Culture medium and culture method for primary cervical cancer cells

Abstract

The present invention provides a culture medium for culturing primary cervical cancer epithelial cells, comprising an MST1 / 2 kinase inhibitor, a ROCK kinase inhibitor, fibroblast growth factor 7, at least one of a B27 additive and an N2 additive, hepatocyte growth factor, insulin-like growth factor 1, CHIR99021, and a TGFβ type I receptor inhibitor. The present invention also relates to a culture method using the primary cell culture medium and its application. The culture method uses the primary cell culture medium to culture primary cells on a culture vessel coated with extracellular matrix gel, resulting in rapid proliferation of the primary cells. The cultured cells can be used to evaluate or screen drugs for treating cervical cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a culture medium and a culture method for culturing or amplifying primary cervical cancer epithelial cells in vitro. Background Art

[0002] Cervical cancer ranks fourth in both incidence and mortality among female malignancies worldwide, and in developing countries it is the second most common malignant tumor, after breast cancer. Worldwide, 500,000 people develop the disease annually, with 130,000 cases in my country, and the age of onset has been trending younger in recent years. Surgery, radiotherapy, and chemotherapy are currently established treatments for cervical cancer. However, satisfactory results have not been achieved for some patients with locally advanced cervical cancer. With the deepening of research on molecular targets for cervical cancer, new targeted therapies are emerging, offering new hope for patients. However, currently, there are only a limited number of targeted therapies with proven efficacy for cervical cancer, and their long-term effects and side effects require further investigation. In addition to genetic testing for the selection of targeted drugs, in vitro culture of primary cells from cervical cancer patient samples has become an important tool for predicting in vitro efficacy and guiding clinical drug use. However, the rapid generation of primary cervical cancer cells in vitro remains a pressing technical challenge.

[0003] Functional testing refers to in vitro testing of the sensitivity of cancer patient cells to anti-tumor drugs. The key to applying this approach lies in developing tumor cell models with a short growth cycle and that represent the biological characteristics of cervical cancer patients themselves. Furthermore, such cell models should be easy to operate and able to quickly and efficiently predict the efficacy of clinical drugs, thereby providing timely and precise medication guidance for cancer patients. However, the success rate of establishing in vitro cell models using primary tumor cells from cancer patients is often low, with long growth cycles and problems such as excessive proliferation of stromal cells such as fibroblasts, which hinder the development of this field. Currently, two technologies for culturing primary epithelial cells / stem cells have developed relatively mature applications in tumor cell functional testing. One is the use of irradiated feeder cells and the ROCK kinase inhibitor Y27632 to promote the growth of primary epithelial cells to assess drug sensitivity in individual patients, namely, cell conditional reprogramming technology (Liu et al., Am J Pathol, 180:599-607, 2012). Another technology is to culture adult stem cells in 3D in vitro to obtain organoids similar to tissues and organs (Hans Clevers et al., Cell, 11, 172(1-2): 373-386, 2018).

[0004] However, both technologies have certain limitations. Cell reprogramming technology is a technology that co-cultures the patient's autologous primary epithelial cells with mouse-derived feeder cells. When the patient's primary cells are tested for drug sensitivity, the presence of these mouse-derived cells will interfere with the drug sensitivity test results of the patient's autologous primary cells; but if the mouse-derived feeder cells are removed, the patient's autologous primary cells will be separated from the reprogramming environment, and the cell proliferation rate and intracellular signaling pathways will undergo significant changes (Liu et al., Am J Pathol, 183(6): 1862-1870, 2013; Liu et al., Cell Death Dis., 9(7): 750, 2018), thereby greatly affecting the response of the patient's autologous primary cells to drugs. Organoid technology is a technology that embeds the patient's autologous primary epithelial cells in an extracellular matrix for in vitro three-dimensional culture. This technology does not require feeder cells, so there is no interference problem with mouse-derived feeder cells. However, the culture medium of organoid technology requires the addition of a variety of specific growth factors (such as Wnt protein and R-spondin family protein), which is expensive and not suitable for widespread clinical application. In addition, the cells of organoids need to be embedded in extracellular matrix gel throughout the culture process. The cell inoculation, passage and plating steps of drug sensitivity testing are cumbersome and time-consuming compared to 2D culture operations. Moreover, the size of the organoids formed by this technology is difficult to control, and some organoids are prone to grow too large and cause internal necrosis. Therefore, compared with 2D culture technology, organoid technology is less operable and applicable, requires professional technicians to operate, and is not suitable for large-scale and widespread application in clinical in vitro drug sensitivity testing (Nick Barker, Nat Cell Biol, 18(3): 246-54, 2016).

[0005] Given the limitations of these technologies, clinical development of a primary cervical cancer epithelial cell culture technology is needed, characterized by a short culture cycle, manageable costs, convenient operation, and immunity to interference from exogenous cells. When this technology is applied to construct primary cervical cancer tumor cell models, the cultured cervical cancer tumor cells can represent the biological characteristics of the cervical cancer patient. By evaluating the sensitivity of anti-tumor drugs in vitro in cell models derived from different individual cancer patients, the response rate of clinical anti-tumor drugs can be improved, reducing the pain caused to patients and the waste of medical resources caused by inappropriate drugs. Summary of the Invention

[0006] The present invention aims to address the shortcomings of existing technologies by providing a culture medium for culturing primary cervical cancer epithelial cells and a method for culturing primary cervical cancer epithelial cells using the culture medium. Using the culture medium and culture method for primary cervical cancer epithelial cells of the present invention for cell culture can achieve the goals of a short in vitro culture cycle, controllable costs, convenient operation, and no interference from exogenous cells. When this technology is applied to construct a primary cervical cancer tumor cell model, primary cervical cancer tumor cells with the biological characteristics of cervical cancer patients can be obtained, and can be used for new drug screening and in vitro drug sensitivity testing.

