CN115975936A - Culture medium and culture method of cervical cancer primary cells - Google Patents

Abstract

The invention provides a culture medium for culturing cervical cancer primary cells, a culture method using the primary cell culture medium and application thereof. The medium comprises an MST1/2 kinase inhibitor; insulin-like growth factor 1; fibroblast growth factor 7; insulin-transferrin-selenium complex; 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 cell culture medium is used for culturing the primary cells on a culture vessel pre-paved with irradiated trophoblasts, so that the primary cells are rapidly proliferated. The cell model obtained by the primary cell culture medium and the primary cell culture method can be used for evaluating and screening the curative effect of the medicine.

Description

Culture medium and culture method of cervical cancer primary cells

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a culture medium and a culture method for in vitro culture or amplification of cervical cancer primary cells.

Background

According to the latest global cancer statistical data "global cancer report" published by international cancer research Institute (IARC) under the world health organization in 2018, 1810 new cancer cases are added globally in 2018, the number of deaths reaches 960 ten thousand, and the global cancer burden is further increased. The Chinese cancer incidence in 2018, TOP10, is breast cancer, lung cancer, colorectal cancer, thyroid cancer, gastric cancer, cervical cancer, liver cancer, esophageal cancer, uterine cancer and brain cancer. Among them, cervical cancer is the 6 th most frequent in women. Worldwide, 20 thousands of women die from cervical cancer each year, and china accounts for about 10%. Because the molecular mechanism of the onset of the cervical cancer is not clear, the treatment means aiming at the cervical cancer, especially for patients in middle and late stages, is still limited, so that the five-year survival rate of the clinical patients is low, and personalized accurate medication guidance is lacked.

Functional testing refers to the in vitro detection of the sensitivity of anti-tumor drugs on cells of cancer patients. The key to the application of this method is the development of a tumor cell model with a short growth cycle that can represent the biological characteristics of a patient with cervical cancer. In addition, the cell model is convenient to operate and can quickly and efficiently predict the curative effect of clinical medication, so that accurate medication guidance is given to cancer patients in time. However, the success rate of establishing a cell model in vitro by using primary tumor cells from cancer patients is often low, the growth cycle is long, and the problems of excessive proliferation of mesenchymal cells such as fibroblasts exist, which restricts the development of the field. There are two techniques currently developed for culturing primary/stem cells that are relatively mature in the field of tumor cell functionality testing applications, one being the technique of using irradiated trophoblasts and ROCK kinase inhibitors to promote the growth of primary cells to investigate drug sensitivity in individual patients, namely the cell conditioning reprogramming technique (Liu et al, am.j. pathol.,180, 599-607, 2012). Another technique is the in vitro 3D culture of adult stem cells to obtain organoid techniques similar to tissues and organs (Hans Clevers et al, cell,11, 172 (1-2): 373-386, 2018).

However, organoid technology is a technology in which patient autologous primary cells are embedded in extracellular matrix for in vitro three-dimensional culture, but the culture medium of the technology needs to be added with a plurality of specific growth factors (such as Wnt proteins and R-spondin family proteins), which is expensive and not suitable for clinical large-scale application. In addition, the organoids need to embed cells in the extracellular matrix gel during the whole culture process, the plating steps of cell inoculation, passage and drug sensitivity test are more complicated and time-consuming compared with the 2D culture operation, and the organoids formed by the technology are not easy to control in size and dimension, so that the situation of internal necrosis caused by over-growth of part of the organoids is easy to occur. Therefore, organoid techniques are less operable and adaptable than 2D culture techniques, require specialized technical personnel, and are not suitable for large-scale widespread use in clinical in vitro drug sensitivity testing (Nick Barker, nat. Cell biol.,18 (3): 246-54, 2016).

The cell reprogramming technology is a technology for co-culturing autologous primary cells of a patient and murine feeder cells, but no test for cervical cancer samples is reported in the literature, so that whether the culture medium components reported in the literature can quickly amplify the cervical cancer primary tumor cells is unknown.

In view of the limitations of the above technologies, there is a clinical need to develop a cervical cancer primary cell culture technology, which has a short culture period, controllable cost and convenient operation, and 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 a cervical cancer patient. By evaluating the sensitivity of the antitumor drug on cell models derived from different cancer patients in vitro, the response rate of the antitumor drug in clinic is improved, and the pain of the patients and the waste of medical resources caused by inappropriate drugs are reduced.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a culture medium for culturing cervical cancer primary cells and a method for culturing the cervical cancer primary cells by using the culture medium. By adopting the cervical cancer primary cell culture medium and the culture method, the purposes of short in-vitro culture period, controllable cost and convenient and fast operation can be achieved. When the technology is applied to the construction of a primary cervical cancer tumor cell model, the primary cervical cancer tumor cell with the biological characteristics of a cervical cancer patient can be obtained, and the technology can be applied to new drug screening and in-vitro drug sensitivity detection.

One aspect of the invention is to provide a primary cell culture medium for culturing cervical cancer primary cells, comprising an MST1/2 kinase inhibitor; insulin-like growth factor 1; fibroblast growth factor 7; insulin-transferrin-selenium complex; 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.

Wherein the content of the first and second substances,

R ₁ selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R ₆ Substituted aryl (e.g., phenyl, naphthyl, and the like), arylC 1-C6 alkyl (e.g., benzyl, and the like), and heteroaryl (e.g., thienyl, and the like);

R ₂ and R ₃ Each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

R ₄ and R ₅ Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, and C3-C6 heterocycloC 1-C6 alkyl (said heterocyclyl is selected from, for example, piperidinyl, tetrahydropyranyl, and the like);

R ₆ selected from the group consisting of halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoromethyl).

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

wherein the content of the first and second substances,

R ₁ selected from C1-C6 alkyl, optionally substituted by 1-2 independent R ₆ Substituted phenyl, optionally substituted with 1-2 independent R ₆ Substituted thienyl, and optionally substituted with 1-2 independent R ₆ Substituted benzyl, R ₁ More preferably optionally substituted with 1-2 independent R ₆ Substituted phenyl;

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

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

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

/>

/>

/>

/>

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

In an embodiment of the invention, the amount of the MST1/2 kinase inhibitor in the culture medium is typically from 2 μ M to 20 μ M, preferably from 5 μ M to 20 μ M, more preferably 5 μ M.

The primary cell culture medium of the invention preferably further contains one or more of the following factors: insulin-like growth factor 1 (IGF-1); fibroblast growth factor 7 (FGF 7); 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. Wherein, in a preferred embodiment, the content of the fibroblast growth factor 7 in the culture medium is preferably 2-40 ng/ml, and more preferably 10-40 ng/ml; the volume ratio of the insulin-transferrin-selenium complex to the culture medium is preferably 1; the content of the insulin-like growth factor 1 is preferably 2-40 ng/ml, and more preferably 10-40 ng/ml; the content of the hepatocyte growth factor is preferably 2-40 ng/ml, and more preferably 10-40 ng/ml; the content of the liothyronine is preferably 2-50 nM, more preferably 2-10 nM; the ROCK kinase inhibitor is preferably Y27632, and the content of the ROCK kinase inhibitor is preferably 2 to 20 μ M, more preferably 2 to 10 μ M.

