Culture medium and culture method of gastric cancer organoids

Abstract

The application provides a culture medium and a culture method of a gastric cancer organoid. The culture medium comprises an MST1 / 2 kinase inhibitor, a fibroblast growth factor 7, R-spondin 1, hydrocortisone, a B27 supplement, an N2 supplement, insulin, a dual regulatory protein, at least one Rho protein kinase inhibitor selected from Y27632, fasudil and H-1152, forskolin, gastrin, fetal bovine serum and cholera toxin. Compared with the existing culture method, the in-vitro culture using the culture medium has higher amplification efficiency; the culture of the gastric cancer organoid using the culture medium can maintain the morphological structure and pathological characteristics of the primary tissue, and improve the success rate and survival rate of the gastric cancer organoid culture.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a culture medium and application thereof, and more particularly to a culture medium for gastric cancer organoids and a method for culturing gastric cancer organoids using the culture medium. BACKGROUND

[0002] Gastric cancer is the most common malignant tumor of the digestive tract. Patients with early gastric cancer have no obvious symptoms, so most patients are diagnosed at an advanced stage. Gastric cancer cells have poor sensitivity to chemotherapeutic drugs, so the effect of chemotherapy is poor, resulting in a low five-year survival rate. In recent years, with the development of science and technology, the diagnosis and treatment technology of gastric cancer has been continuously improved, especially the progress of comprehensive treatment methods such as surgery, endoscopic technology, radiotherapy and targeted therapy, and the treatment effect of gastric cancer has been improved. However, for patients with advanced and metastatic gastric cancer, the prognosis of comprehensive treatment is still not ideal.

[0003] Existing in vitro cultured gastric cancer cell lines are mainly obtained by long-term culture of normal cells to spontaneously immortalize or transfection of cancer genes to promote the immortalization of normal cells. Cell lines established by traditional methods are still the mainstay of cell, molecular and cancer biology research. However, these methods change the genetic background of the cells, and long-term culture of cell lines can also cause genetic instability, which may lead to artificial changes in the phenotype of tumor cell lines and in vivo tumor cells. These cell lines usually lack the complex heterogeneity of primary tumors, which limits the application of these cell lines to predict tumor cell responses and affects the accuracy of scientific research and drug development of gastric cancer. In addition, during the process of obtaining cancer cells from gastric cancer tissue, it is difficult to obtain cancer cells by conventional culture methods, and there are problems such as easy interference by fibroblasts and inability to pass the formed clones during the culture process, which limits the application of human gastric cancer primary cells.

[0004] Organoids are organ-specific cell collections derived from stem cells or precursor cells. In vitro cultured organoids are highly similar to the corresponding organs in terms of cell composition and tissue architecture, and have corresponding functional characteristics. Unlike conventional cell culture, which cultures a single cell population in a two-dimensional environment, organoid culture is a three-dimensional environment that cultures multiple cell populations contained in specific tissues and organs, and its culture system is more similar to the in vivo microenvironment. Therefore, it shows great application prospects in various physiological and pathological basic researches of organs, precision medicine, drug screening and development, gene therapy, regenerative medicine, etc.

[0005] Although various tumor tissues can be successfully cultured into organoids in vitro under different methods and different culture conditions, there are few reports on the culture method of gastric cancer organoids at present, especially the specific experimental procedures, operation steps, culture conditions, and culture medium formula have not been reported much.

[0006] Therefore, there is a need in the art for a gastric cancer organoid culture medium formulation and culture method that has a high success rate in vitro culture and is capable of maintaining the original histopathological characteristics of the organoids. SUMMARY

[0007] To solve the above technical problems, the present application provides a medium and an in vitro culture method for gastric cancer organoids.

[0008] One aspect of the present application is to provide a medium for gastric cancer organoids, the medium comprising an MST1 / 2 kinase inhibitor; R-spondin 1; hydrocortisone; a B27 supplement; an N2 supplement; insulin; dual regulatory protein; at least one Rho protein kinase inhibitor selected from Y27632, fasudil, and H-1152; fetal bovine serum; forskolin; gastrin; fibroblast growth factor 7; and cholera toxin. Wherein, the MST1 / 2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,

[0009]

[0010] wherein,

[0011] R1is selected from C1-C6alkyl, C3-C6cycloalkyl, C4-C8cycloalkylalkyl, C2-C6spirocycloalkyl, and aryl (e.g., phenyl and naphthyl, etc.) optionally substituted with 1-2 independent R6, aryl C1-C6alkyl (e.g., benzyl, etc.), and heteroaryl (e.g., thienyl, etc.);

[0012] R2and R3are each independently selected from C1-C6alkyl, preferably C1-C3alkyl, more preferably methyl;

[0013] R4and R5are each independently selected from hydrogen, C1-C6alkyl, C3-C6cycloalkyl, C4-C8cycloalkylalkyl, C1-C6alkylhydroxy, C1-C6haloalkyl, C1-C6alkylamino C1-C6alkyl, C1-C6alkoxy C1-C6alkyl, and C3-C6heterocyclyl C1-C6alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyranyl, etc.);

[0014] R6is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6alkyl (preferably methyl), C1-C6alkoxy (preferably methoxy), and C1-C6haloalkyl (preferably trifluoromethyl).

