Culture medium, culture method and application of ovarian cancer primary cells

Abstract

The application provides a culture medium and a culture method of ovarian cancer primary cells. The culture medium comprises an MST1 / 2 kinase inhibitor, sodium pyruvate, forskolin, epidermal growth factor, gastrin, fibroblast growth factor 7, nicotinamide, SB431542, and fetal bovine serum. Compared with the existing culture method, the in-vitro culture using the culture medium of the application has higher amplification efficiency; the culture of the ovarian cancer primary cells using the culture medium can maintain the morphological structure and pathological characteristics of the primary tissue, and improve the success rate and survival rate of the ovarian cancer primary cell culture.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a culture medium and application thereof, and more particularly to a culture medium for primary ovarian cancer cells and a culture method and application thereof. BACKGROUND

[0002] Ovarian cancer refers to a malignant tumor disease occurring in the ovary, and is one of the common malignant tumors of the female reproductive organs, with a morbidity rate only next to that of cervical cancer and uterine body cancer. Ovarian cancer is most commonly seen in epithelial carcinoma, followed by malignant germ cell tumors, among which the mortality rate of ovarian epithelial carcinoma ranks first among various gynecological tumors, which can seriously threaten women's lives. Ovarian cancer has no symptoms in the early stage, and in the late stage, symptoms such as lower abdominal discomfort, abdominal distension, and decreased appetite can occur. The main treatment methods include surgical resection, drug treatment, and radiotherapy, and the overall prognosis is poor.

[0003] Chemotherapy is one of the main methods for treating ovarian tumors. Although there are many chemotherapeutic drugs available in clinical practice at present, the clinical treatment efficiency of ovarian tumors is only about 25%. The main reason is that the chemotherapy drug regimen used by patients is mostly based on the experience of clinicians without considering individual differences in patients, and the method of trial and error is used, which not only fails to improve the therapeutic effect of the drug, but also misses the best treatment period, so that the tumor enters the late stage. In addition, during the entire treatment process, patients will bear the burden of drug side effects and high medical expenses.

[0004] Therefore, establishing a primary tumor model in vitro and using it for efficient drug screening experiments is a promising solution. The main method for establishing an in vitro culture model of primary ovarian tumors at present is the patient-derived tumor xenograft (PDX) method, which involves transplanting tumor cells from patients into nude mice and then investigating the therapeutic effect of the anti-tumor drug on them. However, the PDX method also has some disadvantages, such as species differences between humans and mice, long test period (more than 4 weeks), high cost (more than 200,000 yuan), and false positives and false negatives. SUMMARY

[0005] To solve the above technical problems, the present application provides a culture medium and culture method for rapidly expanding primary ovarian cancer cells in vitro.

[0006] One aspect of the present application is to provide a culture medium for primary ovarian cancer cells, which comprises an MST1 / 2 kinase inhibitor, sodium pyruvate, forskolin, epidermal growth factor, gastrin, fibroblast growth factor 7, nicotinamide, SB431542, and fetal bovine serum.

[0007] wherein the MST1 / 2 kinase inhibitor comprises a compound of Formula (I), or a pharmaceutically acceptable salt, or solvate thereof,

[0008]

[0009] wherein,

[0010] R1is selected from C1-C6alkyl, C3-C6cycloalkyl, C4-C8cycloalkylalkyl, C2-C6spirocycloalkyl, and aryl (e.g., phenyl and naphthyl, etc.) optionally substituted with 1-2 independent R6, aryl C1-C6alkyl (e.g., benzyl, etc.) and heteroaryl (e.g., thienyl, etc.) optionally substituted with 1-2 independent R6;

[0011] R2and R3are each independently selected from C1-C6alkyl, preferably C1-C3alkyl, more preferably methyl;

[0012] R4and R5are each independently selected from hydrogen, C1-C6alkyl, C3-C6cycloalkyl, C4-C8cycloalkylalkyl, C1-C6alkylhydroxy, C1-C6haloalkyl, C1-C6alkylamino C1-C6alkyl, C1-C6alkoxy C1-C6alkyl, and C3-C6heterocyclyl C1-C6alkyl (the heterocyclyl being selected from, e.g., piperidinyl, tetrahydropyranyl, etc.);

[0013] R6is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6alkyl (preferably methyl), C1-C6alkoxy (preferably methoxy), and C1-C6haloalkyl (preferably trifluoromethyl).

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

[0015]

[0016] wherein,

[0017] R1is selected from C1-C6alkyl, phenyl optionally substituted with 1-2 independent R6, thienyl optionally substituted with 1-2 independent R6, and benzyl optionally substituted with 1-2 independent R6, R1more preferably being phenyl optionally substituted with 1-2 independent R6;

[0018] R5is selected from hydrogen, C1-C6alkyl, and C3-C6cycloalkyl, R5more preferably being hydrogen;

[0019] R6is each independently selected from halogen, C1-C6alkyl, and C1-C6haloalkyl, R6more preferably being fluorine, methyl or trifluoromethyl.

