Culture medium and culture method of primary cervical cancer cells

Abstract

The present application provides a culture medium for culturing primary cervical cancer cells and a culture method using the primary cell culture medium and applications thereof. The culture medium comprises an MST1 / 2 kinase inhibitor; an insulin-like growth factor 1; a fibroblast growth factor 7; an insulin-transferrin-selenium complex; a hepatocyte growth factor; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152; and liothyronine. In the culture method, the primary cells are cultured on a culture vessel pre-coated with irradiated feeder cells using the primary cell culture medium described above, so that the primary cells proliferate rapidly. The cell model obtained by the primary cell culture medium and the primary cell culture method of the present application can be used for efficacy evaluation and screening of drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and in particular relates to a culture medium and a culture method for culturing or expanding primary cervical cancer cells in vitro. BACKGROUND

[0002] According to the latest global cancer statistics released by the International Agency for Research on Cancer (IARC) under the World Health Organization in 2018, Global Cancer Report shows that there were 1810 million new cancer cases worldwide in 2018, and the number of deaths was 960 million, and the global cancer burden is further aggravated. The top 10 cancer incidence rates in China in 2018 are breast cancer, lung cancer, colorectal cancer, thyroid cancer, gastric cancer, cervical cancer, liver cancer, esophageal cancer, uterine cancer, and brain cancer. Among them, the female incidence rate of cervical cancer is the 6th. Every year, 200,000 women die of cervical cancer worldwide, and China accounts for about 10%. Since the molecular mechanism of cervical cancer is not clear, the treatment means for cervical cancer, especially for patients in the middle and late stages, is still limited, resulting in a low five-year survival rate of patients in clinical practice, and a lack of personalized precise medication guidance.

[0003] Functional test refers to a method for detecting the sensitivity of an anti-tumor drug on cancer patient cells in vitro. The key to applying this method is to develop a tumor cell model with a long growth cycle and short and capable of representing the biological characteristics of the patient's own cervical cancer. In addition, the cell model should be convenient to operate and can quickly and efficiently predict the efficacy of clinical medication, so as to give timely precise medication guidance to cancer patients. However, the success rate of establishing a cell model in vitro from primary tumor cells taken from cancer patients is often very low, the growth cycle is long, and there are problems such as excessive proliferation of fibroblasts and other interstitial cells, which restrict the development of this field. Currently, there are two technologies for culturing primary cells / stem cells that have relatively mature development in the field of tumor cell functional testing, one is a technology using irradiated feeder cells and ROCK kinase inhibitors to promote the growth of primary cells to investigate the drug sensitivity of individual patients, i.e. cell conditional reprogramming technology (Liu et al., Am. J. Pathol., 180: 599-607, 2012). The other technology is a 3D in vitro culture of adult stem cells to obtain organoid technology similar to tissue organs (Hans Clevers et al., Cell, 11, 172(1-2): 373-386, 2018).

[0004] However, the organoid technology is a technology of embedding patient autologous primary cells in extracellular matrix for three-dimensional culture in vitro, but a plurality of specific growth factors (such as Wnt protein and R-spondin family protein) need to be added in the culture medium of the technology, which is expensive and not suitable for popularization to the clinic for large-scale application. In addition, the cells need to be embedded in the extracellular matrix glue during the whole culture process, and the cell inoculation, subculture and drug sensitivity test plating steps are more complicated and time-consuming than 2D culture operation, and the size of the organoids formed by the technology is not easy to control, and some organoids are prone to grow too large to cause internal necrosis. Therefore, compared with the 2D culture technology, the operability and applicability of the organoid technology are not strong, and it needs professional technical personnel to operate, and is not suitable for large-scale and wide application in clinical in vitro drug sensitivity detection (Nick Barker, Nat. Cell Biol., 18(3):246-54, 2016).

[0005] The cell reprogramming technology is a technology of co-culturing patient autologous primary cells with murine feeder cells, but there is no report in the literature for testing cervical cancer samples, so it is unknown whether the culture medium composition reported in the literature can rapidly expand cervical cancer primary tumor cells.

[0006] In view of the limitations of the above technologies, it is necessary to develop a cervical cancer primary cell culture technology in the clinic, which has a short culture period, controllable cost and convenient operation. When the technology is applied to construct a primary cervical cancer tumor cell model, the cultured cervical cancer tumor cells can represent the biological characteristics of the patient's own cervical cancer. By evaluating the sensitivity of antitumor drugs in different cancer patient-derived cell models in vitro, the response rate of antitumor drugs in the clinic can be improved, and the pain and waste of medical resources caused by inappropriate drugs for patients can be reduced. SUMMARY

[0007] The present application aims at the deficiencies of the prior art and provides a culture medium for culturing cervical cancer primary cells and a method for culturing cervical cancer primary cells using the culture medium. The cervical cancer primary cell culture medium and culture method of the present application can achieve the purposes of short in vitro culture period, controllable cost and convenient operation. When the technology is applied to construct a primary cervical cancer tumor cell model, primary cervical cancer tumor cells with the biological characteristics of the patient's own cervical cancer can be obtained, and the technology can be applied to new drug screening and in vitro drug sensitivity detection.

[0008] One aspect of the present application is to provide a primary cell culture medium for culturing primary cells of cervical cancer, which contains an MST1 / 2 kinase inhibitor; an insulin-like growth factor 1; a fibroblast growth factor 7; an insulin-transferrin-selenium complex; a hepatocyte growth factor; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152; and liothyronine, the MST1 / 2 kinase inhibitor comprising 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.) optionally substituted with 1-2 independent R6;

[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 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]

[0017] wherein,

[0018] R1is selected from 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, R1more preferably being phenyl optionally substituted with 1-2 independent R6;

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

[0020] R6is each independently selected from the group consisting of halogen, C1-C6alkyl, and C1-C6haloalkyl, more preferably R6is 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 an embodiment of the present application, the content of the MST1 / 2 kinase inhibitor in the culture medium is generally 2 μM to 20 μM, preferably 5 μM to 20 μM, more preferably 5 μM.