[0007] One aspect of the present invention is to provide a primary cell culture medium for culturing primary cervical cancer epithelial cells, which contains an MST1 / 2 kinase inhibitor; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152; fibroblast growth factor 7 (FGF7); at least one additive selected from B27 additive and N2 additive; hepatocyte growth factor (HGF); insulin-like growth factor 1 (IGF-1); CHIR99021; and at least one TGFβ type I receptor inhibitor selected from A83-01, SB431542, Repsox, SB505124, SB525334, SD208, LY36494, and SJN2511, wherein the MST1 / 2 kinase inhibitor includes a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.

[0008]

[0009] in,

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

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

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

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

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

[0015]

[0016] in,

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

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

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

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

[0021]

[0022]

[0023]

[0024]

[0025]

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

[0027] In an embodiment of the present invention, the content of the MST1 / 2 kinase inhibitor in the culture medium is generally 2 μM to 20 μM, preferably 5 μM to 20 μM.

[0028] In another embodiment, the ROCK kinase inhibitor is preferably Y27632. In another embodiment, the content of the ROCK kinase inhibitor in the culture medium is generally 2 μM to 20 μM, preferably 10 μM.

[0029] In a preferred embodiment, the content of the fibroblast growth factor 7 is 2ng / ml to 40ng / ml, more preferably 10ng / ml to 40ng / ml; the volume concentration of the B27 additive or the N2 additive in the culture medium is 1:25 to 1:100, more preferably 1:50 to 1:100; the content of the hepatocyte growth factor is 2ng / ml to 40ng / ml, more preferably 10ng / ml to 40ng / ml; the content of the insulin-like growth factor 1 is 2ng / ml to 40ng / ml, more preferably 10ng / ml; the content of the CHIR99021 is 1μM to 10μM, more preferably 1μM to 3μM; the TGFβ type I receptor inhibitor is preferably A83-01, and the content of the TGFβ type I receptor inhibitor is 50nM to 500nM, more preferably 100nM to 500nM.

[0030] The culture medium formulation of the present invention further comprises 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 some embodiments, the initial culture medium is preferably DMEM / F12, and the antibiotic is preferably primocin. In further preferred embodiments, the primocin content in the culture medium is 25 to 400 μg / mL, preferably 50 to 200 μg / mL.

[0031] Compared with the components of the cell conditional reprogramming medium and the cervical cancer epithelial cell organoid culture medium, the culture medium formula of the present invention adds MST1 / 2 kinase inhibitors, does not contain uncertain components such as serum and bovine pituitary extract, does not contain niche factors necessary for organoid culture such as Wnt agonists, R-spondin family proteins, and BMP inhibitors, and does not contain nicotinamide and N-acetylcysteine, thereby greatly reducing the cost of the culture medium, simplifying the operational process of preparing the culture medium, and realizing the in vitro culture of primary cervical cancer epithelial cells with controllable cost and convenient operation.

[0032] In the present invention, the primary cervical cancer epithelial cells can be cervical cancer tumor cells, normal cervical cancer epithelial cells, or cervical cancer epithelial stem cells.

[0033] One aspect of the present invention is to provide a method for culturing primary cervical cancer epithelial cells, comprising the following steps:

[0034] (1) Prepare the primary cell culture medium of the present invention according to the above formula.

[0035] (2) Coat the culture vessel with extracellular matrix gel dilution solution.

[0036] Specifically, the extracellular matrix glue uses a low growth factor type extracellular matrix glue, for example, commercially available Matrigel (purchased from Corning) or BME (purchased from Trevigen) can be used. More specifically, the extracellular matrix glue is diluted with serum-free culture medium, and the culture medium can be DMEM / F12 (purchased from Corning). The dilution ratio of the extracellular matrix glue is 1:50-1:400, preferably 1:100-1:200. The coating method is to add the diluted extracellular matrix glue to the culture vessel so that it completely covers the bottom of the culture vessel, and let it stand for more than 30 minutes, preferably at 37°C. The coating time is preferably 30 to 60 minutes. After the coating is completed, the excess extracellular matrix glue dilution is discarded and the culture vessel is ready for use.

[0037] (3) Primary cervical cancer epithelial cells were isolated from cervical cancer tissue.

[0038] Primary cervical cancer epithelial cells can be derived from, for example, cervical cancer surgical specimens or biopsy specimens. Cervical cancer surgical specimens can be derived from, for example, cancer tissue samples removed from cervical cancer patients who have been instructed and consented to undergo surgery. The above tissue samples are collected within half an hour after the patient's surgical resection or biopsy. More specifically, under a sterile environment, tissue samples are cut from non-necrotic areas with a volume of 0.5 cm 3 The tissue transport medium is then placed in 10-15 mL of pre-chilled DMEM / F12 medium in a sterile, plastic centrifuge tube with a lid and transported to the laboratory on ice. The DMEM / F12 medium contains an MST1 / 2 kinase inhibitor of the present invention (e.g., Compound 1) and 0.2-0.4% by volume of Primocin (hereinafter referred to as tissue transport medium). When the MST1 / 2 kinase inhibitor of the present invention is used, the concentration range is 2 μM to 20 μM, preferably 3 μM to 5 μM. When Primocin is used, the concentration range is 25 to 400 μg / mL, preferably 50 to 200 μg / mL, and more preferably 100 μg / mL.

[0039] In the biosafety cabinet, transfer the tissue sample to a cell culture dish, rinse the tissue sample with tissue transport fluid, and wash away the blood cells on the surface of the tissue sample. Transfer the rinsed tissue sample to another new culture dish, add 1-3mL of tissue transport fluid, and use sterile surgical blades and surgical forceps to cut the tissue sample into pieces smaller than 3mm. 3 of tissue fragments.

