Culture medium and culture method for primary gastric cancer cells

Abstract

The present invention provides a culture medium for culturing primary gastric cancer cells, comprising an MST1 / 2 kinase inhibitor, a ROCK kinase inhibitor, at least one of a B27 additive and an N2 additive, basic fibroblast growth factor, CHIR99021, epidermal growth factor, an ITS cell culture additive, SB202190, dexamethasone, N-acetyl-L-cysteine, gastrin, A8301, oncostatin M, and cholera toxin. The present invention also relates to a culture method using the primary cell culture medium. The culture method uses the culture medium to culture primary gastric cancer cells on a culture vessel coated with extracellular matrix glue, thereby rapidly proliferating the primary cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a culture medium for culturing primary gastric cancer cells, and a method for culturing primary gastric cancer cells using the culture medium. Background Art

[0002] Gastric cancer, a malignant tumor originating from the gastric mucosal epithelium, ranks first in incidence among all malignant tumors in my country. It is most common in patients over 50 years old, with a male-to-female incidence ratio of 2:1. Gastric cancer is becoming younger and younger due to factors such as changes in diet, increased work pressure, and Helicobacter pylori infection. Gastric cancer can occur anywhere in the stomach, with over half occurring in the antrum, affecting the greater and lesser curvatures, as well as the anterior and posterior walls. The vast majority of gastric cancers are adenocarcinomas. Early symptoms are asymptomatic, or they may present with nonspecific symptoms such as upper abdominal discomfort and belching, often mimicking those of chronic gastric diseases such as gastritis and gastric ulcers, making them easily overlooked. Consequently, the early diagnosis rate of gastric cancer in my country remains low. Currently, the national diagnosis rate for early gastric cancer remains below 20%, and the five-year survival rate for gastric cancer patients is only 27.4%.

[0003] In recent years, with the rise and development of molecular biology, drug treatment for tumors has shown a trend of diversification. Among them, molecular targeted drugs have become a hot topic of research in the clinical treatment of gastric cancer due to their strong targeting and high safety. However, with so many treatment options in clinical practice, it is particularly important to choose the right one for the patient. Although genetic testing is used as an indicator, some patients do not have gene mutations, or even if some patients have a certain mutation, there are multiple targeted drugs for the mutation. In this case, it is difficult to determine the treatment plan clinically. In addition to gene sequencing, in vitro primary cell culture of gastric cancer patient samples has become an important means to predict efficacy in vitro and guide clinical drug use in the future. However, the rapid acquisition of primary gastric cancer cells in vitro has always been a technical problem that needs to be solved urgently.

[0004] Currently, there are two relatively mature technologies for culturing primary cells. One is the use of irradiated feeder cells and the ROCK kinase inhibitor Y27632 to promote the growth of primary epithelial cells, a technique known as conditional cell reprogramming (Liu et al., Am. J. Pathol., 180:599-607, 2012). The other is the in vitro 3D culture of adult stem cells to produce organoids similar to tissues and organs (Hans Clevers et al., Cell, 11, 172(1-2):373-386, 2018).

[0005] However, both technologies have certain limitations. Cell reprogramming technology is a technology that co-cultures the patient's autologous primary epithelial cells with mouse-derived feeder cells. When the patient's primary cells are tested for drug sensitivity, the presence of these mouse-derived cells will interfere with the drug sensitivity test results of the patient's autologous primary cells; but if the mouse-derived feeder cells are removed, the patient's autologous primary cells will be separated from the reprogramming environment, and the cell proliferation rate and intracellular signaling pathways will change significantly (Liu et al., Am. J. Pathol., 183 (6): 1862-1870, 2013; Liu et al., Cell Death Dis., 9 (7): 750, 2018), thereby greatly affecting the response of the patient's autologous primary cells to drugs. Organoid technology is a technology that embeds the patient's autologous primary epithelial cells in an extracellular matrix for in vitro three-dimensional culture. This technology does not require feeder cells, so there is no interference problem with mouse-derived feeder cells. However, the culture medium of organoid technology requires the addition of multiple specific growth factors (such as Wnt protein and R-spondin family protein), which is expensive and not suitable for large-scale clinical application. In addition, the cells of organoids need to be embedded in extracellular matrix gel throughout the culture process. The cell inoculation, passage and plating steps of drug sensitivity testing are cumbersome and time-consuming compared to 2D culture operations. Moreover, the size of the organoids formed by this technology is difficult to control, and some organoids are prone to grow too large and cause internal necrosis. Therefore, compared with 2D culture technology, organoid technology is less operable and applicable, requires professional technicians to operate, and is not suitable for large-scale and widespread application in clinical in vitro drug sensitivity testing (Nick Barker, Nat. Cell Biol., 18(3): 246-54, 2016).