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

[0021]

[0022]

[0023]

[0024]

[0025]

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

[0027] In an embodiment of the present application, the content of each component in the culture medium of the present application satisfies any one or more or all of the following:

[0028] (1) the content of the MST1 / 2 kinase inhibitor in the culture medium is 2.5-10 μM;

[0029] (2) the content of the sodium pyruvate in the culture medium is 0.25-1 mM;

[0030] (3) the content of the forskolin in the culture medium is 2.5-10 μM;

[0031] (4) the content of the epidermal growth factor in the culture medium is 5-80 ng / mL;

[0032] (5) the content of the gastrin in the culture medium is 3-81 nM;

[0033] (6) the content of the fibroblast growth factor 7 in the culture medium is 5-40 ng / mL;

[0034] (7) the content of the nicotinamide in the culture medium is 1-16 mM;

[0035] (8) the content of the SB431542 in the culture medium is 3.75-30 μM;

[0036] (9) the volume ratio of the fetal bovine serum to the culture medium is 2.5% (v / v) to 40% (v / v).

[0037] In an embodiment of the present application, the culture medium further contains an initial culture medium selected from DMEM / F12, DMEM, F12 or RPMI-1640; and an antibiotic selected from one or more of streptomycin / penicillin, amphotericin B and Primocin.

[0038] In the preferred embodiment, when the antibiotic is selected from streptomycin / penicillin, the streptomycin concentration ranges from 25 to 400 μg / mL, the penicillin concentration ranges from 25 to 400 U / mL, when the antibiotic is selected from amphotericin B, the concentration ranges from 0.25 to 4 μg / mL, and when the antibiotic is selected from Primocin, the concentration ranges from 25 to 400 μg / mL.

[0039] According to a second aspect, the present application also provides an in vitro culture method of ovarian cancer primary cells. In the in vitro culture method of ovarian cancer primary cells of the present application, the ovarian cancer primary cells are cultured in vitro using the ovarian cancer primary cell culture medium of the present application.

[0040] The ovarian cancer primary cell culture method of the present application comprises the following steps:

[0041] 1. Isolation of ovarian cancer primary cells

[0042] (1) Isolate the ovarian cancer tissue sample, add the basal medium and tissue digestion solution at a volume ratio of 1:3 (Note: the amount of tissue digestion solution added is about 5-10 mL for 1 g of tumor tissue), and place it in a constant temperature shaker for digestion. The digestion temperature is 4-37°C, and the digestion rotation speed is 200 rpm-350 rpm;

[0043] (2) The digestion is terminated when no obvious tissue mass is observed, and the digestion time is 3-6 hours;

[0044] (3) After centrifugation, discard the supernatant, the centrifugation speed is 1200-1600 rpm, and the centrifugation time is 2-6 minutes. Add the basal medium for resuspension for standby use.

[0045] 2. Culture using the ovarian cancer primary cell culture medium of the present application

[0046] Resuspend the ovarian cancer primary cells obtained in step 1 above with the ovarian cancer primary cell culture medium of the present application and count them. Seed them into culture dishes at a cell density of 1-10 x 10 4 cells / cm 2 , and add feeder cells at a cell density of 2-3 x 10 4 cells / cm 2 . The cells in the culture dishes can be digested and subcultured when they are more than 90% full.

[0047] Wherein, the formula of the basal culture medium described in step 1 includes an initial culture medium selected from DMEM / F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin / penicillin, amphotericin B and Primocin. The tissue digestion solution formula includes 1640 culture medium, collagenase II (1-2 mg / mL), collagenase IV (1-2 mg / mL), DNA enzyme (50-100 U / mL), hyaluronidase (0.5-1 mg / mL), calcium chloride (1-5 mM), and bovine serum albumin BSA (5-10 mg / mL). The trophoblast cells described in step 2 can be, for example, irradiated NIH-3T3 cells, the irradiation source is X-rays or γ-rays, preferably γ-rays, and the irradiation dose is 20-50 Gy, preferably 30 Gy.

[0048] In yet another aspect, the present invention also provides a method for evaluating or screening a drug for treating ovarian cancer, comprising the following steps:

[0049] (1) Cultivating primary ovarian cancer cells using the culturing method of the present invention for primary ovarian cancer cells for drug screening;

[0050] (2) Select the drug to be tested and dilute it according to the required concentration gradient;

[0051] (3) adding the drug at various concentration gradients to the cells cultured in (1);

[0052] (4) Conduct cell activity test.