[0015] In preferred embodiments, the MST1 / 2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,

[0016] In preferred embodiments, the MST1 / 2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,

[0017] wherein,

[0018] R1is selected from the group consisting of C1-C6alkyl, phenyl optionally substituted with 1-2 independent R6, thienyl optionally substituted with 1-2 independent R6, and benzyl optionally substituted with 1-2 independent R6, R1is more preferably phenyl optionally substituted with 1-2 independent R6;

[0019] R5is selected from the group consisting of hydrogen, C1-C6alkyl, and C3-C6cycloalkyl, R5is more preferably hydrogen;

[0020] each R6is independently selected from the group consisting of halogen, C1-C6alkyl, and C1-C6haloalkyl, R6is more preferably fluorine, methyl or trifluoromethyl.

[0021] Preferably, the MST1 / 2 inhibitor is at least one selected from the group consisting of the following compounds or pharmaceutically acceptable salts, or solvates thereof.

[0022]

[0023]

[0024]

[0025]

[0026]

[0027] Most preferably, the MST1 / 2 kinase inhibitor of the present application is Compound 1.

[0028] In embodiments of the present application, the content of each component in the culture medium of the present application satisfies any one or more or all of the following:

[0029] (1) the concentration of the MST1 / 2 kinase inhibitor is generally in the range of 2.5-40 μM, more preferably 5-20 μM;

[0030] (2) the concentration of the R-spondin 1 is generally in the range of 125-500 ng / mL, more preferably 125-250 ng / mL;

[0031] (3) the concentration of the hydrocortisone is generally in the range of 5-80 ng / mL, more preferably 10-40 ng / mL;

[0032] (5) the volume ratio of the B27 supplement to the culture medium is generally in the range of 1:25-1:400, more preferably 1:25-1:200;

[0033] (5) The volume ratio of the N2 supplement to the culture medium is usually in the range of 1:100 to 1:800, more preferably in the range of 1:100 to 1:200;

[0034] (6) The concentration of the insulin is usually in the range of 1 to 4 μg / mL, more preferably in the range of 1 to 2 μg / mL;

[0035] (7) The concentration of the dual modulator is usually in the range of 1 to 81 ng / mL, more preferably in the range of 3 to 27 ng / mL;

[0036] (8) The concentration of the Rho protein kinase inhibitor is usually in the range of 2.5 to 10 μM;

[0037] (9) The volume concentration of the fetal bovine serum is usually in the range of 0.625% (v / v) to 10% (v / v), more preferably in the range of 2.5% (v / v) to 10% (v / v);

[0038] (10) The concentration of the forskolin is usually in the range of 2.5 to 40 μM, more preferably in the range of 2.5 to 10 μM;

[0039] (11) The concentration of the gastrin is usually in the range of 1 to 81 nM, more preferably in the range of 3 to 27 nM;

[0040] (12) The concentration of the fibroblast growth factor 7 is usually in the range of 5 to 80 ng / mL, more preferably in the range of 10 to 20 ng / mL;

[0041] (13) The concentration of the cholera toxin is usually in the range of 0.05 to 0.8 μg / mL, more preferably in the range of 0.1 to 0.4 μg / mL.

[0042] In the embodiment of the present application, the culture medium further contains an initial medium selected from the group consisting of DMEM / F12, DMEM, F12, or RPMI-1640; and an antibiotic selected from the group consisting of streptomycin / penicillin, amphotericin B, and Primocin.

[0043] In the 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 is in the range of 0.25 to 4 μg / mL, and when the antibiotic is selected from Primocin, the concentration is in the range of 25 to 400 μg / mL.

[0044] According to a second aspect, the present application also provides an in-vitro culture method of a gastric cancer organoid. In the in-vitro culture method of the gastric cancer organoid of the present application, the gastric cancer primary cells are cultured in-vitro using the gastric cancer organoid culture medium of the present application.

