CN115975935A - Culture medium and culture method for primary cervical cancer cells - Google Patents

Abstract

The invention provides a culture medium for culturing primary cervical carcinoma epithelial cells, which contains at least one additive selected from an MST1/2 kinase inhibitor, a ROCK kinase inhibitor, a fibroblast growth factor 7, a B27 additive and an N2 additive, a hepatocyte growth factor, insulin-like growth factor 1, CHIR99021 and a TGF beta type I receptor inhibitor. The invention also relates to a culture method using the primary cell culture medium and application thereof. The culture method uses the primary cell culture medium to culture primary cells on a culture vessel coated with extracellular matrix glue, so that the primary cells are rapidly proliferated, and the cells obtained by culture can be used for evaluating or screening drugs for treating cervical cancer.

Description

Culture medium and culture method for 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 primary cervical carcinoma epithelial cells.

Background

Both the incidence and mortality of cervical cancer are the fourth of female malignancies worldwide, and the second largest malignancy next to breast cancer in developing countries. 50 million people are attacked every year around the world, 13 million people are attacked every year in China, and the annual attack age is about to be younger in recent years. At present, the operation, the radiation therapy and the chemotherapy of the cervical cancer become mature treatment methods. But has not achieved satisfactory curative effect for some patients with locally advanced cervical cancer. With the intensive research on the cervical cancer molecular target, new targeted therapeutic drugs are continuously emerged, and new hopes are brought to patients. However, the existing targeted therapeutic drugs with definite curative effects on cervical cancer are limited, and the long-term effects and toxic and side effects thereof are yet to be further researched. In addition to the selection of targeted drugs for gene detection, the in vitro culture of primary cells of cervical cancer patient samples has become an important means for predicting curative effect and guiding clinical medication in vitro in the future, but the rapid in vitro acquisition of cervical cancer primary cells is a technical problem to be solved urgently.

Functional testing refers to the in vitro detection of the sensitivity of anti-tumor drugs on cells of cancer patients. The key to applying this method is to develop a tumor cell model with a short growth cycle and capable of representing the biological characteristics of the cervical cancer patient. 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 low, the growth period is long, and the problems of excessive proliferation of mesenchymal cells such as fibroblasts exist, which restrict the development of the field. At present, two technologies for culturing primary epithelial cells/stem cells are developed relatively mature in the field of tumor cell functionality test application. One is a technique that uses irradiated feeder cells and the ROCK kinase inhibitor Y27632 to promote growth of primary epithelial cells to investigate drug sensitivity in individual patients, namely a cell conditional reprogramming technique (Liu et al, am J Pathol,180 599-607, 2012. Another technique is 3D culture of adult stem cells in vitro to obtain organoid techniques similar to tissues and organs (Hans Clevers et al, cell,11, 172 (1-2): 373-386, 2018).

However, both of these techniques have certain limitations. The cell reprogramming technology is a technology for co-culturing autologous primary epithelial cells of a patient and murine feeder cells. When primary cells of a patient are subjected to a drug sensitivity test, the existence of the murine cells can interfere with the drug sensitivity detection result of the autologous primary cells of the patient; however, if murine feeder cells are removed, the autologous primary cells of the patient are isolated from the reprogramming environment, and the proliferation rate and intracellular signaling pathways of the cells are significantly altered (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 outcome of the response of the autologous primary cells of the patient to the drug. The organoid technology is a technology for embedding autologous primary epithelial cells of a patient in an extracellular matrix for in vitro three-dimensional culture, and feeder cells are not needed in the technology, so that the problem of interference of mouse-derived feeder cells does not exist. However, the organoid technology requires the addition of several specific growth factors (such as Wnt proteins and R-spondin family proteins) in the culture medium, which is expensive and not suitable for clinical large-scale application. In addition, cells need to be embedded in extracellular matrix gel in the whole culture process of the organoid, the plating steps of cell inoculation, passage and drug sensitivity test are complicated and time-consuming compared with the 2D culture operation, the size of the organoid formed by the technology is not easy to control, and the situation that the inside of the organoid is necrotized due to the fact that the part of the organoid grows too large is easy to occur. Therefore, organoid techniques are less operable and adaptable than 2D culture techniques, require the operation of a skilled technician, and are not suitable for wide application on a large scale in clinical in vitro drug sensitivity assays (Nick Barker, nat Cell Biol,18 (3): 246-54, 2016).