[0053] The technical solution of the present invention can achieve the following technical effects:

[0054] (1) Improve the success rate of primary ovarian cancer cell culture, and be able to culture tumor tissues from multiple sample sources such as epithelial cancer, malignant germ cell tumors, stromal tumors, and metastatic tumors, with a success rate of over 80%;

[0055] (2) Ovarian cancer primary cells cultured in vitro can maintain the patient's pathological characteristics;

[0056] (3) The cultured primary ovarian cancer cells were not interfered with by stromal cells such as fibroblasts and adipocytes;

[0057] (4) High amplification efficiency, as long as 10 5 The number of cells can be successfully expanded to 10 in about a week. 6 The expanded primary ovarian cancer cells can be continuously passaged.

[0058] (5) The culture cost is controllable: the culture medium does not need to add expensive Wnt agonists, R-spondin family proteins, BMP inhibitors, FGF10 and other factors;

[0059] (6) The technology can obtain a large number of primary ovarian cancer cells with high uniformity, which is suitable for high-throughput screening of new candidate compounds and providing high-throughput in vitro sensitivity function test for patients. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 The figure is used to show the effect of different combinations of factors added in the primary ovarian cancer cell culture medium on the growth of primary ovarian cancer cells.

[0061] Figures 2A-2I The figure is used to show the effect of different concentrations of factors added in the primary ovarian cancer cell culture medium on the growth of primary ovarian cancer cells.

[0062] Figures 3A-3J The figure is a photo of the primary ovarian cancer cells cultured by using the primary ovarian cancer cell culture medium of the present application, observed by a microscope.

[0063] Figures 4A-4G The figure is the immunohistochemical result of the primary ovarian cancer tissue cells.

[0064] Figures 5A-5G The figure is the immunohistochemical result of the primary ovarian cancer cells obtained by culturing the primary ovarian cancer tissue cells to the fourth generation by using the primary ovarian cancer cell culture medium of the present application.

[0065] Figures 6A-6D The figure is the cell growth curve of the primary ovarian cancer cells cultured by using the primary ovarian cancer cell culture medium of the present application, the literature culture medium and the commercial culture medium, respectively.

[0066] Figures 7A-7E The figure is the result of the drug screening of the ovarian cancer cells of different generations obtained by using the primary ovarian cancer cell culture medium of the present application. DETAILED DESCRIPTION

[0067] In order to better understand the present application, the present application will be further described below in combination with the embodiments and the drawings, and the following embodiments are only used to illustrate the present application but not to limit it.

[0068] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0069] In the present specification, an MST1 / 2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1 / 2 signaling. Generally, an MST1 / 2 kinase inhibitor, for example, binds to MST1 / 2 kinase and reduces its activity. Since MST1 and MST2 have structural similarity, an MST1 / 2 kinase inhibitor can also be, for example, a compound that binds to MST1 or MST2 and reduces its activity.

[0070] 1. Preparation of MST1 / 2 kinase inhibitor compound 1

[0071] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydrophtalazine-2- yl)amino)benzenesulfonamide 1

[0072]

[0073] 2-amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): In a round bottom flask, 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) was added to methanol (30 mL), followed by dropwise addition of dichlorosulfoxide (1.2 mL) under ice bath. The reaction system was reacted at 85 °C overnight. After the reaction was completed, the system was evaporated under reduced pressure to remove the solvent, and the obtained white solid was directly used in the next step.

[0074] 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetic acid methyl ester (A3): In a round bottom flask, 2-amino-2-(2,6-difluorophenyl)acetic acid methyl ester (2 g) was added to acetone (30 mL) and potassium carbonate (2.2 g), and then the system was cooled to -10 °C using an ice-salt bath, followed by slow addition of a solution of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction system was stirred at room temperature overnight. After the reaction was completed, it was filtered, and the filtrate was evaporated under reduced pressure to remove the solvent. The residue was purified by column chromatography on pressurized silica gel to obtain compound A3. LC / MS: M+H 359.0.

[0075] 2-chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): In a round bottom flask, 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetic acid methyl ester (2.5 g) was added to acetic acid (50 mL) and iron powder (3.9 g). The reaction system was stirred at 60 °C for two hours. After the reaction was completed, the system was evaporated under reduced pressure to remove the solvent, and the obtained product was neutralized to basicity with saturated sodium bicarbonate. Ethyl acetate was extracted, and the organic phase was washed with water and saturated brine, respectively, and then dried with anhydrous sodium sulfate. The organic phase was filtered and evaporated under reduced pressure to obtain a crude product. The crude product was washed with diethyl ether to obtain compound A4. LC / MS: M+H 297.0.

