A culture medium for primary intestinal cancer cells and in vitro culture method and use thereof

Abstract

The present invention provides a culture medium for primary intestinal cancer cells, an in vitro culture method, and applications thereof. The culture medium comprises an MST1 / 2 kinase inhibitor; insulin; a Rho protein kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152; an insulin-transferrin-selenium supplement; a glutamine additive; neuregulin 1; a bovine pituitary extract; basic fibroblast growth factor; R-spondin 1; prostaglandin E2; and B27. Compared to existing culture methods, in vitro culture using the culture medium of the present invention has higher amplification efficiency. Using this culture medium for primary intestinal cancer cells can maintain the morphological structure and pathological characteristics of the primary tissue, thereby improving the success rate and survival rate of primary intestinal cancer cell culture.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a culture medium and applications thereof, and more specifically to a culture medium, a culture method and applications of primary intestinal cancer cells. Background Art

[0002] Colorectal cancer, caused by a combination of genetic and environmental factors, is one of the most common digestive tract tumors worldwide. Survey data (Zheng Shu et al., Prevention of Colorectal Cancer, Chinese Journal of Oncology, 2004, Vol. 13, No. 1, pp. 1-2) show that colorectal cancer is one of the most common tumors, with an overall increasing incidence and mortality rate, making it a serious threat to life and health. Surgery combined with postoperative chemotherapy is currently the main treatment for colorectal cancer. Although recent advances in surgical techniques have improved survival rates for colorectal cancer patients, tumor metastasis and recurrence still pose a poor prognosis. In the precision treatment of colorectal cancer, the emergence of targeted drugs has brought hope to patients with advanced colorectal cancer. Future developments lie in the rational selection of targeted drugs and the development of personalized treatment plans. Continuous innovations in drug sensitivity testing technology have provided strong technical support for predicting the efficacy of targeted drugs, chemotherapy drugs, and combinations of targeted drugs, laying a solid foundation for personalized treatment of colorectal cancer patients.

[0003] Existing in vitro cultured colorectal cancer cell lines are mainly obtained by spontaneously immortalizing normal cells through long-term culture or transfecting them with oncogenes that promote the immortalization of normal cells. Cell lines established by traditional methods are still the mainstay of cellular, molecular and cancer biology research. However, these methods change the genetic background of the cells, and long-term cultured cell lines are also prone to genomic instability, which may lead to artificial changes in the phenotype of tumor cell lines and tumor cells in vivo. These cell lines usually lack the complex heterogeneity of primary tumors, which limits the application of these cell lines in predicting tumor cell responses and affects the accuracy of scientific research and drug development for colorectal cancer. In addition, in the process of culturing cells obtained from colorectal cancer tissue into cancer cells, conventional culture methods are difficult to obtain cancer cells. There are problems such as interference by fibroblasts during the culture process and the inability to propagate the formed clones, which limits the application of primary human colorectal cancer cells.

[0004] In 2017, Xuefeng Liu et al. used irradiated mouse fibroblasts and a Rho-associated kinase inhibitor (Y-27632) to expand epithelial-derived cells. This system has the ability to achieve unlimited growth of epithelial-derived cells without genetic manipulation (Xuefeng Liu et al., Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens. Nat. Protoc. 2017, 12, 439). However, the method established by Xuefeng Liu et al. has a long culture cycle and cannot achieve rapid cell expansion. Co-culture of primary cells with stromal cells is inevitable, and it cannot solve a series of problems existing in culture schemes such as co-culture with stromal cells, which limits the application of this technology. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a culture medium for primary intestinal cancer cells and an in vitro culture method and application.

[0006] One aspect of the present invention is to provide a culture medium for primary intestinal cancer cells, which contains an MST1 / 2 kinase inhibitor; insulin; a Rho protein kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152; an insulin-transferrin-selenium supplement; a glutamine additive; neuregulin 1; a bovine pituitary extract; basic fibroblast growth factor; R-spondin 1; prostaglandin E2; and B27.

[0007] Wherein, the MST1 / 2 kinase inhibitor includes 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 content of MST1 / 2 kinase inhibitor in the culture medium was 1.25-5 μM;

[0029] (2) The insulin content in the culture medium is 1 to 9 μg / mL;

[0030] (3) The Rho protein kinase inhibitor is preferably Y27632, and its content in the culture medium is 1.25-20 μM;

[0031] (4) The volume ratio of insulin-transferrin-selenium supplement to culture medium was 1:400 to 1:100;

[0032] (5) The content of glutamine additive in the culture medium is 0.5-2 mM;

[0033] (6) the content of neuregulin-1 in the culture medium is 2.5 to 40 ng / ml;

[0034] (7) The volume ratio of bovine pituitary extract to culture medium is 1:2000 to 1:125;

[0035] (8) The content of basic fibroblast growth factor in the culture medium is 2.5-20 ng / ml;

[0036] (9) The content of R-spondin1 in the culture medium was 1.25-10 ng / mL;

[0037] (10) The content of prostaglandin E2 in the culture medium is 5-80 nM;

[0038] (11) The volume ratio of B27 to culture medium is 1:200 to 1:12.5.

