CN115975940A - Culture medium and culture method of gastric cancer primary cells - Google Patents

Abstract

The present invention provides a medium for culturing primary gastric cancer cells, which contains MST1/2 kinase inhibitors, ROCK kinase inhibitors, at least one of B27 additives and N2 additives, basic fibroblast growth factor, CHIR99021, Epidermal Growth Factor, ITS Cell Culture Supplement, 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. In the culture method, primary cells of gastric cancer are cultured on a culture vessel coated with extracellular matrigel by using the above-mentioned culture medium, so that the primary cells proliferate rapidly.

Description

Culture medium and culture method for primary gastric cancer cells

Technical Field

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

Gastric cancer is a malignant tumor originating from the gastric mucosal epithelium. It ranks first in incidence among various malignant tumors in my country. It is more common in people over 50 years old, with a male-female incidence ratio of 2:1. Gastric cancer tends to be younger due to changes in dietary structure, increased work pressure, and Helicobacter pylori infection. Gastric cancer can occur in any part of the stomach, with more than half occurring in the gastric antrum, and the greater and lesser curvatures of the stomach and the anterior and posterior walls can be affected. The vast majority of gastric cancers are adenocarcinomas, which have no obvious symptoms in the early stage, or have non-specific symptoms such as upper abdominal discomfort and belching, which are often similar to the symptoms of chronic gastric diseases such as gastritis and gastric ulcers and are easily overlooked. Therefore, the early diagnosis rate of gastric cancer in my country is still low. At present, the national diagnosis rate of early gastric cancer is still less than 20%, and the 5-year survival rate of gastric cancer patients is only 27.4%.

In recent years, with the rise and development of molecular biology, tumor drug treatment has shown a diversified trend. Among them, molecular targeted drugs have become a hot topic in the clinical treatment of gastric cancer due to their strong targeting and high safety. However, in clinical practice, it is particularly important to choose a suitable treatment for the patient for many treatment options. Although genetic testing is used as an indicator, some patients do not have gene mutations, or even if some patients have certain mutations, there are multiple targeted drugs for the mutation. At this time, 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 of in vitro prediction of efficacy and guidance of 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.

At present, there are two main technologies for culturing primary cells that have developed relatively maturely. One is to use irradiated feeder cells and ROCK kinase inhibitor Y27632 to promote the growth of primary epithelial cells, namely cell conditional reprogramming technology (Liu et al., Am. J. Pathol., 180: 599-607, 2012). The other technology is to culture adult stem cells in 3D in vitro to obtain organoid technology similar to tissues and organs (Hans Clevers et al., Cell, 11, 172 (1-2): 373-386, 2018).

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 out of 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 from mouse-derived feeder cells. However, the culture medium of organoid technology needs to add a variety of 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 during the entire culture process. The cell inoculation, subculture and drug sensitivity test plating steps are cumbersome and time-consuming compared with 2D culture operations. 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 not as operable and applicable, and requires professional technicians to operate, which is not suitable for large-scale and widespread application in clinical in vitro drug sensitivity detection (Nick Barker, Nat. Cell Biol., 18 (3): 246-54, 2016).

In view of the limitations of the above technologies, it is clinically necessary to develop a primary gastric cancer cell culture technology with a short culture cycle, controllable cost, convenient operation, and no interference from exogenous cells. When this technology is applied to the construction of a primary gastric cancer tumor cell model, the cultured gastric cancer tumor cells can represent the biological characteristics of the gastric cancer patient himself. By evaluating the sensitivity of anti-tumor drugs in vitro on cell models derived from different cancer patients, the response rate of clinical anti-tumor drugs can be improved, reducing the pain caused to patients by inappropriate drugs and the waste of medical resources.

Summary of the invention

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

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; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152; at least one of B27 additives and N2 additives; basic fibroblast growth factor; CHIR99021; epidermal cell 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,

in,

R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl (e.g., phenyl and naphthyl, etc.), aryl C1-C6 alkyl (e.g., benzyl, etc.) and heteroaryl (e.g., thienyl, etc.) optionally substituted by 1-2 independently R ₆ ;

_R2 and _R3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

_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.);

R ₆ 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).

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

in,

R ₁ is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R ₆ , thienyl optionally substituted by 1-2 independently R ₆ , and benzyl optionally substituted by 1-2 independently R ₆ , and R ₁ is more preferably phenyl optionally substituted by 1-2 independently R ₆ ;

R ₅ is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, and R ₅ is more preferably hydrogen;

R ₆ is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R ₆ is more preferably fluorine, methyl or trifluoromethyl.