[0006] Given the limitations of these technologies, clinical development of a primary gastric cancer cell culture technology is needed, characterized by a short culture cycle, manageable costs, convenient operation, and immunity to interference from exogenous cells. When this technology is applied to construct primary gastric cancer tumor cell models, the cultured gastric cancer tumor cells can represent the biological characteristics of the gastric cancer patient. By evaluating the sensitivity of anti-tumor drugs in vitro in cell models derived from different individual cancer patients, the response rate of clinical anti-tumor drugs can be improved, reducing the pain caused to patients and the waste of medical resources by inappropriate drugs. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a culture medium and a culture method for rapidly expanding primary gastric cancer cells in vitro.

[0008] One aspect of the present invention is to provide a culture medium for primary gastric cancer cells, the culture medium comprising an MST1 / 2 kinase inhibitor; at least one ROCK kinase inhibitor selected from Y27632, fasudil, and H-1152; at least one additive selected from B27 additive and N2 additive; basic fibroblast growth factor; CHIR99021; epidermal growth factor; ITS cell culture additive; SB202190; dexamethasone; N-acetyl-L-cysteine; gastrin; A8301; oncostatin M; and cholera toxin. The MST1 / 2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.

[0009] in,

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

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

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

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

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

[0015]

[0016] in,

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

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

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

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

[0021]

[0022]

[0023]

[0024]

[0025]

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

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

[0028] (1) The concentration of the MST1 / 2 kinase inhibitor is 1.25 to 20 μM, more preferably 2.5 to 10 μM;

[0029] (2) The volume ratio of the B27 or N2 cell culture additive to the culture medium is 1:25 to 1:400, more preferably 1:50 to 1:200;

[0030] (3) The concentration of basic fibroblast growth factor is 2.5 to 40 ng / mL, more preferably 5 to 20 ng / mL;

[0031] (4) The volume ratio of the ITS cell culture additive to the culture medium is 1:25 to 1:400, more preferably 1:50 to 1:200;

[0032] (5) The concentration of the ROCK kinase inhibitor is 2.5 to 40 μM, more preferably 5 to 20 μM;

[0033] (6) The concentration of dexamethasone is 25 to 400 nM, more preferably 50 to 200 nM;

[0034] (7) The concentration of CHIR99021 is 1.25 to 20 μM, more preferably 2.5 to 10 μM;

[0035] (8) The concentration of the epidermal growth factor is 2.5 to 40 ng / mL, more preferably 2.5 to 20 ng / mL;

[0036] (9) The concentration of the cholera toxin is 1.25 to 20 ng / mL, more preferably 2.5 to 10 ng / mL;

[0037] (10) The concentration of gastrin is 1.25 to 20 nM, more preferably 5 to 20 nM;

[0038] (11) The concentration of SB202190 is 50 to 800 nM, more preferably 50 to 200 nM;

[0039] (12) The concentration of N-acetyl-L-cysteine ​​is 0.25 to 4 mM, more preferably 1 to 4 mM;

[0040] (13) The concentration of oncostatin M is 2.5 to 40 ng / mL, more preferably 2.5 to 10 ng / mL;

[0041] (14) The concentration of A8301 is 1.25 to 20 nM, more preferably 2.5 to 20 nM.

[0042] 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.

[0043] 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.

[0044] The present invention also provides a method for culturing primary gastric cancer cells. In the method for culturing primary gastric cancer cells of the present invention, the primary gastric cancer cells are cultured using the primary gastric cancer cell culture medium of the present invention.

[0045] The method for culturing primary gastric cancer cells of the present invention comprises the following steps.