[0039] 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.

[0040] 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.

[0041] According to a second aspect, the present invention further provides an in vitro culture method for primary intestinal cancer cells. In the in vitro culture method for primary intestinal cancer cells of the present invention, the primary intestinal cancer cells are cultured in vitro using the primary intestinal cancer cell culture medium of the present invention.

[0042] The in vitro culture method of primary intestinal cancer cells of the present invention comprises the following steps:

[0043] 1. Isolation of Primary Colorectal Cancer Cells

[0044] (1) Isolate the colorectal cancer tissue sample, 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 it in a constant temperature shaker for digestion at a temperature of 4-37°C and a speed of 200-350 rpm;

[0045] (2) Digestion can be terminated when no obvious tissue blocks are observed. The digestion time is 3 to 6 hours.

[0046] (3) After centrifugation, the supernatant is discarded. The centrifugal speed is 1200-1600 rpm and the centrifugation time is 2-6 minutes. The primary intestinal cancer cell culture medium of the present invention is added and resuspended for later use.

[0047] 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).

[0048] 2. Cultivation using the intestinal cancer primary cell culture medium of the present invention

[0049] Use basal culture medium and matrigel ( 356231) were mixed evenly on ice, plated, and incubated in a 37°C, 5% CO2 incubator for 25-35 minutes, and the supernatant was discarded. The primary intestinal cancer cells obtained in step 1 above were resuspended in the primary intestinal cancer cell culture medium of the present invention and counted, and the cell density was adjusted to 2-4×10 4 cells / mL, add the primary intestinal cancer cell culture medium of the present invention, and culture in an incubator.

[0050] In another aspect, the present invention further provides a method for evaluating or screening a drug for treating intestinal cancer, comprising the following steps:

[0051] (1) Cultivating primary intestinal cancer cells using the method for culturing primary intestinal cancer cells of the present invention;

[0052] (2) Select the drug to be tested and dilute it according to the required concentration gradient;

[0053] (3) adding the drug in various concentration gradients to the primary intestinal cancer cells cultured in (1);

[0054] (4) Conduct cell activity test.

[0055] The technical solution of the present invention can achieve the following technical effects:

[0056] (1) Improve the success rate of primary intestinal cancer cell culture, and be able to culture tumor tissues from multiple sample sources such as colon, rectum, and duodenal cancer, with a success rate of over 80%;

[0057] (2) Primary intestinal cancer cells cultured in vitro can maintain the patient's pathological characteristics;

[0058] (3) The cultured primary intestinal cancer cells are not interfered with by stromal cells such as fibroblasts and adipocytes;

[0059] (4) High amplification efficiency, primary colorectal cancer cells can be successfully cultured within about a week, and the amplified primary colorectal cancer cells can be continuously passaged;

[0060] (5) The culture cost is controllable, and the culture medium does not need to be added with expensive Wnt agonists and other factors;

[0061] (6) The culture medium does not contain serum and can avoid the co-culture of primary cells and stromal cells, which also solves a series of problems existing in traditional culture schemes such as serum-containing and co-culture with stromal cells;

[0062] (7) The primary intestinal cancer cells cultured using the technology are large in number and highly homogenized, making them suitable for high-throughput screening of new candidate compounds and providing patients with high-throughput in vitro drug sensitivity functional testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is a graph showing the effects of different combinations of added factors in the culture medium of primary intestinal cancer cells on the growth of primary intestinal cancer cells.

[0064] Figures 2A-2K The figure shows the effects of different concentrations of added factors to the culture medium of primary intestinal cancer cells on the growth of primary intestinal cancer cells.

[0065] Figures 3A-3F The photographs are obtained by observing, under a microscope, primary intestinal cancer cells cultured using the intestinal cancer primary cell culture medium of the present invention.

[0066] Figures 4A-4D These are the immunohistochemical results of primitive intestinal cancer tissue cells.

[0067] Figures 5A-5D The present invention provides immunohistochemical results of primary intestinal cancer cells obtained by culturing original intestinal cancer tissue cells to the fifth passage using the primary intestinal cancer cell culture medium of the present invention.

[0068] Figure 6 The figure shows the cell growth curve of in vitro culture of primary intestinal cancer cells using the primary intestinal cancer cell culture medium of the present invention.

[0069] Figure 7 The results show the comparison of the proliferation efficiency of primary intestinal cancer cells cultured using the primary intestinal cancer cell culture medium of the present invention and the culture medium of the literature of the prior art.