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

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

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

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

(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;

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

(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;

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

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

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

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

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

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

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

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

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

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

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.

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

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.

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

(1) The primary cell culture medium of the present invention is prepared according to the above formula.

(2) Coat the culture vessel with diluted extracellular matrix gel.

Specifically, the extracellular matrix glue uses a low growth factor type extracellular matrix glue, for example, commercially available Matrigel (purchased from BD Biotechnology) or BME (purchased from Trevigen) can be used. More specifically, the extracellular matrix glue is diluted with a 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.

(3) Isolate samples from gastric cancer solid tumor tissues to obtain gastric cancer primary cells.

Primary gastric cancer cells can be derived from gastric cancer surgical samples and biopsy endoscopic samples, for example. Gastric cancer tissue samples are derived from surgically removed cancer tissue samples of gastric cancer patients who have been explained and consented, and endoscopic samples are 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, in a sterile environment, a tissue sample of a non-necrotic part is cut, the volume of which is more than 5 mm ³ , and it is transported to the laboratory on ice.

In the biosafety cabinet, transfer the tissue sample to a cell culture dish and wash off 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 tissue fragments with a volume of less than 3 ^mm3 .

Transfer the tissue sample fragments to 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 preparation method of tissue digestion solution is: 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, 5-10 mg/mL bovine serum albumin are dissolved in 1640 culture medium), mark the sample number, seal with sealing film, and digest in a constant temperature shaker (Zhichu Instrument ZQLY-180N) at 37°C and 200-300 rpm. Observe whether the digestion is complete every half an hour or one hour; if no obvious tissue blocks are seen, the digestion can be terminated, otherwise, the digestion is continued until the digestion is sufficient, and the digestion time ranges from 4 to 8 hours. After digestion is completed, the undigested tissue mass is filtered out by a cell filter (cell sieve aperture is, for example, 70-100 μm), the tissue mass on the filter is rinsed with basal culture medium, the residual 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 whether it contains blood cells. If there are blood cells, blood cell lysis solution (purchased from Sigma) is added, mixed, lysed at 4°C for 10-20 minutes, shaken and mixed once for 5 minutes, taken out after lysis is completed, and centrifuged at 1000-3000 rpm for 3-5 minutes.

(4) Inoculating the primary gastric cancer cells isolated in step (3) into the coated culture vessel, and culturing them using the primary cell culture medium in step (1).

More specifically, primary gastric cancer tumor cells are inoculated at a density of 2×10 ⁴ to 8×10 ⁴ cells/cm ² (e.g., 4×10 ⁴ cells/cm ² ) in one well of a multi-well plate, and an appropriate amount of primary gastric cancer cell culture medium is added. The cells are cultured in a cell culture incubator at, for example, 37°C and 5% CO ₂ for 8-16 days, during which time 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 about 80% to 90% of the bottom area of the multi-well plate, digestion and passage are performed.

This inoculation step does not require the use of feeder cells. Compared with cell conditional reprogramming technology, it eliminates the steps of culturing and irradiating feeder cells. Compared with organoid technology, this step 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. The pre-coated culture vessels can be directly used for primary cell inoculation. In addition, the coated culture vessels only require a small amount of diluted extracellular matrix gel, which saves the use of expensive extracellular matrix gel compared to organoid technology and simplifies the operation steps.

Optionally, after the inoculated primary gastric cancer cells are cultured for 8 to 16 days, when the cell clones formed in the culture container converge to 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 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, 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 obtained cell suspension is placed in a T25 cell culture flask coated with extracellular matrix glue to continue to expand the culture. The coating operation of the T25 cell culture flask is the same as step (2).

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.

The beneficial effects of the present invention include:

(1) Improve the success rate of primary gastric cancer cell culture to more than 90%;

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

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

(4) The culture medium does not contain serum, so it is not affected by the quality and quantity of serum from different batches;

(5) High amplification efficiency, which can rapidly culture primary gastric cancer cells, and the amplified primary gastric cancer cells can be continuously passaged;

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

(7) The culture cost is controllable, and the culture medium does not need to add expensive Wnt agonists, R-spondin family proteins, Noggin proteins, BMP inhibitors and other factors;

(8) The technology described above 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

1A-1N are graphs showing the effects of different concentrations of factors added to the culture medium for primary gastric cancer cells of the present invention on the proliferation of primary gastric cancer cells.