[0046] (1) Prepare the primary cell culture medium of the present invention according to the above formula.

[0047] (2) Coat the culture vessel with extracellular matrix gel dilution solution.

[0048] Specifically, the extracellular matrix glue uses a low growth factor type extracellular matrix glue, for example, commercially available Matrigel (purchased from BD Biosciences) or BME (purchased from Trevigen). More specifically, the extracellular matrix glue is diluted with serum-free culture medium, and the culture medium can be DMEM / F12 (purchased from Corning). The dilution ratio of the extracellular matrix glue is 1:50-1:400, preferably 1:100-1:200. The coating method is to add the diluted extracellular matrix glue to the culture vessel so that it completely covers the bottom of the culture vessel, and let it stand for more than 30 minutes, preferably at 37°C. The coating time is preferably 30 to 60 minutes. After the coating is completed, the excess extracellular matrix glue dilution is discarded and the culture vessel is ready for use.

[0049] (3) Samples were isolated from solid gastric cancer tissues to obtain primary gastric cancer cells.

[0050] Primary gastric cancer cells can be derived from gastric cancer surgical specimens and biopsy endoscopic specimens. Gastric cancer tissue samples can be derived from surgically removed cancer tissue samples of gastric cancer patients who have explained and obtained consent, and endoscopic specimens can be collected from gastric lesions under endoscopic guidance. The above tissue samples are collected within half an hour after the patient's surgical resection or biopsy. More specifically, under a sterile environment, tissue samples are cut from non-necrotic areas with a volume of 5 mm 3 It was transported on ice to the laboratory.

[0051] In the biosafety cabinet, transfer the tissue sample to a cell culture dish and wash away the blood cells on the surface of the tissue sample. Transfer the rinsed tissue sample to another new culture dish and use a sterile surgical blade and surgical forceps to cut the tissue sample into pieces smaller than 3mm. 3 of tissue fragments.

[0052] Transfer the tissue sample fragments into a centrifuge tube and centrifuge at 1000-3000 rpm for 3-5 minutes using a tabletop centrifuge (Sigma 3-18K). Discard the supernatant and add basal culture medium (e.g., DMEM / F12 culture medium containing 100 μg / mL) at a ratio of 1:3. Primocin) and tissue digestion solution (the tissue digestion solution is prepared by dissolving 1-2 mg / mL collagenase II, 1-2 mg / mL collagenase IV, 50-100 U / mL deoxyribonuclease, 0.5-1 mg / mL hyaluronidase, 1-5 mM calcium chloride, and 5-10 mg / mL bovine serum albumin in 1640 culture medium). Label the sample number, seal with parafilm, and digest in a thermostatic shaker (Zhichu Instrument ZQLY-180N) at 37°C and 200-300 rpm. Observe the digestion completion every half an hour or one hour. If no obvious tissue fragments are observed, terminate the digestion. Otherwise, continue digestion until complete, which lasts for 4-8 hours. After digestion is complete, undigested tissue clumps are filtered out using a cell strainer (e.g., with a pore size of 70-100 μm). The tissue clumps on the strainer are rinsed with basal culture medium, and the remaining cells are flushed into a centrifuge tube and centrifuged at 1000-3000 rpm for 3-5 minutes using a desktop centrifuge. The supernatant is discarded, and the remaining cell mass is observed to see if it contains blood cells. If so, a blood cell lysis buffer (available from Sigma) is added, mixed, and lysed at 4°C for 10-20 minutes, shaking once every 5 minutes to mix. After lysis is complete, the cell is removed and centrifuged at 1000-3000 rpm for 3-5 minutes.

[0053] (4) The primary gastric cancer cells isolated in step (3) are inoculated into the coated culture vessel and cultured using the primary cell culture medium in step (1).

[0054] More specifically, 2 × 10 4 ~8×10 4 pieces / cm 2 (For example 4×10 4 pieces / cm 2 ) and inoculate primary gastric cancer cells at a density of 100 μg / mL. An appropriate amount of primary gastric cancer cell culture medium is added and cultured in a cell culture incubator at, for example, 37°C and 5% CO2 for 8-16 days. During this period, the culture medium is replaced with fresh primary cell culture medium every 4 days. When the primary gastric cancer cells grow to a cell density that occupies approximately 80% to 90% of the bottom area of ​​the multi-well plate, digestion and passage are performed.