Compared with the components of a cell condition reprogramming culture medium and an organoid culture medium, the components of the culture medium formula are added with an MST1/2 kinase inhibitor, but the culture medium does not contain uncertain components such as serum, bovine pituitary extract and the like, does not contain niche factors necessary for culturing organoids such as Wnt agonist, R-spondin family protein, BMP inhibitor and the like, and does not contain nicotinamide and N-acetylcysteine, thereby greatly reducing the cost of the culture medium, simplifying the operation flow for preparing the culture medium, and realizing the in-vitro culture of the cervical cancer primary cells with controllable cost and convenient operation.

In the invention, the cervical cancer primary cells can be cervical cancer tumor cells, normal cervical cancer primary cells and cervical cancer epithelial stem cells.

One aspect of the present invention provides a method for culturing primary cervical cancer cells, comprising the steps of:

(1) Preparing the primary cell culture medium according to the formula;

(2) Pre-laying culture container with irradiated trophoblast.

Specifically, the trophoblasts may be irradiated NIH-3T3 cells, with X-ray or gamma-ray radiation, preferably gamma-ray radiation, in the dosage of 30-50 Gy, preferably 35Gy, and in the irradiation period of 5-20 min. Specifically, the irradiated NIH-3T3 cells are treated according to the ratio of 2-5 × 10 ⁴ Per cm ² The cells are seeded in a culture vessel such as a 48-well plate, 24-well plate, 12-well plate, 6-well plate or T25 cell culture flask, and are ready for use after the cells are adherent.

(3) Cervical cancer primary cells were isolated from cervical cancer tissue.

Cervical cancer primary cells can be derived, for example, from cervical cancer tissue samples and para-carcinoma tissue samples. The cervical cancer tissue sample is derived from a cancer tissue sample which has been surgically removed from a patient having a cervical cancer tumor and which has been prescribed and approved, and the paracancerous tissue sample is collected from tissue at a distance of at least 5cm from the cervical cancer tissue. The collection of the tissue sample is performed within half an hour after surgical resection of the patient. More specifically, a tissue sample is excised from a non-necrotic area in a volume of 0.5cm under sterile conditions ³ The cells were placed in pre-cooled 3-5mL DMEM/F12 medium, which was contained inConveying the plastic sterile centrifuge tube with a cover to a laboratory on ice; wherein the DMEM/F12 medium contains 1-2 vol% of penicillin/streptomycin and/or 0.2-0.4 vol% of Primocin (hereinafter referred to as tissue transfusion solution). When streptomycin/penicillin is used, the concentration of streptomycin is 25 to 400. Mu.g/mL, preferably 50 to 200. Mu.g/mL, more preferably 200. Mu.g/mL, and the concentration of penicillin is 25 to 400U/mL, preferably 50 to 200U/mL, more preferably 200U/mL; when Primocin is used, the concentration ranges from 25 to 400. Mu.g/mL, preferably from 50 to 200. Mu.g/mL, more preferably 100. Mu.g/mL.

In the biological safety cabinet, the tissue sample is transferred to a cell culture dish, the tissue sample is rinsed by using a transport solution, and blood cells on the surface of the tissue sample are cleaned. Transferring the tissue sample after the moistening into another new culture dish, adding 1-3mL of transport solution, and cutting the tissue sample into pieces with the volume less than 3mm by using a sterile surgical blade and surgical forceps ³ A fragment of the tissue of (a).

Transferring the fragment of the tissue sample into a centrifuge tube, and centrifuging for 3-5 minutes at 1000-3000 r/min by using a desktop centrifuge (3-18K of Sigma company); discarding the supernatant, adding tissue transport fluid and tissue digestive fluid (5 mL tissue digestive fluid is used per 10mg tissue according to 1:1, wherein the preparation method of the tissue digestive fluid is that 1-2 mg/mL collagenase II, 1-2 mg/mL collagenase IV, 50-100U/mL deoxyribonucleic acid I, 0.5-1 mg/mL hyaluronidase, 0.1-0.5 mg/mL calcium chloride, 5-10 mg/mL bovine serum albumin are dissolved in HBSS and RPMI-1640 with the volume ratio of 1:1), marking the sample number, sealing the sealing film, digesting with 37 ℃ and 200-300 rotary constant temperature shaking table (known as ZQLY-180N), and observing whether the digestion is completed or not every 1 hour; if no obvious tissue block is found, the digestion can be stopped, otherwise, the digestion is continued until the digestion is full, and the digestion time range is 4 to 8 hours. After digestion is complete, the undigested tissue pellet is filtered off with a cell strainer (cell mesh size, for example, 70 μm), the tissue pellet on the strainer is rinsed with a tissue transfer fluid, the remaining cells are rinsed into a centrifuge tube, and centrifuged with a tabletop centrifuge at 1000 to 3000 rpm for 3 to 5 minutes. Discarding the supernatant, observing whether the residual cell mass contains blood cells, adding 1-5 mL of blood cell lysate (purchased from Sigma company) if the blood cells exist, uniformly mixing, cracking at 4 ℃ for 10-20 minutes, shaking for 5 minutes, uniformly mixing once, taking out after cracking, and centrifuging at 1000-3000 r/min for 3-5 minutes. The supernatant was discarded, the primary cell culture medium of the present invention was added to resuspend the cells, and the total number of cells was counted using a flow cytometer (jiamboo FIL, jiangsu microbial technology ltd).

(4) Inoculating the primary cervical cancer cells separated in the step (3) into a culture vessel pre-inoculated with trophoblasts, and culturing by adopting the primary cell culture medium in the step (1).

More specifically, after the trophoblasts adhere to the wall, the thickness is 2X 10 ⁴ ～8×10 ⁴ Per cm ² (e.g., 4X 10) ⁴ Per cm ² ) Inoculating the primary cervical cancer tumor cells at a density of 0.5 to 2mL per well of the primary cell culture medium, at, e.g., 37 ℃ and 5% CO ₂ Culturing in a cell culture box for 8-16 days, changing into a fresh primary cell culture medium every 4 days, and carrying out digestion passage when the cervical cancer primary cells grow to a cell density of about 80-90% of the bottom area of the multi-well plate.

Compared with organoid technology, the step does not need to mix the primary cells and the matrigel uniformly on ice to form gel drops, and adds the culture medium after the gel drops are solidified. In addition, the use amount of the expensive extracellular matrix glue is saved, and the operation steps are simplified.