DETAILED DESCRIPTION

In order to better understand the present invention, the present invention is further described below in conjunction with the embodiments and drawings. The following embodiments are only for illustrating the present invention but not for limiting it.

[Preparation Example of MST1/2 Kinase Inhibitor]

In the present specification, an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signal transduction. Generally speaking, an MST1/2 kinase inhibitor, for example, binds to MST1/2 kinase and reduces its activity. Since MST1 and MST2 have similar structures, an MST1/2 kinase inhibitor may also be, for example, a compound that binds to MST1 or MST2 and reduces its activity.

1. Preparation of MST1/2 kinase inhibitor compound 1

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

2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): Add 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) to a round-bottom flask, then add methanol (30 ml), and then add dichlorothionyl (1.2 ml) dropwise under ice bath. The reaction system is reacted at 85°C overnight. After the reaction is completed, the solvent is evaporated under reduced pressure, and the obtained white solid is directly used in the next step.

2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetic acid methyl ester (A3): Add 2-amino-2-(2,6-difluorophenyl)acetic acid methyl ester (2 g) to a round-bottom flask, then add acetone (30 ml) and potassium carbonate (2.2 g), then cool the system to -10°C with an ice-salt bath, and then slowly add 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction system is stirred at room temperature overnight. After the reaction is completed, filter, remove the solvent from the filtrate under reduced pressure, and purify the residue by pressurized silica gel column chromatography to obtain compound A3. LC/MS: M+H 359.0.

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

2-Chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (A5): Add 2-chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (2 g) and N,N-dimethylacetamide (10 ml) to a round-bottom flask, cool to -35°C, add iodomethane (0.9 ml), then add sodium hydride (615 mg), and continue stirring the reaction system for two hours. After the reaction is completed, water is added to quench, and ethyl acetate is extracted. The organic phase is washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. The organic phase is filtered and evaporated to dryness under reduced pressure to obtain a crude product. The crude product is washed with ether to obtain compound A5. LC/MS: M+H325.0.

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-butyl alcohol (5 ml) were added to a round-bottom flask. The reaction system was stirred at 120°C overnight. After the reaction was completed, the mixture was filtered and washed with methanol and ether to obtain compound 1. LC/MS: M+H 461.1.

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

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.

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

(1) Preparation of gastric cancer primary cell culture medium

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 culture medium (purchased from Corning) + 100 μg/mL Primocin (purchased from InvivoGen, 0.2% (v/v), commercially available product concentration 50 mg/ml).

Different types of additives (see Table 1) were added to the basic culture medium to prepare gastric cancer primary cell culture medium containing different added ingredients.

(2) Isolation and treatment of primary gastric cancer cells

1. Sample selection

Gastric cancer solid tumor tissue samples (intraoperatively) were obtained from patients by professional medical staff of professional medical institutions, and all patients signed informed consent. The intraoperative samples were 0.25 cm ³ and were stored and transported in commercial tissue preservation solution (manufacturer: Miltenyi Biotec).

2. Material Preparation

After surface disinfection, place the 15mL sterile centrifuge tube, pipette, 10mL pipette, sterile pipette tip, etc. in a clean bench and irradiate with UV light for 30 minutes. Take out the basal culture medium from the 4℃ refrigerator 30 minutes in advance, and take out the tissue digestion solution from the -20℃ refrigerator 30 minutes in advance.

Tissue digestion fluid 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).

The collagenase II, collagenase IV, DNA enzyme and hyaluronidase mentioned above were all purchased from Sigma; calcium chloride was purchased from Shanghai Biotech Co., Ltd.; and BSA was purchased from Biofroxx.

3. Sample separation

3.1 Take tissue samples from the clean bench and place them in a culture dish. Remove the tissue with blood, rinse it twice with basal culture medium, transfer the tissue to another culture dish, and use a sterile scalpel to perform mechanical separation to divide the tissue blocks into 1*1* ^1mm3 size;

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

3.3 Discard the supernatant, add basal culture medium and tissue digestion solution in a ratio of 1:3 (Note: the amount of tissue digestion solution added is about 10 mL of tissue digestion solution for 1 g of tumor tissue), mark the sample name and number, seal with sealing film, and digest at 37°C in a 300 rpm shaker (Zhichu Instrument ZQLY-180N). During this period, observe whether the digestion is complete every 30 minutes. The judgment basis is the absence of visible particles;