[0029] The primary cell culture medium of the present application preferably further contains one or more of the following factors: insulin-like growth factor 1 (IGF-1); fibroblast growth factor 7 (FGF7); insulin-transferrin-selenium complex (ITS); hepatocyte growth factor (HGF); liothyronine; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152. Among them, in a preferred embodiment, the content of the fibroblast growth factor 7 in the culture medium is preferably 2 to 40 ng / ml, more preferably 10 to 40 ng / ml; the insulin-transferrin-selenium complex is preferably 1:100 to 1:25 relative to the volume of the culture medium; the content of the insulin-like growth factor 1 is preferably 2 to 40 ng / ml, more preferably 10 to 40 ng / ml; the content of the hepatocyte growth factor is preferably 2 to 40 ng / ml, more preferably 10 to 40 ng / ml; the content of the liothyronine is preferably 2 to 50 nM, more preferably 2 to 10 nM; and the ROCK kinase inhibitor is preferably Y27632, and the content of the ROCK kinase inhibitor is preferably 2 to 20 μM, more preferably 2 to 10 μM.

[0030] Compared with the cell condition reprogramming medium and organoid medium, the medium formula of the application adds MST1 / 2 kinase inhibitor, but does not contain serum, bovine pituitary extract and other uncertain components, does not contain Wnt agonists, R-spondin family proteins, BMP inhibitors and other necessary factors for organoid culture, and does not contain nicotinamide and N-acetylcysteine, thereby greatly reducing the cost of the medium, simplifying the operation process of preparing the medium, and realizing the in-vitro culture of cervical cancer primary cells with controllable cost and convenient operation.

[0031] In the application, the cervical cancer primary cells can be cervical cancer tumor cells, normal cervical cancer primary cells, or cervical cancer epithelial stem cells.

[0032] One aspect of the application is to provide a method for culturing cervical cancer primary cells, which comprises the following steps:

[0033] (1) preparing the primary cell culture medium of the application according to the above formula;

[0034] (2) using irradiated trophoblast cells to precoat the culture vessel.

[0035] Specifically, the trophoblast cells can be, for example, irradiated NIH-3T3 cells, the irradiation source is X-ray or gamma ray, preferably gamma ray, the irradiation dose is 30-50 Gy, preferably 35 Gy, and the irradiation time is 5-20 minutes. Specifically, the irradiated NIH-3T3 cells are inoculated in the culture vessel at a concentration of 2-5 x 10 4 cells / cm 2 The cells are inoculated in a culture vessel such as a 48-well plate, a 24-well plate, a 12-well plate, a 6-well plate or a T25 cell culture flask, and are ready for use after adhering to the culture vessel.

[0036] (3) isolating cervical cancer primary cells from cervical cancer tissue.

[0037] The cervical cancer primary cells can be derived from cervical cancer tissue samples and paracancerous tissue samples. The cervical cancer tissue sample is derived from a cervical cancer tumor patient who has undergone surgery and has given consent, and the paracancerous tissue sample is collected from tissue at least 5 cm away from the cervical cancer tissue. The collection of the above-mentioned tissue samples is performed within half an hour after the patient's surgery. More specifically, in a sterile environment, the tissue sample is cut from a non-necrotic part, and the volume is 0.5 cm 3The above is placed in pre-cooled 3-5 mL DMEM / F12 medium, the medium is in a plastic sterile capped centrifuge tube, transported to the laboratory on ice; wherein the DMEM / F12 medium contains 1-2% by volume penicillin / streptomycin, and / or 0.2-0.4% by volume Primocin (hereinafter referred to as tissue transport fluid). When using streptomycin / penicillin, the streptomycin concentration ranges from 25 to 400 μg / mL, preferably 50 to 200 μg / mL, more preferably 200 μg / mL, and the penicillin concentration ranges from 25 to 400 U / mL, preferably 50 to 200 U / mL, more preferably 200 U / mL; when using Primocin, the concentration ranges from 25 to 400 μg / mL, preferably 50 to 200 μg / mL, more preferably 100 μg / mL.

[0038] In a biological safety cabinet, the tissue sample is transferred to a cell culture dish, the tissue sample is rinsed with the transport fluid, and the blood cells on the surface of the tissue sample are washed away. The rinsed tissue sample is transferred to another new culture dish, 1-3 mL of transport fluid is added, and the tissue sample is divided into tissue fragments with a volume of less than 3 mm 3 using a sterile scalpel and surgical forceps.

[0039] The tissue sample pieces are transferred into centrifuge tubes and centrifuged at 1000-3000 rpm for 3-5 minutes using a bench centrifuge (Sigma 3-18K). The supernatant is discarded, and tissue transport liquid and tissue digestion liquid are added at a ratio of 1:1 (the amount used is about 5 mL of tissue digestion liquid per 10 mg of tissue, and the tissue digestion liquid is prepared by dissolving 1-2 mg / mL collagenase II, 1-2 mg / mL collagenase IV, 50-100 U / mL deoxyribonuclease I, 0.5-1 mg / mL hyaluronidase, 0.1-0.5 mg / mL calcium chloride, and 5-10 mg / mL bovine serum albumin in HBSS and RPMI-1640 at a volume ratio of 1:1). The sample is labeled, sealed with a sealing film, and digested at 37°C, 200-300 rpm on a constant temperature shaker (Zhichu Instruments ZQLY-180N). The digestion is observed every 1 hour to determine whether it is complete. If no obvious tissue pieces are observed, the digestion is terminated. Otherwise, the digestion is continued until it is complete, and the digestion time is 4-8 hours. After the digestion is complete, the undigested tissue pieces are filtered out using a cell filter (cell screen pore size, for example, 70 μm), and the tissue pieces on the filter are washed with tissue transport liquid. The remaining cells are washed into the centrifuge tube, and centrifuged at 1000-3000 rpm for 3-5 minutes using a bench centrifuge. The supernatant is discarded, and the remaining cell pieces are observed to determine whether they contain blood cells. If blood cells are present, 1-5 mL of blood cell lysis liquid (purchased from Sigma) is added, mixed, and lysed at 4°C for 10-20 minutes. The mixture is mixed once every 5 minutes, and then removed after the lysis is complete. The mixture is centrifuged at 1000-3000 rpm for 3-5 minutes. The supernatant is discarded, and the primary cell culture medium of the present application is added to resuspend the cells. The cells are counted using a flow image counter (Jiangsu Zhuo Microbiological Technology Co., Ltd. JIMBIO FIL), and the total number of cells is obtained.