[0076] 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (A5): In a round bottom flask, 2-chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (2 g) and N,N-dimethylacetamide (10 mL) were added and cooled to -35 °C, iodomethane (0.9 mL) was added, followed by sodium hydride (615 mg), the reaction system was continued to stir for two hours. After the reaction was completed, water was added to quench, extracted with ethyl acetate, the organic phase was washed with water, saturated brine respectively, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain compound A5. LC / MS: M+H 325.0.

[0077] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino) benzenesulfonamide (1): In a round bottom flask, 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) were added. The reaction system was stirred at 120 °C overnight. After the reaction was completed, it was filtered and washed with methanol and diethyl ether to obtain compound 1. LC / MS: M+H 461.1.

[0078] 2. Preparation of other MST1 / 2 inhibitor compounds of the present application

[0079] Other MST1 / 2 inhibitor compounds of the present application were synthesized according to the similar method as compound 1, the structures and mass spectrometry data of which are shown in the following table.

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Example 1 Effect of each additive factor in the culture medium of ovarian cancer primary cells on the proliferation of ovarian cancer primary cells

[0086] (1) Preparation of the culture medium of ovarian cancer primary cells

[0087] First, the base medium containing the initial medium was prepared. The initial medium can be selected from DMEM / F12, DMEM, F12 or RPMI-1640 commonly used in the art. In this embodiment, the formula of the base medium is: DMEM / F12 medium (purchased from Corning) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available product concentration 50 mg / ml). Different types of additives were added into the base medium respectively (see Table 1) to prepare the primary ovarian cancer cell culture medium containing different additives.

[0088] (2) Isolation and processing of ovarian cancer primary cells

[0089] 1 Sample selection

[0090] Ovarian cancer solid tumor tissue samples (intraoperative) were obtained from patients by professional medical staff of professional medical institutions, and the patients all signed informed consent. The intraoperative sample was 0.25 cm 3 , the endoscopic sample was 0.025 cm 3 ; stored and transported using commercial tissue preservation solution (manufacturer: Miltenyi Biotec).

[0091] 2 Material preparation

[0092] 15 mL sterile centrifuge tube, pipette, 10 mL pipette, sterile tip, etc. were surface sterilized and placed in the ultraclean workbench for ultraviolet irradiation for 30 minutes. The washing medium was taken out from the 4°C refrigerator 30 minutes in advance, and the tissue digestion solution was taken out from the -20°C refrigerator 30 minutes in advance.

[0093] Base medium: DMEM / F12 medium containing 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available product concentration 50 mg / ml).

[0094] Tissue digestion solution: 1640 medium (Corning, 10-040-CVR), collagenase II (2 mg / mL), collagenase IV (2 mg / mL), DNAase (50 U / mL), hyaluronidase (0.75 mg / mL), calcium chloride (3.3 mM), BSA (10 mg / mL).

[0095] The above-mentioned collagenase II, collagenase IV, DNAase, and hyaluronidase were purchased from Sigma Company; calcium chloride was purchased from Shengong Bioengineering (Shanghai) Co., Ltd.; BSA was purchased from Biofroxx Company.

[0096] 3. Isolation of ovarian cancer primary cells

[0097] 3.1 Take the tissue sample in a culture dish, remove the blood-containing tissue, rinse twice with basal medium, transfer the tissue to another culture dish and mechanically separate it using a sterile scalpel to divide the tissue block into 1x1x1mm 3 size;

[0098] 3.2 Absorb the cut intraoperative tissue into a 15mL centrifuge tube, add 5mL of basal medium, mix well, and centrifuge at 1500rpm for 4 minutes;

[0099] 3.3 Discard the supernatant, add basal medium and tissue digestion solution at a ratio of 1:3 (Note: the amount of tissue digestion solution added is about 10mL for 1g of tumor tissue), label the sample name and number, seal with sealing film, and digest at 37°C in a 300rpm shaker (ZQLY-180N, Zichen Instrument) for 4 hours. Observe the digestion every 30 minutes during the period, and the judgment basis is no visible particles under the naked eye. The digestion time is 4 hours.

[0100] 3.4 After digestion is complete, filter out the undigested tissue clumps through a 100μm filter screen, rinse the tissue clumps on the filter screen into a centrifuge tube with basal medium to reduce cell loss, and centrifuge at 1500rpm for 4 minutes at 25°C.

[0101] 3.5 Discard the supernatant, observe whether there are blood cells, if there are blood cells, add 8mL of blood cell lysis solution (purchased from Sigma Company), mix well, lyse at 4°C for 20 minutes, and mix well once during the period, centrifuge at 1500rpm for 4 minutes at 25°C.

[0102] 3.6 Discard the supernatant, add 2mL of basal medium to resuspend the cells, and reserve for use.

[0103] 4 Cell counting and processing

[0104] 4.1 Microscopic observation: take a small amount of resuspended cells and spread them on a culture dish, and observe the cancer cell density and morphology under a microscope (CNOPTEC, BDS400).