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

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, lyse at 4°C for 20 minutes, invert once during mixing, and centrifuge at 1500 rpm for 4 minutes at 25°C;

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

4. Cell Counting and Processing

4.1 Observation under microscope: Pipette a small amount of resuspended cells and spread them on a culture dish, and observe the density and morphology of cancer cells under a microscope (CNOPTEC, BDS400);

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

(3) Culture of primary gastric cancer cells

(BD生物科技公司制)使用无血清DMEM/F12培养基按1:100比例稀释，配制成细胞外基质稀释液，在48孔培养板内加入500μl/孔的细胞外基质稀释液使其完全覆盖培养板孔的底部。在37℃培养箱内静置1小时。1小时后，移除细胞外基质稀释液，得到包被有Matrigel的培养板。Extracellular matrix glue

(BD Biotech) Serum-free DMEM/F12 medium was diluted at a ratio of 1:100 to prepare an extracellular matrix diluent, and 500 μl/well of the extracellular matrix diluent was added to a 48-well culture plate to completely cover the bottom of the culture plate wells. The plate was left to stand in a 37°C incubator for 1 hour. After 1 hour, the extracellular matrix diluent was removed to obtain a culture plate coated with Matrigel.

The primary gastric cancer cells obtained in the above steps were resuspended and counted with precooled DMEM/F12. The culture medium with different components (Table 1) was added to a 48-well plate coated with extracellular matrix gel (Matrigel) at a volume of 500 μl/well. The counted primary gastric cancer cells were inoculated in a 48-well culture plate coated with Matrigel at a cell density of 2×10 ⁴ /cm ² , and the surface was sterilized and placed in a 37°C, 5% CO ₂ incubator (purchased from Thermo Fisher), and the same number of freshly isolated gastric cancer tumor cells (No. GC-001) were cultured under different culture medium formulations. The culture medium was replaced every 4 days after the start of culture. After 12 days of culture, the cells were counted to compare the promoting effect of each factor on the proliferation of primary gastric cancer cells. Among them, as an experimental control, a basic culture medium without any additives was used, and the experimental results are shown in Table 1.

Table 1 Additives in the culture medium and their effects on promoting organoid proliferation

Among them, "+" means 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; "-" means that the culture medium added with the additive shows an inhibitory effect on at least one of the primary gastric cancer cells isolated from gastric cancer tissue; "○" means 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.

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 additives and N2 additives, 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.

Example 2 Effects of different concentrations of culture medium added factors on the proliferation of primary gastric cancer cells

According to the method of Example 1 (2), primary gastric cancer cells were obtained from intraoperative tissue samples (numbered GC-002 and GC-003), and the culture medium formula in Table 2 below was used for primary cell culture.

Table 2 Culture medium formula (concentration is final concentration)

When using the culture medium of Formula 1, 200 μL of compound 1 was added to each well of a 48-well plate seeded with primary cells on the basis of Formula 1, and the final concentrations of compound 1 were 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, respectively; and the control well (BC) was set using the culture medium of Formula 1. The final concentrations of other added factors in this series of culture media were the same as those of GC-2.2 culture medium. The experiments of the following formulas 1-14 were also carried out in the same way and will not be repeated here.

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, and 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.

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, and 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.

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

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, and 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.

When using the culture medium of Formula 6, 200 μL of prepared epidermal growth factor was added to each well of a 48-well plate seeded with primary cells based on Formula 6, and the final concentrations of epidermal growth factor 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 up using the culture medium of Formula 6.

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

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, and the final concentrations of SB202190 were 50 nM, 100 nM, 200 nM, 400 nM, and 800 nM, respectively; and control wells (BC) were set up using the culture medium of Formula 8.

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, and the final concentrations of dexamethasone were 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM, respectively; and control wells (BC) were set using the culture medium of Formula 9.

When using the culture medium of Formula 10, 200 μL of prepared cholera toxin was added to each well of a 48-well plate seeded with primary cells based on Formula 10, and 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.

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, and the final concentrations of N-acetyl-L-cysteine were 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.

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, and the final concentrations of gastrin were 1.25 nM, 2.5 nM, 5 nM, 10 nM, and 20 nM, respectively; and control wells (BC) were set using the culture medium of Formula 12.

When using the culture medium of Formula 13, 200 μL of prepared oncostatin M was added to each well of a 48-well plate seeded with primary cells based on Formula 13, and 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.

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, and the final concentrations of A8301 were 1.25 nM, 2.5 nM, 5 nM, 10 nM, and 20 nM, respectively; and control wells (BC) were set up using the culture medium of Formula 14.

