, and most preferably 3 ng / mL; the concentration range of Y27632 is preferably 1.25 to 20 μM, more preferably 2. 5-20 μM, most preferably 10 μM; the volume ratio of fetal bovine serum to culture medium is preferably in the range of 1.25% (v / v) to 20% (v / v), more preferably 1.25% (v / v) to 2.5% (v / v), and most preferably 2.5% (v / v); the concentration of basic fibroblast growth factor is preferably in the range of 2.5-40 ng / mL, more preferably 10-20 ng / mL, and most preferably 10 ng / mL; the concentration of insulin-like growth factor-1 is preferably in the range of 12.5-100 ng / mL, more preferably 12.5-25 ng / mL, and most preferably 12.5 ng / mL; the concentration range of Forsklin is preferably 0.625-2.5 μM, more preferably 0.625-1.25 μM, and most preferably 1.25 μM; the volume ratio of bovine pituitary extract to culture medium is preferably 1:2000-1:500, and most preferably 1:1000; the concentration range of fibroblast growth factor 7 is preferably 0.625-2.5 ng / mL, and most preferably 0.625 ng / mL; the concentration range of compound 1 is preferably 1.25-10 μM, more preferably 2.5-10 μM, and most preferably 5 μM.

[0153] The optimal concentration of each added factor in the above-mentioned culture medium was used as the intestinal cancer organoid culture medium of the present invention used in the following examples, which contained: basal culture medium BM, 5 μM compound 1, 1:50 (v / v) B27, 1:200 (v / v) N2, 0.5 μg / mL insulin, 3 nM A8301, 10 ng / mL human fibroblast growth factor-10, 5 ng / mL neuregulin-1, 3 ng / mL amphiregulin, 10 μM Y-27632, 2.5% (v / v) fetal bovine serum, 10 ng / mL basic fibroblast growth factor, 12.5 ng / mL insulin-like growth factor-1, 1.25 μM Forsklin, 1:1000 (v / v) bovine pituitary extract, and 0.625 ng / mL fibroblast growth factor 7.

[0154] Example 4: Culture and Passaging of Intestinal Cancer Organoids

[0155] (1) Colorectal cancer organoid culture

[0156] According to the method of step (2) of Example 1, primary colorectal cancer cells were obtained from endoscopic tissue samples (numbered OE(E)050, OE(E)071, OE(E)078, OE(E)120, OE(O)012, and OE(O)015), resuspended in basal culture medium, counted, and mixed with matrigel ( 356231) were mixed evenly on ice, and the final cell density was 5×10 5 50 μL of matrix gel and cell suspension were taken to form a solidified droplet in the center of each well of a 24-well culture plate. The culture plate was placed at 37°C for 30 minutes to allow the matrix gel to completely solidify. 1 mL of the intestinal cancer organoid culture medium of the present invention at room temperature was gently added to each well along the side wall of the well using a pipette. After the surface of the 24-well culture plate was disinfected, it was placed at 37°C and 5% CO 2 The cells were cultured in an incubator (purchased from Thermo Fisher Scientific). After 3 to 8 days of culture, the cultured intestinal cancer organoids were observed using a microscope (Invitrogen, EVOS M500). Figures 3A-3F This photo shows a colon cancer organoid taken with a 10x objective lens. Under the microscope, the colon cancer organoid appears spherical with a smooth surface, resembling an intestinal structure.

[0157] (2) Passaging of colorectal cancer organoids

[0158] According to the method of step (2) of step 3 of Example 1, primary colorectal cancer cells were obtained from endoscopic tissue samples OE(E)113 and OE(E)120, and resuspended in basal culture medium and counted, and then mixed with matrix gel ( 356231) were mixed evenly on ice, and the final cell density was 5×10 550 μL of Matrigel and cell suspension were placed in the center of each well of a 24-well culture plate to form a solidified droplet. The culture plate was incubated at 37°C for 30 minutes to allow the Matrigel to completely solidify. 1 mL of the intestinal cancer organoid culture medium of the present invention was added, and the cells were cultured for 8 days before passage.

[0159] Primary intestinal cancer cells were obtained from the first generation organoids cultured in the above steps, resuspended in basal culture medium and counted, and then mixed with matrix gel ( 356231) were mixed evenly on ice, and the final cell density was 5×10 5 50 μL of Matrigel and cell suspension were placed in the center of each well of a 24-well culture plate to form a solidified droplet. The plate was incubated at 37°C for 30 minutes to allow the Matrigel to completely solidify. 1 mL of the intestinal cancer organoid culture medium of the present invention was added, and culture was continued for 8 days to obtain the next generation of organoids. The third to fifth generations of organoids were obtained using the same method.