In view of the limitations of the above technologies, there is a need to develop a primary cervical cancer epithelial cell culture technology in clinical practice, which has a short culture period, controllable cost, and convenient operation, and is not interfered by exogenous cells. When the technology is applied to the construction of 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 primary cervical carcinoma epithelial cells and a culture method of the primary cervical carcinoma epithelial cells by using the culture medium. The primary cervical carcinoma epithelial cell culture medium and the culture method are adopted for cell culture, and the purposes of short in-vitro culture period, controllable cost, convenient operation and no interference of exogenous cells can be realized. 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 primary cervical cancer epithelial cells, comprising an MST1/2 kinase inhibitor; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152; fibroblast growth factor 7 (FGF 7); at least one additive selected from the group consisting of B27 additives and N2 additives; hepatocyte Growth Factor (HGF); insulin-like growth factor 1 (IGF-1); CHIR99021; and at least one TGF beta type I receptor inhibitor selected from A83-01, SB431542, repsox, SB505124, SB525334, SD208, LY36494, and SJN2511, 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 first and the second end of the pipe are connected with each other,

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 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.

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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 between 2. Mu.M and 20. Mu.M, preferably between 5. Mu.M and 20. Mu.M.

In yet another embodiment, the ROCK kinase inhibitor is preferably Y27632. Further preferably, the ROCK kinase inhibitor is contained in the medium in an amount of usually 2 μ M to 20 μ M, preferably 10 μ M.

In a preferred embodiment, the fibroblast growth factor 7 is present in an amount of 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; the content of the hepatocyte growth factor is 2ng/ml to 40ng/ml, and more preferably 10ng/ml to 40ng/ml; the content of the insulin-like growth factor 1 is 2 ng/ml-40 ng/ml, and more preferably 10ng/ml; the content of the CHIR99021 is 1-10 mu M, and more preferably 1-3 mu M; the TGF beta I type receptor inhibitor is preferably A83-01, and the content of the TGF beta I type receptor inhibitor is 50 nM-500 nM, more preferably 100 nM-500 nM.

The medium formulation of the invention also contains a starting medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and an antibiotic selected from one or more of streptomycin/penicillin, amphotericin B, and Primocin. In some embodiments, the initial medium is preferably DMEM/F12 and the antibiotic is preferably Primocin. In a further preferred embodiment, the Primocin is present in the culture medium in an amount of 25 to 400. Mu.g/mL, preferably 50 to 200. Mu.g/mL.

Compared with the cell condition reprogramming culture medium and the cervical cancer epithelial cell organoid culture medium, the culture medium formula of the invention adds the MST1/2 kinase inhibitor, 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 (Nicotinamide) and N-Acetylcysteine (N-acetyl cysteine), 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 primary cervical cancer epithelial cells with controllable cost and convenient operation.

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

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

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

(2) The culture vessels were coated with extracellular matrix gel diluent.

Specifically, the extracellular matrix gel is a low-growth factor type extracellular matrix gel, and for example, commercially available Matrigel (available from corning corporation) or BME (available from Trevigen corporation) can be used. More specifically, the extracellular matrix glue was diluted with serum-free medium, which may be DMEM/F12 (from Corning). The dilution ratio of the extracellular matrix glue is 1. The coating method comprises the steps of adding the diluted extracellular matrix glue into a culture vessel, enabling the diluted extracellular matrix glue to completely cover the bottom of the culture vessel, standing and coating for more than 30 minutes, preferably standing and coating at 37 ℃, and preferably coating for 30-60 minutes. And (4) after the coating is finished, sucking and discarding the redundant extracellular matrix glue diluent, and using a culture vessel for later use.

(3) Separating primary cervical carcinoma epithelial cells from cervical carcinoma tissues.

Primary cervical cancer epithelial cells can be derived, for example, from a cervical cancer surgical sample or a biopsy sample. Cervical cancer surgical specimens are derived, for example, from cancer tissue specimens surgically excised from patients who have undergone and obtained consenting cervical cancer tumor. The tissue sample is collected within half an hour after surgical resection or biopsy sampling 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 Placing the culture medium into a precooled 10-15mL DMEM/F12 culture medium, placing the culture medium into a plastic sterile centrifuge tube with a cover, and transporting the culture medium to a laboratory on ice; wherein the DMEM/F12 medium contains the MST1/2 kinase inhibitor of the present invention (e.g., compound 1) and 0.2 to 0.4 vol% Primocin (hereinafter referred to as tissue transport fluid). When the MST1/2 kinase inhibitor of the invention is used, the concentration ranges from 2 μ M to 20 μ M, preferably from 3 μ M to 5 μ M; 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, and the tissue sample is rinsed by using a tissue transport solution to wash away blood cells on the surface of the tissue sample. Transferring the rinsed tissue sample to another new culture dishAdding 1-3mL of tissue transfusion solution, and cutting the tissue sample into pieces with volume less than 3mm by using sterile surgical blade and surgical forceps ³ The tissue fragment of (a).

Transferring the tissue sample fragment 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 3-5 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 epithelial cells separated in the step (3) into a coated culture vessel, and culturing by adopting the primary cell culture medium in the step (1).