[0040] (4) The primary cervical cancer cells separated in step (3) are inoculated in a culture vessel pre-seeded with trophoblast cells, and the primary cell culture medium in step (1) is used for culture.

[0041] More specifically, after the trophoblast cells adhere, the primary cervical cancer tumor cells are inoculated at a density of 2 x 10 4 - 8 x 10 4 cells / cm 2 (for example, 4 x 10 4 cells / cm 2 ), 0.5-2 mL of the primary cell culture medium is added to each well, and the cells are cultured in a cell culture incubator at, for example, 37°C, 5% CO2 for 8-16 days. Fresh primary cell culture medium is added every 4 days during the culture, and the primary cervical cancer cells are passaged when they grow to a cell density of 80%-90% of the bottom area of the multi-well plate.

[0042] Compared with the organoid technology, the step does not need to mix the primary cells and the Matrigel on ice to form a gel drop, and wait for the gel drop to solidify before adding the culture medium. In addition, the use of the expensive extracellular matrix gel is also saved, and the operation steps are simplified.

[0043] Optionally, after inoculation, the cervical cancer primary cells are cultured for 8-16 days, when the cell clones formed in the culture container reach 80% of the bottom area, the supernatant is discarded, 1-2 mL of 0.25% trypsin (purchased from Thermo Fisher Company) is added for digestion for 1 minute, then 0.25% trypsin is aspirated, and 1-2 mL of 0.05% trypsin is added for cell digestion, and incubated at room temperature for 5-20 minutes; then 2-4 mL of culture solution containing, for example, 5% (v / v) fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin is used to resuspend the digested cells, and the single cells are centrifuged at 1000-3000 rpm for 3-5 minutes, and the primary cell culture medium of the application is used to resuspend the digested single cells, and the obtained cell suspension is placed into a culture vessel pre-coated with feeder cells for further expansion culture. The pre-treatment operation of the culture vessel is synchronized with step (2).

[0044] The expanded cervical cancer primary cells grow in 2D, avoiding the uneven size of the organoids and the internal necrosis of the overgrown organoids that occur during the expansion of the organoid technology.

[0045] The application also provides a method for evaluating or screening drugs for treating cervical cancer diseases, which comprises the following steps:

[0046] (1) culturing cervical cancer primary cells using the culture method of cervical cancer primary cells of the application;

[0047] (2) selecting the drug to be tested and diluting it according to the required concentration gradient;

[0048] (3) adding the diluted drug to the cervical cancer primary cells cultured in (1);

[0049] (4) testing the cell activity.

[0050] The beneficial effects of the application also include:

[0051] (1) improving the success rate of culturing cervical cancer primary cells, with a success rate of more than 85%;

[0052] (2) ensuring that the in vitro cultured cervical cancer primary cells can maintain the pathological phenotype and heterogeneity of the patient from which the primary cells are derived;

[0053] (3) the culture medium does not contain serum, so it is not affected by the quality and quantity of different batches of serum;

[0054] (4) The efficiency of amplifying cervical cancer primary cells is high, and only 10 4 orders of magnitude of cervical cancer primary cells can be successfully amplified in about two weeks, and the amplified cervical cancer primary cells can be continuously passaged. 6

[0055] (5) The passage step does not require ice operation and dissociation of the substrate glue, and the digestion and passage of cells can be completed in 10-15 minutes.

[0056] (6) The culture cost is controllable, and the cervical cancer primary cell culture medium does not need to add expensive Wnt agonists, R-spondin family proteins, and BMP inhibitors, thereby saving the cost of cell culture.

[0057] (7) The operation is convenient, and compared with the organoid technology, the cells do not need to be embedded in the substrate glue, and the operation steps of the technology are simple and easy to operate.

[0058] (9) The number of cervical cancer primary cells obtained by the technology is large, and the degree of uniformity is high, which is suitable for high-throughput screening of new candidate compounds, and provides high-throughput drug in vitro sensitivity function test for patients.

[0059] The cell culture medium of the embodiment can culture cervical cancer primary cells derived from human or other mammals, including cervical cancer tumor cells, normal cervical cancer primary cells, cervical cancer epithelial stem cells, or tissues containing at least any of these cells. At the same time, the culture medium of the present technology can also be used to develop a kit for in vitro cervical cancer primary cell amplification culture.

[0060] In addition, the cells obtained by the culture method of the embodiment can be applied to regenerative medicine, basic medical research of cervical cancer primary cells, screening of drug responses, and development of new drugs derived from cervical cancer diseases. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figures 1A-1G is a graph showing the effect of the concentration of each added factor on the proliferation of cervical cancer primary cells.

[0062] Figure 2A and 2B is a photograph taken under an inverted microscope of cervical cancer tumor cells cultured to day 4 and day 12 using the culture medium CM of the present application from cells isolated from a clinical tissue sample of 1 case of cervical cancer.

[0063] Figure 3 is a view comparing the total number of cells collected after 7 days of culture of cells isolated from a surgical resection sample of 1 case of cervical cancer using 3 different culture media.

[0064] ​Figure 4 is a comparison chart of cell proliferation effects of cells isolated from 6 surgical resection samples of cervical cancer after being cultured for 7 days under three different medium conditions.

[0065] Figure 5A and 5B is a picture of staining of cervical cancer tumor cells obtained by culturing cells isolated from 1 surgical resection sample of cervical cancer using the medium CM of the present application, using non-specific nuclear dye DAPI and cervical cancer specific antibody p16, respectively.

[0066] Figure 6 is a comparison chart of immunohistochemical results of original tissue cells of 1 surgical resection sample of cervical cancer and cervical cancer tumor cells obtained by culturing the cells using the medium CM of the present application.

[0067] Figure 7 shows cell activity curves of cervical cancer tumor cells obtained by culturing cells isolated from 2 surgical resection cancer tissue samples of cervical cancer patients according to the method of the present application under the influence of 8 different drugs. DETAILED DESCRIPTION

[0068] In the present specification, primary cells include differentiated primary cells and epithelial stem cells obtained from epithelial tissues. The "epithelial stem cell" refers to a cell having long-term self-renewal ability and differentiating into a primary cell, and refers to a stem cell derived from an epithelial tissue. As the epithelial tissue, there can be exemplified, for example, cornea, oral mucosa, skin, conjunctiva, bladder, renal tubule, kidney, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), large intestine (including colon)), liver, pancreas, mammary gland, salivary gland, lacrimal gland, prostate, hair root, trachea, lung, etc. Among them, the cell culture medium of the present embodiment is preferably a medium for cervical cancer primary cells.