[0040] Transfer the tissue sample fragments into a centrifuge tube and centrifuge at 1000-3000 rpm for 3-5 minutes using a tabletop centrifuge (Sigma 3-18K). Discard the supernatant and add tissue transport solution and tissue digestion solution in a 1:1 ratio (the usage amount is about 5 mL of tissue digestion solution for every 10 mg of tissue. The preparation method of tissue digestion solution is as follows: 1-2 mg / mL collagenase II, 1-2 mg / mL collagenase IV, 50-100 U / mL deoxyribonucleic acid I, 0.5-1 mg / mL hyaluronidase, 0.1-0.5mg / mL calcium chloride, 5-10mg / mL bovine serum albumin dissolved in HBSS and RPMI-1640 with a volume ratio of 1: 1), mark the sample number, seal the tube with a sealing film, and digest it at 37 ° C, 200-300 rpm constant temperature shaker (Zhichu Instrument ZQLY-180N). Observe whether the digestion is complete every 1 hour; if no obvious tissue block is seen, the digestion can be terminated, otherwise the digestion is continued until the digestion is sufficient. The digestion time range is 4-8 hours. After the digestion is completed, the cell strainer (cell sieve pore size is, for example, 70 μm) is used to filter out the undigested tissue agglomerates, the tissue agglomerates on the filter are rinsed with tissue transport fluid, and 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 was discarded and the remaining cell mass was observed to see if it contained blood cells. If so, 3-5 mL of blood cell lysis buffer (purchased from Sigma) was added, mixed, and lysed at 4°C for 10-20 minutes, shaking and mixing once every 5 minutes. After lysis, the cells were removed and centrifuged at 1000-3000 rpm for 3-5 minutes. The supernatant was discarded, the primary cell culture medium of the present invention was added and resuspended, and the total number of cells was obtained by counting using a flow cytometer (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL).

[0041] (4) The primary cervical cancer epithelial cells isolated in step (3) are inoculated into the coated culture vessel and cultured using the primary cell culture medium in step (1).

[0042] More specifically, 2 × 10 4 ~8×10 4 pieces / cm 2 (For example 4×10 4 pieces / cm 2 ) and inoculate primary cervical cancer tumor cells at a density of 100 μg / mL. Add an appropriate amount, such as 2-3 mL, of primary epithelial cell culture medium and culture in a cell culture incubator at, for example, 37°C and 5% CO2 for 8-16 days. During this period, replace the culture medium with fresh primary cell culture medium every 4 days. Digest and passage the primary cervical cancer epithelial cells when they grow to a cell density that occupies approximately 80% to 90% of the bottom area of ​​the multi-well plate.

[0043] This inoculation step does not require the use of feeder cells, and compared to cell conditional reprogramming technology, it eliminates the steps of culturing and irradiating feeder cells. Compared to organoid technology, this step also does not require mixing primary cells and matrix gel on ice to form gel droplets, and then waiting for the gel droplets to solidify before adding culture medium. Pre-coated culture vessels can be directly used for primary cell inoculation. In addition, only a small amount of diluted extracellular matrix gel is required to coat the culture vessels. Compared to organoid technology, this saves the use of expensive extracellular matrix gel and simplifies the operation steps.

[0044] Optionally, after culturing the inoculated primary cervical cancer epithelial cells for 8 to 16 days, when the cell clones formed in the culture container are confluent to 80% of the bottom area, the supernatant is discarded, and 0.5 to 2 mL of 0.05% trypsin (purchased from Thermo Fisher) is added for cell digestion, and the cells are incubated at room temperature for 5 to 20 minutes; then, the digested cells are resuspended in 1 to 4 mL of DMEM / F12 culture medium containing, for example, 5% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and the cells are centrifuged at 1000 to 3000 rpm for 3 to 5 minutes. The digested single cells are resuspended in the primary cell culture medium of the present invention, and the resulting cell suspension is placed in a T25 cell culture flask coated with extracellular matrix glue for further expansion and culture. The coating operation of the T25 cell culture flask is the same as step (2).

[0045] The expanded cervical cancer epithelial cells grow in 2D, avoiding the uneven size of organoids and internal necrosis of overgrown organoids that occur when organoid technology is expanded.

[0046] The present invention also provides a method for evaluating or screening drugs for treating cervical cancer, comprising the following steps:

[0047] (1) Culturing cervical cancer epithelial cells using the culturing method of primary cervical cancer epithelial cells of the present invention;

[0048] (2) Select the drug to be tested and dilute it according to the required concentration gradient;

[0049] (3) adding the diluted drug to the cervical cancer epithelial cells cultured in (1);

[0050] (4) Conduct cell activity test.

[0051] The beneficial effects of the present invention include:

[0052] (1) Improve the success rate of primary cervical cancer epithelial cell culture to over 80%;

[0053] (2) Ensure that the primary cultured cervical cancer epithelial cells in vitro can maintain the pathological phenotype and heterogeneity of the patient from whom the primary cells were derived;

[0054] (3) The cultured primary cervical cancer epithelial cells are not interfered with by fibroblasts, and purified cervical cancer epithelial cells can be obtained;

[0055] (4) The culture medium does not contain serum and is therefore not affected by the quality and quantity of serum from different batches;

[0056] (5) The efficiency of amplifying cervical cancer epithelial cells is high, as long as there are 10 4 The number of cells can be successfully expanded to 10 in about two weeks. 6 The amplified cervical cancer epithelial cells can be continuously passaged.

[0057] (6) The cell culture process does not require ice operation or dissociation of the matrix gel, and cell digestion and cell culture can be completed within 10-15 minutes;

[0058] (7) The culture cost is controllable. The primary cervical cancer cell culture medium does not need to add expensive Wnt agonists, R-spondin family proteins, BMP inhibitors and other factors. It is a simplification and improvement of the existing primary cervical cancer epithelial cell or organoid culture medium. Cell inoculation does not require the use of a high concentration of extracellular matrix to mix with primary cells to form gel droplets. Instead, only a small amount of diluent prepared by extracellular matrix glue is needed, which saves the amount of expensive extracellular matrix.