[0070] Figures 8A-8F The results of drug screening using primary intestinal cancer cells cultured to different passages using the primary intestinal cancer cell culture medium of the present invention are shown. DETAILED DESCRIPTION

[0071] 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.

[0072] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0073] 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.

[0074] 1. Preparation of MST1 / 2 kinase inhibitor compound 1

[0075] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide 1

[0076]

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 2. Preparation of other MST1 / 2 inhibitor compounds of the present invention

[0083] 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.

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] Example 1 Effects of various added factors in the culture medium of primary intestinal cancer cells on the growth of primary intestinal cancer cells

[0090] (1) Preparation of primary intestinal cancer cell culture medium

[0091] First, a basal 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 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 culture medium for primary intestinal cancer cells containing different additives.

[0092] (2) Isolation and processing of primary colorectal cancer cells

[0093] 1. Sample selection

[0094] 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.

[0095] 2. Material Preparation

[0096] After disinfecting the surfaces of sterile 15mL centrifuge tubes, pipettes, 10mL pipettes, and sterile pipette tips, place them in a clean bench and irradiate with 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 from the -20°C refrigerator 30 minutes in advance.

[0097] 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).

[0098] 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.

[0099] 3. Isolation of Primary Colorectal Cancer Cells

[0100] 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;

[0101] 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;

[0102] 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 parafilm, 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 4 hours.

[0103] 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.

[0104] 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.

[0105] 3.6 Discard the supernatant and add 2 mL of basal culture medium to resuspend the cells for later use.

[0106] 4. Cell Counting and Processing

[0107] 4.1 Microscopic observation: 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).

[0108] 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%.

[0109] (3) Culture of primary colorectal cancer cells

[0110] The primary colorectal cancer cells isolated from three colorectal cancer tissues (numbered OE(O)001, OE(O)002, and OE(E)003) according to the above step (2) were resuspended in basal culture medium and counted. The basal culture medium was mixed with matrigel ( 356231) were mixed on ice, 300 μL was taken and plated, incubated in a 37°C, 5% CO2 incubator for 25 to 35 minutes, preferably 30 minutes, and the supernatant was discarded. The culture medium containing the different components in Table 1 containing primary intestinal cancer cells was added at a rate of 4 × 10 4 Cells were added to a 48-well plate at a volume of 500 μL / well. As a control, basal medium without any additives was used. After 7-10 days of culture, when the cells reached 85% confluence, the medium was discarded and the cells were rinsed once with 100 μL of 0.05% trypsin (Gibco) per well. After aspiration, 200 μL of 0.05% trypsin was added to each well. The cells were incubated at 37°C and 5% CO₂ for 10 minutes. Complete digestion of the cells was observed under a microscope (CNOPTEC, BDS400). Digestion was terminated by adding 300 μL of DMEM / F12 medium supplemented with 10% serum (ExcellBio, FND500). 20 μL of the medium was then added to a cell counting plate (Countstar, specification: 50 plates / box) and the total number of cells was counted using a cell counter (Countstar, IC1000). The results are shown in Table 1.

[0111]

[0112] Among them, "+" indicates that compared with the basic culture medium, the culture medium with the addition of this additive has a proliferation-promoting effect on two cases of primary intestinal cancer cells isolated from intestinal cancer tissue; "-" indicates that the culture medium with the addition of this additive shows an inhibitory effect on at least one case of primary intestinal cancer cells isolated from intestinal cancer tissue; "○" indicates that the culture medium with the addition of this additive has no obvious effect on the proliferation of at least two cases of primary intestinal cancer cells isolated from intestinal cancer tissue.

[0113] Based on the above results, factors such as A8301, insulin, Y-27632, insulin-transferrin-selenium supplement, glutamine additive, neuregulin-1, bovine pituitary extract, basic fibroblast growth factor, noggin, R-spondin1, compound 1, prostaglandin E2, B27, and Forsklin were selected for further culture experiments.

[0114] Example 2 Effects of different combinations of added factors in the culture medium of primary intestinal cancer cells on the proliferation of primary intestinal cancer cells

[0115] According to the ingredients in Table 2, culture medium for primary colorectal cancer cells with different combinations of added factors were prepared to investigate the proliferation-promoting effects of different combinations of added factors on primary colorectal cancer cells.

[0116] Table 2 Preparation of different components of culture medium (concentration is final concentration)

[0117]

[0118] Primary colorectal cancer cells were obtained from colorectal cancer tissues (numbered OE(O)001, OE(O)002, OE(E)060, and OE(E)071) according to the method of step (2) of Example 1. The obtained cell suspension was divided into 16 equal parts and centrifuged at 1500 rpm for 4 minutes. After centrifugation, 200 μL of BM and No. 1 to 15 culture medium were used to resuspend the cells. The basal culture medium was mixed with matrigel ( 356231) were mixed on ice, 300 μL was plated on a 48-well plate, and incubated in a 37°C, 5% CO2 incubator for 25 to 35 minutes, preferably 30 minutes, and the supernatant was discarded. 4 pieces / cm 2 Cells were seeded into a 48-well plate (20,000 cells per well), and the volume of each well was filled to 1 mL with the corresponding culture medium. The cells were mixed thoroughly. After surface disinfection, the cells were cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific).