[0069] Further, in the present specification, the "epithelial tumor cell" refers to a cell obtained by tumorigenesis of a cell derived from the above-mentioned epithelial tissue.

[0070] In the present specification, the "organoid" refers to a three-dimensional, organoid cell tissue body formed by spontaneously organizing and aggregating cells in a controlled space at high density.

[0071] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0072] 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.

[0073] 1. Preparation of MST1 / 2 kinase inhibitor compound 1

[0074] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2- yl)amino)benzoic acid Sulfonamide 1

[0075]

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 2. Preparation of other MST1 / 2 inhibitor compounds of the present invention

[0082] 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.

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] [Example 1]

[0089] Isolation of primary human cervical cancer cells

[0090] The cervical cancer tissue samples were obtained from surgically removed cancer tissue samples of three cervical cancer patients who explained and obtained consent. They are sample numbers CCA2, CCA3, and CCA4. One of the samples (number CCA2) is used for explanation below.

[0091] The above tissue samples were collected within half an hour after the patient's surgical resection. More specifically, under a sterile environment, tissue samples were cut from non-necrotic areas with a volume of 0.5 cm 3 The above tissues were placed in 4 mL of pre-cooled tissue transport solution (see Table 1 for specific preparation), and the transport solution was placed in a 5 mL plastic sterile capped cryotube (purchased from Guangzhou Jiete Biotechnology) and transported to the laboratory under cold chain (0-10°C).

[0092] Table 1 Tissue transport fluid formula

[0093]

[0094]

[0095] Table 2 Tissue digestion solution formula

[0096] Composition of Tissue Digestion Solution Supplier Final Concentration HBSS Gibco 50% (by volume) RPMI-1640 Corning 50% (by volume) Collagenase II Sigma 2 mg / mL Collagenase IV Sigma 2 mg / mL Deoxyribonuclease I Sigma 50 U / mL Hyaluronidase Sigma 0.5 mg / mL Calcium Chloride Shanghai Genomics 0.33 mg / mL Bovine Serum Albumin Shanghai Genomics 10 mg / mL

[0097] In a biosafety cabinet, transfer the tissue sample (number CCA2) to a 100mm cell culture dish (purchased from NEST). Rinse the tissue sample with tissue transport fluid to remove residual blood from the surface of the tissue sample and remove excess tissue such as fat from the surface of the tissue sample. Transfer the rinsed tissue sample to another new 100mm culture dish, add 2mL of transport fluid, and use a sterile surgical blade and surgical forceps to cut the tissue sample into pieces smaller than 3mm. 3 of tissue fragments.

[0098] Transfer the tissue sample fragments to a 15 mL centrifuge tube and centrifuge at 1500 rpm for 4 minutes using a tabletop centrifuge (Sigma 3-18K). Discard the supernatant and add tissue transport solution and tissue digestion solution in a 1:1 ratio (approximately 5 mL of tissue digestion solution per 10 mg of tissue; see Table 2 for the specific preparation). Label the sample number, seal with sealing film, and digest in a thermostatic shaker (Zhichu Instrument ZQLY-180N) at 37°C and 300 rpm. Observe the digestion completion every 1 hour.

[0099] After digestion, undigested tissue clumps were filtered out through a 70 μm filter. The tissue clumps on the filter were rinsed with tissue transport fluid, and the remaining cells were flushed into a centrifuge tube and centrifuged at 1500 rpm for 4 minutes.

[0100] Discard the supernatant and observe whether the remaining cell mass contains blood cells. If blood cells are present, add 3 mL of blood cell lysis solution (purchased from Sigma) and mix well. Incubate at 4°C for 15 minutes, and shake well once every 5 minutes. After the lysis is complete, remove the mixture and centrifuge at 1500 rpm for 4 minutes. Discard the supernatant to obtain the primary cervical cancer cells after digestion and separation. Add the base medium (BM) to resuspend the cells, wherein the base medium is prepared by adding 0.2% (v / v) Primocin (purchased from Invivogen, with a concentration of 50 mg / mL) to a commercially available DMEM / F-12 medium to obtain a final concentration of 100 μg / mL.

[0101] Two other cervical cancer tumor tissue samples were separated according to the same method as above, and the total number of cells obtained was 1.97 million (CCA3) and 2.32 million (CCA4), respectively.

[0102] [Example 2]

[0103] Optimization of the medium for culturing primary cervical cancer cells

[0104] (1) Effects of different factors

[0105] The cultured NIH-3T3 cells (purchased from ATCC, cultured using DMEM medium containing 10% fetal bovine serum) were digested with 0.25% trypsin (purchased from Thermo Fisher), and the digestion was terminated using DMEM medium containing 5% (v / v) fetal bovine serum (purchased from Ecoscience), 100 U / mL penicillin, and 100 μg / mL streptomycin, and collected into a 15 mL centrifuge tube. After centrifugation at 1500 rpm for 4 minutes, the supernatant was discarded. The cell pellet after centrifugation was resuspended using the above-mentioned DMEM medium containing 10% fetal bovine serum, and counted using a flow image counter (JIMBIO FIL, Jiangsu). The cells were then irradiated with a 35 Gy dose of gamma rays for 10 minutes, and then seeded into culture vessels at a density of 2 x 10 4 2 The cells were cultured in a 37°C incubator until they adhered. Before seeding the primary cells, the culture medium in the culture vessels was removed.

[0106] Prepare the base medium (abbreviated as BM): add 0.2% (v / v) Primocin (purchased from Invivogen, with a concentration of 50 mg / mL) to a commercially available DMEM / F-12 medium to obtain a final concentration of 100 μg / mL to prepare the BM.

[0107] ​Next, different kinds and different concentrations of additive factors (Table 3) were added into the basal medium (BM) respectively to prepare the primary cervical cancer cell culture medium containing different additive components.