[0105] 4.2 Live cell counting: take 12μL of resuspended cell suspension, mix thoroughly with 12μL of trypan blue dye solution (Shanghai Genechem Biotech Co., Ltd.), and then take 20μL and add it to a cell counting plate (Countstar, specification: 50 pieces / box), and calculate the percentage of live large cells (cell diameter >10μm) = live cell number / total cell number x 100% under a cell counter (Countstar, IC1000).

[0106] (3) Culture of ovarian cancer primary cells

[0107] The culture medium with different components in Table 1 was added into a 48-well plate at a volume of 1 mL / well.

[0108] The primary ovarian cancer cells were cultured at 4×10 4 The cells were seeded at a density of 10 cells / well in a 48-well culture plate and 2×10 4 NIH-3T3 cells (purchased from ATCC and resuspended in basal medium) irradiated with gamma rays (irradiation dose of 30 Gy) were cultured at 37°C and 5% CO₂. After 7-10 days of culture, when the cells reached 85% confluence, the medium was discarded and the cells were rinsed once with 100 μL of 0.05% trypsin (purchased from Gibco) per well. The cells were then aspirated and 200 μL of 0.05% trypsin was added to each well. The cells were incubated in a 37°C, 5% CO₂ incubator for 10 minutes. Complete digestion was observed under a microscope (CNOPTEC, BDS400). Digestion was terminated by adding 300 μL of DMEM / F12 medium supplemented with 10% serum (Excell Bio, FND500). 20 μL of the culture medium was then added to a cell counting plate (Countstar, specification: 50 plates / box) and the total number of cells was counted using a cell counter (Countstar, IC1000). As an experimental control, a basal medium without any additives was used. The experimental results are shown in Table 1.

[0109] Table 1 Additives in culture medium and their effects on promoting cell proliferation

[0110]

[0111] Among them, "+" indicates that compared with the basal medium, the culture medium to which the additive is added has a proliferation-promoting effect on at least two of the primary ovarian cancer cells isolated from ovarian cancer tissue; "-" indicates that the culture medium to which the additive is added has a proliferation-promoting effect on one of the primary ovarian cancer cells isolated from ovarian cancer tissue; and "○" indicates that the culture medium to which the additive is added has no significant effect on the proliferation of at least two of the primary ovarian cancer cells isolated from ovarian cancer tissue.

[0112] Based on the above results, factors such as SB202190, compound 1, hydrocortisone, SB431542, epidermal growth factor, insulin, insulin-transferrin-selenium supplement, nicotinamide, non-essential amino acids, Y-27632, fetal bovine serum, forskolin, sodium pyruvate, gastrin, fibroblast growth factor 7, and cholera toxin were selected for further culture experiments.

[0113] Example 2 Effects of different combinations of added factors in the culture medium for primary ovarian cancer cells on the proliferation of primary ovarian cancer cells

[0114] The ovarian cancer primary cell culture medium was prepared according to the composition in Table 2 with different additive factor combinations, and the effects of different additive factor combinations on the proliferation of ovarian cancer primary cells were investigated.

[0115] Table 2 Preparation of different component culture media (concentrations are final concentrations)

[0116]

[0117]

[0118] According to the method of step (2) of Example 1, ovarian cancer primary cells were obtained from ovarian cancer tissues (Nos. L37, L40, L43, L44), and the obtained cell suspension was evenly divided into 18 parts and centrifuged at 1500 rpm for 4 minutes. After centrifugation, 200 μL of BM and culture media Nos. 1-17 were used for resuspension, respectively, and the cells were inoculated in 48-well plates at a cell density of 4 x 10 4 cells / cm 2 After inoculation in 48-well plates (40,000 cells per well), 2 x 10 4 cells / cm 2 γ-ray irradiated (irradiation dose: 30 Gy) NIH-3T3 cells (purchased from ATCC and resuspended with basal medium (BM)) were added, and the volume of each well of the 48-well plate was finally adjusted to 1 mL with the corresponding culture medium, and mixed thoroughly. After surface sterilization, the plate was incubated in a 37°C, 5% CO2 incubator (purchased from Thermo).

[0119] When the cells in the 48-well plate reached more than 85%, the culture medium was discarded, and 100 μL of 0.05% trypsin (purchased from Gibco) was used for rinsing once, and then 200 μL of 0.05% trypsin was added to each well. The plate was incubated in a 37°C, 5% CO2 incubator for 10 minutes, and the cells were observed under a microscope (CNOPTEC, BDS400) to determine whether they had been completely digested. Then, 300 μL of DMEM / F12 medium containing 10% serum (Excell Bio, FND500) was added to terminate the digestion, and 20 μL of the cell suspension was added to a cell counting plate (Countstar, specification: 50 pieces / box), and the total number of cells was counted using a cell counter (Countstar, IC1000). The results obtained from the ovarian cancer primary cells isolated from the intraoperative samples L37, L40, L43, and L44 are shown in Figure 1 .