[0119] When the cells in the 48-well plate grow to more than 85%, the culture medium is discarded and rinsed once with 100 μL of 0.05% trypsin (purchased from Gibco). After absorbing the solution, 200 μL of 0.05% trypsin is added to each well. The cells are placed in a 37°C, 5% CO2 incubator for 10 minutes. The cells are observed under a microscope (CNOPTEC, BDS400) to ensure complete digestion. 300 μL of DMEM culture medium containing 10% fetal bovine serum is added to terminate the digestion. 20 μL is taken and added to a cell counting plate (Countstar, specification: 50 plates / box). The total number of cells is counted using a cell counter (Countstar, IC1000). The results obtained from the primary intestinal cancer cells of samples OE(O)001, OE(O)002, OE(E)060, and OE(E)071 isolated from intraoperative and endoscopic tissues are shown in Figure 2. Figure 1 .

[0120] according to Figure 1 The results show that compared with the basal culture medium, the use of the above-mentioned No. 1 to No. 15 culture media can promote the proliferation of primary intestinal cancer cells to varying degrees. When the added factors A8301 (No. 2), noggin (No. 12), and Forsklin (No. 15) are omitted, the proliferation-promoting effect of the culture medium formula is more obvious. Therefore, when the culture medium containing insulin, Y-27632, insulin-transferrin-selenium supplement, glutamine additive, neuregulin-1, bovine pituitary extract, basic fibroblast growth factor, R-spondin1, compound 1, prostaglandin E2, B27 and other added ingredients is used to culture primary intestinal cancer cells, the proliferation effect is the best.

[0121] Example 3 Effects of Different Concentrations of Additive Factors in the Culture Medium for Primary Intestinal Cancer Cells on the Proliferation of Primary Intestinal Cancer Cells

[0122] Primary colorectal cancer cells were obtained from endoscopic tissue samples (numbered OE(E)060, OE(E)078, OE(O)014, OE(O)015, and OE(E)071) according to the method of step (2) of Example 1. The cells were cultured using the combined culture medium of the effective factors in Example 2. The basal culture medium was mixed with matrigel ( 356231) on ice, mix well, take 4 mL and plate on a T12.5 culture flask, incubate in a 37°C, 5% CO2 incubator for 25 to 35 minutes, preferably 30 minutes, and discard the supernatant.

[0123] The obtained primary colorectal cancer cells were cultured at a viable cell density of 2.4×10 4 pieces / cm 2The cells were seeded into T12.5 well plates (300,000 cells per well), and after surface disinfection, they were cultured in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher Scientific). Cells were cultured and expanded in the combination of culture media (containing basal medium BM, 3 μg / mL insulin, 10 μM Y-27632, 1:400 insulin-transferrin-selenium supplement, 2 mM glutamine supplement, 27 ng / mL neuregulin-1, 1:500 bovine pituitary extract, 20 ng / mL basic fibroblast growth factor, 5 ng / mL R-spondin 1, 5 μM Compound 1, 20 nM prostaglandin E2, and 1:100 B27) based on the effective factor combination identified in Example 2. Cells were cultured and expanded until they reached over 85% growth. 500 μL of 0.05% trypsin (purchased from Gibco) was added for 1 minute, then aspirated and 500 μL of 0.05% trypsin was added to each well. The cells were incubated at 37°C, 5% CO2 for 2-10 minutes, or until the cells were completely digested. After centrifugation at 1500 rpm for 4 minutes, the supernatant was discarded. The cell pellet was resuspended in DMEM / F12. 20 μL of the solution was added to a cell counting plate (manufacturer: Countstar, specification: 50 plates / box), and the total number of cells was counted using a cell counter (Countstar, IC1000). The resulting cells were used in the following culture experiments.

[0124] The following 11 culture media were prepared for the experiment.

[0125] Recipe 1: The above-mentioned primary intestinal cancer cell culture medium components do not contain insulin;

[0126] Recipe 2: The above-mentioned primary colorectal cancer cell culture medium components do not contain Y-27632;

[0127] Recipe 3: The above primary intestinal cancer cell culture medium components do not contain insulin-transferrin-selenium supplement;

[0128] Recipe 4: The above-mentioned primary intestinal cancer cell culture medium components do not contain glutamine additive;

[0129] Recipe 5: The above-mentioned primary intestinal cancer cell culture medium components do not contain neuregulin-1;

[0130] Formula 6: The above-mentioned primary intestinal cancer cell culture medium components do not contain bovine pituitary extract;

[0131] Recipe 7: The above-mentioned primary intestinal cancer cell culture medium components do not contain basic fibroblast growth factor;

[0132] Recipe 8: The above-mentioned primary intestinal cancer cell culture medium components do not contain R-spondin 1;

[0133] Formulation 9: The above-mentioned primary intestinal cancer cell culture medium components do not contain compound 1;

[0134] Formulation 10: The above-mentioned primary intestinal cancer cell culture medium components do not contain prostaglandin E2;

[0135] Recipe 11: The above-mentioned primary intestinal cancer cell culture medium components do not contain B27;

[0136] Add 20 μl of 1x10 4 For each cell resuspension, 1 mL of the culture medium of formulas 1 to 11 was used to dilute the cell suspension.