More specifically, the ratio of 2X 10 in one well of a multi-well plate ⁴ ～8×10 ⁴ Per cm ² (e.g., 4X 10) ⁴ Per cm ² ) Inoculating primary cervical cancer tumor cells at a certain density, and adding appropriate amount of the cellsE.g. 2-3mL of primary epithelial cell culture medium, at e.g. 37 ℃, 5% ₂ Culturing in a cell culture box for 8-16 days, changing into a fresh primary cell culture medium every 4 days, and performing digestion passage when the primary cervical carcinoma epithelial cells grow to a cell density of about 80-90% of the bottom area of the porous plate.

The inoculation step does not need feeder cells, and compared with a cell condition reprogramming technology, the operation steps of culturing and irradiating the feeder cells are omitted. Compared with organoid technology, the step does not need to uniformly mix primary cells and matrigel on ice to form gel drops, and adds culture medium after the gel drops are solidified, and a pre-coated culture vessel can be directly used for primary cell inoculation. In addition, only a small amount of diluted extracellular matrix glue is needed for coating the culture vessel, compared with organoid technology, the use amount of the extracellular matrix glue with high price is saved, and the operation steps are simplified.

Optionally, after the inoculated primary cervical cancer epithelial cells are cultured for 8-16 days, when cell clones formed in a culture container are converged to reach a bottom area of 80%, discarding the supernatant, adding 0.5-2mL of 0.05% pancreatin (purchased from Thermo Fisher company) for cell digestion, and incubating at room temperature for 5-20 minutes; then 1 to 4mL of a DMEM/F12 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 primary cell culture medium of the present invention is used to resuspend the digested single cells, and the resulting cell suspension is placed in an extracellular matrix gel-coated T25 cell culture flask for further expansion culture. The coating operation of the T25 cell culture bottle is the same as the step (2).

The epithelial cells of the amplified cervical cancer grow in 2D, thereby avoiding the conditions of non-uniform size of organoid, necrosis of the grown organoid and the like caused by organoid technical amplification.

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

(1) The cervical cancer epithelial cells are cultured by using the primary cervical cancer epithelial cell culture method;

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

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

(4) Cell viability assays were performed.

The beneficial effects of the invention include:

(1) The success rate of primary cervical carcinoma epithelial cell culture is improved and reaches over 80 percent;

(2) The cervical cancer epithelial cells subjected to in vitro primary culture can keep the pathological phenotype and heterogeneity of patients from which primary cells are derived;

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

(4) 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;

(5) The efficiency of amplifying the cervical cancer epithelial cells is high, only 10 ⁴ The cell number of the grade can be successfully amplified to 10 within about two weeks ⁶ The cervical cancer epithelial cells of the order of magnitude, the cervical cancer epithelial cells amplified can also be continuously passed;

(6) 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;

(7) The culture cost is controllable, the primary cervical carcinoma cell culture medium does not need to add factors such as expensive Wnt agonist, R-spondin family protein, BMP inhibitor and the like, the existing primary cervical carcinoma epithelial cell or organoid culture medium is simplified and improved, cell inoculation does not need to use extracellular matrix with higher concentration to be mixed with the primary cells to form colloidal drops, only needs to use a small amount of diluent prepared by extracellular matrix glue, and saves the using amount of the extracellular matrix with higher cost;

(8) Compared with the conditional reprogramming technology, the technology has the advantages that the feeder cells do not need to be cultured and radiated, the problem that the quality and the quantity of the feeder cells in different batches influence the culture efficiency of the primary cells is solved, and the planking and the detected object of drug screening are only the primary cervical carcinoma epithelial cells and are not interfered by the feeder cells in a co-culture system required by the cell conditional reprogramming technology; compared with organoid technology, the method for coating the extracellular matrix glue, provided by the invention, has the advantages that a culture vessel can be prepared in advance, cells are not required to be embedded in the matrix glue like organoid technology, and the technical operation steps are simple, convenient and feasible;

(9) The cervical cancer epithelial cells obtained by the technical culture 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 epithelial cells derived from a human or other mammal, including cervical cancer tumor cells, normal cervical epithelial 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 primary cervical cancer cells in vitro.

The cells obtained by the culture method of the present embodiment can be applied to regenerative medicine, basic medicine studies of cervical cancer epithelial cells, screening of drug responses, development of new drugs derived from cervical cancer diseases, and the like.

Drawings

FIGS. 1A-1H 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 obtained by culturing cells isolated from 1 clinical tissue specimen for cervical cancer to day 4 and day 12, respectively, using the medium FCM of the present invention.

FIGS. 3A-3C are photographs taken under an inverted microscope of cells isolated from 1 surgically excised sample of cervical cancer after 15 days of culture in three different media.