[0059] (8) Easy operation. Compared with conditional reprogramming technology, this technology does not require the culture of feeder cells and the radiation of feeder cells, thus avoiding the problem that the quality and quantity of different batches of feeder cells affect the efficiency of primary cell culture. The objects of drug screening and detection are only primary cervical cancer epithelial cells, without interference from feeder cells in the co-culture system required by cell conditional reprogramming technology. Compared with organoid technology, the extracellular matrix gel coating method adopted in the present invention allows the culture vessels to be prepared in advance, without the need to embed cells in matrix gel as in organoid technology. The operation steps of the technology are simple and easy.

[0060] (9) The cervical cancer epithelial cells cultured using the technology are large in number and highly homogenized, making them suitable for high-throughput screening of new candidate compounds and providing patients with high-throughput in vitro drug sensitivity functional testing.

[0061] The cell culture medium of this embodiment can be used to culture cervical cancer epithelial cells derived from humans or other mammals, including cervical cancer tumor cells, normal cervical epithelial cells, cervical cancer epithelial stem cells, or tissues containing at least one of these cells. The culture medium of the present invention can also be used to develop a kit for in vitro expansion and culture of primary cervical cancer cells.

[0062] Furthermore, the cells obtained by the culture method of this embodiment can be applied to regenerative medicine, basic medical research on cervical cancer epithelial cells, screening of drug responses, and development of new drugs for cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figures 1A-1H This is a graph showing the effect of the concentration of each added factor on the proliferation of primary cervical cancer cells.

[0064] Figure 2A and 2B The photographs are taken under an inverted microscope of cervical cancer tumor cells isolated from a clinical tissue sample of cervical cancer, cultured for 4 days and 12 days respectively using the culture medium FCM of the present invention.

[0065] Figures 3A-3C These are photos taken under an inverted microscope of cells isolated from a surgical resection specimen of cervical cancer after being cultured under three different culture medium conditions for 15 days.

[0066] Figure 4 This is a comparison of the cell proliferation effects of cells isolated from 9 cervical cancer surgical resection specimens after culturing for 16 days under three different culture medium conditions.

[0067] Figure 5 This is a comparison chart of cell growth curves obtained by culturing cells isolated from a clinical tissue sample of cervical cancer using three different culture medium conditions.

[0068] Figure 6 This is a comparison chart of immunohistochemical results of cervical cancer tumor cells obtained by culturing cells isolated from a cervical cancer surgical resection specimen using the culture medium FCM of the present invention.

[0069] Figure 7 The cell activity curves of cervical cancer epithelial cells isolated from 3 cervical cancer surgical resection specimens and cultured according to the method of the present invention under the influence of 8 different drugs are shown. DETAILED DESCRIPTION

[0070] In this specification, epithelial cells include differentiated epithelial cells and epithelial stem cells obtained from epithelial tissue. "Epithelial stem cells" refers to cells with long-term self-renewal ability and differentiation into epithelial cells, and refers to stem cells derived from epithelial tissue. As epithelial tissue, for example, cervix, cornea, oral mucosa, skin, conjunctiva, bladder, renal tubules, 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. can be cited. Among them, the cell culture medium of the present embodiment is preferably a culture medium for culturing epithelial cells derived from cervical epithelial cells.

[0071] In addition, in this specification, "epithelial tumor cells" refer to cells derived from the above-mentioned epithelial tissues that have become tumors.

[0072] As used herein, “organoid” refers to a three-dimensional, organ-like cellular tissue formed by spontaneously organizing and aggregating cells at high density in a controlled space.

[0073] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0074] 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.

[0075] 1. Preparation of MST1 / 2 kinase inhibitor compound 1

[0076] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide 1

[0077]

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 2. Preparation of other MST1 / 2 inhibitor compounds of the present invention

[0084] 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.

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] [Example 1]

[0091] Isolation of primary human cervical cancer epithelial cells

[0092] Cervical cancer tissue samples were obtained from surgically removed cervical cancer tissue samples from patients who provided information and consent. The following example uses one sample (number CCa2) for illustration.

[0093] The above tissue samples are collected within half an hour after the patient's surgical resection or biopsy. More specifically, under a sterile environment, tissue samples are cut from non-necrotic areas with a volume of 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).

[0094] Table 1 Tissue transport fluid formula

[0095] Tissue transport fluid components supplier Final concentration DMEM / F12 Corning 99.8% by volume Primocin Invivogen 0.2 volume % (commercial product concentration 50 mg / ml) Compound 1 self made 3μM

[0096] Table 2 Tissue digestion solution formula

[0097] Tissue digestive fluid components supplier Final concentration HBSS Gibco 50% (volume) RPMI-1640 Corning 50% (volume) Collagenase II Sigma 2mg / mL Collagenase IV Sigma 2mg / mL DNA I Sigma 50 U / mL Hyaluronidase Sigma 0.5mg / mL calcium chloride Shanghai Bioengineering 0.33 mg / mL Bovine serum albumin Shanghai Bioengineering 10 mg / mL

[0098] In a biosafety cabinet, transfer the tissue sample (No. CCa2) to a 100mm cell culture dish (purchased from NEST). Rinse the tissue sample with tissue transport fluid to remove residual blood on the surface of the tissue sample and remove excess tissue such as fat on 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.

[0099] 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 is used for every 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.

[0100] 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.

[0101] The supernatant was discarded and the remaining cell mass was observed to see if it contained blood cells. If so, 3 mL of blood cell lysis solution (purchased from Sigma) was added, mixed, and lysed at 4°C for 15 minutes. The cells were shaken and mixed once for 5 minutes. After the lysis was completed, the cells were taken out and centrifuged at 1500 rpm for 4 minutes. The supernatant was discarded to obtain the primary cervical cancer cells after digestion and separation. The cells were resuspended in basal medium (BM), wherein the basal medium was a commercially available DMEM / F-12 medium with 0.2% by volume of Primocin (purchased from Invivogen at a concentration of 50 mg / mL) added to obtain a final concentration of 100 μg / mL. A flow cytometer (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL) was used for counting, and the total number of cells was 1.62 million.