[0120] According to Figure 1The results show that compared with the basal medium (BM), when using the above-mentioned No. 1 to No. 17 culture media, the proliferation effect is best when ovarian cancer primary cells are cultured using culture media containing added ingredients such as sodium pyruvate, forskolin, epidermal growth factor, gastrin, fibroblast growth factor 7, nicotinamide, SB431542, compound 1, fetal bovine serum, etc.

[0121] Example 3 Effects of different concentrations of factors added to ovarian cancer culture medium on the proliferation of primary ovarian cancer cells

[0122] Primary ovarian cancer cells were obtained from tissue samples (numbered L53, L55, and L56) according to the method of step (2) of Example 1. The obtained primary ovarian cancer cells were cultured at a viable cell density of 3×10 4 pieces / cm 2 The cells were seeded in 6-well plates (300,000 cells per well) at a cell density of 2×10 4 pieces / cm 2 NIH-3T3 cells irradiated with gamma rays (irradiation dose 30 Gy) were added and mixed. After surface disinfection, the cells were cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific). The cells were then cultured and expanded using the combination of effective factors identified in Example 2 (containing basal medium BM, 1 mM sodium pyruvate, 10 μM forskolin, 20 ng / mL epidermal growth factor, 27 nM gastrin, 5 ng / mL fibroblast growth factor 7, 4 mM nicotinamide, 15 μM SB431542, 10 μM compound 1, and 10% (v / v) fetal bovine serum). When cells have grown to over 85%, add 500 μL of 0.05% trypsin (Gibco) and rinse for 1 minute. After aspiration, add 1 mL of 0.05% trypsin to each well. Incubate in a 37°C, 5% CO2 incubator for 2-10 minutes until cells are completely digested. Add 1 mL of DMEM / F12 medium containing 10% serum (ExcellBio, FND500) to terminate digestion. Centrifuge at 1500 rpm for 4 minutes and discard the supernatant. Resuspend the cell pellet in DMEM / F12. Take 20 μL of the culture and add it to a cell counting plate (Countstar, size: 50 plates / box). Count the total number of cells using a cell counter (Countstar, IC1000). The resulting cells will be used in the following culture experiments.

[0123] Next, the following 9 culture medium formulations were prepared for the experiment:

[0124] Recipe 1: The above-mentioned ovarian cancer primary cell culture medium components do not contain sodium pyruvate;

[0125] Recipe 2: The above-mentioned primary ovarian cancer cell culture medium components do not contain forskolin;

[0126] Formulation 3: The above primary ovarian cancer cell culture medium component does not contain epidermal growth factor;

[0127] Formulation 4: The above primary ovarian cancer cell culture medium component does not contain gastrin;

[0128] Formulation 5: The above primary ovarian cancer cell culture medium component does not contain fibroblast growth factor 7;

[0129] Formulation 6: The above primary ovarian cancer cell culture medium component does not contain nicotinamide;

[0130] Formulation 7: The above primary ovarian cancer cell culture medium component does not contain SB431542;

[0131] Formulation 8: The above primary ovarian cancer cell culture medium component does not contain Compound 1;

[0132] Formulation 9: The above primary ovarian cancer cell culture medium component does not contain fetal bovine serum.

[0133] 20 μl of cell resuspension containing 4 x 10 4 The cell suspension was diluted with 1 mL of the above Formulations 1 to 9, respectively.

[0134] When using the medium of Formulation 1, 1 mL of prepared sodium pyruvate was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of sodium pyruvate was 0.25 mM, 0.5 mM, 1 mM, 2 mM, and 4 mM, respectively; and a control well (BC) was set using the medium of Formulation 1.

[0135] When using the medium of Formulation 2, 1 mL of prepared forskolin was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of forskolin was 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively; and a control well (BC) was set using the medium of Formulation 2.

[0136] When using the medium of Formulation 3, 1 mL of prepared epidermal growth factor was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of epidermal growth factor was 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL, respectively; and a control well (BC) was set using the medium of Formulation 3.

[0137] When using the medium of Formulation 4, 1 mL of prepared gastrin was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of gastrin was 1 nM, 3 nM, 9 nM, 27 nM, and 81 nM, respectively; and a control well (BC) was set using the medium of Formulation 4.

[0138] In the medium using formula 5, 1 mL of prepared fibroblast growth factor 7 was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of fibroblast growth factor 7 was 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively; and a control well (BC) was set using the medium of formula 5.