[0045] The in-vitro culture method of the gastric cancer organoid of the present application comprises the following steps:

[0046] 1. Isolation of gastric cancer primary cells

[0047] (1) Isolate the gastric cancer tissue sample, add the basic culture medium and the 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 per 1 g of tumor tissue), and place it in a constant temperature shaker for digestion. The digestion temperature ranges from 4 to 37℃, and the digestion rotation speed ranges from 200 rpm to 350 rpm.

[0048] (2) The digestion is terminated when no obvious tissue mass is observed, and the digestion time is 3-6 hours.

[0049] (3) After centrifugation, discard the supernatant. The centrifugation speed ranges from 1200 to 1600 rpm, and the centrifugation time is 2-6 minutes. Add the basic culture medium and resuspend for use.

[0050] The basic culture medium formula includes an initial culture medium selected from DMEM / F12, DMEM, F12 or RPMI-1640, and an antibiotic selected from one or more of streptomycin / penicillin, amphotericin B and Primocin. The tissue digestion solution formula includes 1640 culture medium, collagenase II (1-2 mg / mL), collagenase IV (1-2 mg / mL), DNAase (50-100 U / mL), hyaluronidase (0.5-1 mg / mL), calcium chloride (1-5 mM), and bovine serum albumin BSA (5-10 mg / mL).

[0051] 2. Culture using the gastric cancer organoid culture medium of the present application

[0052] Resuspend the gastric cancer primary cells obtained in step 1 above with the basic culture medium and count them. Mix the cells with the Matrigel evenly on ice at a volume ratio of 1:1, and the final cell density is 1-8 x 10 5 mL. Take the Matrigel and cell suspension to form a coagulation droplet in the culture plate, and place the culture plate at 4-37℃ for 10-60 minutes to allow the Matrigel to completely coagulate. Add the gastric cancer organoid culture medium of the present application and place it in the incubator for culture.

[0053] The technical solution of the present application can achieve the following technical effects:

[0054] (1) The success rate of culturing the gastric organoid is improved, and the success rate is more than 80%;

[0055] (2) The gastric cancer organoid cultured in vitro can maintain the pathological characteristics of the patient;

[0056] (3) The cultured gastric cancer organoid is not interfered by interstitial cells such as fibroblasts and adipocytes;

[0057] (4) The amplification efficiency is high, and the gastric cancer organoid can be successfully cultured in about one week, and the amplified gastric cancer organoid can be continuously passaged;

[0058] (5) The culture cost is controllable, and the culture medium does not need to add expensive Wnt agonists. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 The figure is used to show the effect of different combinations of additives in the gastric cancer organoid culture medium on the growth of the gastric cancer organoid.

[0060] Figures 2A-2M The figure is used to show the effect of different concentrations of additives in the gastric cancer organoid culture medium on the growth of the gastric cancer organoid.

[0061] Figures 3A-3F The figure is a photograph of the gastric cancer organoid obtained by using the gastric cancer organoid culture medium of the present application to culture the gastric cancer organoid.

[0062] Figures 4A-4D The figure is the immunohistochemical result of the original gastric cancer tissue cells.

[0063] Figures 5A-5D The figure is the immunohistochemical result of the gastric cancer organoid obtained by using the gastric cancer organoid culture medium of the present application to culture the original gastric cancer tissue cells to the third generation.

[0064] Figure 6 The figure is a microscope photograph of the gastric cancer organoid obtained by using the gastric cancer organoid culture medium of the present application, the literature culture medium, and the commercial culture medium to culture the gastric cancer organoid, respectively. DETAILED DESCRIPTION

[0065] In order to better understand the present application, the present application will be further described below in conjunction with the embodiments and the drawings, and the following embodiments are only used to illustrate the present application but not to limit it.

[0066] [Preparation example of MST1 / 2 kinase inhibitor]

[0067] In the present specification, 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 an MST1 / 2 kinase and reduces its activity. Since the structures of MST1 and MST2 are similar, an MST1 / 2 kinase inhibitor can also be a compound that, for example, binds to MST1 or MST2 and reduces its activity.

[0068] 1. Preparation of MST1 / 2 kinase inhibitor compound 1

[0069] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropyrimidin-2-yl)amino)

[0070] benzenesulfonamide 1

[0071]

[0072] 2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): To a round bottom flask was added 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) followed by methanol (30 mL) and then dropwise dichlorosulfoxide (1.2 mL) with ice bath cooling. The reaction was stirred at 85 °C overnight. After the reaction was complete, the solvent was removed under reduced pressure and the resulting white solid was used directly in the next step.