Optionally, after the inoculated cervical cancer primary cells are cultured for 8-16 days, when cell clones formed in a culture container are converged to reach bottom area of 80%, discarding supernatant, adding 1-2mL0.25% pancreatin (purchased from Thermo Fisher company) for digestion for 1 minute, then sucking out 0.25% pancreatin, adding 1-2mL 0.05% pancreatin for cell digestion, and incubating for 5-20 minutes at room temperature; then, 2 to 4mL of a culture medium containing, for example, 5% (v/v) fetal bovine serum, 100U/mL penicillin, and 100. Mu.g/mL streptomycin is used to resuspend the digested cells, centrifuged at 1000 to 3000 rpm for 3 to 5 minutes, the digested single cells are resuspended using the primary cell culture medium of the present invention, and the resulting cell suspension is placed in a culture vessel on which feeder cells have been pre-spread for continuous expansion culture. The pretreatment operation of the culture vessel is the same as the step (2).

The primary cervical carcinoma cells are grown in 2D, so that the conditions of non-uniform size of the organoid, necrosis of the grown organoid and the like caused by organoid technical amplification are avoided.

The present invention also provides a method for evaluating or screening a drug for treating a cervical cancer disease, comprising the steps of:

(1) Culturing the primary cervical cancer cells by using the culture method of the primary cervical cancer cells;

(2) Selecting a medicine to be detected and diluting according to a required concentration gradient;

(3) Adding the diluted medicine to the cervical cancer primary cells obtained by culturing in the step (1);

(4) Cell viability assays were performed.

The beneficial effects of the invention also include:

(1) The success rate of cervical cancer primary cell culture is improved and reaches more than 85 percent;

(2) The cervical cancer primary cells cultured in vitro can be ensured to maintain the pathological phenotype and heterogeneity of patients from which the primary cells are derived;

(3) The components of the culture medium do not contain serum, so the culture medium is not influenced by the quality and quantity of serum of different batches;

(4) The primary cell for amplifying the cervical cancer has high efficiency as long as 10 ⁴ The cell number of the grade can be successfully amplified to 10 within about two weeks ⁶ The cervical cancer primary cells with the magnitude order can be continuously passed;

(5) In the passage step, the operation and dissociation of matrigel are not needed, and the digestion passage of the cells can be completed within 10-15 minutes;

(6) The culture cost is controllable, factors such as expensive Wnt agonist, R-spondin family protein, BMP inhibitor and the like do not need to be added into the cervical cancer primary cell culture medium, and the cell culture cost is saved;

(7) Compared with organoid technology, the technology does not need to embed cells in matrigel like organoid technology, and the technical operation steps are simple and easy to implement;

(9) The cervical cancer primary cells obtained by the culture of the technology have large quantity and high homogenization degree, are suitable for screening new candidate compounds at high flux, and provide high-flux medicament in-vitro sensitivity function test for patients.

With the cell culture medium of the present embodiment, cervical cancer primary cells derived from a 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, can be cultured. Meanwhile, the culture medium of the technology can also be used for developing a kit for amplification culture of in-vitro cervical cancer primary cells.

In addition, the cells obtained by the culture method of the present embodiment can be applied to regenerative medicine, basic medicine research of cervical cancer primary cells, screening of drug response, development of new drugs derived from cervical cancer diseases, and the like.

Drawings

FIGS. 1A to 1G are graphs showing the effect of the concentration of each additive factor on the proliferation of cervical cancer primary cells.

FIGS. 2A and 2B are photographs taken under an inverted microscope of cervical cancer tumor cells in which cells isolated from 1 clinical tissue specimen for cervical cancer were cultured to day 4 and day 12, respectively, using the culture medium CM of the present invention.

FIG. 3 is a view showing a comparison of the total number of cells collected from cells isolated from 1 sample obtained by surgical excision of cervical cancer, which were cultured in 3 different media for 7 days.

FIG. 4 is a graph showing the comparison of cell proliferation effects obtained after cells isolated from 6 cases of surgical resection specimens of cervical cancer were cultured for 7 days under three different medium conditions.

FIGS. 5A and 5B are photographs showing staining using a nonspecific nuclear dye DAPI and a cervical cancer-specific antibody p16, respectively, after cervical cancer tumor cells were obtained by culturing cells isolated from 1 sample obtained by surgical resection of cervical cancer using the culture medium CM of the present invention.

FIG. 6 is a graph showing a comparison between immunohistochemical results of primary tissue cells of 1 example of a sample obtained by surgical excision of cervical cancer and cervical cancer tumor cells obtained by culturing the cells in the medium CM of the present invention.

FIG. 7 shows cell activity curves of cervical cancer tumor cells isolated from surgically excised cancer tissue samples of 2 patients with cervical cancer cultured according to the method of the present invention under the influence of 8 different drugs.

Detailed Description

In the present specification, the primary cells include differentiated primary cells and epithelial stem cells obtained from epithelial tissue. "epithelial stem cells" refer to cells that have long-term self-renewal ability and differentiate into primary cells, and refer to stem cells derived from epithelial tissues. Examples of the epithelial tissue include 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, breast, salivary gland, lacrimal gland, prostate, hair root, trachea, and lung. Among them, the cell culture medium of the present embodiment is preferably a medium for primary cells derived from cervical cancer.

In the present specification, the term "epithelial tumor cell" refers to a cell obtained by tumorigenization of a cell derived from the above-mentioned epithelial tissue.

In the present specification, the term "organoid" refers to a three-dimensional solid tissue body similar to an organ, which is formed by spontaneously organizing and aggregating cells at a high density in a controlled space.

[ preparation example of MST1/2 kinase inhibitor ]

In the present specification, an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly down-regulates MST1/2 signaling. In general, MST1/2 kinase inhibitors, for example, bind to and reduce the activity of MST1/2 kinase. Due to the structural similarity of MST1 and MST2, MST1/2 kinase inhibitors may also be compounds that bind to and reduce the activity of MST1 or MST2, for example.

Preparation of MST1/2 kinase inhibitor Compound 1

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

Methyl 2-amino-2- (2,6-difluorophenyl) acetate (A2): after 2-amino-2- (2,6-difluorophenyl) acetic acid (2.0 g) was added to the round bottom flask, methanol (30 ml) was added followed by thionyl chloride (1.2 ml) dropwise under ice-bath. The reaction system was allowed to react overnight at 85 ℃. After the reaction was complete, the solvent was evaporated to dryness under reduced pressure to give a white solid which was used directly in the next step.

Methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2,6-difluorophenyl) acetate (A3): to a round bottom flask was added methyl 2-amino-2- (2,6-difluorophenyl) acetate (2 g), followed by acetone (30 ml) and potassium carbonate (2.2 g), then the system was cooled to-10 ℃ with an ice salt bath, followed by the slow addition of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction was stirred at room temperature overnight. After the reaction, the reaction mixture was filtered, the solvent was removed from the filtrate under reduced pressure, and the residue was purified by pressure silica gel column chromatography to obtain Compound A3.LC/MS: m + H359.0.