[0108] Table 3 Preparation of different component culture medium (concentration is final concentration)

[0109]

[0110]

[0111] Different component culture medium was added into the 48-well culture plate pre-paved with gamma ray irradiated NIH-3T3 cells at a volume of 500 μl / well. Cervical cancer tumor cells (No. CCA12) isolated from cervical cancer tissue according to the same method of Example 1 were inoculated into the 48-well culture plate pre-paved with gamma ray irradiated NIH-3T3 cells at a cell number of 4 x 10 4 After surface sterilization, the same number of freshly isolated cervical cancer tumor cells (No. CCA12) were cultured under different culture medium formula conditions in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher). Medium replacement and feeder cell replenishment were performed every 4 days after the start of culture. After 7 days of culture, cell counting was performed. As an experimental control, basal medium (BM) without any additive was used. The results are shown in Table 3. "-" represents that the additive has no effect on cell proliferation, and "+" represents that the additive has a promoting effect on cell proliferation. Different concentrations of different factors added to BM have different effects on cell proliferation. Among them, B27 additive, N2 additive, insulin-transferrin-selenium complex, hepatocyte growth factor, insulin-like growth factor 1, fibroblast growth factor 7, compound 1, Y27632 and iodothyronine have a certain promoting effect on cell proliferation within a certain concentration range.

[0112] (2) Different concentrations of added factors on the proliferation of cervical cancer primary cells obtained by the present patent

[0113] The cervical cancer primary cell culture medium of this example was prepared as follows: fibroblast growth factor 7 (FGF7) was added to the basal medium (BM) at a final concentration of 20 ng / ml, insulin-like growth factor 1 (IGF-1) was added at a final concentration of 20 ng / ml, hepatocyte growth factor (HGF) was added at a final concentration of 20 ng / ml, insulin-transferrin-selenium complex (ITS) stock solution was added at a dilution ratio of 1:50 (the final concentration of insulin in the culture medium was 10 μg / ml, the final concentration of transferrin in the culture medium was 5 μg / ml, and the final concentration of sodium selenite in the culture medium was 5 ng / ml), compound 1 was added at a final concentration of 5 μM, Y27632 was added at a final concentration of 10 μM, and liothyronine was added at a final concentration of 10 nM to prepare a cervical cancer primary cell culture medium.

[0114] The same method as in Example 1 was used to isolate primary cervical cancer cells from the cancer tissue of a cervical cancer patient (sample number CCA8). Subsequently, the cervical cancer tumor cells derived from the cancer tissue were counted using a flow cytometer (Jiangsu Zhuowei Biotechnology Co., Ltd. JIMBIO FIL) to obtain the total number of cells. The cells were then counted using a 4×10 4 pieces / cm 2 The cells were seeded into a 12-well plate pre-plated with gamma-irradiated NIH-3T3 cells at a high density. 2 mL of the prepared culture medium for primary cervical cancer cells was added to the 12-well plate and cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific). When the cells in the culture plate grew to about 80% of the bottom area, the culture supernatant in the 12-well plate was discarded, and 0.5 mL of 0.25% trypsin (purchased from Thermo Fisher) was added for digestion for 1 minute. The 0.25% trypsin was then aspirated and 0.5 mL of 0.05% trypsin was added for cell digestion. The cells were incubated at room temperature for 5 to 20 minutes until the cells were completely digested as observed under a microscope (Invitrogen EVOS M500). The digestion was terminated with 1 mL of DMEM / F12 culture medium containing 5% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were collected into a 15 mL centrifuge tube and centrifuged at 1500 rpm for 4 minutes. The supernatant was discarded. The cell pellet after centrifugation was resuspended in basal medium BM and counted using a flow cytometer (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL) to obtain the total number of cells. The obtained cells were used in the following culture experiments.

[0115] Next, the following 8 culture media were prepared for the experiment:

[0116] Recipe 1: The above culture medium components do not contain fibroblast growth factor 7;

[0117] Formulation 2: The above medium components do not contain insulin-transferrin-selenium complex;

[0118] Formulation 3: The above medium components do not contain insulin-like growth factor 1;

[0119] Formulation 4: The above medium components do not contain hepatocyte growth factor;

[0120] Formulation 5: The above medium components do not contain Y27632;

[0121] Formulation 6: The above medium components do not contain Compound 1;

[0122] Formulation 7: The above medium components do not contain liothyronine.

[0123] The above digested cell suspension was diluted with the above Formulations 1-7, respectively, and seeded into 48-well plates pre-coated with gamma-irradiated NIH-3T3 cells at a density of 10,000 cells per well in a volume of 250 microliters.

[0124] In the case of using the medium of Formulation 1, fibroblast growth factor 7 was added to the 48-well plates seeded with primary cells at a final concentration of 40 ng / ml, 10 ng / ml, and 2 ng / ml, respectively, in a volume of 250 microliters per well, and a control well (BC) was set using the medium of Formulation 1.

[0125] In the case of using the medium of Formulation 2, insulin-transferrin-selenium complex was added to the 48-well plates seeded with primary cells at a final concentration of 1:100, 1:50, and 1:25 (corresponding to a final concentration of insulin / transferrin / sodium selenite of 5 μg / ml-2.5 μg / ml-2.5 ng / ml; 10 μg / ml-5 μg / ml-5 ng / ml; 20 μg / ml-10 μg / ml-10 ng / ml, respectively) in a volume of 250 microliters per well, and a control well (BC) was set using the medium of Formulation 2.

[0126] In the case of using the medium of Formulation 3, insulin-like growth factor 1 was added to the 48-well plates seeded with primary cells at a final concentration of 40 ng / ml, 10 ng / ml, and 2 ng / ml, respectively, in a volume of 250 microliters per well, and a control well (BC) was set using the medium of Formulation 3.

[0127] When using the medium of Formula 4, 250 microliters of prepared hepatocyte growth factor was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of hepatocyte growth factor was 40 ng / ml, 10 ng / ml, and 2 ng / ml, respectively; and the control well (BC) was set using the medium of Formula 4.

[0128] When using the medium of Formula 5, 250 microliters of prepared Y27632 was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of Y27632 was 20 μM, 10 μM, and 2 μM, respectively; and the control well (BC) was set using the medium of Formula 5.

[0129] When using the medium of Formula 6, 250 microliters of prepared compound 1 was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of compound 1 was 20 μM, 5 μM, and 2 μM, respectively; and the control well (BC) was set using the medium of Formula 6.

[0130] When using the medium of Formula 7, 250 microliters of prepared iodothyronine was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of iodothyronine was 50 nM, 10 nM, and 2 nM, respectively; and the control well (BC) was set using the medium of Formula 7.