[0137] When using the culture medium of Formula 1, 1 mL of prepared insulin was added to each well of a 48-well plate seeded with primary cells. The final concentrations of insulin were 1 μg / mL, 3 μg / mL, 9 μg / mL, 27 μg / mL, and 81 μg / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 1.

[0138] When using the culture medium of Formula 2, 1 mL of prepared Y-27632 was added to each well of a 48-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 up using the culture medium of Formula 2.

[0139] When using the culture medium of Formula 3, 1 mL of the prepared insulin-transferrin-selenium supplement was added to each well of a 48-well plate seeded with primary cells. The final concentrations of the insulin-transferrin-selenium supplement were 1:800 (V / V), 1:400 (V / V), 1:200 (V / V), 1:100 (V / V), and 1:50 (V / V), respectively; and control wells (BC) were set using the culture medium of Formula 3.

[0140] When using the culture medium of Formula 4, 1 mL of the prepared glutamine additive was added to each well of a 48-well plate seeded with primary cells. The final concentrations of the glutamine additive were 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM, respectively. Control wells (BC) were set up using the culture medium of Formula 4.

[0141] When using the culture medium of Formula 5, 1 mL of prepared neuregulin-1 was added to each well of a 48-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 up using the culture medium of Formula 5.

[0142] When using the culture medium of Formula 6, 1 mL of the prepared bovine pituitary extract was added to each well of a 48-well plate seeded with primary cells. The final concentrations of the bovine pituitary extract were 1:2000 (V / V), 1:1000 (V / V), 1:500 (V / V), 1:250 (V / V), and 1:125 (V / V), respectively; and control wells (BC) were set using the culture medium of Formula 6.

[0143] When using the culture medium of Formula 7, 1 mL of prepared basic fibroblast growth factor was added to each well of a 48-well plate seeded with primary cells. The final concentrations of basic fibroblast growth factor were 1.25 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 7.

[0144] When using the culture medium of Formula 8, 1 mL of prepared R-spondin 1 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of R-spondin 1 were 1.25 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 8.

[0145] When using the culture medium of Formula 9, 1 mL of the prepared compound 1 was added to each well of a 48-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 9.

[0146] When using the culture medium of Formula 10, 1 mL of prepared prostaglandin E2 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of prostaglandin E2 were 5 nM, 10 nM, 20 nM, 40 nM, and 80 nM, respectively. Control wells (BC) were set up using the culture medium of Formula 10.

[0147] When using the culture medium of Formula 11, 1 mL of prepared B27 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of B27 were 1:200 (V / V), 1:100 (V / V), 1:50 (V / V), 1:25 (V / V), and 1:12.5 (V / V), respectively. Control wells (BC) were set up using the culture medium of Formula 11.

[0148] When the cells were expanded to about 85% of the 48 wells, the cells were digested and counted, and the proliferation times were calculated by referring to the number of cells in the control wells (BC). The results were shown in Figures 2A to 2K . Figures 2A to 2KThe ratio is the ratio of the number of cells cultured in each culture medium for one generation to the number of cells cultured in the corresponding control well for one generation. A ratio greater than 1 indicates that the culture medium containing the factor or small molecule compound at different concentrations promotes cell proliferation more effectively than the culture medium in the control well. A ratio less than 1 indicates that the culture medium containing the factor or small molecule compound at different concentrations promotes cell proliferation less effectively than the culture medium in the control well.