2-chloro-7- (2,6-difluorophenyl) -7,8-dihydropteridin-6 (5H) -one (A4): to a round bottom flask was added 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 was stirred at 60 ℃ for two hours. After the reaction was completed, the solvent was evaporated under reduced pressure, and the obtained product was neutralized to be alkaline with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. Filtering the organic phase, and evaporating to dryness under reduced pressure to obtain a crude product. Washing the crude product with diethyl ether to obtain a compound A4.LC/MS: m + H297.0.

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, cooled to-35 deg.C, iodomethane (0.9 mL) was added followed by sodium hydride (615 mg) and the reaction was stirred for an additional two hours. After the reaction, water was added to quench, ethyl acetate was extracted, and the organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. Filtering the organic phase, and evaporating to dryness under reduced pressure to obtain a crude product. Washing the crude product with diethyl ether to obtain a compound A5.LC/MS: m + H325.0.

4- ((7- (2,6-difluorophenyl) -5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl) amino) benzenesulfonamide (1): into a round bottom flask was added 2-chloro-7- (2,6-difluorophenyl) -5,8-dimethyl-7,8-dihydropteridin-6 (5H) -one (100 mg), sulfanilamide (53 mg), p-toluenesulfonic acid (53 mg), and sec-butanol (5 ml). The reaction was stirred at 120 ℃ overnight. After the reaction is finished, filtering, and washing by methanol and ether to obtain the compound 1.LC/MS: m + H461.1.

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

Other MST1/2 inhibitor compounds of the invention were synthesized in analogy to compound 1 and their structural and mass spectral data are shown in the table below.

/>

/>

/>

/>

[ example 1]

Isolation of primary cells for human cervical cancer

Cervical cancer tissue samples were obtained from three patients who had undergone surgery to remove cancer tissue from a cervical cancer tumor and had received consent, and they were sample numbers CCA2, CCA3, CCA4, and one of the samples (number CCA 2) is described below.

The collection of the tissue sample is performed within half an hour after surgical resection of the patient. More specifically, under sterile conditions, a tissue sample of a non-necrotic area is excised and its volume is 0.5cm ³ The resulting mixture was placed in a pre-chilled 4mL tissue infusion solution (see Table 1 for specific formulations) contained in a 5mL plastic sterile lidded cryopreservation tube (purchased from Jiety, guangzhou), and transported to the laboratory in the cold chain (0-10 ℃).

TABLE 1 tissue transport fluid formulations

TABLE 2 tissue digestive juice formulation

Tissue digestive juice component	Suppliers of goods	Final concentration
			HBSS	Gibco
	50% (by volume)
		RPMI-1640	Corning	50% (volume)
Collagenase II	Sigma				2mg/mL
		Collagenase IV	Sigma	2mg/mL
Deoxyribonucleic acid I	Sigma				50U/mL
		Hyaluronidase	Sigma	0.5mg/mL
Calcium chloride	Shanghai worker				0.33mg/mL
		Bovine serum albumin	Shanghai worker	10mg/mL

In the biosafety cabinet, the tissue specimen (No. CCA 2) was transferred to a 100mm cell culture dish (purchased from NEST corporation), rinsed with a tissue transport solution, and the residual blood on the surface of the tissue specimen was washed away, and excess tissue such as fat on the surface of the tissue specimen was removed. Transferring the tissue sample after being moistened into another new 100mm culture dish, adding 2mL of transport solution, and cutting the tissue sample into pieces with the volume less than 3mm by using a sterile surgical blade and surgical forceps ³ The tissue fragment of (a).

Transfer the tissue sample pieces to a 15mL centrifuge tube and centrifuge at 1500rpm for 4 minutes using a bench top centrifuge (Sigma Co., 3-18K); discarding the supernatant, adding the tissue transport fluid and the tissue digestive fluid (5 mL of tissue digestive fluid is used per 10mg of tissue, see Table 2 for specific preparation) according to the proportion of 1:1, marking the sample number, sealing with a sealing film, digesting with a constant temperature shaking table (ZQLY-180N) at 37 ℃ and 300 revolutions, and observing whether the digestion is completed every 1 hour.

After digestion was complete, undigested tissue pellet was filtered through a 70 μm filter, the tissue pellet on the filter was rinsed with tissue transfusion solution, the remaining cells were flushed into a centrifuge tube and centrifuged at 1500rpm for 4 minutes.

The supernatant was discarded and the remaining cell pellet was observed for the presence of blood cells, and 3mL of a hemocyte lysate (purchased from Sigma) was added thereto and mixed, lysed at 4 ℃ for 15 minutes, and mixed by shaking for 5 minutes. After the cleavage, the cells were removed and centrifuged at 1500rpm for 4 minutes. The supernatant was discarded to obtain primary cells of cervical cancer after digestion and separation, and resuspended in Basal Medium (BM) prepared by adding 0.2 vol% Primocin (obtained from Invivogen at a concentration of 50 mg/mL) in commercially available DMEM/F-12 medium to obtain a final concentration of 100. Mu.g/mL. The total number of cells was 208 ten thousand by counting using a flow cytometer (JIMBIO FIL, ohio).

Two other cervical cancer tumor tissue samples were isolated in the same manner as above, and the total number of cells obtained was 197 ten thousand (CCA 3) and 232 ten thousand (CCA 4), respectively.

[ example 2]

Optimization of cervical cancer primary cell culture medium

(1) Effect of different factors

Cultured NIH-3T3 cells (purchased from ATCC using DMEM medium containing 10% fetal bovine serum) were digested with 0.25% trypsin (purchased from Thermo Fisher), the digestion was stopped with DMEM medium containing 5% (v/v) fetal bovine serum (purchased from Eikey), 100U/mL penicillin and 100. Mu.g/mL streptomycin (purchased from Corning), and collected in 15mL centrifuge tubes, centrifuged at 1500rpm for 4 minutes, and the supernatant was discarded. The above 10% fetal calf blood is usedThe cell pellet after the clear DMEM culture solution was resuspended and centrifuged, counted using a flow cytometer (JIMBIO FIL, ohio, jiangsu, zhang microbial technology Co., ltd.), irradiated with gamma rays at an irradiation dose of 35Gy for 10 minutes, and then irradiated at 2X 10 ⁴ Per cm ² Is inoculated into a culture vessel. Culturing in an incubator at 37 ℃ until the cells adhere to the wall. Prior to seeding the primary cells, the medium in the culture vessel was removed.

Basal medium (abbreviated BM): BM was prepared by adding 0.2 vol% Primocin (purchased from Invivogen at 50 mg/mL) to a commercially available DMEM/F-12 medium to give a final concentration of 100. Mu.g/mL.

Then, different kinds and different concentrations of additive factors (table 3) were added to the Basal Medium (BM) to prepare primary cervical cancer cell culture media containing different additive components.