[0073] 2-((2-Chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetic acid methyl ester (A3): To a round bottom flask was added 2-amino-2-(2,6-difluorophenyl)acetic acid methyl ester (2 g) followed by acetone (30 mL) and potassium carbonate (2.2 g) and then the system was cooled to -10 °C with an ice-salt bath followed by the slow addition of a solution of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction was stirred at room temperature overnight. After the reaction was complete, it was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on pressurized silica gel to give compound A3. LC / MS: M+H 359.0.

[0074] 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): To a round bottom flask was added 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetic acid methyl ester (2.5 g) followed by acetic acid (50 mL) and iron powder (3.9 g). The reaction was stirred at 60 °C for two hours. After the reaction was complete, the solvent was removed under reduced pressure and the resulting material was neutralized to basic with saturated sodium bicarbonate. The ethyl acetate extract was washed with water, saturated brine and then dried over anhydrous sodium sulfate. The organic phase was filtered and concentrated under reduced pressure to give the crude product. The crude product was washed with diethyl ether to give compound A4. LC / MS: M+H 297.0.

[0075] 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (A5): In a round bottom flask, 2-chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (2 g) and N,N-dimethylacetamide (10 mL) were added, cooled to -35 °C, iodomethane (0.9 mL) was added, followed by sodium hydride (615 mg), the reaction system was continuously stirred for two hours. After the reaction was completed, water was added to quench, extracted with ethyl acetate, the organic phase was washed with water, saturated brine respectively, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated under reduced pressure to obtain the crude product. The crude product was washed with ether to obtain compound A5. LC / MS: M+H 325.0.

[0076] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino) benzenesulfonamide (1): In a round bottom flask, 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (100 mg), sulfanilamide (53 mg), p-toluenesulfonic acid (53 mg) and sec-butanol (5 mL) were added. The reaction system was stirred at 120 °C overnight. After the reaction was completed, it was filtered and washed with methanol and ether to obtain compound 1. LC / MS: M+H 461.1.

[0077] 2. Preparation of other MST1 / 2 inhibitor compounds of the present application

[0078] Other MST1 / 2 inhibitor compounds of the present application were synthesized according to a method similar to that of compound 1, and their structures and mass spectrometry data are shown in the following table.

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Example 1 Effect of each additive factor in gastric cancer organoid culture medium on the growth of gastric cancer organoids

[0086] (1) Preparation of gastric cancer organoid culture medium

[0087] First, the base medium containing the initial medium was prepared. The initial medium can be selected from DMEM / F12, DMEM, F12 or RPMI-1640 commonly used in the art. In this embodiment, the formula of the base medium is: DMEM / F12 medium (purchased from Corning Company) + 100 μg / mL Primocin (purchased from InvivoGen Company, 0.2% (v / v), commercially available product concentration 50 mg / ml). Different types of additives were added into the base medium respectively (see Table 1) to prepare the gastric cancer organoid culture medium containing different additive components.

[0088] (2) Isolation and processing of gastric cancer primary cells

[0089] 1 Sample selection

[0090] Gastric cancer solid tumor tissue samples (intraoperative / endoscopic) were obtained from patients by professional medical personnel of professional medical institutions, and the patients all signed the informed consent form. The intraoperative sample was 0.25 cm 3 , and the endoscopic sample was 0.025 cm 3 ; the commercialized tissue preservation solution (manufacturer: Miltenyi Biotec) was used for storage and transportation.

[0091] 2 Material preparation

[0092] 15 mL sterile centrifuge tube, pipette, 10 mL pipette, sterile tip, etc. were surface sterilized and placed in the ultraclean bench for ultraviolet irradiation for 30 minutes. The base medium was taken out from the 4℃ refrigerator 30 minutes in advance, and the tissue digestion solution (formula as follows) was taken out from the -20℃ refrigerator 30 minutes in advance.

[0093] Tissue digestion solution: 1640 medium (Corning, 10-040-CVR), collagenase II (2 mg / mL), collagenase IV (2 mg / mL), DNAase (50 U / mL), hyaluronidase (0.75 mg / mL), calcium chloride (3.3 mM), BSA (10 mg / mL).

[0094] The above-mentioned collagenase II, collagenase IV, DNAase, and hyaluronidase were purchased from Sigma Company; calcium chloride was purchased from Shengong Bioengineering (Shanghai) Co., Ltd.; BSA was purchased from Biofroxx Company.

[0095] 3. Isolation of gastric cancer primary cells

[0096] 3.1 The tissue sample was taken in a culture dish, the tissue with blood was removed, and the tissue was transferred to another culture dish for mechanical separation with a sterile scalpel, and the tissue block was divided into 1×1×1 mm 3 size.