[0102] [Example 2]

[0103] Optimization of culture medium for primary cervical cancer epithelial cells

[0104] (1) The effects of different factors

[0105] Extracellular matrix glue Prepare an extracellular matrix diluent (BD Biosciences) using serum-free DMEM / F12 medium at a 1:100 ratio. Add 500 μl / well of the extracellular matrix diluent to a 48-well culture plate, completely covering the bottom of the wells. Incubate in a 37°C incubator for 1 hour. After 1 hour, remove the extracellular matrix diluent to obtain a Matrigel-coated culture plate.

[0106] Preparation of basal medium (abbreviated as BM): 0.2% by volume of Primocin (purchased from Invivogen, concentration of 50 mg / mL) was added to commercially available DMEM / F-12 medium to obtain a final concentration of 100 μg / mL to prepare BM.

[0107] Next, different types and concentrations of additive factors (Table 3) were added to the basal medium (BM) to prepare cervical cancer epithelial cell culture media containing different additive components.

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

[0109]

[0110]

[0111] The cervical cancer tumor cells (numbered CCa5) obtained by separating from cervical cancer tissue according to the same method as in Example 1 were seeded in a 384-well culture plate at a cell density of 3000 cells / well, and culture media of different components were added at 50 μl / well. After surface disinfection, the cells were placed in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific) to culture the same number of freshly isolated cervical cancer tumor cells (numbered CCa5) under different culture medium formulation conditions. After 6 days of culture, CCK8 detection reagent (purchased from MCE) was added at 5 μl / well, placed in an incubator and incubated for 2-4 hours, and the absorbance at OD450 was measured using a multifunctional microplate reader (purchased from PE). Among them, as an experimental control, a basal medium (BM) without any additives was used. The results are shown in Table 3. The ratio refers to the ratio of the absorbance obtained by adding CCK8 to the absorbance obtained by culturing in different culture media and the absorbance obtained by culturing in the basal medium BM. As shown in the table, adding different factors in Table 3 to BM produces different effects on cell proliferation. Among them, within a specific concentration range, B27 additive, N2 additive, fibroblast growth factor 7, CHIR99021, hepatocyte growth factor, insulin-like growth factor 1, compound 1, Y27632 and A83-01 all had a more significant effect on promoting cell proliferation.

[0112] (2) Effects of different concentrations of added factors on the proliferation of primary cervical cancer cells obtained in this patent

[0113] Extracellular matrix glue ( Prepare an extracellular matrix diluent (BD Biosciences) by diluting serum-free DMEM / F12 medium at a ratio of 1:100. Add 200 μl / well of the extracellular matrix diluent to a 48-well culture plate, completely covering the bottom of the wells. Incubate in a 37°C incubator for 1 hour. After 1 hour, remove the extracellular matrix diluent to obtain a Matrigel-coated culture plate.

[0114] The primary cervical cancer epithelial 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 40 ng / ml, hepatocyte growth factor (HGF) was added to a final concentration of 40 ng / ml, insulin-like growth factor 1 (IGF-1) was added to a final concentration of 40 ng / ml, B27 additive was added to a final concentration of 1:50 volume ratio, compound 1 was added to a final concentration of 5 μM, Y27632 was added to a final concentration of 10 μM, TGFβ1 inhibitor A83-01 was added to a final concentration of 500 nM, and CHIR99021 was added to a final concentration of 10 μM to prepare the primary cervical cancer epithelial cell culture medium.

[0115] The same method as in Example 1 was used to isolate cervical cancer epithelial cells from the cancer tissue of a cervical cancer patient (No. CCa8). Next, the cervical cancer epithelial cells from the cancer tissue were counted using a flow cytometer (Jiangsu Zhuowei Biotechnology Co., Ltd. JIMBIO FIL) to obtain the total number of cells. Then, 4×10 4 pieces / cm 2 Density inoculation to 2 mL of prepared primary cervical cancer epithelial cell culture medium (purchased from BD Biosciences) was added to the 48-well plate and cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific). When the cells reached approximately 80% of the bottom area of ​​the culture plate, the supernatant was discarded from the 48-well plate and the cells were digested with 500 μL of 0.05% trypsin (purchased from Gibco). The cells were incubated at 37°C for 10 minutes until complete digestion was observed under a microscope (Invitrogen EVOS M500). Digestion was terminated with 1 mL of DMEM / F12 culture medium containing 5% (v / v) fetal bovine serum (purchased from Ecosine), 100 U / mL penicillin (purchased from Corning), and 100 μg / mL streptomycin (purchased from Corning). The cells were collected into a 15 mL centrifuge tube and centrifuged at 1500 rpm for 4 minutes, and the supernatant was discarded. The cell pellet after centrifugation was resuspended in basal medium (BM) and counted using a flow cytometer (Jiangsu Zhuowei Microbiology Technology Co., Ltd., JIMBIO FIL) to obtain the total cell count. The resulting cells were used in the following culture experiments.

[0116] Next, the following 8 culture media were prepared for the experiment:

[0117] Formulation 1: The above medium components do not contain B27 supplement;

[0118] Formulation 2: The above culture medium components do not contain fibroblast growth factor 7;

[0119] Recipe 3: The above culture medium components do not contain insulin-like growth factor 1;

[0120] Recipe 4: The above culture medium components do not contain hepatocyte growth factor;

[0121] Formulation 5: The above culture medium components do not contain Y27632;

[0122] Formulation 6: The above culture medium components do not contain compound 1;

[0123] Formulation 7: The above culture medium components do not contain A83-01;

[0124] Recipe 8: The above culture medium components do not contain CHIR99021.

[0125] The above-mentioned formulas 1 to 8 were used to dilute the above-mentioned digested cell suspension, respectively, and 10,000 cells were seeded into a 48-well plate in a volume of 250 μl per well.

[0126] When using the medium of Formula 1, add 250 μl of the prepared B27 additive to each well of a 48-well plate seeded with primary cells, with the final concentrations of B27 additive being 1:100, 1:50, and 1:25, respectively; and set up control wells (BC) using the medium of Formula 1.