[0055] This inoculation step does not require the use of feeder cells, and compared to cell conditional reprogramming technology, it eliminates the steps of culturing and irradiating feeder cells. Compared to organoid technology, this step also does not require mixing primary cells and matrix gel on ice to form gel droplets, and then waiting for the gel droplets to solidify before adding culture medium. Pre-coated culture vessels can be directly used for primary cell inoculation. In addition, only a small amount of diluted extracellular matrix gel is required to coat the culture vessels. Compared to organoid technology, this saves the use of expensive extracellular matrix gel and simplifies the operation steps.

[0056] Optionally, after culturing the inoculated primary gastric cancer cells for 8 to 16 days, when the cell clones formed in the culture container have reached 80% of the bottom area, the supernatant is discarded, 0.5 to 2 mL of 0.05% trypsin (purchased from Thermo Fisher) is added for cell digestion, and the cells are incubated at room temperature for 5 to 20 minutes; then, the digested cells are resuspended in 1 to 4 mL of DMEM / F12 culture medium containing, for example, 5% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and the cells are centrifuged at 1000 to 3000 rpm for 3 to 5 minutes, and the digested single cells are resuspended in the primary cell culture medium of the present invention, and the resulting cell suspension is placed in a T25 cell culture flask coated with extracellular matrix glue for further expansion and culture. The coating operation of the T25 cell culture flask is the same as step (2).

[0057] The expanded primary gastric cancer cells grow in 2D, avoiding the uneven size of organoids and internal necrosis of overgrown organoids caused by organoid technology expansion.

[0058] The beneficial effects of the present invention include:

[0059] (1) Improve the success rate of primary gastric cancer cell culture to over 90%;

[0060] (2) Ensure that the primary gastric cancer cells cultured in vitro can maintain the patient's pathological characteristics;

[0061] (3) The cultured primary gastric cancer epithelial cells are not interfered with by fibroblasts, and purified gastric cancer epithelial cells can be obtained;

[0062] (4) The culture medium does not contain serum and is therefore not affected by the quality and quantity of serum from different batches;

[0063] (5) High amplification efficiency, capable of rapidly culturing primary gastric cancer cells, and the amplified primary gastric cancer cells can be continuously passaged;

[0064] (6) The cell culture process does not require ice operation or dissociation of the matrix gel, and the cell digestion and cell culture can be completed within 10-15 minutes;

[0065] (7) The culture cost is controllable, and the culture medium does not need to be added with expensive factors such as Wnt agonists, R-spondin family proteins, Noggin proteins, and BMP inhibitors;

[0066] (8) The technology described herein can culture a large number of primary gastric cancer cells, which are suitable for high-throughput screening of candidate compounds and providing high-throughput in vitro drug sensitivity functional testing for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figures 1A-1N This is a graph showing the effects of different concentrations of factors added to the gastric cancer primary cell culture medium of the present invention on the proliferation of gastric cancer primary cells. DETAILED DESCRIPTION

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

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

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

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

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

[0073]

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

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

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

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

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

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

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

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Example 1 Effects of various added factors in the culture medium of primary gastric cancer cells on the proliferation of primary gastric cancer cells

[0087] (1) Preparation of culture medium for primary gastric cancer cells

[0088] 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, which are commonly used in the art. In this embodiment, the basal medium is formulated as follows: DMEM / F12 medium (purchased from Corning) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available at a concentration of 50 mg / ml).

[0089] Different types of additives (see Table 1) were added to the basal culture medium to prepare gastric cancer primary cell culture medium containing different additives.

[0090] (2) Isolation and processing of primary gastric cancer cells

[0091] 1. Sample selection

[0092] Gastric cancer solid tumor tissue samples (intraoperative) were obtained from patients by professional medical staff of professional medical institutions, and all patients signed informed consent. 3 Commercial tissue preservation solution (manufacturer: Miltenyi Biotec) was used for storage and transportation.

[0093] 2. Material Preparation

[0094] 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.