[0160] Figures 4A-4B Images of colorectal cancer organoids at different passages (P1 to P5) taken with a 10x objective lens show that the OE(E)113 and OE(E)120 colorectal cancer organoids maintained stable growth and proliferation after continuous passage to the fifth passage.

[0161] Example 5 Histochemical Identification of Cultured Intestinal Cancer Organoids

[0162] A 0.25 cm spherical ... 3 The cancer tissue of the size was immersed in 1 mL of 4% paraformaldehyde for fixation. The sample OE(O)015 was continuously cultured to the 6th generation using the intestinal cancer organoid culture medium of the present invention using the method of Example 4. The intestinal cancer organoids fixed with 4% paraformaldehyde were embedded in paraffin and cut into 4 μm thick tissue sections using a microtome. Conventional immunohistochemistry was then performed (for specific steps, see Li et al., Nature Communication, (2018) 9: 2983). The primary antibodies used were ki-67, CK20, CDX-2, and villin (all purchased from CST).

[0163] Figures 5A-5D and Figures 6A-6D The figures show the comparison of immunohistochemical results of original tissue cells and intestinal cancer organoids obtained by culturing the cells to the sixth passage using the intestinal cancer organoid culture medium of the present invention. Figure 5A and Figure 6A These are pictures of the Ki-67 antibody labeled on the original colorectal cancer tissue and the cultured colorectal cancer organoids. Figure 5B and Figure 6BThese are pictures of the CK20 antibody labeled on the original colorectal cancer tissue and the cultured colorectal cancer organoids. Figure 5C and Figure 6C These are pictures of CDX-2 antibody labeled on original intestinal cancer tissue and cultured intestinal cancer organoids. Figure 5D and Figure 6D These images show the villin antibody, which is used to label the original colorectal cancer tissue and the cultured colorectal cancer organoids. This confirms that the expression of colorectal cancer-related biomarkers in the colorectal cancer organoids (sample number OE(O)015) cultured using the technology of this invention at passage 6 is essentially consistent with that in the original tissue sections from which the organoids were derived. This demonstrates that the organoids cultured using the technology of this invention retain the original pathological characteristics of colorectal cancer tissue from colorectal cancer patients.

[0164] Comparison of culture effects of Example 6 with literature culture medium and commercial culture medium

[0165] (1) Preparation of culture medium

[0166] Literature culture medium (Seungil Kim et al., Original Research, 2020, Vol. 25(7) 744–754): DMEM / F12 + 10% (v / v) fetal bovine serum + 1% penicillin / streptomycin + 1% (v / v) glutamine supplement (purchased from thermo) + 1% (v / v) HEPES (purchased from Gibco) + 1:100 (v / v) N2 (purchased from Gibco) + 1:50 (v / v) B-27 (purchased from Gibco) + 1 mM N-acetylcysteine ​​(purchased from Taosu Biochemical) + 50 ng / mL epidermal growth factor (purchased from R&D) + 100 ng / mL Noggin (purchased from R&D) + 10 mM nicotinamide (purchased from MCE) + 500 nM A8301 (purchased from MCE) + 10 μM SB202190 (purchased from MCE) + 0.01 μM prostaglandin E2 (purchased from Tocris).

[0167] Commercial culture medium: IntestiCult TM Organoid Growth Medium (Human) (available from STEMCELL, 06010).

[0168] (2) Obtaining primary colorectal cancer cells and culturing colorectal cancer organoids

[0169] According to the method of step (2) of step 3 of Example 1, primary colorectal cancer cells were obtained from an endoscopic tissue sample (numbered OE(E)122), resuspended in basal culture medium and counted, and then mixed with matrix gel ( 356231) were mixed evenly on ice, and the final cell density was 5×10 5 50 μL of matrigel and cell suspension were taken to form a solidified droplet in the center of each well of a 24-well culture plate. The culture plate was placed at 37°C for 30 minutes to allow the matrigel to completely solidify. 1 mL of the intestinal cancer organoid culture medium of the present invention, the culture medium from the literature, and the commercial culture medium at room temperature were gently added to each well along the side wall of the well using a pipette. After the surface of the 24-well culture plate was disinfected, it was placed at 37°C and 5% CO 2 After 5 days of culture, the primary colorectal cancer cells were observed using a microscope (Invitrogen EVOS M500). Figure 7 This is a photo of intestinal cancer organoids taken under a 10x objective lens.