[0149] according to Figures 2A to 2K The results show that insulin, Y-27632, insulin-transferrin-selenium supplement, glutamine additive, neuregulin-1, bovine pituitary extract, basic fibroblast growth factor, R-spondin1, compound 1, prostaglandin E2, and B27 have a significant proliferative effect on primary intestinal cancer cells. According to the results of this embodiment, the content of insulin is preferably 1 μg / mL to 9 μg / mL, and the cell proliferation effect is most obvious when the concentration is 9 μg / mL; the content of Y-27632 is preferably 1.25 μM to 20 μM, more preferably 5 μM to 20 μM, and the cell proliferation effect is most obvious when the concentration is 10 μM; the content of insulin-transferrin-selenium supplement is preferably 1:400 (V / V) to 1:100 (V / V), and the concentration is 1:200 (V / V). The cell proliferation effect is most obvious when the concentration of glutamine additive is 0.5mM~2mM, and the cell proliferation effect is most obvious when the concentration is 2mM; the content of neuregulin-1 is preferably 2.5ng / ml~40ng / ml, and the cell proliferation effect is most obvious when the concentration is 10ng / mL; the content of bovine pituitary extract is preferably 1:2000(V / V)~1:125(V / V), more preferably 1:1000(V / V)~1:250(V / V), and the cell proliferation effect is most obvious when the concentration is 1:500(V / V); the content of basic fibroblast growth factor is preferably 2.5ng / ml~20ng / ml, and the cell proliferation effect is most obvious when the concentration is 5ng / mL; the content of R-spondin1 is preferably 1.25ng / ml~10ng / ml, more preferably 1.25ng / ml~5ng / ml, and the cell proliferation effect is most obvious when the concentration is 2.5ng / mL; the content of compound 1 is preferably The content of prostaglandin E2 is preferably 5nM to 80nM, more preferably 20nM to 80nM, and the cell proliferation effect is most obvious at a concentration of 40nM; the content of B27 is preferably 1:200 (V / V) to 1:12.5 (V / V), more preferably 1:50 (V / V) to 1:25 (V / V), and the cell proliferation effect is most obvious at a concentration of 1:50 (V / V).

[0150] The optimal concentrations of each added factor in the above-mentioned culture medium were used as the culture medium for the primary intestinal cancer cells of the present invention used in the following examples, which contained: basal culture medium BM, 9 μg / mL insulin, 10 μM Y-27632, 1:200 insulin-transferrin-selenium supplement, 2 mM glutamine additive, 10 ng / mL neuregulin-1, 1:500 bovine pituitary extract, 5 ng / mL basic fibroblast growth factor, 2.5 ng / mL R-spondin1, 2.5 μM compound 1, 40 nM prostaglandin E2, and 1:50 B27 (hereinafter referred to as "CA-2" culture medium).

[0151] Example 4 Culture of primary intestinal cancer cells

[0152] Primary colorectal cancer cells were obtained from endoscopic tissue samples (numbered OE(E)088, OE(E)089, OE(E)099, OE(E)100, OE(E)101, and OE(E)104) according to the method of step (2) of Example 1 and cultured in CA-2 medium. The basal medium was mixed with matrigel ( 356231) were mixed evenly on ice, 2 mL was plated on a 6-well culture plate, and incubated in a 37°C, 5% CO2 incubator for 25 to 35 minutes, preferably 30 minutes, and the supernatant was discarded. The obtained primary intestinal cancer cells were cultured at a viable cell density of 2×10 4 pieces / cm 2 The cells were seeded into 6-well plates (250,000 cells per well), and 4 ml of CA-2 medium was added. After surface disinfection, the cells were cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific).

[0153] The cultured primary intestinal cancer cells were observed using a microscope (Invitrogen EVOS M500). Figures 3A to 3F This is a photo taken under a 10x objective lens. The cells are closely arranged under the microscope and have slightly irregular shapes.

[0154] Example 5 Histochemical Identification of Primary Intestinal Cancer Cell Culture

[0155] A 0.25 cm spherical ... 3Cancer tissues of different sizes were immersed in 1 mL of 4% paraformaldehyde for fixation. Sample OE(O)015 was continuously cultured to the fifth generation using the intestinal cancer primary cell culture medium CA-2 of the present invention using the method of Example 4. The intestinal cancer primary cells 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).

[0156] Figures 4A-4D 5A-5D are comparative diagrams of the immunohistochemical results of the original tissue cells and the primary intestinal cancer cells obtained by culturing the cells with the intestinal cancer primary culture medium CA-2 of the present invention. Figure 4A and Figure 5A These are pictures of colorectal cancer tissue and cultured primary colorectal cancer cells labeled with ki-67 antibodies. Figure 4B and Figure 5B These are pictures of colorectal cancer tissue and cultured primary colorectal cancer cells labeled with CK20 antibodies. Figure 4C and Figure 5C These are pictures of colorectal cancer tissue and cultured primary colorectal cancer cells labeled with CDX-2 antibody. Figure 4D and Figure 5D The images show villin antibodies labeled on colorectal cancer tissue and cultured primary colorectal cancer cells. This confirms that the expression of colorectal cancer-related biomarkers in primary colorectal cancer cells cultured using the technology of the present invention at passage 5 is essentially consistent with that in the original tissue sections from which they were derived. This demonstrates that primary colorectal cancer cells cultured using the technology of the present invention retain the original pathological characteristics of colorectal cancer tissue from patients.