[0139] In the medium using formula 6, 1 mL of prepared nicotinamide was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of nicotinamide was 1 mM, 2 mM, 4 mM, 8 mM, and 16 mM, respectively; and a control well (BC) was set using the medium of formula 6.

[0140] In the medium using formula 7, 1 mL of prepared SB431542 was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of SB431542 was 3.75 μM, 7.5 μM, 15 μM, 30 μM, and 60 μM, respectively; and a control well (BC) was set using the medium of formula 7.

[0141] In the medium using formula 8, 1 mL of prepared compound 1 was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of compound 1 was 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively; and a control well (BC) was set using the medium of formula 8.

[0142] In the medium using formula 9, 1 mL of prepared fetal bovine serum was added to each well of the 48-well plate inoculated with primary cells, and the addition ratio of fetal bovine serum was 2.5% (v / v), 5% (v / v), 10% (v / v), 20% (v / v), and 40% (v / v), respectively; and a control well (BC) was set using the medium of formula 9.

[0143] After the cells were expanded to about 85% of the 48-well plate, the cell count was digested, and the proliferation fold was calculated by referring to the cell number of the control well (BC), and the results are shown in Table 1, Table 2, Table 3, Table 4, and Table 5, respectively. Figures 2A-2I . Figures 2A-2I In Table 1, Table 2, Table 3, Table 4, and Table 5, the ratio is the ratio of the number of cells obtained by using each medium for one generation to the number of cells obtained by culturing the corresponding control well for one generation. If the ratio is greater than 1, it means that the prepared medium containing different concentrations of factors or small molecule compounds has a better proliferation effect than the control well medium; if the ratio is less than 1, it means that the prepared medium containing different concentrations of factors or small molecule compounds has a weaker proliferation effect than the control well medium.

[0144] According to the results of the above experiments, the following conclusions can be drawn: Figures 2A-2IAs a result, the content of sodium pyruvate is preferably 0.25 to 1 mM, and the cell proliferation effect is most obvious at a concentration of 0.5 mM; the content of forskolin is preferably 2.5 to 10 μM, and the cell proliferation effect is most obvious at a concentration of 2.5 μM; the content of epidermal growth factor is preferably 5 to 80 ng / mL, more preferably 5 to 20 ng / mL, and the cell proliferation effect is most obvious at a concentration of 10 ng / mL; the content of gastrin is preferably 3 to 81 nM, more preferably 9 to 81 nM, and the cell proliferation effect is most obvious at a concentration of 27 nM; the content of fibroblast growth factor 7 is preferably 5 to 40 ng / ml, more preferably 5 to 20 ng / ml, and the cell proliferation effect is most obvious at a concentration of 10 ng / ml; the content of nicotinamide in the culture medium is preferably 1 to 16 mM, more preferably 1 to 4 mM, and the cell proliferation effect is most obvious at a concentration of 1 mM; the content of SB431542 is preferably 3.75 to 30 μM, more preferably 3.75 to 15 μM, and the cell proliferation effect is most obvious at a concentration of 7.5 μM; the content of compound 1 is preferably 2.5 to 10 μM, more preferably 2.5 to 5 μM, and the cell proliferation effect is most obvious at a concentration of 5 μM; and the volume content of fetal bovine serum is preferably 2.5 to 40% (v / v), more preferably 5 to 20% (v / v), and the cell proliferation effect is most obvious at a concentration of 10% (v / v).

[0145] The optimal concentrations of the above-mentioned culture medium additives were used as the primary ovarian cancer cell culture medium of the present application used in the following examples, which contained: basal medium BM, 0.5 mM sodium pyruvate, 2.5 μM forskolin, 10 ng / mL epidermal growth factor, 27 nM gastrin, 10 ng / ml fibroblast growth factor 7, 1 mM nicotinamide, 7.5 μM SB431542, 5 μM compound 1, and 10% (v / v) fetal bovine serum (hereinafter referred to as "OC-1" medium).

[0146] Example 4 Primary ovarian cancer cell culture and identification

[0147] Primary ovarian cancer cells were obtained from 10 tissue samples (Nos. L53, L54, L56, L58, L60, L62, L65, L66, L69, L74) according to the method of step (3) of procedure (2) of Example 1, and cultured using the OC-1 medium of Example 3. The obtained primary ovarian cancer cells were cultured in a 6-well plate (30 million cells per well) at a cell density of 3 x 105cells / cm2. 4 2 Inoculation was performed in a 6-well plate (30 million cells per well) at a cell density of 2 x 105cells / cm2. 4 2 NIH-3T3 cells irradiated with γ-rays (irradiation dose 30 Gy) were added and mixed. After surface sterilization, the cells were cultured in a 37°C, 5% CO2incubator (purchased from Thermo Fisher).​​

[0148] The cultured ovarian cancer primary cells were observed using a microscope (Invitrogen EVOS M500), Figures 3A-3J are photos taken under 10x objective lens, the cells under the microscope are closely arranged, and the morphology is slightly irregular.