FIG. 4 is a graph showing the comparison of cell proliferation effects obtained after culturing cells isolated from 9 surgical resection specimens of cervical cancer for 16 days under three different medium conditions.

FIG. 5 is a graph showing a comparison of cell growth curves obtained by culturing cells isolated from 1 clinical tissue specimen for cervical cancer in three different medium conditions, respectively.

FIG. 6 is a graph showing a comparison of immunohistochemical results of cervical cancer tumor cells obtained by culturing cells isolated from 1 sample obtained by surgical resection of cervical cancer using the medium FCM of the present invention.

FIG. 7 shows cell activity curves of epithelial cells of cervical cancer obtained by culturing cells isolated from 3 specimens surgically excised from cervical cancer according to the method of the present invention under the influence of 8 different drugs.

Detailed Description

In the present specification, the epithelial cells include differentiated epithelial cells and epithelial stem cells obtained from epithelial tissues. "epithelial stem cells" refer to cells having long-term self-renewal ability and differentiation into epithelial cells, and refer to stem cells derived from epithelial tissues. Examples of the epithelial tissue include cervix, cornea, oral mucosa, skin, conjunctiva, bladder, renal tubule, kidney, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), large intestine (including colon)), liver, pancreas, breast, salivary gland, lacrimal gland, prostate, hair root, trachea, lung, and the like. Among them, the cell culture medium of the present embodiment is preferably a medium for culturing cervical epithelial cells.

In the present specification, the term "epithelial tumor cell" refers to a cell derived from the above-mentioned epithelial tissue and made into a tumor.

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 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 reacted at 85 ℃ overnight. 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 to dryness under reduced pressure, and the resultant 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 used for extraction, 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.

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[ example 1]

Isolation of human primary cervical carcinoma epithelial cells

The cervical cancer tissue sample is derived from a cancer tissue sample surgically removed from a patient having a cervical cancer tumor who has been prescribed and agreed upon. An example of the sample (reference number CCa 2) will be described below.

The collection of the tissue sample is performed within half an hour after surgical resection or biopsy 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

Tissue transfusion component	Suppliers of goods	Final concentration
			DMEM/F12	Corning	99.8% by volume
Primocin	Invivogen	0.2 vol% (commercial product concentration 50 mg/ml)
			Compound 1	Self-made	3μM

TABLE 2 tissue digestive juice formulation

Tissue digestive juice component	Suppliers of goods	Final concentration
			HBSS	Gibco
	50% (by volume)
		RPMI-1640	Corning	50% (by 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 (reference number CCa 2) was transferred to a 100mm cell culture dish (purchased from NEST) and rinsed with a tissue transfer solution to wash away residual blood on the surface of the tissue specimen and remove excess tissue such as fat on the surface of the tissue specimen. Transferring the tissue sample after the moistening 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).

The tissue sample fragment was transferred to a15 mL centrifuge tube and centrifuged at 1500rpm for 4 minutes using a bench top centrifuge (Sigma 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, specifically shown in Table 2) 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 to see whether or not it contained blood cells, and if any, 3mL of a blood cell lysate (purchased from Sigma) was added, mixed well, lysed at 4 ℃ for 15 minutes, shaken once for 5 minutes, taken out after lysis, and centrifuged at 1500rpm for 4 minutes. The supernatant was discarded to obtain primary cells of cervical cancer after digestion and isolation, and the cells were resuspended in a Basal Medium (BM) prepared by adding 0.2 vol% of Primocin (obtained from Invivogen at a concentration of 50 mg/mL) in a commercially available DMEM/F-12 medium to obtain a final concentration of 100. Mu.g/mL. The total number of cells was 162 ten thousand by counting using a flow image counter (JIMBIO FIL, zhang Zong Biotech Co., ltd.).

[ example 2]

Optimization of primary cervical carcinoma epithelial cell culture medium

(1) Effect of different factors

Mixing extracellular matrix glue

An extracellular matrix diluent was prepared by diluting the culture medium (manufactured by BD Biotech) at a ratio of 1:100 using serum-free DMEM/F12, and 500. Mu.l/well of the extracellular matrix diluent was added to a 48-well plate so as to completely cover the bottom of the well of the plate. The mixture was allowed to stand in an incubator at 37 ℃ for 1 hour. After 1 hour, the extracellular matrix diluent was removed to give a Matrigel-coated culture plate.

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 cervical cancer epithelial cell culture media containing different additive components.