[0157] Example 6: Culture cycle and cell number statistics of primary intestinal cancer cells and calculation of Population Doubling (PD) value

[0158] Primary colorectal cancer cells were obtained from four colorectal cancer endoscopic specimens (numbered OE(E)067, OE(E)071, OE(E)090, and OE(E)099) according to the method of step (2) of Example 1. The obtained primary colorectal cancer cells were cultured at a viable cell density of 2.4×10 4 pieces / cm 2Inoculate in T12.5 culture flasks (300,000 cells per flask). Use a pipette to gently add 4 mL of the intestinal cancer primary cell culture medium CA-2 of the present invention at room temperature to each T12.5 culture flask. After surface disinfection, place in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific) for culture. After 5 to 9 days of cell culture, subculture and count, and record the number of days in culture until subculture, and the number of days in culture until subculture is regarded as a culture cycle. Continue to culture under the experimental conditions, and amplify the amplified cells for different generations. After each generation, count and record the corresponding culture cycle, and calculate PD according to the formula Population Doubling (PD) = 3.32 × log10 (total number of cells after digestion / initial number of cells seeded). For the formula, see Chapman et al., Stem Cell Research & Therapy 2014, 5: 60.

[0159] Figure 6 The growth curves of four primary cells cultured using the intestinal cancer primary cell culture medium CA-2 of the present invention, drawn using Graphpad Prism software, are shown. The horizontal axis represents the number of days of cell culture, and the vertical axis represents the cumulative cell proliferation multiple, which represents the multiple of cell expansion during the culture cycle. The larger the value, the more times the cells expand within a certain cycle, that is, the more cells are expanded. The slope represents the rate of cell expansion. Figure 6 It can be confirmed that when the primary intestinal cancer cells cultured in the culture medium of the present invention are continuously cultured and expanded for at least 80 days, the cell expansion rate remains basically unchanged and still has the ability to continue to expand.

[0160] Comparison of culture effects of Example 7 with literature culture medium

[0161] (1) Preparation of culture medium

[0162] Literature culture medium (Seungil Kim et al., Original Research, 2020, Vol. 25 (7) 744–754), whose formula is 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) + N2 1:100 (v / v) (purchased from Gibco) + B27 1:50 (v / v) (purchased from Gibco) + 1mM N-acetylcysteine ​​(purchased from Taosu Biochemical) + 50ng / mL epidermal growth factor (purchased from R&D) + 100ng / mL Noggin (purchased from R&D) + 10mM nicotinamide (purchased from MCE) + 500nM A8301 (purchased from MCE) + 10μM SB202190 (purchased from MCE) + 0.01 μM prostaglandin E2 (purchased from Tocris) (hereinafter referred to as "literature" culture medium).

[0163] (2) Acquisition and culture of primary colorectal cancer cells

[0164] Primary colorectal cancer cells were obtained from an endoscopic tissue sample (numbered OE(E)124) according to the method of step (2) of Example 1. The basal medium was mixed with matrigel ( 356231) were mixed evenly on ice, 4 mL was plated on a T12.5 culture flask, and incubated in a 37°C, 5% CO2 incubator for 25 to 35 minutes, preferably 30 minutes, and the supernatant was discarded. The obtained primary intestinal cancer cells were cultured at a viable cell density of 2.4 × 10 4 pieces / cm 2 Inoculate into T12.5 culture flasks (300,000 cells per flask). Use a pipette to gently add 4 mL of the intestinal cancer primary cell culture medium CA-2 of the present invention or the culture medium from the literature at room temperature to each T12.5 culture flask. After surface disinfection, place in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific) for culture. After 7 days of culture, count the cells. Figure 7 The proliferation efficiency of primary cells cultured using two different media is shown.

[0165] according to Figure 7 The results show that compared with the culture medium in the literature, the intestinal cancer primary cell culture medium of the present invention has a significantly better proliferation efficiency in culturing intestinal cancer cells in vitro.

[0166] Example 8: Primary intestinal cancer cells amplified using the culture medium of the present invention are used for drug screening and efficacy evaluation

[0167] 1. Cell Culture and Plating

[0168] According to the method of step (2) 3 of Example 1, primary colorectal cancer cells (numbered OE(E)113) were isolated and cultured as the first generation using the colorectal cancer primary cell culture medium CA-2 of the present invention. After the cells expanded to 85%, they were passaged. According to step (2) 4 of Example 1, the cell passages were counted and the cells were plated at a viable cell density of 1×10 5 After the cells / mL were placed in a sample addition tank (purchased from Corning) and fully mixed, they were cultured in a 384-well opaque white cell culture plate (purchased from Corning), with a volume of 50 μL per well and 5000 cells per well. The intestinal cancer primary cell culture medium of the present invention was added from the edge of the well plate to seal the plate, and the sample name, drug addition time and CellTiter-Glo (purchased from Promega) detection time were marked on the plate. The surface was disinfected with 75% alcohol (purchased from Lierkang), placed in a 37°C, 5% CO2 incubator for culture, and the drug was added after 24 hours. The first, second, third, fourth and fifth generation cells were obtained for drug screening, and the drug sensitivity of the intestinal cancer primary cells cultured using the culture medium of the present invention was tested.