[0127] When using the culture medium of Formula 2, 250 μL of prepared fibroblast growth factor 7 was added to each well of a 48-well plate seeded with primary cells, with final concentrations of fibroblast growth factor 7 of 40 ng / mL, 10 ng / mL, and 2 ng / mL, respectively; and control wells (BC) were set up using the culture medium of Formula 2.

[0128] When using the culture medium of Formula 3, 250 μL of prepared insulin-like growth factor 1 was added to each well of a 48-well plate seeded with primary cells, with final concentrations of insulin-like growth factor 1 of 40 ng / mL, 10 ng / mL, and 2 ng / mL, respectively; and control wells (BC) were set up using the culture medium of Formula 3.

[0129] When using the culture medium of Formula 4, 250 μL of prepared hepatocyte growth factor was added to each well of a 48-well plate seeded with primary cells, with final concentrations of hepatocyte growth factor of 40 ng / mL, 10 ng / mL, and 2 ng / mL, respectively; and control wells (BC) were set up using the culture medium of Formula 4.

[0130] When using the culture medium of Formula 5, 250 μL of the prepared Y27632 was added to each well of a 48-well plate seeded with primary cells, with the final concentrations of Y27632 being 20 μM, 10 μM, and 2 μM, respectively; and control wells (BC) were set up using the culture medium of Formula 5.

[0131] When using the culture medium of Formula 6, 250 μL of prepared compound 1 was added to each well of a 48-well plate seeded with primary cells, with final concentrations of compound 1 of 20 μM, 5 μM, and 2 μM, respectively; and control wells (BC) were set using the culture medium of Formula 6.

[0132] When using the medium of Formula 7, 250 μL of prepared A83-01 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of A83-01 were 500 nM, 100 nM, and 50 nM, respectively. Control wells (BC) were set up using the medium of Formula 7.

[0133] When using the culture medium of Formula 8, 250 μL of prepared CHIR99021 was added to each well of a 48-well plate seeded with primary cells, with final concentrations of CHIR99021 of 10 μM, 3 μM, and 1 μM, respectively; and control wells (BC) were set up using the culture medium of Formula 8.

[0134] When the cells are expanded to about 85% of the 48 wells, the digestion counts are calculated and the ratios are calculated with reference to the cell numbers in the control wells (BC). The results are shown in Figures 1A to 1H . Figures 1A to 1H The ratio is the ratio of the number of cells cultured in each culture medium for one generation to the number of cells cultured in the corresponding control well for one generation. A ratio greater than 1 indicates that the culture medium containing the factor or small molecule compound at different concentrations promotes cell proliferation more effectively than the culture medium in the control well. A ratio less than 1 indicates that the culture medium containing the factor or small molecule compound at different concentrations promotes cell proliferation less effectively than the culture medium in the control well.

[0135] according to Figures 1A to 1H The results show that the volume concentration of B27 additive in the culture medium is preferably 1:25 to 1:100, more preferably 1:50 to 1:100; the content of fibroblast growth factor 7 is preferably 2 ng / ml to 40 ng / ml, more preferably 10 ng / ml to 40 ng / ml; the content of insulin-like growth factor 1 is preferably 2 ng / ml to 40 ng / ml, more preferably 10 ng / ml to 40 ng / ml; the content of hepatocyte growth factor is preferably 2 ng / ml to 40ng / ml, more preferably 10ng / ml~40ng / ml; the content of Y27632 is preferably 2μM~20μM, more preferably 2μM~10μM; the content of compound 1 is preferably 2μM~20μM, more preferably 5μM~20μM; the content of A83-01 is preferably 50nM~500nM, more preferably 100nM~500nM; the content of CHIR99021 is preferably 1μM~10μM, more preferably 1μM~3μM.

[0136] According to the preferred concentrations of the above components, the preferred culture medium formula FCM of the present invention is prepared, 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 B27 additive, 5 μM compound 1, 10 μM Y27632, 500 nM A83-01, and 3 μM CHIR99021.

[0137] [Example 3]

[0138] Culture of primary cervical cancer cells derived from cervical cancer tissue

[0139] The same method as in Example 1 was used to isolate cervical cancer epithelial cells from the cancer tissue of a cervical cancer patient (sample number CCa15). The cervical cancer epithelial cells were then 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 Density inoculation to 2 mL of prepared primary cervical cancer epithelial cell culture medium FCM was added to the 12-well plate and cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific).

[0140] Figure 2A This embodiment is based on 4×10 4 pieces / cm 2 Microscopic photographs (photographed using a 10x inverted phase-contrast microscope) of cells seeded at high density into Matrigel-coated 6-well plates on day 4 of culture from initiation of inoculation showed that the cultured primary cervical cancer cells, derived from cancerous tissue, were of high purity and lacked fibroblasts. Figure 2B This is a photo taken on the 12th day after inoculation (photographed with a 40x inverted phase contrast microscope). 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 cervical cancer epithelial cells in vitro.

[0141] [Example 4]

[0142] Effects of different culture media on the proliferation of primary cervical cancer cells derived from cervical cancer tissue

[0143] (1) Comparison of the effects of different culture media on primary cell clone formation and proliferation

[0144] The primary cervical cancer epithelial cell culture medium FCM and the basal culture medium BM as a control were prepared using the same method as in Example 2. In addition, the literature culture medium RM was prepared as another control example. The preparation steps are shown in (Ma Liping et al., Jilin Medicine, 42(6):1289-1293, 2021). The culture medium formula is shown in Table 4.

[0145] Table 4 Literature culture medium (RM) composition

[0146] Culture medium components supplier Final concentration DMEM / F12 medium Corning 100% by volume Fibroblast Growth Factor Beijing Yiqiao 10ng / ml Epidermal Growth Factor Beijing Yiqiao 20ng / ml

[0147] 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:

[0148] A. The present invention's technology: According to 4×10 4 pieces / cm 2 Primary cervical cancer cells were inoculated at a density of 2 mL of the primary cervical cancer epithelial cell culture medium FCM of the present invention was used to culture the cells in a 24-well plate coated with BD Biosciences.