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

Cervical cancer tumor cells (accession number CCa 5) isolated from cervical cancer tissue according to the same method as in example 1 were seeded at a cell density of 3000 cells/well in 384-well culture plates, different components of culture medium were added at 50. Mu.l/well, surface sterilized, and then placed at 37 ℃ for 5% CO ₂ Incubators (purchased from semer fly) cultured the same number of freshly isolated cervical cancer tumor cells (accession number CCa 5) under different media formulations. After 6 days of incubation, CCK8 detection reagent (from MCE) was added at 5. Mu.l/well, incubated in an incubator for 2-4 hours, and the absorbance at OD450 was measured using a multifunctional microplate reader (from PE). As 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 after different culture media are cultured and the absorbance obtained by detecting after the basic culture medium BM is cultured. As shown in the table, the addition of different factors from Table 3 on the basis of BM produces different effects on cell proliferation. Wherein, under a specific concentration range, the B27 additive, the N2 additive, the fibroblast growth factor 7, the CHIR99021, the hepatocyte growth factor, the insulin-like growth factor 1, the compound 1, the Y27632 and the A83-01 have more remarkable promotion effect on cell proliferation.

(2) Proliferation of cervical cancer primary cells obtained in this patent by different concentrations of the added factor

Mixing extracellular matrix glue (A)

BD bioscience) was diluted at a ratio of 1:100 using serum-free DMEM/F12 medium to prepare an extracellular matrix diluent, and 200 μ l/well of the extracellular matrix diluent was added to a 48-well plate so as to completely cover the bottom of the well of the plate. The mixture was allowed to stand in an incubator at 37 ℃ for 1 hour. After 1 hour, the extracellular matrix diluent was removed to give a Matrigel-coated culture plate.

Preparation of primary cervical cancer epithelial cell culture medium of this example: fibroblast growth factor 7 (FGF 7) was added to Basal Medium (BM) at a final concentration of 40ng/ml, hepatocyte Growth Factor (HGF) was added to Basal Medium (BM) at a final concentration of 40ng/ml, insulin-like growth factor 1 (IGF-1) was added to Basal Medium (BM) at a final concentration of 40ng/ml, B27 additive was added to Basal Medium (BM) at a final concentration of 1.

Cervical cancer epithelial cells derived from a cancer tissue (accession number CCa 8) of a cervical cancer patient were isolated and obtained from the cancer tissue by the same method as in example 1. Subsequently, the cervical cancer epithelial 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 Density inoculation to

(available from BD biosciences) were coated in treated 48-well plates. 2mL of the prepared primary cervical carcinoma epithelial cell culture medium was added to a 48-well plate, and the mixture was incubated at 37 ℃ and 5% CO ₂ Incubators (purchased from Saimeri fly) were used for culturing. When the cells grown in the culture plates reached about 80% of the basal area, the culture supernatant in 48-well plates was discarded, 500. Mu.L of 0.05% pancreatin (purchased from Gibco) was added to digest the cells, and the cells were incubated at 37 ℃ for 10 minutes until complete digestion of the cells was observed under a microscope (EVOS M500, invitrogen), i.e., 1mL of a DMEM/F12 culture containing 5% (v/v) fetal bovine serum (purchased from Eicosa), 100U/mL penicillin (purchased from Corning) and 100. Mu.g/mL streptomycin (purchased from Corning) was terminated and collected in 15mL centrifuge tubes, and the supernatant was discarded after centrifugation at 1500rpm for 4 minutes. Resuspending the centrifuged cell pellet using a basal medium BM, and counting the cell pellet using a flow image counter (JIMBIO FIL, ohio, jiangsu, zhang Zolmi technologies, ltd.) to obtain the total number of cells. The resulting cells were used in the following culture experiments.

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

formula 1: the components of the culture medium do not contain B27 additive;

and (2) formula: the components of the culture medium do not contain fibroblast growth factor 7;

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) 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 A83-01;

and (4) formula 8: the components of the culture medium do not contain CHIR99021.

The above-described formulations 1 to 8 were used to dilute the above-described digested cell suspensions, respectively, and the cells were seeded into 48-well plates in a volume of 250. Mu.l per well.

When the culture medium of formula 1 is used, 250 microliters of the prepared B27 additive is added to each well of a 48-well plate inoculated with primary cells, and the final concentrations of the B27 additive are respectively 1; and control wells (BC) were set using medium of formula 1.

When the culture medium of formula 2 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 the medium of formulation 2.

When the culture medium of formula 3 is used, 250 microliters of prepared insulin-like growth factor 1 is added into 48-well plates inoculated with primary cells respectively, and the final concentrations of the insulin-like growth factor 1 are 40ng/mL, 10ng/mL and 2ng/mL respectively; and control wells (BC) were set using medium of formula 3.

When the culture medium of formula 4 is used, 250 microliters of the prepared hepatocyte growth factor per well 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 formulation 5.

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

When the culture medium of formula 7 is used, 250 microliters of the prepared A83-01 per well are respectively added into a 48-well plate inoculated with primary cells, and the final concentrations of the A83-01 are respectively 500nM, 100nM and 50nM; and control wells (BC) were set using medium of formula 7.