[0169] 2. Screening drug preparation

[0170] Six drugs (cytarabine, doxorubicin, bortezomib, panobinostat, azacitidine, homoharringtonine; all purchased from MCE) with six concentration gradients were prepared according to the table below, 30 μL was added to each well of a 384-well drug plate (purchased from Thermo Fisher Scientific), and stored for use.

[0171] Table 3 Drug action concentration settings

[0172]

[0173] 3. High-throughput dosing

[0174] Remove the prepared drug plate, place it at room temperature, and centrifuge it in a Beckman centrifuge at 1000 rpm for 1 minute. High-throughput drug addition was performed using a high-throughput automated sample delivery system (Perkin Elmer JANUS). 0.1 μL of the selected drug at the corresponding concentration was added to each well of a 384-well plate containing primary human colorectal cancer cells. After drug addition, the 384-well plate was surface-disinfected and moved to an incubator. Cell viability was measured 72 hours later.

[0175] 4. Cell activity test

[0176] Remove CellTiter-Glo luminescent reagent (purchased from Promega) from a 4°C refrigerator and place 10 mL of the reagent in a sample reservoir. Remove the 384-well plate to be tested from the incubator and add 10 μL of CellTiter-Glo luminescent reagent to each well. Let it stand for 10 minutes, then mix thoroughly and analyze using a multi-function microplate reader (Perkin Elmer Envision).

[0177] 5. Data processing

[0178] The cell inhibition rate after different drugs acted on cells was calculated according to the formula: cell inhibition rate (%) = 100% - chemiluminescence value of drug-treated wells / chemiluminescence value of control wells × 100%. The half inhibition rate (IC) of drug action on cells was calculated using GraphPad Prism software. 50 The results are shown in Figures 8A-8F .

[0179] Depend on Figures 8A-8F It was confirmed that drug screening using primary colorectal cancer cells cultured in the CA-2 culture medium of the present invention showed essentially consistent inhibitory effects of the same drug on cells of different passages (inhibition curves remained essentially the same). Cells from the same patient exhibited varying sensitivities to different drugs at their maximum blood concentrations in the human body. These results can be used to assess the clinical effectiveness of this drug in colorectal cancer patients and demonstrate the stable drug sensitivity of tumor cells of different passages cultured using the present method.

[0180] Industrial Applicability

[0181] The present invention provides a primary cell culture medium and a culture method for culturing primary intestinal cancer cells in vitro. The cultured cells can be used for evaluating and screening drug efficacy. Therefore, the present invention is suitable for industrial application.

[0182] 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 primary intestinal cancer cells, characterized in that: Made with the following ingredients: MST1 / 2 kinase inhibitor; insulin; Y27632; insulin-transferrin-selenium supplement; glutamine supplement; neuregulin 1; bovine pituitary extract; basic fibroblast growth factor; R-spondin 1; prostaglandin E2; B27; 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 content of the MST1 / 2 kinase inhibitor in the culture medium is 1.25 to 5 μM; (2) The content of insulin in the culture medium is 1 to 9 μg / mL; (3) the content of Y27632 in the culture medium is 1.25-20 μM; (4) the volume ratio of the insulin-transferrin-selenium supplement to the culture medium is 1:400 to 1:100; (5) The content of the glutamine additive in the culture medium is 0.5 to 2 mM; (6) the content of neuregulin-1 in the culture medium is 2.5 to 40 ng / ml; (7) The volume ratio of the bovine pituitary extract to the culture medium is 1:2000 to 1:125; (8) The content of basic fibroblast growth factor in the culture medium is 2.5-20 ng / ml; (9) The content of R-spondin1 in the culture medium is 1.25-10 ng / mL; (10) The content of prostaglandin E2 in the culture medium is 5 to 80 nM; (11) The volume ratio of B27 to culture medium is 1:200 to 1:12.

5.

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

3. A method for culturing primary intestinal cancer cells, characterized in that: The following steps are involved: (1) preparing a culture medium for primary intestinal cancer cells according to claim 1 or 2; (2) Obtaining primary colorectal cancer cells from colorectal cancer tissue samples; (3) Adding the culture medium of the primary intestinal cancer cells obtained in step (1) to the primary intestinal cancer cells obtained in step (2) for culturing.

4. A method for screening drugs for treating intestinal cancer, comprising the following steps: (1) Culturing primary intestinal cancer cells using the culture method according to claim 3; (2) Select the drug to be tested and dilute it according to the required concentration gradient; (3) adding the drug in various concentration gradients to the primary intestinal cancer cells cultured in (1); (4) Conduct cell activity test.

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