When the cells were expanded to about 85% of the 48 wells, the digestion counts were performed, and the proliferation times were calculated by referring to the cell numbers of the control wells (BC), and the data collected from the two samples were summarized and shown in Figures 1A to 1N. In Figures 1A to 1N, the ratio is the ratio of the number of cells obtained by culturing one generation using each culture medium to the number of cells obtained by culturing one generation using the corresponding control wells. A ratio greater than 1 indicates that the culture medium containing different concentrations of factors or small molecule compounds has a better proliferation-promoting effect than the control well culture medium; a ratio less than 1 indicates that the culture medium containing different concentrations of factors or small molecule compounds has a weaker proliferation-promoting effect than the control well culture medium.

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

Industrial Applicability

The present invention provides a culture medium and a culture method for culturing primary gastric cancer cells, and 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.

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 (10)

1. A culture medium for primary gastric cancer cells, characterized in that it comprises an MST1/2 kinase inhibitor; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152; at least one of a B27 additive and an 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, Wherein, the MST1/2 kinase inhibitor includes a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,

in, R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl, aryl C1-C6 alkyl and heteroaryl optionally substituted by 1-2 independently R ₆ ; _R2 and _R3 are each independently selected from C1-C6 alkyl; _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; _R6 is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl. 2. The culture medium of claim 1, wherein R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and phenyl, naphthyl, benzyl and thienyl optionally substituted by 1-2 independently R ₆ ; _R2 and _R3 are each independently selected from C1-C3 alkyl; _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, piperidinylC1-C6 alkyl, and tetrahydropyranylC1-C6 alkyl; _R6 is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl. 3. The culture medium of claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,

in, R ₁ is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R ₆ , thienyl optionally substituted by 1-2 independently R ₆ , and benzyl optionally substituted by 1-2 independently R ₆ ; _R5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; R ₆ is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl. 4. The culture medium as claimed in claim 3, wherein R ₁ is phenyl optionally substituted by 1-2 independently R ₆ ; _R5 is hydrogen; _R6 is preferably fluorine, methyl or trifluoromethyl. 5. The culture medium according to claim 1, wherein the MST1/2 kinase inhibitor is selected from at least one of the following compounds or pharmaceutically acceptable salts thereof:

6. The culture medium according to any one of claims 1 to 5, characterized in that the content of each component in the culture medium satisfies any one or more or all of the following: The concentration of the MST1/2 kinase inhibitor is 1.25-20 μM; The volume ratio of the B27 or N2 cell culture additive to the culture medium is 1:25 to 1:400; The concentration of the basic fibroblast growth factor is 2.5-40 ng/mL; The volume ratio of the ITS cell culture additive to the culture medium is 1:25 to 1:400; The concentration of the ROCK kinase inhibitor is 2.5-40 μM; The concentration of dexamethasone is 25-400 nM; The concentration of CHIR99021 is 1.25-20 μM; The concentration of the epidermal growth factor is 2.5-40 ng/mL; The concentration of the cholera toxin is 1.25-20 ng/mL; The concentration of gastrin is 1.25-20 nM; The concentration of SB202190 is 50-800 nM; The concentration of N-acetyl-L-cysteine is 0.25-4 mM; The concentration of oncostatin M is 2.5-40 ng/mL; The concentration of A8301 is 1.25-20 nM. 7. The culture medium according to any one of claims 1 to 5, characterized in that the content of each component in the culture medium satisfies any one or more or all of the following: The concentration of the MST1/2 kinase inhibitor is 2.5 to 10 μM; The volume ratio of the B27 or N2 cell culture 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 ROCK kinase inhibitor was 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 2.5-20 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 N-acetyl-L-cysteine is 1 to 4 mM; The concentration of oncostatin M is 2.5-10 ng/mL; The concentration of A8301 is 2.5-20 nM. 8. The culture medium according to any one of claims 1 to 5, further comprising: 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. 9. The culture medium according to any one of claims 1 to 5, characterized in that the culture medium does not contain Wnt agonists, R-spondin family proteins, Noggin proteins, or BMP inhibitors. 10. A method for culturing primary gastric cancer cells, characterized by comprising the following steps: (1) preparing a culture medium according to any one of claims 1 to 9; (2) coating the culture vessel with a diluted 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 are inoculated into a culture vessel coated with extracellular matrix gel, and cultured using the culture medium described in step (1).

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