[0097] 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;

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 3.6 Discard the supernatant and add 2 mL of basal culture medium to resuspend the cells for later use.

[0102] 4. Cell Counting and Processing

[0103] 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).

[0104] 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.). After thorough mixing, 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%.

[0105] (3) Culture of gastric cancer organoids

[0106] The primary gastric cancer cells isolated from two gastric cancer tissues (numbered LYH and HCY) 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 5A 7 μL aliquot of Matrigel was mixed with 100 μL of cell suspension in the center of each well of a 96-well plate to form a solidified droplet, and the plate was left to stand at 37°C for 30 minutes to allow the Matrigel to completely solidify. The medium of different components in Table 1 was added to the 96-well plate at a volume of 100 μL / well. Among them, as an experimental control, the basal medium without any added components was used. After 7 to 10 days of culture, 50 μL of CellTiter-Glo (purchased from Promega Corporation) luminescent reagent was added to each well, mixed after standing for 10 minutes, and detected using a multifunctional enzyme label instrument (Envision of PerkinElmer Corporation). The relative cell viability (%) was calculated according to the formula: experimental well chemiluminescence value / control well chemiluminescence value x 100%, and the promoting effect of different added components on the growth of gastric cancer organoids was inferred. The experimental results are shown in Table 1.

[0107] Table 1: Added components in the medium and cell proliferation effect

[0108]

[0109]

[0110] Among them, "+" means that the medium added with the component promotes the proliferation of at least two cases of gastric cancer primary cells isolated from gastric cancer tissues compared with the basal medium; "-" means that the medium added with the component shows a promoting effect on the proliferation of one case of gastric cancer primary cells isolated from gastric cancer tissues; "O" means that the medium added with the component has no obvious effect on the proliferation of gastric cancer primary cells isolated from gastric cancer tissues.

[0111] According to the above results, compounds 1, noggin, R-spondin1, hydrocortisone, B27, N2, insulin, dual regulatory protein, nicotinamide, Y-27632, fetal bovine serum, forskolin, sodium pyruvate, gastrin, fibroblast growth factor 7, cholera toxin, etc. were selected for further culture experiments.

[0112] Example 2: Effect of different combinations of added factors in gastric cancer organoid culture medium on the growth of gastric cancer organoids

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

[0114] Table 2: Preparation of different component media (concentration is the final concentration)

[0115]

[0116]

[0117] Primary gastric cancer cells were obtained from gastric cancer tissues (numbered LYH, HCY) 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. 17 to the 48-well plate respectively. After culturing for 7 to 10 days, add 20μL 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 the cells is calculated = chemiluminescence value of the experimental well / chemiluminescence value of the control well × 100%, and the promotion effect of different component culture media on the growth of gastric cancer organoids is obtained. The experimental results are shown in Figure 1 .

[0118] according to Figure 1 The results show that, compared with the basal medium, the above-mentioned No. 1 to No. 17 media were able to promote the proliferation of primary gastric cancer cells to varying degrees. When the added factors noggin (No. 3), nicotinamide (No. 10), and sodium pyruvate (No. 14) were omitted, the proliferation-promoting effect of the medium formulation was even more pronounced. Therefore, in subsequent examples, further studies were conducted using a medium formulation containing factors such as Compound 1, R-spondin 1, hydrocortisone, B27, N2, insulin, amphiregulin, Y-27632, fetal bovine serum, forskolin, gastrin, fibroblast growth factor 7, and cholera toxin as a culture medium for culturing gastric cancer organoids.

[0119] Example 3 Effects of Different Concentrations of Factors Added to Gastric Cancer Organoid Culture Medium on the Proliferation of Gastric Cancer Organoids

[0120] Primary gastric cancer cells were obtained from gastric cancer tissues (numbered YHG, GD11) 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.

[0121] Next, experiments were performed with a gastric cancer organoid culture medium containing basal medium BM, 5 μΜ Compound 1, 125 ng / mL R-spondin 1, 10 ng / mL hydrocortisone, 1:100 (v / v) B27, 1:200 (v / v) N2, 2.5 μg / mL insulin, 9 ng / mL noggin, 2.5 μΜ Y-27632, 5% (v / v) fetal bovine serum, 5 μΜ forskolin, 9 nM gastrin, 10 ng / mL fibroblast growth factor 7, 0.2 μg / mL cholera toxin, and the following 13 formulae.