[0131] After the cells were expanded to about 85% of the 48-well digestion count, the ratio was calculated by referring to the cell number of the control well (BC), and the results are shown in Table 1, Table 2, Table 3, and Table 4, respectively. Figures 1A-1G . Figures 1A-1G The ratio is the ratio of the number of cells obtained by culturing one generation using each medium to the number of cells obtained by culturing one generation in the corresponding control well. If the ratio is greater than 1, it indicates that the prepared medium containing different concentrations of factors or small molecule compounds has a better proliferation-promoting effect than the control well medium; if the ratio is less than 1, it indicates that the prepared medium containing different concentrations of factors or small molecule compounds has a weaker proliferation-promoting effect than the control well medium.

[0132] According to Figures 1A-1GAs a result, the content of fibroblast growth factor 7 in the culture medium is preferably 2-40 ng / ml, more preferably 10-40 ng / ml; the volume concentration of the insulin-transferrin-selenium complex is preferably 1:25-1:100, more preferably 1:25-1:50 (corresponding to the final concentrations of insulin / transferrin / sodium selenite of 5-20 μg / ml-2.5-10 μg / ml-2.5-10 ng / ml, respectively; preferably 10-20 μg / ml-5-10 μg / ml-5-10 ng / ml); the content of insulin-like growth factor 1 is preferably 2 ng / ml-40 ng / ml, more preferably 10 ng / ml-40 ng / ml; the content of hepatocyte growth factor is preferably 2 ng / ml-40 ng / ml, more preferably 10 ng / ml-40 ng / ml; the content of Y27632 is preferably 2 μM-20 μM, more preferably 2 μM-10 μM; the content of compound 1 is preferably 2 μM-20 μM, more preferably 5 μM-20 μM; the content of liothyronine is preferably 2 nM-50 nM, more preferably 2 nM-10 nM.

[0133] According to the preferred concentrations of the above components, a preferred medium formulation CM of the application is formulated, which comprises: basal medium (BM), 10 ng / ml fibroblast growth factor 7 (FGF7), 10 ng / ml hepatocyte growth factor (HGF), 10 ng / ml insulin-like growth factor 1 (IGF-1), 1:50 volume ratio of insulin-transferrin-selenium complex, 5 μM compound 1, 10 μM Y27632 and 10 nM liothyronine.

[0134] [Example 3]

[0135] Culture of primary cervical cancer tumor cells derived from human cervical cancer tissue

[0136] Cancer tissue-derived cervical cancer primary cells were isolated from cancer tissue of a cervical cancer patient (sample No. CCA5) using the same method as in Example 1. Next, the cancer tissue-derived cervical cancer tumor cells were counted using a flow image cytometer (JIMBIO FIL, Jiangsu, China) to obtain the total number of cells. Then, 4 x 10 4 cells / cm 2 were seeded into a 12-well plate pre-coated with γ-ray irradiated NIH-3T3 cells. 2 mL of the prepared cervical cancer primary cell culture medium CM was added to the 12-well plate, which was then incubated in a 37°C, 5% CO2 incubator (purchased from Thermo).

[0137] Figure 2A In this example, 4 x 10 4 cells / cm 2Microscopic photographs (taken with a 100x inverted phase contrast microscope) of a 12-well plate inoculated with primary cervical cancer cells at a high density on day 4 of culture from the start of inoculation. Microscopic observation revealed that the cultured primary cervical cancer cells derived from cancer tissue had formed relatively large colonies. Figure 2B This is a photo taken under a microscope (100x inverted phase contrast microscope) on the 12th day after inoculation in this example. The cells have grown completely in the field of view. Figure 2A and 2B As can be seen from the two figures, after the separation, the primary cervical cancer cells were cultured in vitro for 4 days, and obvious clone formation could be seen under the microscope. Moreover, after 12 days of expansion, the number of cells was significantly increased, indicating that the technology of the present invention is an efficient technology for expanding primary cervical cancer cells in vitro.

[0138] [Example 4]

[0139] Effects of different culture media on the proliferation of primary cervical cancer cells derived from cervical cancer tissue

[0140] (1) Comparison of the effects of different culture media on primary cell clone formation and proliferation

[0141] The same method as in Example 2 was used to prepare CM, a culture medium for primary cervical cancer cells, and BM, a basal culture medium for control. In addition, FM, a culture medium used in the literature on conditional cell reprogramming, was prepared as another control. The preparation steps are described in (Liu et al., Nat. Protoc., 12(2):439-451, 2017). The culture medium formula is shown in Table 4.

[0142] Table 4 Components of culture medium (FM) used in literature on cell conditional reprogramming technology

[0143]

[0144]

[0145] The same method as in Example 1 was used to obtain primary cervical cancer tumor cells (number CCA10) derived from cervical cancer tissue. 4 pieces / cm 2 ) were cultured under the following three culture conditions:

[0146] A. The present invention's technology: According to 4×10 4 pieces / cm 2 Seeding density: Primary cervical cancer tumor cells were inoculated into a 24-well plate pre-plated with gamma-ray irradiated NIH-3T3 cells (purchased from ATCC) and cultured using 1 mL of the cervical cancer primary cell culture medium CM of the present invention;

[0147] B. Cell Condition Reprogramming Technology: 4 x 10 4 cells / cm 2 The primary cervical cancer tumor cells were inoculated at a seeding density of 4 x 10

[0148] C. 4 x 10 4 cells / cm 2 The primary cervical cancer tumor cells were inoculated at a seeding density of 4 x 10

[0149] In the above three cultures, the cells cultured under the three culture conditions were changed every 5 days. At the same time, the formation of clones and the proliferation state of the cells in the 24-well plates were observed, and the growth status of the cells was recorded by taking pictures using a microscope (Invitrogen EVOS M500).