[0095] Tissue digestion solution formula: 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 bovine serum albumin (BSA) (10 mg / mL).

[0096] The above-mentioned collagenase II, collagenase IV, DNA enzyme, and hyaluronidase were all purchased from Sigma; calcium chloride was purchased from Sangon Biotech (Shanghai) Co., Ltd.; and BSA was purchased from Biofroxx.

[0097] 3. Sample separation

[0098] 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 to cut the tissue into 1*1*1mm blocks. 3 size;

[0099] 3.2 Pipette the cut tissue into a 15 mL centrifuge tube, add 5 mL of basal culture medium, mix well, and centrifuge at 1500 rpm for 4 minutes;

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 3.6 Discard the supernatant and add 2 mL of basal culture medium to resuspend the cells for later use.

[0104] 4. Cell Counting and Processing

[0105] 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).

[0106] 4.2 Viable cell counting: Take 12 μL of the resuspended cell suspension and mix thoroughly with 12 μL of trypan blue dye (manufacturer: Sangon Biotech (Shanghai) Co., Ltd.). Then, take 20 μL and add it to a cell counting plate (manufacturer: Countstar, specification: 50 plates / box). Using a cell counter (Countstar, IC1000), calculate the percentage of viable large cells (cell size >10 μm) = number of viable cells / total number of cells*100%.

[0107] (3) Culture of primary gastric cancer cells

[0108] Extracellular matrix glue Prepare an extracellular matrix diluent (BD Biosciences) using serum-free DMEM / F12 medium at a 1:100 ratio. Add 500 μl / well of the extracellular matrix diluent to a 48-well culture plate, completely covering the bottom of the wells. Incubate in a 37°C incubator for 1 hour. After 1 hour, remove the extracellular matrix diluent to obtain a Matrigel-coated culture plate.

[0109] The primary gastric cancer cells obtained in the above steps were resuspended in pre-cooled DMEM / F12 and counted. Different culture media (Table 1) were added to a 48-well plate coated with extracellular matrix (Matrigel) at a volume of 500 μl / well. The counted primary gastric cancer cells were plated at 2×10 4 pieces / cm 2 Cells were seeded at a density of 100 μg / mL in a Matrigel-coated 48-well culture plate. After surface disinfection, the plate was placed in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific). Equal numbers of freshly isolated gastric cancer cells (code GC-001) were cultured under different culture medium formulations. The culture medium was replaced every four days after initiation of the culture. After 12 days of culture, cell counts were performed to compare the effects of various factors on the proliferation of primary gastric cancer cells. As a control, a basal culture medium without any additives was used. The experimental results are shown in Table 1.

[0110] Table 1 Additives in culture medium and their effects on promoting organoid proliferation

[0111]

[0112]

[0113] Among them, "+" indicates that compared with the basic culture medium, the culture medium added with the additive has a proliferation-promoting effect on at least two of the primary gastric cancer cells isolated from gastric cancer tissue; "-" indicates that the culture medium added with the additive shows an inhibitory effect on the proliferation of at least one of the primary gastric cancer cells isolated from gastric cancer tissue; "○" indicates that the culture medium added with the additive has no obvious effect on the proliferation of at least two of the primary gastric cancer cells isolated from gastric cancer tissue.

[0114] Based on the above results, it is planned to select MST1 / 2 kinase inhibitor compound 1, ROCK kinase inhibitor Y27632, at least one of B27 additive and N2 additive, basic fibroblast growth factor, CHIR99021, epidermal growth factor, ITS cell culture additive, SB202190, dexamethasone, N-acetyl-L-cysteine, gastrin, A8301, oncostatin M, cholera toxin and other factors for further culture experiments.

[0115] Example 2 Effects of different concentrations of culture medium additives on the proliferation of primary gastric cancer cells

[0116] Primary gastric cancer cells were obtained from intraoperative tissue samples (numbered GC-002 and GC-003) according to the method of Example 1 (2), and the primary cells were cultured using the culture medium formula in Table 2 below.