[0122] Formula 1: The above gastric cancer organoid culture medium components did not contain Compound 1;

[0123] Formula 2: The above gastric cancer organoid culture medium components did not contain R-spondin 1;

[0124] Formula 3: The above gastric cancer organoid culture medium components did not contain hydrocortisone;

[0125] Formula 4: The above gastric cancer organoid culture medium components did not contain B27;

[0126] Formula 5: The above gastric cancer organoid culture medium components did not contain N2;

[0127] Formula 6: The above gastric cancer organoid culture medium components did not contain insulin;

[0128] Formula 7: The above gastric cancer organoid culture medium components did not contain noggin;

[0129] Formula 8: The above gastric cancer organoid culture medium components did not contain Y-27632;

[0130] Formula 9: The above gastric cancer organoid culture medium components did not contain fetal bovine serum;

[0131] Formula 10: The above gastric cancer organoid culture medium components did not contain forskolin;

[0132] Formula 11: The above gastric cancer organoid culture medium components did not contain gastrin;

[0133] Formula 12: The above gastric cancer organoid culture medium components did not contain fibroblast growth factor 7;

[0134] Formula 13: The above gastric cancer organoid culture medium components did not contain cholera toxin.

[0135] When using the medium of Formula 1, 100 μL of prepared compound 1 was added to each well of the 96-well plate inoculated with primary cells, and the final concentration of compound 1 was 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, respectively; and the control well (BC) was set using the medium of Formula 1.

[0136] When using the medium of Formula 2, 100 μL of prepared R-spondin 1 was added to each well of the 96-well plate inoculated with primary cells, and the final concentration of R-spondin 1 was 125 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, respectively; and the control well (BC) was set using the medium of Formula 2.

[0137] When using the medium of Formula 3, 100 μL of prepared hydrocortisone was added to each well of the 96-well plate inoculated with primary cells, and the final concentration of hydrocortisone was 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 50 ng / mL, respectively; and the control well (BC) was set using the medium of Formula 3.

[0138] When using the medium of Formula 4, 100 μL of prepared B27 was added to each well of the 96-well plate inoculated with primary cells, and the volume ratio of B27 to the medium was 1:400, 1:200, 1:100, 1:50, 1:25, respectively; and the control well (BC) was set using the medium of Formula 4.

[0139] When using the medium of Formula 5, 100 μL of prepared N2 was added to each well of the 96-well plate inoculated with primary cells, and the volume ratio of N2 to the medium was 1:1600, 1:800, 1:400, 1:200, 1:100, respectively; and the control well (BC) was set using the medium of Formula 5.

[0140] When using the medium of Formula 6, 100 μL of prepared insulin was added to each well of the 96-well plate inoculated with primary cells, and the final concentration of insulin was 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, respectively; and the control well (BC) was set using the medium of Formula 6.

[0141] When using the medium of Formula 7, 100 μL of prepared dual regulatory protein was added to each well of the 96-well plate inoculated with primary cells, and the final concentration of dual regulatory protein was 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, 81 ng / mL, respectively; and the control well (BC) was set using the medium of Formula 7.

[0142] When using the medium of Formula 8, 100 μL of prepared Y-27632 was added to each well of the 96-well plate inoculated with primary cells, and the final concentration of Y-27632 was 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively; and a control well (BC) was set using the medium of Formula 8.

[0143] When using the medium of Formula 9, 100 μL of prepared fetal bovine serum was added to each well of the 96-well plate inoculated with primary cells, and the volume ratio of fetal bovine serum to the medium was 0.625%, 1.25%, 2.5%, 5%, and 10%, respectively; and a control well (BC) was set using the medium of Formula 9.

[0144] When using the medium of Formula 10, 100 μL of prepared forskolin was added to each well of the 96-well plate inoculated with primary cells, and the final concentration of forskolin was 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively; and a control well (BC) was set using the medium of Formula 10.

[0145] When using the medium of Formula 11, 100 μL of prepared gastrin was added to each well of the 96-well plate inoculated with primary cells, and the final concentration of gastrin was 1 nM, 3 nM, 9 nM, 27 nM, and 81 nM, respectively; and a control well (BC) was set using the medium of Formula 11.

[0146] When using the medium of Formula 12, 100 μL of prepared fibroblast growth factor 7 was added to each well of the 96-well plate inoculated with primary cells, and the final concentration of fibroblast growth factor 7 was 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL, respectively; and a control well (BC) was set using the medium of Formula 12.

[0147] When using the medium of Formula 13, 100 μL of prepared cholera toxin was added to each well of the 96-well plate inoculated with primary cells, and the final concentration of cholera toxin was 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL, and 0.8 μg / mL, respectively; and a control well (BC) was set using the medium of Formula 13.