TABLE 3 preparation of media of different composition (final concentration)

The culture media with different components are added into a 48-hole culture plate pre-paved with NIH-3T3 cells after gamma ray irradiation according to the volume of 500 mul/hole. Cervical cancer tumor cells (accession No. CCA 12) isolated from cervical cancer tissue according to the same method as in example 1 were cultured at 4X 10 ⁴ The cell number per well was seeded in the above 48-well culture plate preplated with NIH-3T3 cells after gamma-ray irradiation, surface-sterilized and placed at 37 ℃ 5% by CO ₂ Incubators (purchased from siemer fly) were used to culture the same number of freshly isolated cervical cancer tumor cells (No. CCA 12) under different media formulations. The medium replacement and feeder cell supplementation were performed every 4 days after the start of the culture. After 7 days of culture, cell counts were performed. Wherein, as experimental control, basal Medium (BM) without any additives was used. The results are shown in Table 3. The "-" sign indicates that the supplement had no pro-proliferative effect on the cells, and the "+" sign indicates that the supplement had a pro-proliferative effect on the cells. Different factors in Table 3 were added at different concentrations on the basis of BM to produce different effects on cell proliferation. Wherein, under a specific concentration range, the B27 additive, the N2 additive, the insulin-transferrin-selenium compound, the hepatocyte growth factor, the insulin sample growth factor 1, the fibroblast growth factor 7, the compound 1, the Y27632 and the liothyronine have certain promotion effects on cell proliferation.

(2) The proliferation effect of different concentrations of the added factors on the cervical cancer primary cells obtained by the method

Preparation of cervical cancer primary cell culture medium of this example: fibroblast growth factor 7 (FGF 7) was added to a Basal Medium (BM) at a final concentration of 20ng/ml, insulin-like growth factor 1 (IGF-1) was added to a Basal Medium (BM) at a final concentration of 20ng/ml, hepatocyte Growth Factor (HGF) was added to a basal medium at a final concentration of 20ng/ml, insulin-transferrin-selenium complex (ITS) stock solution (10 μ g/ml for insulin, 5 μ g/ml for transferrin, and 5ng/ml for sodium selenite) was added to a Basal Medium (BM) at a final concentration of 1.

Cervical cancer primary cells derived from a cancer tissue were isolated from a cancer tissue (sample No. CCA 8) of a cervical cancer patient by the same method as in example 1. Subsequently, the cervical cancer tumor cells derived from the cancer tissue were counted by a flow image counter (JIMBIO FIL, zerk microbial technologies, ltd) to obtain the total number of cells. Then press 4X 10 ⁴ Per cm ² The cells were densely seeded in 12-well plates pre-plated with NIH-3T3 cells after gamma irradiation. 2mL of the prepared cervical cancer primary cell culture medium was added to a 12-well plate, and the mixture was allowed to stand at 37 ℃ and 5% CO ₂ Incubators (purchased from Saimeri fly) were used for culturing. When the cell growth in the culture plate reaches about 80% of the basal area, the culture medium supernatant in the 12-well plate is discarded0.5mL of 0.25% pancreatin (from Thermo Fisher) was added and digested for 1 minute, then 0.25% pancreatin was aspirated, 0.5mL of 0.05% pancreatin was added and cell digestion was performed, incubation was carried out at room temperature for 5 to 20 minutes until complete cell digestion could be observed under a microscope (EVOS M500 from Invitrogen), digestion was stopped with 1mL of DMEM/F12 medium containing 5% (v/v) fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin, and collected in a 15mL centrifuge tube, and after centrifugation at 1500rpm for 4 minutes, the supernatant was discarded. The centrifuged cell pellet was resuspended in a basal medium BM and counted by a flow cytometer (JIMBIO FIL, ohio, jiangsu, ohio, ltd.) to obtain the total number of cells. The cells obtained were used in the following culture experiments.

Next, the following 8 media formulations were prepared for the experiments:

formula 1: the components of the culture medium do not contain fibroblast growth factor 7;

and (2) formula: the culture medium contains no insulin-transferrin-selenium compound;

and (3) formula: the components of the culture medium do not contain insulin-like growth factor 1;

and (4) formula: the components of the culture medium do not contain hepatocyte growth factors;

and (5) a formula: the components of the culture medium do not contain Y27632;

and (6) formula: the components of the culture medium do not contain the compound 1;

and (3) formula 7: the components of the culture medium do not contain liothyronine.

The above-mentioned formulations 1 to 7 were used to dilute the above-mentioned digested cell suspensions, respectively, and the cells were seeded into a 48-well plate pre-plated with NIH-3T3 cells after gamma-ray irradiation in a volume of 250. Mu.l per well of 1 ten thousand cells.

When the culture medium of the formula 1 is used, 250 microliters of prepared fibroblast growth factor 7 per well is respectively added into a 48-well plate inoculated with primary cells, and the final concentrations of the fibroblast growth factor 7 are respectively 40ng/ml, 10ng/ml and 2ng/ml; and control wells (BC) were set using medium of formula 1.

When the culture medium of formula 2 is used, 250 microliters of the prepared insulin-transferrin-selenium complex per well is added to 48-well plates inoculated with primary cells, respectively, and the final concentrations of the insulin-transferrin-selenium complex stock solutions are 1; and control wells (BC) were set using medium of formula 2.

In the case of the formulation 3 medium, 250. Mu.l of formulated insulin-like growth factor 1 was added to 48-well plates inoculated with primary cells, respectively, at final concentrations of 40ng/ml, 10ng/ml and 2ng/ml of insulin-like growth factor 1, and control wells (BC) were set using the formulation 3 medium.

When the culture medium of formula 4 is used, 250 microliters of the prepared hepatocyte growth factor is respectively added into a 48-well plate inoculated with primary cells, and the final concentrations of the hepatocyte growth factor are respectively 40ng/ml, 10ng/ml and 2ng/ml; and control wells (BC) were set using medium of formula 4.

When the culture medium of formula 5 is used, 250 microliters of prepared Y27632 per well are respectively added into a 48-well plate inoculated with primary cells, and the final concentrations of Y27632 are respectively 20 muM, 10 muM and 2 muM; and control wells (BC) were set using medium of formula 5.

When the culture medium of formula 6 is used, 250 microliters of the prepared compound 1 per well is added to 48-well plates inoculated with primary cells, respectively, and the final concentrations of the compound 1 are 20 μ M, 5 μ M and 2 μ M, respectively; and control wells (BC) were set using medium of formulation 6.

When the culture medium of formula 7 is used, 250 microliters of prepared liothyronine per well are respectively added into a 48-well plate inoculated with primary cells, and the final concentrations of the liothyronine are respectively 50nM, 10nM and 2nM; and control wells (BC) were set using medium of formula 7.

When the cells were expanded to about 85% of the 48 wells and digested, the ratio was calculated with reference to the number of cells in the control well (BC), and the results are shown in FIGS. 1A to 1G, respectively. In FIGS. 1A to 1G, the ratio is the ratio of the number of cells obtained by one-pass culture using each medium to the number of cells obtained by one-pass culture using the corresponding control well. The ratio is more than 1, which indicates that the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is better than that of a control Kong Peiyang medium; if the ratio is less than 1, the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is weaker than that of the culture medium in the control hole.