When the culture medium of formula 8 is used, 250 microliters of prepared CHIR99021 is respectively added into 48-well plates inoculated with primary cells, and the final concentrations of the CHIR99021 are respectively 10 muM, 3 muM and 1 muM; and control wells (BC) were set using medium of formula 8.

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 1H, respectively. In FIGS. 1A to 1H, 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 the factors or the small molecular compounds with different concentrations is better than that of a control Kong Peiyang medium, and the ratio is less than 1, which indicates that the proliferation promoting effect of the prepared culture medium containing the factors or the small molecular compounds with different concentrations is weaker than that of the control hole culture medium.

According to the results of fig. 1A to 1H, the volume concentration of the B27 additive in the medium is preferably 1; the content of the fibroblast growth factor 7 is preferably 2ng/ml to 40ng/ml, and more preferably 10ng/ml to 40ng/ml; 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 A83-01 is preferably 50 nM-500 nM, more preferably 100 nM-500 nM; the content of CHIR99021 is preferably 1. Mu.M to 10. Mu.M, more preferably 1. Mu.M to 3. Mu.M.

According to the preferred concentrations of the above components, a preferred medium formulation FCM according to 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 B27 additive, 5 μ M Compound 1, 10 μ M Y27632, 500nM A83-01, and 3 μ M CHIR99021.

[ example 3]

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

Cervical cancer epithelial cells derived from a cancer tissue were isolated from a cancer tissue (sample No. CCa 15) of a cervical cancer patient by the same method as in example 1. Subsequently, the cervical cancer epithelial 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 Density inoculation to

(available from BD biosciences) were coated in treated 12-well plates. 2mL of the prepared primary cervical carcinoma epithelial cell culture medium FCM was added to a 12-well plate, and the mixture was concentrated at 37 ℃ 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 The density was inoculated into a Matrigel-coated 6-well plate, and an under-lens photograph (photographed by a 10-fold inverted phase contrast microscope) was taken from the time of incubation to day 4 after the start of inoculation. As can be seen by observation under a mirror, the purity of the primary cervical cancer tumor cells from the cultured cancer tissues is higher, and the primary cervical cancer tumor cells do not contain fibroblasts. FIG. 2B is an under-lens photograph (40-fold inverted phase contrast microscope photograph) of the present example from day 12 of post-inoculation culture. As can be seen from the two graphs of FIGS. 2A and 2B, the isolated cervical cancer primary cells were cultured in vitro for 4 days to see significant colony formation under a microscope, and 12 days laterThe cell number is obviously amplified, which indicates that the technology of the invention is a high-efficiency technology for amplifying the cervical cancer epithelial 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

Primary cervical cancer epithelial cell culture medium FCM and basal medium BM as a control were prepared in the same manner as in example 2. In addition, as a further control, a literature culture medium RM was prepared, the formulation steps being described in (Ma Liping et al, jilin medicine 42 (6): 1289-1293, 2021) and the medium formulation in Table 4.

TABLE 4 literature culture Medium (RM) composition

Media composition	Suppliers of goods	Final concentration
			DMEM/F12 medium	Corning		100% by volume
Fibroblast growth factor	Beijing Yinqiao	10ng/ml
			Epidermal growth factor	Beijing Yiqiao	20ng/ml

Primary cervical cancer tumor cells derived from cervical cancer tissue (accession number CCa 10) were obtained in the same manner as in example 1. Then, the same density (4X 10) was used ⁴ Per cm ² ) The culture is carried out under the following three 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 to

(manufactured by BD Biotech Co., ltd.) in the coated 24-well plate, 2mL of the primary cervical cancer epithelial cell culture medium FCM of the present invention was used for culture;

B. by 4X 10 ⁴ Per cm ² Inoculating the primary cervical cancer tumor cells to

(manufactured by BD Biotech Co., ltd.) in the treated 24-well plate, 2mL of the literature culture medium RM was used to culture the cells in the 24-well plate.

C. By 4X 10 ⁴ Per cm ² Inoculation Density Primary cervical cancer tumor cells were inoculated to

(manufactured by BD Biotech Co., ltd.) in the treated 24-well plate, 2mL of the basal medium BM was used to culture the cells in the 24-well plate.

In the three cultures, the cells cultured under the three culture conditions were changed every 4 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 (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, 500. Mu.L of 0.05% pancreatin (purchased from GIBCO) is added to digest the cells, the cells are incubated at 37 ℃ for 10 minutes until the cells are completely digested under a microscope (EVOS M500, invitrogen), 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 to stop the digestion, the cells are collected in a15 mL centrifuge tube, and the cells are centrifuged at 1500rpm for 4 minutes, and then the supernatant is discarded. The cell pellet after centrifugation was resuspended in the culture medium of the present invention, and the total number of cells was counted by using a flow cytometer (JIMBIO FIL, jiangsu Zong microbial technology Co., ltd.) to obtain 19 ten thousand cells. The cells cultured under the other two culture conditions were digested and counted in the same manner as described above, and the total number of cells cultured using medium FM and CM was 4.5 ten thousand and 4.6 ten thousand, respectively.