[0148] After the organoids were cultured for 7-10 days, the relative viability was calculated by referring to the cell number of the control well (BC), and the results are shown in Figures 2A-2M . Figures 2A-2MIn the data, relative viability is the ratio of gastric cancer organoid viability after 7–10 days of culture in each culture medium to the corresponding control well viability after 7–10 days of culture. A ratio greater than 1 indicates that the culture medium containing the factor or small molecule at a different concentration is more effective in promoting proliferation than the control well culture medium; a ratio less than 1 indicates that the culture medium containing the factor or small molecule at a different concentration is less effective in promoting proliferation than the control well culture medium.

[0149] according to Figures 2A-2M According to the results, the concentration range of compound 1 is preferably 2.5-20 μM, and most preferably 5 μM; the concentration range of R-spondin 1 is preferably 125-500 ng / mL, and more preferably 125 ng / mL; the concentration range of hydrocortisone is preferably 5-80 ng / mL, and most preferably 20 ng / mL; the volume ratio of B27 additive to culture medium is preferably 1:25-1:400, and most preferably 1:100; the volume ratio of N2 additive to culture medium is preferably 1:100-1:800, and most preferably 1:200; the concentration range of insulin is preferably 1-2 μg / mL, and most preferably 2 μg / mL; the concentration range of amphiregulin is preferably 3-81 ng / mL, and most preferably 9 ng / mL; the concentration range of Y27632 is preferably 2.5-10 μM, The most preferred concentration is 5 μM; the volume ratio of fetal bovine serum to culture medium is preferably in the range of 0.625% (v / v) to 10% (v / v), and the most preferred concentration is 5% (v / v); the concentration range of forskolin is preferably 2.5 to 40 μM, and the most preferred concentration is 2.5 μM; the concentration range of gastrin is preferably 1 to 81 nM, and the most preferred concentration is 27 nM; the concentration range of fibroblast growth factor 7 is preferably 5 to 80 ng / mL, and the most preferred concentration is 20 ng / mL; the concentration range of cholera toxin is preferably 0.05 to 0.8 μg / mL, and the most preferred concentration is 0.2 μg / mL.

[0150] The optimal concentrations of each added factor in the above-mentioned culture medium were used as the gastric cancer organoid culture medium of the present invention used in the following examples, which contained: basal medium BM, 5 μM compound 1, 125 ng / mL R-spondin 1, 20 ng / mL hydrocortisone, 1:100 (v / v) B27, 1:200 (v / v) N2, 2 μg / mL insulin, 9 ng / mL amphiregulin, 5 μM Y-27632, 5% (v / v) fetal bovine serum, 2.5 μM forskolin, 27 nM gastrin, 20 ng / mL fibroblast growth factor 7, and 0.2 μg / mL cholera toxin.

[0151] Example 4 Gastric Cancer Organoid Culture

[0152] Primary cells of gastric cancer were obtained from 6 intraoperative tissue samples (coded as LYH, WHY, DWF, WSY, GD6, JXH) according to the method of step (2) of Example 1, resuspended with basal medium and counted, and then mixed with Matrigel (BD) at a volume ratio of 1:1 on ice, with a final cell density of 5x10 356231) ice, with a final cell density of 5x10 5 6L Matrigel and cell suspension in the center of each well of a 24-well culture plate to form a coagulation droplet, and the culture plate was placed at 37°C for 30 minutes to allow the Matrigel to completely coagulate. Then, 1 mL of the gastric cancer organoid culture medium of the present application 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 sterilized, it was placed in a 37°C, 5% CO2 incubator (purchased from Thermo) for culture. After 3-8 days of culture, the gastric cancer organoids obtained by culture were observed using a microscope (Invitrogen, EVOS M500), and the pictures were recorded under a 10x objective lens. Figures 3A-3F

[0153] Example 5 Histological identification of gastric cancer organoids cultured

[0154] About 0.25 cm 3 size of cancer tissue was taken from the intraoperative tissue of a gastric cancer patient (sample number GD20) and fixed by immersion in 1 mL of 4% paraformaldehyde. The sample GD20 was continuously cultured to the 3rd generation using the gastric cancer organoid culture medium of the present application according to the method of Example 4. The gastric cancer organoids fixed by 4% paraformaldehyde were paraffin-embedded and cut into 4 μm thick tissue sections using a microtome. Subsequently, conventional immunohistochemical detection was performed (for specific steps, see Li et al., Nature Communication, (2018) 9:2983). The primary antibodies used were ki-67, CK7, CDX-2, and villin (all purchased from CST).