According to the results of FIGS. 1A to 1G, the content of fibroblast growth factor 7 in the medium is preferably 2 to 40ng/ml, more preferably 10 to 40ng/ml; the volume concentration of the insulin-transferrin-selenium complex is preferably 1; the content of the insulin-like growth factor 1 is preferably 2ng/ml to 40ng/ml, and more preferably 10ng/ml to 40ng/ml; the content of the hepatocyte growth factor is preferably 2ng/ml to 40ng/ml, and more preferably 10ng/ml to 40ng/ml; the content of Y27632 is preferably 2 to 20. Mu.M, more preferably 2 to 10. Mu.M; the content of the compound 1 is preferably 2 to 20. Mu.M, more preferably 5 to 20. Mu.M; the content of liothyronine is preferably 2nM to 50nM, more preferably 2nM to 10nM.

According to the preferred concentrations of the above components, a preferred medium formulation CM of the invention is formulated, comprising: basal Medium (BM), 10ng/ml fibroblast growth factor 7 (FGF 7), 10ng/ml Hepatocyte Growth Factor (HGF), 10ng/ml insulin-like growth factor 1 (IGF-1), 1 volume ratio of insulin-transferrin-selenium complex, 5 μ M compound 1, 10 μ MY27632 and 10nM liothyronine.

[ example 3]

Culture of primary cervical cancer tumor cells from human cervical cancer tissue

Cervical cancer primary cells derived from a cancer tissue were isolated and obtained from a cancer tissue (sample No. CCA 5) of a cervical cancer patient by the same method as in example 1. Subsequently, the cervical cancer tumor cells derived from the cancer tissue were counted by a flow image counter (JIMBIO FIL, zerk microbial technologies, ltd) to obtain the total number of cells. Then press 4X 10 ⁴ Per cm ² Inoculating to 12 wells pre-plated with NIH-3T3 cells after gamma irradiationInside the plate. Adding 2mL of prepared cervical cancer primary cell culture medium CM to a 12-well plate, incubating at 37 deg.C and 5% CO ₂ Incubators (purchased from Saimeri fly) were used for culturing.

FIG. 2A shows a schematic view of the present embodiment at 4X 10 ⁴ Per cm ² 12-well plates of primary cervical cancer cells were densely inoculated, and an under-lens photograph (photographed by a 100-fold inverted phase contrast microscope) was taken from the culture to day 4 after the start of inoculation. It can be seen from microscopic observation that the cultured cancer tissue-derived primary cervical cancer tumor cells have formed larger clones. FIG. 2B is an under-the-lens photograph (photographed by a 100-fold inverted phase contrast microscope) of the present example from the 12 th day of culture after inoculation, and the cells were overgrown in the visual field. As can be seen from the two graphs of FIGS. 2A and 2B, the separated cervical cancer primary cells can be obviously cloned under a microscope after being cultured in vitro for 4 days, and the cell number is obviously amplified after being amplified for 12 days, which indicates that the technology of the invention is a high-efficiency technology for amplifying the cervical cancer primary cells in vitro.

[ example 4]

Proliferation promoting effect of different culture media on primary cervical cancer tumor cells derived from cervical cancer tissues

(1) Comparison of Effect of different media on Primary cell clonogenic and proliferative Effect

A cervical cancer primary cell culture medium CM and a basal medium BM as a control were prepared in the same manner as in example 2. In addition, as another control example, medium FM used in the technical literature of cell conditional reprogramming was prepared, and the formulation steps are shown in (Liu et al, nat. Protocol., 12 (2): 439-451, 2017), and the medium formulation is shown in Table 4.

TABLE 4 Medium (FM) Components used in the technical literature for the conditional reprogramming of cells

Use andprimary cervical cancer tumor cells (No. CCA 10) derived from cervical cancer tissue were obtained in the same manner as in example 1. Then, the same density (4X 10) was applied ⁴ Per cm ² ) The culture was carried out under the following 3 culture conditions, respectively:

A. the technology of the invention is as follows: by 4X 10 ⁴ Per cm ² Inoculating the primary cervical cancer tumor cells into a 24-well plate pre-paved with NIH-3T3 cells (purchased from ATCC company) after gamma-ray irradiation at an inoculation density, and culturing by adopting 1mL of the cervical cancer primary cell culture medium CM of the invention;

B. cell condition reprogramming techniques: by 4X 10 ⁴ Per cm ² Inoculating density primary cervical cancer tumor cells to NIH-3T3 cells (purchased from ATCC company) pre-plated with gamma-ray irradiation, and culturing in a 24-well plate by using 1mL of cell condition reprogramming technical culture medium FM (see (Liu et al, nat. Protoc.,12 (2): 439-451, 2017);

C. by 4X 10 ⁴ Per cm ² Inoculation Density Primary cervical cancer tumor cells were inoculated into 24-well plates pre-plated with gamma-irradiated NIH-3T3 cells (purchased from ATCC) and cultured in 24-well plates using 1mL of basal medium BM.

In the above three cultures, the cells cultured under the three culture conditions were changed every 5 days. The state of cell formation cloning and cell proliferation under culture in each medium in a 24-well plate was observed at the same time, and the growth of cells was recorded by photographing using a microscope (EVOS M500, invitrogen).

For primary cervical cancer tumor cells (No. CCA 10) cultured by the technique of the present invention, when the cell growth in the culture plate reaches about 80% of the basal area, the culture medium supernatant in a 24-well plate is discarded, 0.5mL of 0.25% pancreatin (purchased from Thermo Fisher) is added for digestion for 1 minute, then 0.25% pancreatin is aspirated, 0.5mL of 0.05% pancreatin is added for cell digestion, incubation is performed at 37 ℃ for 10 minutes until the cells are completely digested under the microscope (EVOS M500 from Invitrogen), i.e., 1mL of a DMEM/F12 culture solution containing 5% (v/v) fetal bovine serum, 100U/mL of penicillin and 100. Mu.g/mL of streptomycin is used for terminating the digestion, the cells are collected in a 15mL centrifuge tube, and the supernatant is discarded after centrifugation at 1500rpm for 4 minutes. The cell pellet after centrifugation was resuspended in the culture medium of the present invention, and the total number of cells was counted by a flow cytometer (JIMBIO FIL, jiangsu Zong microbial technology Co., ltd.) to obtain 61.7 ten thousand cells. The cells cultured under the other 2 culture conditions were digested and counted in the same manner as described above, and the total number of cells cultured using medium FM and BM was 23.3 ten thousand and 12.9 ten thousand, respectively.

Figure 3 is a plot of the total number of cells expanded by numbered CCA10 cells under different conditions.