[0117] Table 2 Culture medium formula (concentration is final concentration)

[0118]

[0119] When using the medium of Formula 1, 200 μL of compound 1 prepared in Formula 1 was added to each well of a 48-well plate seeded with primary cells, with final concentrations of 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, respectively. A control well (BC) was set up using the medium of Formula 1. The final concentrations of other additives in this series of culture media were the same as those in GC-2.2 medium. The following experiments for Formulas 1-14 were conducted in the same manner and are not further described.

[0120] When using the culture medium of Formula 2, 200 μL of prepared Y27632 was added to each well of a 48-well plate seeded with primary cells based on Formula 2. The final concentrations of Y27632 were 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively; and control wells (BC) were set using the culture medium of Formula 2.

[0121] When using the culture medium of Formula 3, 200 μL of prepared B27 was added to each well of a 48-well plate seeded with primary cells based on Formula 3. The final concentrations of B27 were 1:25, 1:50, 1:100, 1:200, and 1:400, respectively; and control wells (BC) were set up using the culture medium of Formula 3.

[0122] When using the culture medium of Formula 4, 200 μL of the prepared basic fibroblast growth factor was added to each well of a 48-well plate seeded with primary cells based on Formula 4. The final concentrations of the basic fibroblast growth factor were 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively. Control wells (BC) were set using the culture medium of Formula 4.

[0123] When using the culture medium of Formula 5, 200 μL of prepared CHIR99021 was added to each well of a 48-well plate seeded with primary cells based on Formula 5. The final concentrations of CHIR99021 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 5.

[0124] When using the culture medium of Formula 6, 200 μL of the prepared epidermal growth factor was added to each well of a 48-well plate seeded with primary cells based on Formula 6. The final concentrations of the epidermal growth factor were 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 6.

[0125] When using the medium of Formula 7, 200 μL of the prepared ITS cell culture supplement was added to each well of a 48-well plate seeded with primary cells based on Formula 7. The final concentrations of the ITS cell culture supplement were 1:25, 1:50, 1:100, 1:200, and 1:400, respectively. Control wells (BC) were set up using the medium of Formula 7.

[0126] When using the culture medium of Formula 8, 200 μL of prepared SB202190 was added to each well of a 48-well plate seeded with primary cells based on Formula 8. The final concentrations of SB202190 were 50 nM, 100 nM, 200 nM, 400 nM, and 800 nM, respectively. Control wells (BC) were set up using the culture medium of Formula 8.

[0127] When using the culture medium of Formula 9, 200 μL of prepared dexamethasone was added to each well of a 48-well plate seeded with primary cells based on Formula 9. The final concentrations of dexamethasone were 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM, respectively. Control wells (BC) were set up using the culture medium of Formula 9.

[0128] When using the culture medium of Formula 10, 200 μL of the prepared cholera toxin was added to each well of a 48-well plate seeded with primary cells based on Formula 10. The final concentrations of cholera toxin were 1.25 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL, respectively; and control wells (BC) were set up using the culture medium of Formula 10.

[0129] When using the culture medium of Formula 11, 200 μL of prepared N-acetyl-L-cysteine ​​was added to each well of a 48-well plate seeded with primary cells based on Formula 11, with final concentrations of N-acetyl-L-cysteine ​​of 0.25 mM, 0.5 mM, 1 mM, 2 mM, and 4 mM, respectively; and control wells (BC) were set using the culture medium of Formula 11.

[0130] When using the culture medium of Formula 12, 200 μL of prepared gastrin was added to each well of a 48-well plate seeded with primary cells based on Formula 12. The final concentrations of gastrin were 1.25 nM, 2.5 nM, 5 nM, 10 nM, and 20 nM, respectively. Control wells (BC) were set using the culture medium of Formula 12.

[0131] When using the culture medium of Formula 13, 200 μL of the prepared oncostatin M was added to each well of a 48-well plate seeded with primary cells based on Formula 13. The final concentrations of oncostatin M were 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively; and control wells (BC) were set using the culture medium of Formula 13.

[0132] When using the culture medium of Formula 14, 200 μL of prepared A8301 was added to each well of a 48-well plate seeded with primary cells based on Formula 14. The final concentrations of A8301 were 1.25 nM, 2.5 nM, 5 nM, 10 nM, and 20 nM, respectively. Control wells (BC) were set up using the culture medium of Formula 14.