[0150] For the primary cervical cancer tumor cells (No. CCA10) cultured using the technology of the present application, when the cells in the culture plates grew to about 80% of the bottom area, the culture medium supernatant in the 24-well plates was discarded, 0.5 mL of 0.25% trypsin (purchased from Thermo Fisher) was added for digestion for 1 minute, then the 0.25% trypsin was aspirated, 0.5 mL of 0.05% trypsin was added for cell digestion, and incubated at 37°C for 10 minutes until the cells were completely digested under a microscope (Invitrogen EVOS M500), i.e. the digestion was stopped by adding 1 mL of DMEM / F12 culture medium containing 5% (v / v) fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and collected into a 15 mL centrifuge tube, centrifuged at 1500 rpm for 4 minutes, and the supernatant was discarded. The cell pellet after centrifugation was resuspended using the culture medium of the present application, and counted using a flow image counter (Jiangsu Zhuomi Biotechnology Co., Ltd. JIMBIO FIL), and the total number of cells was 617,000. The cells cultured under the other two culture conditions were digested and counted in the same way as described above, and the total number of cells cultured using the culture medium FM and BM was 233,000 and 129,000, respectively.

[0151] Figure 3 is a plot of the total number of cells expanded from No. CCA10 cells under different conditions.

[0152] Figure 4Figure 1 is a comparison chart of cell proliferation effects of 6 cervical cancer patient samples (No. CCA8, CCA6, CCA2, CCA3, CCA10, CG2) obtained according to the method of Example 1, and then cultured in the above three different culture medium conditions for 7 days, wherein represents a certain colony formation ability and proliferation effect, represents a more obvious colony formation ability and proliferation effect, represents a very obvious colony formation ability and proliferation effect, and represents no colony formation. From the figure, it can be confirmed that the CM culture medium has obvious advantages in colony formation ability and cell proliferation effect in the culture of primary cells obtained from cervical cancer tissue sources compared with the other two culture conditions.

[0153] [Example 5]

[0154] Identification of primary cervical cancer tumor cells from cancer tissue sources

[0155] (1) Immunofluorescence identification of primary cervical cancer tissue and cervical cancer cells after subculture

[0156] Using the same method as Example 1, cancer tissue-derived primary cervical cancer cells were isolated from the cancer tissue of a cervical cancer patient (sample No. CG2). Then, the cancer tissue-derived cervical cancer tumor cells were counted using a flow image counter (JIMBIO FIL, Jiangsu Zhumo Biotechnology Co., Ltd.). The total number of cells was obtained. Then, 4 x 10 4 cells / cm 2 were inoculated into a 24-well plate pre-coated with γ-ray irradiated NIH-3T3 cells, and a circular cell fragment (purchased from Thermo) for immunofluorescence staining was placed in the 24-well plate. 1 mL of prepared cervical cancer primary cell culture medium CM was added to the 24-well plate, and the plate was incubated in a 37°C, 5% CO2 incubator (purchased from Thermo).

[0157] When the cells in the 24-well plate reached 80% of the bottom area, the culture medium was discarded, and the cells were fixed with 4% formaldehyde on ice for 30 minutes. PBS (purchased from Shanghai Generay Biotech) was washed for 5 minutes x 3 times. The PBS was discarded, and permeabilization solution was added, avoiding light, and the membrane was broken on a shaker (100 rpm) for 30 minutes, and PBS was washed for 5 minutes x 3 times. Then, a 5% volume concentration of BSA (purchased from Shanghai Generay Biotech) solution was used for blocking with PBS + 0.3% Triton X-100 (purchased from Shanghai Generay Biotech), and the blocking was performed at 37°C for 30 minutes.

[0158] Prepare PBS + 0.3% Triton X-100 in advance for dilution of antibodies, dilute cervical cancer specific antibody p16 (purchased from CST company) at a ratio of 1:50, discard the blocking solution, add the prepared primary antibody diluent, and incubate overnight in a 4°C refrigerator. Take out at 4°C, equilibrate to room temperature, continue to incubate at 37°C for 1 hour, and wash with PBS for 5 minutes x 3 times.

[0159] Prepare PBS + 0.3% Triton X-100 in advance for dilution of secondary antibodies, dilute fluorescent secondary antibody (purchased from Thermo Fisher) with excitation light of 488 nm and species of rabbit at a ratio of 1:1000, incubate at room temperature in the dark for 1 hour, and wash with PBS for 5 minutes x 3 times.

[0160] Dilute nonspecific fluorescent dye DAPI (purchased from Sigma company) with PBS at a ratio of 1:1000 by volume, stain at room temperature in the dark for 5 minutes, and wash with PBS for 5 minutes x 3 times. Image under a microscope (Invitrogen EVOS M500) and take pictures for record.

[0161] Figure 5A and 5B are pictures of fluorescence under different fields of view under a 10x objective, wherein Figure 5A is a picture of staining of cell nuclei using nonspecific fluorescent dye DAPI, Figure 5B is a picture of staining using cervical cancer specific antibody p16. As shown in the figure, the position of the cell nuclei marked in Figure 5A is all stained darkly (gray part in the figure) in Figure 5B , indicating that the cells after culture are cervical cancer cells, which are consistent with the clinical pathological diagnosis.

[0162] (2) Immunohistochemical identification of primary cervical cancer tissue and cervical cancer cells after subculture

[0163] Take out a soybean-sized cancer tissue (sample number CG2) from a clinical surgical resection sample of a cervical cancer patient, and soak it in 1 mL of 4% paraformaldehyde for fixation. The remaining cancer tissue is used to obtain cervical cancer primary cells (sample number CG2) using the same method as in Example 1. The sample CG2 is continuously cultured to the fourth generation using the culture medium CM of the application using the method of Example 3.

[0164] The expression of important biomarkers related to cervical cancer in the CG2 original tissue and the primary cells obtained by culturing the cells to passage 4 was detected by immunohistochemistry. The 4% paraformaldehyde-fixed tissues were embedded in paraffin and cut into 4-μm-thick tissue sections using a microtome. Subsequently, conventional immunohistochemical detection was performed (for details, see Li et al., Nature Communication, (2018) 9:2983). The primary antibodies used were cytokeratin 7 (CK7) (purchased from CST), p16 antibody (purchased from CST), Ki67 antibody (purchased from CST), and p53 antibody (purchased from CST).

[0165] Figure 6 FIG. 4 is a comparison chart of the immunohistochemical results of the original tissue cells and the cervical cancer tumor cells obtained by culturing the cells in the culture medium CM of the present application. The results of the immunohistochemical detection of the original tissue cells and the cervical cancer tumor cells obtained by culturing the cells in the culture medium CM of the present application are shown in FIG. 4. Figure 6 It can be confirmed that the expression of the biomarkers related to cervical cancer on the cervical cancer tumor cells (sample No. CG2) cultured to passage 4 using the technology of the present application is basically consistent with the marker expression of the original tissue section of the cell source. It is shown that the cells cultured using the technology of the present application maintain the original pathological characteristics of the cancer tissue of the cervical cancer patient.