[0149] B. Press 4×10 4 pieces / cm 2 Primary cervical cancer cells were inoculated at a density of The cells were cultured in 24-well plates coated with 2 mL of RM culture medium (manufactured by BD Biosciences).

[0150] C. Press 4×10 4 pieces / cm 2 Primary cervical cancer cells were inoculated at a density of The cells were cultured in a 24-well plate coated with 2 mL of basal medium (BM) in the 24-well plate.

[0151] In the three cultures described above, the medium was changed every 4 days for the cells cultured under the three culture conditions. The clone formation and cell proliferation of cells cultured in each culture medium in the 24-well plate were observed simultaneously, and the cell growth status was recorded by taking photos using a microscope (Invitrogen EVO SM500).

[0152] For primary cervical cancer tumor cells (numbered CCa10) cultured using the technology of the present invention, when the cell growth in the culture plate reached about 80% of the bottom area, the culture supernatant in the 24-well plate was discarded, 500 μL of 0.05% trypsin (purchased from GIBCO) was added to digest the cells, and incubated at 37°C for 10 minutes until the cells could be observed to be completely digested under a microscope (Invitrogen EVOSM500). 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, and the cells were collected into a 15 mL centrifuge tube and 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 invention and counted using a flow cytometer (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL), resulting in a total cell count of 190,000. The cells cultured under the other two culture conditions were digested and counted using the same procedures as above. The total number of cells obtained by culturing using culture medium FM and culture medium CM was 45,000 and 46,000, respectively.

[0153] Figures 3A-3C The cell photos are taken under a microscope (10x inverted phase contrast microscope) of sample number CCa10 on the 15th day after culture under three different culture conditions: Figure 3A This is a microscopic photograph of CCa10 cultured in basal medium BM on day 15; Figure 3B This is a microscopic photograph of CCa10 cultured using our patented culture medium FCM on day 15; Figure 3C This is a microscopic photograph of CCa10 cultured on day 15 using the literature culture medium RM. As can be seen from the figure, the sample CCa10 was cultured using the basic culture medium BM ( Figure 3A ) could not form cell clones after 15 days of culture; using the literature culture medium RM ( Figure 3C ) could not form cell clones after 15 days of culture, and the cell state was poor; using the culture medium FCM ( Figure 3B ) After 15 days of culture, cells formed clones and the proliferation-promoting effect was obvious.

[0154] Figure 4 This is a comparison of the cell proliferation effects of primary cervical cancer cells obtained from 9 cervical cancer patient samples according to the method of Example 1 after culturing for 16 days under the above three different culture medium conditions, where √ represents general clone formation ability and proliferation promoting effect, √√ represents relatively obvious clone formation ability and proliferation promoting effect, √√√ represents relatively strong clone formation ability and proliferation promoting effect, and × represents no clone formation. Figure 6It can be confirmed that the culture medium of the present invention has obvious advantages over the other two culture conditions in terms of clone formation ability, cell proliferation promoting effect, and culture success rate when culturing primary cells obtained from cervical cancer tissue.

[0155] (2) Continuous culture of primary cervical cancer cells using different culture media and plotting of growth curves

[0156] The same method as in Example (1) was used to obtain the primary cervical cancer epithelial cell culture medium FCM, as well as the culture media BM and RM as controls.

[0157] The same method as in Example (1) was used to culture primary cervical cancer tumor cells (numbered CCa5) derived from cervical cancer tissue under three culture medium conditions, and digested, passaged, and counted.

[0158] When the cells after passage grow to about 80% of the bottom area of ​​the culture plate again, the cells obtained by culture are digested and collected again according to the above operation method and counted. 4 The cells were seeded at a density of 100 cells / well and cultured continuously.

[0159] The following is the calculation formula for the population doubling of primary cervical cancer epithelial cells under different culture conditions:

[0160] Population Doubling (PD) = 3.32*log10 (total number of cells after digestion / initial number of cells seeded), the formula is as follows (Chapman et al. Stem Cell Research & Therapy 2014, 5: 60).

[0161] Figure 5 The following are growth curves of CCa5 cells plotted using Graphpad Prism software under three different culture conditions. The horizontal axis represents the number of days in culture, while the vertical axis represents the cumulative cell proliferation factor, which indicates the number of times the cells expanded during the culture cycle. A larger value indicates a greater number of cell proliferations within a given cycle, i.e., a greater number of cells obtained through amplification. The slope represents the rate of cell expansion. The graph confirms that the proliferation rate of cervical cancer epithelial cells cultured in the FCM culture medium of the present invention is superior to that in the other two culture conditions.

[0162] [Example 5]

[0163] Immunohistochemical identification of primary cervical cancer tissues and cervical cancer cells after subculture

[0164] A mung bean-sized piece of cancerous tissue (sample number CCa14) was removed from a clinical surgical resection specimen from a cervical cancer patient and fixed in 1 mL of 4% paraformaldehyde. Cervical cancer epithelial cells (sample number CCa14) were obtained from the remaining cancerous tissue using the same method as in Example 1. Sample CCa14 was cultured to the third passage using the culture medium FCM of the present invention, using the method of Example 3.

[0165] Immunohistochemistry was used to detect the expression of important biomarkers associated with cervical cancer in the original CCa14 tissue and primary cells obtained by continuous culture to the third generation. The tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm thick tissue sections using a microtome. Conventional immunohistochemistry was then performed (for specific steps, see Li et al., Nature Communications, (2018) 9: 2983). The primary antibodies used were P16 antibody (purchased from Affinit), P63 antibody (purchased from CST), and Ki67 antibody (purchased from CST).