[0133] 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 cell number of the control well (BC). The data collected from the two samples were summarized and shown in Figures 1A to 1N . Figures 1A to 1NThe 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.

[0134] according to Figures 1A to 1N The results show that the content of MST1 / 2 kinase inhibitor compound 1 is preferably 1.25-20 μM, more preferably 2.5-10 μM; the concentration of Y27632 is preferably 2.5-40 μM, more preferably 5-20 μM; the volume concentration of B27 is preferably 1:25-1:400, more preferably 1:50-1:200; the concentration of basic fibroblast factor is preferably 2.5-40 ng / mL, more preferably 5-20 ng / mL; the concentration of CHIR99021 is preferably 1.25-20 μM, more preferably 2.5-10 μM; the concentration of epidermal growth factor is preferably 2.5-40 ng / mL, more preferably 2.5-20 ng / mL; the volume concentration of ITS cell culture additive relative to the culture medium is preferably 1:25 The concentration of SB202190 is preferably 50-800 nM, more preferably 50-200 nM; the concentration of dexamethasone is preferably 25-400 nM, more preferably 50-200 nM; the concentration of cholera toxin is preferably 1.25-20 ng / mL, more preferably 2.5-10 ng / mL; the concentration of N-acetyl-L-cysteine ​​is preferably 0.25-4 mM, more preferably 1-4 mM; the concentration of gastrin is preferably 1.25-20 nM, more preferably 5-20 nM; the concentration of oncostatin M is preferably 2.5-40 ng / mL, more preferably 2.5-10 ng / mL; the concentration of A8301 is preferably 1.25-20 nM, more preferably 2.5-20 nM.

[0135] Industrial Applicability

[0136] The present invention provides a culture medium and a culture method for culturing primary gastric cancer cells. The cultured primary gastric cancer cells can be used for evaluating and screening the efficacy of drugs. Therefore, the present invention is suitable for industrial application.

[0137] 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 gastric cancer cells, characterized in that The invention is prepared from the following components: 1.25-20 μM MST1 / 2 kinase inhibitor; 2.5-40 μM Y27632; B27 supplement at a volume ratio of 1:25-1:400 relative to the culture medium; 2.5-40 ng / mL basic fibroblast growth factor; 1.25-20 μM CHIR99021; 2.5-40 ng / mL epidermal growth factor; ITS cell culture supplement at a volume ratio of 1:25-1:400 relative to the culture medium; 50-800 nM SB202190; and 25-400 nM dexamethasone. 0.25-4 mM N-acetyl-L-cysteine; 1.25-20 nM gastrin; 1.25~20nM A8301; 2.5~40ng / mL oncostatin M; 1.25~20ng / mL cholera toxin, 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, 2. The culture medium according to claim 1, wherein The content of each component in the culture medium is: The concentration of the MST1 / 2 kinase inhibitor is 2.5 to 10 μM; The volume ratio of the B27 additive to the culture medium is 1:50 to 1:200; The concentration of the basic fibroblast growth factor is 5 to 20 ng / mL; The volume ratio of the ITS cell culture additive to the culture medium is 1:50 to 1:200; The concentration of Y27632 is 5 to 20 μM; The concentration of dexamethasone is 50-200 nM; The concentration of CHIR99021 is 2.5-10 μM; The concentration of the epidermal growth factor is 2.5 to 20 ng / mL; The concentration of the cholera toxin is 2.5 to 10 ng / mL; The concentration of gastrin is 5 to 20 nM; The concentration of SB202190 is 50-200 nM; The concentration of the N-acetyl-L-cysteine ​​is 1 to 4 mM; The concentration of oncostatin M is 2.5 to 10 ng / mL; The concentration of A8301 is 2.5-20 nM.

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

4. A method for culturing primary gastric cancer cells, characterized in that The following steps are involved: (1) preparing a culture medium according to any one of claims 1 to 3; (2) coating the culture vessel with a dilution of an extracellular matrix glue, wherein the extracellular matrix glue is selected from at least one of Matrigel and BME; (3) Primary gastric cancer epithelial cells isolated from gastric cancer tissue were inoculated into a culture vessel coated with extracellular matrix gel and cultured using the culture medium described in step (1).

Images