[0166] [Example 6]

[0167] Drug sensitivity function test of cervical cancer tumor cells from cancer tissue

[0168] The following is an example of a cervical cancer patient surgical resection sample to illustrate that the cervical cancer tumor cells cultured from the cervical cancer tumor sample of the patient can be used to detect the sensitivity of the patient's tumor cells to different drugs.

[0169] I. Plating of primary cervical cancer tumor cells: The suspension of the separated cervical cancer tumor cells (No. CCa5 and No. CCa9) obtained according to the method in Example 1 was plated at a density of 4 x 10 4 cells / cm 2Density seeding into 12-well plates pre-paved with gamma-ray irradiated NIH-3T3 cells. Add 2 mL of prepared cervical cancer primary cell culture medium CM to the 12-well plates and place them in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher) for culture. When the cells in the culture plate grow to about 80% of the bottom area, discard the culture supernatant in the 12-well plate, add 0.5 mL of 0.25% trypsin (purchased from Thermo Fisher) for 1 minute of digestion, then aspirate the 0.25% trypsin, and then add 0.5 mL of 0.05% trypsin for cell digestion, incubate at 37°C for 10 minutes, until the cells are completely digested under a microscope (Invitrogen EVOS M500), stop the digestion with 1 mL of DMEM / F12 culture medium containing 5% (v / v) fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and collect into a 15 mL centrifuge tube, centrifuge at 1500 rpm for 4 minutes, and discard the supernatant. Resuspend the cell pellet after centrifugation with CM medium, count using a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microbiological Technology Co., Ltd.), and obtain a total of 800,000 and 760,000 cells, respectively. Seed at a density of 2000-4000 cells / well in a 384-well plate, and allow the cells to adhere overnight.

[0170] II. Drug gradient experiment

[0171] (1) Prepare the drug storage plate using the concentration gradient dilution method: take 40 μL of 10 μM test drug stock solution as the highest concentration, and then take 10 μL from it and add to a 0.5 mL EP tube containing 20 μL of DMSO, and then take 10 μL from the above EP tube to a second 0.5 mL EP tube containing 20 μL of DMSO, i.e. dilute the drug by 1:3. Repeat the above method for sequential dilution, and finally obtain the required 7 concentrations for drug addition. Add different concentrations of drugs to the 384-well drug storage plate. Add an equal volume of DMSO to each well of the solvent control group as a control. In this example, the test drugs are anlotinib (purchased from MCE), pazopanib (purchased from MCE), apatinib (purchased from MCE), bortezomib (purchased from MCE), cisplatin (purchased from MCE), paclitaxel (purchased from MCE), 5-fluorouracil (purchased from MCE), and topotecan (purchased from MCE).

[0172] (2) Use a high-throughput automated workstation (Perkin Elmer JANUS) to add different concentrations of drugs and solvent controls in the 384-well drug storage plate to the 384-well cell culture plate paved with cervical cancer tumor cells, with 3 replicate wells for each drug group and solvent control group. Add 100 nL of drug to each well.

[0173] (3) Cell activity detection: 72 hours after drug administration, the chemiluminescence value of the cells after drug administration was detected by Cell Titer-Glo detection reagent (purchased from Promega Company), and the size of the chemiluminescence value reflected the cell activity and the influence of the drug on the cell activity. 10 μL of prepared Cell Titer-Glo detection solution was added to each well, mixed, and then the chemiluminescence value was detected by using an enzyme-labeled instrument (Perkin Elmer Envision). According to the formula cell survival rate (%) = chemiluminescence value of drug administration hole / chemiluminescence value of control hole*100%, the cell survival rate after the action of different drugs on the cells was calculated, and the Graphpad Prism software was used for plotting and calculating the half inhibition rate IC 50 .

[0174] (4) The drug sensitivity test results are shown in Figure 7 . Figure 7 indicate the sensitivity results of the cervical cancer tumor cells obtained by culturing the surgical resection cancer tissue samples (number CCa5 and number CCa9) from two different cervical cancer patients to the target drugs anlotinib, pazopanib, apatinib, bortezomib, and the chemotherapeutic drugs cisplatin, paclitaxel, 5-fluorouracil, and topotecan. The results show that the sensitivity of the cells of the same patient to different concentrations of drugs is different, and the sensitivity of the cells of different patients to the same drug is also different, and according to the results, the effectiveness of the cervical cancer patient in the clinical use of the drug can be judged.

[0175] Industrial Applicability

[0176] The present application provides a culture medium and a culture method for culturing or expanding cervical cancer primary cells in vitro, and the cells obtained by culture can be applied to the efficacy evaluation and screening of drugs. Thus, the present application is suitable for industrial application.

[0177] 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, any modification 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 culturing primary cervical cancer cells, characterized in that: Made with the following ingredients: MST1 / 2 kinase inhibitor; insulin-like growth factor 1; fibroblast growth factor 7; insulin-transferrin-selenium complex; hepatocyte growth factor; Y27632; liothyronine; an initial culture medium selected from DMEM / F12; and primocin; Wherein, the MST1 / 2 kinase inhibitor is compound 1 or a pharmaceutically acceptable salt or solvate thereof, in, The content of the MST1 / 2 kinase inhibitor is 2 to 20 μM; The content of the insulin-like growth factor 1 is 2-40 ng / ml; The content of fibroblast growth factor 7 is 2-40 ng / ml; The volume ratio of the insulin-transferrin-selenium complex to the culture medium for culturing primary cervical cancer cells is 1:100 to 1:25; The content of the hepatocyte growth factor is 2-40 ng / ml; The content of Y27632 is 2 to 20 μM; The content of liothyronine is 2-50 nM.

2. A method for culturing primary cervical cancer cells, characterized in that: The following steps are involved: (1) preparing the culture medium according to claim 1; (2) Pre-plating culture vessels with trophoblast cells irradiated with X-rays or gamma rays; (3) Cervical cancer primary cells isolated from cervical cancer tissue are inoculated into a culture dish pre-coated with trophoblast cells, and cultured using the culture medium in step (1).

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

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