[0170] according to Figure 7 The results show that compared with the culture medium in literature and commercial culture medium, the intestinal cancer organoid culture medium of the present invention has a short culture cycle, a large number of organoids and a complete organoid structure when culturing intestinal cancer organoids in vitro, and its effect is significantly better than the culture medium in literature and commercial culture medium.

[0171] Industrial Applicability

[0172] The present invention provides a culture medium and method for culturing intestinal cancer organoids. The cultured organoids can be used for evaluating and screening drug efficacy. Therefore, the present invention is suitable for industrial applications.

[0173] Although the present invention is described in detail herein, the present invention is not limited thereto. Those skilled in the art may make modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the scope of protection of the present invention.

Claims

1. A culture medium for intestinal cancer organoids, characterized in that: Made with the following ingredients: MST1 / 2 kinase inhibitor; B27 supplement; N2 supplement; insulin; A8301; human fibroblast growth factor-10; neuregulin-1; amphiregulin; Y27632; fetal bovine serum; basic fibroblast growth factor; insulin-like growth factor-1; forsklin; bovine pituitary extract; fibroblast growth factor 7; and an initial culture medium selected from DMEM / F12, DMEM, F12, or RPMI-1640; and antibiotic; Wherein, the MST1 / 2 kinase inhibitor is compound 1 or a pharmaceutically acceptable salt or solvate thereof, in, (1) The concentration of the MST1 / 2 kinase inhibitor is 1.25 to 10 μM; (2) The volume ratio of the B27 additive to the culture medium is 1:12.5 to 1:200; (3) The volume ratio of the N2 additive to the culture medium is 1:25 to 1:400; (4) The concentration of the insulin is 0.25 to 1 μg / mL; (5) The concentration of A8301 is 1 to 9 nM; (6) The concentration of human fibroblast growth factor-10 is 2.5 to 40 ng / mL; (7) the concentration of neuregulin-1 is 2.5 to 40 ng / mL; (8) the concentration of the amphiregulin is 1 to 27 ng / mL; (9) The concentration of Y27632 is 1.25-20 μM; (10) The volume concentration of the fetal bovine serum is 1.25% (v / v) to 20% (v / v); (11) The concentration of the basic fibroblast growth factor is 2.5 to 40 ng / mL; (12) The concentration of the insulin-like growth factor-1 is 12.5 to 100 ng / mL; (13) The concentration of Forsklin is 0.625 to 2.5 μM; (14) The volume ratio of the bovine pituitary extract to the culture medium is 1:2000 to 1:500; (15) The concentration of the fibroblast growth factor 7 is 0.625 to 2.5 ng / mL.

2. The culture medium according to claim 1, wherein: (1) The concentration of the MST1 / 2 kinase inhibitor is 2.5 to 10 μM; (2) The volume ratio of the B27 additive to the culture medium is 1:50 to 1:100; (3) The volume ratio of the N2 additive to the culture medium is 1:100 to 1:400; (4) The concentration of the insulin is 0.25 to 0.5 μg / mL; (5) The concentration of A8301 is 1 to 3 nM; (6) The concentration of human fibroblast growth factor-10 is 5 to 20 ng / mL; (7) the concentration of neuregulin-1 is 2.5 to 10 ng / mL; (8) the concentration of the amphiregulin is 1 to 3 ng / mL; (9) The concentration of Y27632 is 2.5-20 μM; (10) The volume concentration of the fetal bovine serum is 1.25% (v / v) to 2.5% (v / v); (11) The concentration of the basic fibroblast growth factor is 10 to 20 ng / mL; (12) The concentration of the insulin-like growth factor-1 is 12.5 to 25 ng / mL; (13) The concentration of Forsklin is 0.625-1.25 μM; (14) The volume ratio of the bovine pituitary extract to the culture medium is 1:2000 to 1:500; (15) The concentration of the fibroblast growth factor 7 is 0.625 to 2.5 ng / mL.

3. The culture medium according to claim 1, wherein The antibiotic is selected from one or more of streptomycin / penicillin, amphotericin B and primocin.

4. A method for culturing intestinal cancer organoids, characterized in that: The following steps are involved: (1) preparing a culture medium for intestinal cancer organoids according to any one of claims 1 to 3; (2) obtaining primary colorectal cancer cells from colorectal cancer tissue samples, and mixing the obtained suspension of primary colorectal cancer cells with matrigel; (3) Adding the culture medium of the intestinal cancer organoids obtained in step (1) to the mixture of the primary intestinal cancer cells and matrigel obtained in step (2) for culturing.

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