FIG. 4 is a graph showing the comparison of the cell proliferation effect obtained after 6 specimens of cervical cancer patients (accession No. CCA8, CCA6, CCA2, CCA3, CCA10, CG 2) were cultured for 7 days according to the method of example 1, wherein √ represents a certain clonogenic capacity and proliferation promoting effect, √ represents a more significant clonogenic capacity and proliferation promoting effect, √ represents a very significant clonogenic capacity and proliferation promoting effect, and x represents a failure to form a clone. From the figure, it was confirmed that the CM medium had significant advantages in clonogenic capacity and cell proliferation promoting effect in primary cell culture obtained from the cervical cancer tissue source over the other 2 culture conditions.

[ example 5]

Identification of cancer tissue-derived primary cervical cancer tumor cells

(1) Immunofluorescence identification of primary cervical cancer tissue and cervical cancer cell after subculture

Cervical cancer primary cells derived from a cancer tissue were isolated from a cancer tissue (sample No. CG 2) of a cervical cancer patient by the same method as in example 1. Then, the cervical cancer tumor cells derived from the cancer tissue were counted by a flow cytometer (jiamboo FIL, jiangsu microbial technologies, ltd) to obtain the total number of cells. Then press 4X 10 ⁴ Per cm ² The cells were densely seeded in 24-well plates pre-plated with NIH-3T3 cells after gamma irradiation, while circular cell fragments for immunofluorescent staining (purchased from Sameran Federation) were pre-placed in the 24-well plates. Adding 1mL of prepared cervical cancer primary cell culture medium into a 24-well plateCM, at 37 ℃ and 5% CO ₂ Incubators (purchased from Saimeri fly) were used for culturing.

When 80% of the basal area of the cells in the 24-well plate was expanded, the culture solution was discarded, and the cells were fixed on 4% formaldehyde ice for 30 minutes. PBS (from shanghai bio-workers) for 5 min x 3 times. Discard PBS, add the permeation solution, avoid light, break the membrane for 30 minutes by shaking table (about 100 rpm), wash 5 minutes x 3 times with PBS. Subsequently, a 5% strength by volume BSA (from Shanghai works) solution was prepared for blocking using PBS +0.3% Triton X-100 (from Shanghai works), and blocked at 37 ℃ for 30 minutes.

Triton X-100 was prepared in advance for diluting the antibody, the cervical cancer-specific antibody p16 (purchased from CST Co.) was diluted at a ratio of 1. The cells were removed at 4 ℃ and allowed to equilibrate to room temperature, incubated for an additional 1 hour at 37 ℃ and washed 5 minutes x 3 times with PBS.

PBS +0.3% triton X-100 was prepared in advance for secondary antibody dilution, fluorescent secondary antibody (purchased from sequoyifei) with excitation light of 488nm and belonging to rabbit species was diluted at a ratio of 1.

The nonspecific fluorescent dye DAPI (purchased from Sigma) was diluted with PBS at a volume ratio of 1. Images were taken under a microscope (EVOS M500, invitrogen) and recorded by photography.

Fig. 5A and 5B are pictures of fluorescence photographs taken under 10-fold objective lenses in different fields, respectively, in which fig. 5A is a picture of staining a cell nucleus with a nonspecific fluorescent dye DAPI, and fig. 5B is a picture of staining with a cervical cancer-specific antibody p 16. As shown, the locations of the marker nuclei in FIG. 5A are all heavily stained in FIG. 5B (gray portion of the figure), indicating that the cultured cells were cervical cancer cells, consistent with a clinical pathological diagnosis.

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

Cancer tissue (sample No. CG 2) having a size of about a mung bean grain was taken from a clinical surgical resection sample of one cervical cancer patient and fixed by immersing in 1mL of 4% paraformaldehyde. The remaining cancer tissues were subjected to cervical cancer primary cells (sample No. CG 2) using the same method as in example 1. The CG2 sample was continuously cultured to the fourth passage using the culture medium CM of the present invention using the method of example 3.

And detecting the expression of important biomarkers related to cervical cancer in the CG2 original tissue of the sample and primary cells obtained by continuously culturing the primary tissue of the sample to the 4 th generation by using an immunohistochemical method. The tissue fixed with 4% paraformaldehyde was embedded in paraffin and cut into 4 μm thick tissue sections with a microtome. Conventional immunohistochemical detection was then performed (see, for specific steps, li et al, nature communication, (2018) 9. The primary antibodies used were cytokeratin 7 (CK 7) (purchased from CST), p16 antibody (purchased from CST), ki67 antibody (purchased from CST), and p53 antibody (purchased from CST).

FIG. 6 is a graph comparing immunohistochemical results of primary tissue cells and cervical cancer tumor cells obtained by culturing the cells in the culture medium CM of the present invention. As can be confirmed from FIG. 6, when the cervical cancer tumor cells (sample No. CG 2) cultured by the technique of the present invention were cultured up to the 4 th generation, the expression of the biomarker associated with cervical cancer on the cells substantially coincided with the expression of the marker in the original tissue section from which the cells were derived. It is demonstrated that the cells cultured by the technology of the invention maintain the original pathological characteristics of the cancer tissues of cervical cancer patients.

[ example 6]

Functional test for drug sensitivity of cervical cancer tumor cells derived from cancer tissue

Taking the sample of the cervical cancer patient after surgical excision as an example, the cervical cancer tumor cells cultured from the cervical cancer tumor sample from the patient can be used for detecting the sensitivity of the cervical cancer tumor cells to different drugs.

1. Plating primary cervical cancer tumor cells: the isolated cervical cancer tumor cell suspensions (accession No. CCa5 and accession No. CCa 9) obtained according to the method of example 1 were prepared at 4X 10 ⁴ Per cm ² The cells were densely seeded in 12-well plates pre-plated with NIH-3T3 cells after gamma irradiation. 2mL of the prepared cervical cancer primary cell culture medium CM was added to a 12-well plate, incubated at 37 ℃ and 5% CO ₂ Incubators (purchased from Saimeri fly) were used for culturing.When the cells grown in the culture plate reached about 80% of the basal area, the culture medium supernatant in the 12-well plate was discarded, 0.5mL of 0.25% trypsin (purchased from Thermo Fisher) was added and digested for 1 minute, then 0.25% trypsin was aspirated, 0.5mL of 0.05% trypsin was added and the cells were digested, incubated at 37 ℃ for 10 minutes until complete digestion of the cells was observed under a microscope (Invitrogen EVOS M500), and digestion was terminated with 1mL of DMEM/F12 culture medium containing 5% (v/v) fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin and collected in a 15mL centrifuge tube, and after centrifugation at 1500rpm for 4 minutes, the supernatant was discarded. The centrifuged cell pellet was suspended by CM culture medium, and counted by a flow image counter (JIMBIO FIL, jejuno microbial technologies, ltd.) to obtain 80 million and 76 million total cells, respectively. Inoculating the cells in 384-well plate at 2000-4000/well density to make the cells adhere to the wall overnight.