[0166] Depend on Figure 6 It was confirmed that the expression of cervical cancer-related biomarkers in cervical cancer cells (sample number CCa14) cultured in the medium of the present invention at passage 3 was essentially consistent with that in the original tissue sections from which the cells were derived. This indicates that cells cultured in the medium of the present invention retain the original pathological characteristics of cervical cancer tissue from patients.

[0167] [Example 6]

[0168] Functional testing of drug sensitivity of cervical cancer cells derived from cancer tissue

[0169] The following uses surgical resection samples from cervical cancer patients as an example to illustrate that cervical cancer tumor cells cultured from patient-derived cervical cancer tumor samples can be used to detect the sensitivity of patient tumor cells to different drugs.

[0170] 1. Plating of primary cervical cancer cells: The suspension of cervical cancer cells (numbered CCa5, CCa6, and CCa9) isolated according to the method in Example 1 was plated at 4×10 4 pieces / cm 2Cells were seeded at a high density into 12-well plates. 2 mL of the prepared FCM culture medium for primary cervical cancer epithelial cells was added to the 12-well plates and cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific). When the cells reached approximately 80% of the bottom area of ​​the culture plate, the supernatant was discarded and 0.5 mL of 0.25% trypsin (purchased from Thermo Fisher Scientific) 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 37°C for 10 minutes until complete digestion was observed under a microscope (Invitrogen EVOS M500). 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, and the supernatant was discarded. The centrifuged cell pellets were resuspended in FCM medium and counted using a flow cytometer (Jiangsu Zhuowei Microbiology Technology Co., Ltd., JIMBIO FIL). The total cell counts were 530,000, 780,000, and 630,000, respectively. Cells were seeded at a density of 1,000 to 2,000 cells / well in 384-well plates and allowed to adhere overnight.

[0171] 2. Drug gradient experiment:

[0172] (1) Prepare the drug storage plate using the concentration gradient dilution method: aspirate 40 μL of 10 μM stock solution of the drug to be tested as the highest concentration, then aspirate 10 μL from each of the 40 μL stock solutions and add them to a 0.5 mL EP tube containing 20 μL of DMSO, then aspirate 10 μL from the above EP tubes and add them to a second 0.5 mL EP tube containing 20 μL of DMSO, i.e., dilute the drug at a ratio of 1:3. Repeat the above method and dilute in sequence to obtain the 7 concentrations required for drug addition. Add drugs of different concentrations 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 embodiment, the drugs to be tested are cisplatin (purchased from MCE), paclitaxel (purchased from MCE), 5-fluorouracil (5-F, purchased from MCE), topotecan (purchased from MCE), bortezomib (purchased from MCE), anlotinib (purchased from MCE), pazopanib (purchased from MCE), and apatinib (purchased from MCE).

[0173] (2) Using a high-throughput automated workstation (Perkin Elmer JANUS), different concentrations of drug and solvent controls from a 384-well drug reservoir plate were added to a 384-well cell culture plate containing cervical cancer cells. Three replicate wells were set up for each drug group and solvent control group. The volume of drug added to each well was 100 nL.

[0174] (3) Cell viability detection: 72 hours after drug administration, the chemiluminescence value of the cells after drug addition and culture was detected using Cell Titer-Glo detection reagent (purchased from Promega). The size of the chemiluminescence value reflects the cell viability and the effect of the drug on cell viability. 10 μL of the prepared Cell Titer-Glo detection solution was added to each well, mixed, and the chemiluminescence value was detected using a microplate reader (Perkin Elmer Envision). According to the formula cell viability (%) = chemiluminescence value of the drug-added well / chemiluminescence value of the control well * 100%, the cell viability of the cells after different drugs were treated was calculated, and the half inhibition rate IC was calculated using Graphpad Prism software. 50 .

[0175] (4) Drug sensitivity test results such as Figure 7 shown.

[0176] Figure 7 The following table shows the sensitivity of cervical cancer cells cultured from surgically resected cancer tissue samples from three different cervical cancer patients (numbered CCa5, CCa6, and CCa9) to four chemotherapy drugs: cisplatin, paclitaxel, 5-fluorouracil, and topotecan; and to four targeted drugs: bortezomib, anlotinib, pazopanib, and apatinib. The results show that cells from the same patient have varying sensitivities to different drug concentrations, and that cells from different patients have varying sensitivities to the same drug. These results can be used to assess the effectiveness of these drugs in clinical practice for cervical cancer patients.

[0177] Industrial Applicability

[0178] The present invention provides a culture medium and a culture method for culturing primary cervical cancer epithelial cells. The cultured cells can be used for evaluating and screening drug efficacy. Therefore, the present invention is suitable for industrial application.

[0179] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications or improvements may be made based on this description. Therefore, such modifications or improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A culture medium for culturing primary cervical cancer epithelial cells, characterized in that: Made with the following ingredients: MST1 / 2 kinase inhibitor; Y27632; fibroblast growth factor 7; B27 supplement; hepatocyte growth factor; insulin-like growth factor 1; CHIR99021; A83-01; initial culture medium selected from DMEM / F12, DMEM, F12, or RPMI-1640; and antibiotics; Wherein, the MST1 / 2 kinase inhibitor is compound 1 or a pharmaceutically acceptable salt or solvate thereof, in, The content of the MST1 / 2 kinase inhibitor is 2 μM to 20 μM; The content of Y27632 is 2 μM to 20 μM; The content of fibroblast growth factor 7 is 2ng / ml to 40ng / ml; The volume concentration of the B27 additive in the primary cervical cancer epithelial cell culture medium is 1:25 to 1:100; The content of the hepatocyte growth factor is 2ng / ml to 40ng / ml; The content of the insulin-like growth factor 1 is 2ng / ml to 40ng / ml; The content of CHIR99021 is 1 μM to 10 μM; The content of A83-01 is 50nM to 500nM.

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

3. The culture medium according to claim 1, wherein: The primary cervical cancer epithelial cells are selected from cervical cancer tumor cells, normal cervical cancer epithelial cells, and cervical cancer epithelial stem cells.

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

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

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