The cell photographs of FIGS. 3A-3C are under-lens photographs (under a 10-fold inverted phase contrast microscope) of the sample of accession number CCa10 cultured to day 15 under three different culture conditions: wherein FIG. 3A is an under-the-lens photograph of CCa10 grown to day 15 using basal medium BM; FIG. 3B is an under-the-mirror photograph of CCa10 cultured up to day 15 using the culture medium FCM of this patent; FIG. 3C is an under-the-lens photograph of CCa10 cultured up to day 15 using the literature culture medium RM. As can be seen, the sample CCa10 could not form cell clones after 15 days of culture using the basal medium BM (FIG. 3A); cell clones could not be formed and the cell state was poor by 15 days of culture using the literature medium RM (fig. 3C); the cells are cultured for 15 days by using the culture medium FCM (figure 3B) to form clones, and the proliferation promoting effect is obvious.

FIG. 4 is a graph showing a comparison of cell proliferation effects obtained after 9 samples of cervical cancer patients were cultured for 16 days in the above three different culture media, wherein V represents general clonogenic ability and proliferation promoting effects, V represents relatively significant clonogenic ability and proliferation promoting effects, V represents relatively strong clonogenic ability and proliferation promoting effects, and X represents inability to form a clone, in cervical cancer primary cells obtained by the method of example 1. From FIG. 6, it can be confirmed that the culture medium of the present invention has significant advantages in terms of clonality, cell proliferation promoting effect, and culture success rate when used for culturing primary cells obtained from cervical cancer tissue sources, compared to other two culture conditions.

(2) Continuous culture of different culture media on primary cervical cancer tumor cells and drawing of growth curve

Primary cervical cancer epithelial cell culture medium FCM, and culture media BM and RM as controls were obtained using the same method as in this example (1).

Primary cervical cancer tumor cells (accession number CCa 5) derived from cervical cancer tissue were cultured in the same manner as in example (1) under the three medium conditions, and subjected to digestion passaging and counting.

When the cells after passaging grow again in the culture plate to about 80% of the plate bottom area, the cultured cells were again collected by digestion and counted as described above. Similarly by 4X 10 ⁴ The cells were inoculated at a density per well and cultured continuously.

The following formula is the calculation formula of the amplification factor (amplification Doubling) of primary cervical carcinoma epithelial cells under different culture conditions:

poultion Doubling (PD) =3.32 log10 (total number of cells after digestion/initial number of seeded cells), formula see (Chapman et al Stem Cell Research & Therapy 2014, 60.

FIG. 5 is a graph of the growth of cells CCa5 in three different culture conditions using Graphpad Prism software. The abscissa represents the number of days in which the cells are cultured, and the ordinate represents the cumulative cell proliferation fold, which represents the fold of the cells amplified during the culture period, wherein a larger value represents a larger number of times the cells are amplified during a certain period, i.e., a larger number of cells are amplified, and the slope represents the rate of cell amplification. From the figure, it was confirmed that the growth rate of the epithelial cells of cervical cancer cultured by the medium FCM of the present invention was superior to those of the other two culture conditions.

[ example 5]

Immunohistochemical identification of primary cervical cancer tissue and cervical cancer cells after subculture

Cancer tissue (sample No. CCa 14) having a size of about mung bean grain was taken from a clinical surgical resection sample of one example of cervical cancer patient, and fixed by immersing in 1mL of 4% paraformaldehyde. The remaining cancer tissues were obtained from the cervical cancer epithelial cells (sample No. CCa 14) by the same method as in example 1. The sample CCa14 was continuously cultured to the third generation using the medium FCM of the present invention using the method of example 3.

The expression of important biomarkers related to cervical cancer in the original tissue of the sample CCa14 and primary cells obtained by continuous culture to the third generation is detected by 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 P16 antibody (purchased from Affinit), P63 antibody (purchased from CST), and Ki67 antibody (purchased from CST).

As can be confirmed from FIG. 6, when the cervical cancer tumor cells (sample No. CCa 14) cultured with the medium of the present invention were cultured up to the 3 rd 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 culture medium 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

The following takes the example of a sample obtained by surgical excision of a cervical cancer patient as an example, and the cervical cancer tumor cells obtained by culturing the cervical cancer tumor sample from the patient can be used for detecting the sensitivity of the tumor cells of the patient to different drugs.