2. Drug gradient experiments:

(1) Preparing a drug storage plate by adopting a concentration gradient dilution method: the drug was diluted in 1:3 by pipetting 40. Mu.L of each 10. Mu.M of the test drug stock solution as the highest concentration, pipetting 10. Mu.L of each stock solution, adding the pipetted stock solution to a 0.5mL EP tube containing 20. Mu.L of DMSO, and pipetting 10. Mu.L of each stock solution from the above EP tube to a second 0.5mL EP tube containing 20. Mu.L of DMSO. Repeating the above method, sequentially diluting to obtain 7 concentrations required by dosing. Drugs at different concentrations were added to 384-well drug storage plates. Solvent control an equal volume of DMSO was added to each well as a control. In this example, the drugs to be tested were An Luoti ni (from MCE), pazopanib (from MCE), apatinib (from MCE), bortezomib (from MCE), cisplatin (from MCE), paclitaxel (from MCE), 5-fluorouracil (from MCE), and topotecan (from MCE).

(2) Different concentrations of drug and solvent controls in 384-well drug storage plates were added to 384-well cell culture plates plated with cervical cancer tumor cells using a high throughput automated workstation (Perkin Elmer JANUS), each with 3 replicates of the drug and solvent controls. The volume of drug added per well was 100nL.

(3) And (3) detecting the activity of the cells: administration of drugsAfter 72 hours, the chemiluminescence values of the cells after drug addition culture were measured with a Cell Titer-Glo detection reagent (available from Promega), the chemiluminescence values reflect the Cell viability and the effect of the drug on the Cell viability, 10 μ L of the prepared Cell Titer-Glo detection solution was added to each well, and the chemiluminescence values were measured with an enzyme-linked immunosorbent assay (Envision, perkin Elmer). Calculating the cell survival rate after obtaining the cells acted by different drugs according to the formula of cell survival rate (%) = adding hole chemiluminescence value/control hole chemiluminescence value 100%, drawing by using Graphpad Prism software, and calculating the half inhibition rate IC ₅₀ 。

(4) The results of the drug susceptibility testing are shown in figure 7. FIG. 7 shows the results of the sensitivity of cervical cancer tumor cells cultured from surgically resected cancer tissue samples (accession number CCa5 and accession number CCa 9) from two different cervical cancer patients to the targeted drugs An Luoti ni, pazopanib, apatinib, bortezomib, and the chemotherapeutic drugs cisplatin, paclitaxel, 5-fluorouracil, topotecan. The results show that the sensitivity of the cells of the same patient to the action of the drugs with different concentrations is different, and the sensitivity of the cells of different patients to the same drug is also different, so that the effectiveness of the cervical cancer patients in clinical use of the drug can be judged according to the results.

Industrial applicability

The invention provides a culture medium and a culture method for in vitro culture or amplification of cervical cancer primary cells, and the cells obtained by culture can be applied to the curative effect evaluation and screening of medicaments. Thus, the present invention is suitable for industrial applications.

Although the present invention has been described in detail herein, the present invention is not limited thereto, and modifications can be made by those skilled in the art based on the principle of the present invention, and thus, it is to be understood that various modifications made in accordance with the principle of the present invention are within the scope of the present invention.

Claims (11)

1. A culture medium for culturing primary cells of cervical cancer, comprising:

contains an MST1/2 kinase inhibitor; insulin-like growth factor 1; fibroblast growth factor 7; insulin-transferrin-selenium complex; hepatocyte growth factor; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152, and liothyronine,

wherein the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof,

wherein the content of the first and second substances,

R ₁ selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R ₆ Substituted aryl, arylC 1-C6 alkyl and heteroaryl;

R ₂ and R ₃ Each independently selected from C1-C6 alkyl;

R ₄ and R ₅ Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, and C3-C6 heterocycloC 1-C6 alkyl;

R ₆ selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.

2. The culture medium of claim 1, wherein

R ₁ Selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R ₆ Substituted phenyl, naphthyl, benzyl and thienyl;

R ₂ and R ₃ Each independently selected from C1-C3 alkyl;

R ₄ and R ₅ Each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, piperidinyl C1-C6 alkyl, and tetrahydropyranyl C1-C6An alkyl group;

R ₆ selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.

3. The culture medium of claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,

wherein the content of the first and second substances,

R ₁ selected from C1-C6 alkyl, optionally substituted with 1-2 independent R ₆ Substituted phenyl, optionally substituted with 1-2 independent R ₆ Substituted thienyl, and optionally substituted with 1-2 independent R ₆ A substituted benzyl group;

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

R ₆ each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl.

4. The culture medium of claim 3, wherein

R ₁ Is optionally substituted by 1-2 independent R ₆ Substituted phenyl;

R ₅ is hydrogen;

R ₆ preferably fluorine, methyl or trifluoromethyl.

5. The culture medium of claim 1, wherein the MST1/2 kinase inhibitor is selected from at least one of the following compounds or a pharmaceutically acceptable salt thereof:

/>

/>

/>

/>

6. the culture medium according to any one of claims 1 to 5, wherein:

the content of the MST1/2 kinase inhibitor is 2-20 mu M.

7. A culture medium according to any one of claims 1 to 5, wherein the medium meets any one or more or all of the following:

the content of the insulin-like growth factor 1 is 2-40 ng/ml;

the content of the fibroblast growth factor 7 is 2-40 ng/ml;

the volume ratio of the insulin-transferrin-selenium compound to the culture medium is 1;

the content of the hepatocyte growth factor is 2-40 ng/ml;

the content of the ROCK kinase inhibitor is 2-20 mu M;

the content of the liothyronine is 2-50 nM.

8. The culture medium according to any one of claims 1 to 5, wherein:

is free of serum, bovine pituitary extract, wnt agonists, R-spondin family proteins, BMP inhibitors, nicotinamide, and N-acetylcysteine.

9. The culture medium according to any one of claims 1 to 5, wherein:

the cervical cancer primary cell is selected from cervical cancer tumor cells, normal cervical cancer primary cells and cervical cancer epithelial stem cells.

10. A method for culturing primary cervical cancer cells is characterized by comprising the following steps:

(1) Preparing a culture medium according to any one of claims 1 to 9;

(2) Pre-laying a culture vessel with the trophoblasts irradiated by X rays or gamma rays;

(3) Inoculating a culture vessel pre-paved with trophoblasts to obtain cervical cancer primary cells separated from cervical cancer tissues, and culturing by using the culture medium in the step (1).

11. A method for evaluating or screening a drug for treating a cervical cancer disease, comprising the steps of:

(1) Culturing the cervical cancer primary cell using the method of culturing the cervical cancer primary cell of claim 10;

(2) Selecting a medicine to be detected and diluting according to a required concentration gradient;

(3) Adding the diluted medicine to the cervical cancer primary cells obtained by culturing in the step (1);

(4) Cell viability assays were performed.

Images