[0155] Figures 4A-4D and Figures 5A-5D are comparison pictures of immunohistochemical results of the original tissue cells and the gastric cancer organoids obtained by culturing the cells to the 3rd generation using the gastric cancer organoid culture medium of the present application, respectively. Figure 4A and Figure 5A are pictures of the marker ki-67 antibody of the original tissue of gastric cancer and the cultured gastric cancer organoids, respectively, Figure 4B and Figure 5B are pictures of the marker CK7 antibody of the original tissue of gastric cancer and the cultured gastric cancer organoids, respectively, Figure 4C and Figure 5C are pictures of the marker CDX-2 antibody of the original tissue of gastric cancer and the cultured gastric cancer organoids, respectively,​Figure 4D and Figure 5D These images show the villin antibody, which is used to label gastric cancer primary tissue and cultured gastric cancer organoids. This confirms that the expression of gastric cancer-related biomarkers in gastric cancer organoids (sample number GD20) cultured using the technology of this invention at passage 3 is essentially consistent with that in the original tissue sections from which the organoids were derived. This demonstrates that organoids cultured using the technology of this invention retain the original pathological characteristics of gastric cancer tissue from patients.

[0156] Comparison of culture effects of Example 6 with literature culture medium and commercial culture medium

[0157] (1) Preparation of culture medium

[0158] 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).

[0159] Commercial culture medium: IntestiCult TM Organoid Growth Medium (Human) (available from STEMCELL, 06010).

[0160] (2) Obtaining primary gastric cancer cells and culturing gastric cancer organoids

[0161] According to the method of step (2) of step 3 of Example 1, primary gastric cancer cells were obtained from an endoscopic tissue sample (numbered OE(E)122), 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 5The 50 μL Matrigel and the cell suspension are taken to the center of each well of a 24-well culture plate to form a coagulation droplet, the culture plate is placed at 37°C for 30 minutes to allow the Matrigel to completely coagulate, and 1 mL of the gastric cancer organoid culture medium of the present application, the literature culture medium and the commercial culture medium at room temperature are gently added into each well along the side wall of the well using a pipette. The surface of the 24-well culture plate is sterilized and placed in a 37°C, 5% CO2 incubator (purchased from Thermo) for culture. After 5 days of culture, the gastric cancer primary cells obtained by culture are observed using a microscope (Invitrogen EVOS M500), and the results are shown in FIG. 1. Figure 6 is a gastric cancer organoid recorded by a 10-fold objective lens.

[0162] According to the results, compared with the literature culture medium and the commercial culture medium, the gastric cancer organoid culture medium of the present application has a short culture period, a large organoid volume and an intact organoid structure when culturing a gastric cancer organoid in vitro, and its effect is obviously better than that of the literature culture medium and the commercial culture medium. Figure 6

[0163] Industrial applicability

[0164] The present application provides a culture medium and a culture method for gastric cancer organoid culture, and the organoid obtained by culture can be applied to drug efficacy evaluation and screening. Thus, the present application is suitable for industrial application.

[0165] Although the present application is described in detail herein, the present application is not limited thereto, and those skilled in the art can make modifications according to the principles of the present application, therefore, all kinds of modifications made according to the principles of the present application should be understood as falling within the scope of protection of the present application.​

Claims

1. A culture medium for gastric cancer organoids, characterized in that Made with the following ingredients: MST1 / 2 kinase inhibitor; R-spondin 1; hydrocortisone; B27 supplement; N2 supplement; insulin; amphiregulin; Y27632; fetal bovine serum; forskolin; gastrin; fibroblast growth factor 7; cholera toxin; An initial culture medium selected from DMEM / F12, DMEM, F12, or RPMI-1640; and antibiotics; 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 2.5 to 20 μM; (2) the concentration of R-spondin1 is 125 to 500 ng / mL; (3) The concentration of hydrocortisone is 5 to 80 ng / mL; (4) The volume ratio of the B27 additive to the culture medium is 1:25 to 1:400; (5) The volume ratio of the N2 additive to the culture medium is 1:100 to 1:800; (6) The concentration of the insulin is 1 to 2 μg / mL; (7) the concentration of the amphiregulin is 1 to 81 ng / mL; (8) The concentration of Y27632 is 2.5 to 10 μM; (9) The volume concentration of the fetal bovine serum is 0.625% (v / v) to 10% (v / v); (10) The concentration of forskolin is 2.5 to 40 μM; (11) The concentration of gastrin is 1 to 81 nM; (12) The concentration of fibroblast growth factor 7 is 5 to 80 ng / mL; (13) The concentration of the cholera toxin is 0.05 to 0.8 μg / mL.

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

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

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