1. Plating primary cervical cancer tumor cells: the suspensions of the isolated cervical cancer tumor cells (accession No. CCa5, accession No. CCa6 and accession No. CCa 9) obtained according to the method of example 1 were prepared at 4X 10 ⁴ Per cm ² Density inoculation into 12-well plates. 2mL of the prepared primary cervical carcinoma epithelial cell culture medium FCM was added to a 12-well plate, and the mixture was concentrated at 37 ℃ and 5% CO ₂ Incubators (purchased from Saimeri fly) were used for culturing. When the cell growth 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 Co., ltd.) was added and digested for 1 minute, followed by aspiration of 0.25% trypsin and further0.5mL of 0.05% trypsin was added for cell digestion and incubated at 37 ℃ for 10 minutes until complete digestion of the cells could be observed under a microscope (EVOS M500, invitrogen), using 1mL of DMEM/F12 medium containing 5% (v/v) fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin to stop digestion, collected in a15 mL centrifuge tube, centrifuged at 1500rpm for 4 minutes, and the supernatant was discarded. The centrifuged cell pellet was suspended by FCM medium and counted by a flow cytometer (JIMBIO FIL, jejuno microbial technologies, ltd.) to obtain total cell numbers of 53 ten thousand, 78 ten thousand and 63 ten thousand, respectively. Inoculating the cells into 384-well plates at a density of 1000-2000 cells/well, and allowing the cells to adhere 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 were added at different concentrations 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 cisplatin (from MCE), paclitaxel (from MCE), 5-fluorouracil (5-F from MCE), topotecan (from MCE), bortezomib (from MCE), an Luoti ni (from MCE), pazopanib (from MCE), and apatinib (from MCE), and the like.

(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: 72 hours after administration, the chemiluminescence values of the cells after the addition of the drug and the culture were measured with Cell Titer-Glo assay reagent (available from Promega corporation), the magnitude of the chemiluminescence values reflecting the Cell viability and drug-to-fineThe Cell viability was influenced by adding 10. Mu.L of the prepared Cell Titer-Glo detection solution to each well, mixing well, and then detecting the chemiluminescence value using an enzyme-linked immunosorbent assay (Envision, perkin Elmer). Calculating the cell survival rate after different drug action cells according to the formula of cell survival rate (%) = adding hole chemiluminescence value/control hole chemiluminescence value 100%, plotting by using Graphpad Prism software and calculating half inhibition rate IC ₅₀ 。

(4) The results of the drug susceptibility testing are shown in figure 7.

FIG. 7 shows the drug sensitivity of cervical cancer tumor cells cultured from surgically excised cancer tissue samples (accession number CCa5, accession number CCa6 and accession number CCa 9) of three different cervical cancer patients to four chemotherapeutic drugs cisplatin, paclitaxel, 5-fluorouracil and topotecan and to four targeted drugs bortezomib, an Luoti ni, pazopanib and apatinib, respectively. 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 culturing primary cervical carcinoma epithelial cells, which can be used for evaluating and screening the curative effect of medicaments by using the cultured cells. Thus, the present invention is suitable for industrial applications.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the description. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Claims (13)

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

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; at least one additive of B27 additive and N2 additive; hepatocyte growth factor; insulin-like growth factor 1; CHIR99021; and at least one TGF beta type I receptor inhibitor selected from A83-01, SB431542, repsox, SB505124, SB525334, SD208, LY36494, and SJN2511,

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 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-C6 alkyl;

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:

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

the content of the MST1/2 kinase inhibitor in the culture medium 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 ROCK kinase inhibitor in the culture medium is 2-20 mu M;

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

the volume concentration of the B27 additive or the N2 additive in the culture medium is 1;

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

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

the content of the CHIR99021 is 1-10 mu M;

the content of the TGF beta I type receptor inhibitor is 50 nM-500 nM.

8. 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 MST1/2 kinase inhibitor is compound 1;

the ROCK kinase inhibitor is Y27632;

the TGF beta type I receptor inhibitor is A83-01.

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

a starting medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and

an antibiotic selected from one or more of streptomycin/penicillin, amphotericin B, and Primocin.

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

is free of serum, bovine pituitary extract, wnt agonist, R-spondin family protein, BMP inhibitor, nicotinamide and N-acetylcysteine.

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

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

12. A method for culturing primary cervical carcinoma epithelial cells is characterized by comprising the following steps:

(1) Preparing a culture medium according to any one of claims 1 to 11;

(2) Coating a culture vessel with an extracellular matrix gel diluent selected from at least one of Matrigel and BME;

(3) Inoculating primary cervical cancer epithelial cells separated from cervical cancer tissues into a culture vessel coated with extracellular matrix glue, and culturing by using the culture medium in the step (1).

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

(1) Culturing the cervical cancer epithelial cells using the method of culturing primary cervical cancer epithelial cells of claim 12;

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

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

(4) Cell viability assays were performed.

Images