[0149] About 0.25 cm3of cancer tissue was taken from the intraoperative tissue (sample No. L62) of an ovarian cancer patient, 3 fixed in 1 mL of 4% paraformaldehyde. The sample L62 was continuously cultured to the 4th generation using the culture medium OC-1 of the present application by the method of Example 3. The tissue or cells fixed by 4% paraformaldehyde were paraffin-embedded and cut into 4 pm-thick tissue sections using a microtome. Subsequently, conventional immunohistochemical detection was performed (see Li et al., Nature Communication, (2018) 9:2983 for specific steps). The primary antibodies used were ER, PR, P53, NapsinA, Pax-8, WT-1, and Ki-67 (all purchased from CST).

[0150] Figures 4A-4G and 5A-5G are comparison charts of immunohistochemical results of the original tissue cells and the ovarian cancer primary cells obtained by culturing the cells using the ovarian cancer primary culture medium OC-1 of the present application. Figure 4A and Figure 5A are pictures of the marker ER antibody of the ovarian cancer tissue and the cultured ovarian cancer primary cells, respectively, Figure 4B and Figure 5B are pictures of the marker PR antibody of the ovarian cancer tissue and the cultured ovarian cancer primary cells, respectively, Figure 4C and Figure 5C are pictures of the marker P53 antibody of the ovarian cancer tissue and the cultured ovarian cancer primary cells, respectively, Figure 4D and Figure 5D are pictures of the marker NapsinA antibody of the ovarian cancer tissue and the cultured ovarian cancer primary cells, respectively, Figure 4E and Figure 5E are pictures of the marker Pax-8 antibody of the ovarian cancer tissue and the cultured ovarian cancer primary cells, respectively, Figure 4F and Figure 5F are pictures of the marker WT-1 antibody of the ovarian cancer tissue and the cultured ovarian cancer primary cells, respectively, Figure 4G and Figure 5GThe pictures are the marker Ki-67 antibody of ovarian cancer tissue and cultured ovarian cancer primary cells, respectively. It can be confirmed that the expression of the biomarker related to ovarian cancer on the ovarian cancer primary cells cultured by the technology of the application to the 4th generation is basically consistent with the marker expression of the original tissue section of the ovarian cancer primary cells. This shows that the ovarian cancer primary cells cultured by the technology of the application maintain the original pathological characteristics of the cancer tissue of the ovarian cancer patient.

[0151] Example 5 Comparison of culture effect with existing medium, and calculation of culture cycle and cell number statistics and Population Doubling (PD) value of ovarian cancer primary cells

[0152] Literature medium (Xuefeng Liu et al., Nat. Protoc., 12(2): 439-451, 2017), the formula of which is DMEM / F12 medium + 250 ng / ml amphotericin B (purchased from Selleck Company) + 10 μg / ml gentamicin (purchased from MCE Company) + 0.1 nM cholera toxin + 0.125 ng / ml EGF + 25 ng / ml hydrocortisone + 10 μM Y27632 + 10% FBS.

[0153] Commercial medium: Defined K-SFM, Keratinocyte Serum Medium (purchased from gibco company, 10744-019)

[0154] According to the method of step (2) of Example 1, ovarian cancer primary cells were obtained from 4 ovarian cancer tissue samples (No. BNVT1083, L57, L58, L60). For the obtained ovarian cancer primary cells, the literature medium, the commercial medium and the OC-1 medium in Example 3 were used for culture, and the cell density was 3×10 4 2 The cells were inoculated in a 6-well plate and cultured, and after the cells were expanded to 95%, they were digested and counted, and the number of days of culture until digestion was recorded, which was taken as a culture cycle. Under the experimental conditions, the cells obtained by expansion were expanded for different generations, and after each generation was digested and counted, the corresponding culture cycle was recorded, and the PD was calculated according to the formula Population Doubling (PD) = 3.32*log10 (total number of cells after digestion / initial number of cells inoculated). The formula is referred to in Chapman et al., Stem Cell Research & Therapy 2014, 5:60.

[0155] Figures 6A-6D ​The growth curves of four primary cells cultured using the literature medium, the commercial medium and the ovarian cancer primary cell culture medium OC-1 of the present application are shown, which are plotted using Graphpad Prism software, the abscissa represents the days of cell culture, and the ordinate is the cumulative cell proliferation fold, which represents the fold of cell expansion in the culture period, and the larger the value is, the more times the cells are expanded in a certain period, that is, the more cells are obtained by expansion, and the slope represents the cell expansion rate.

[0156] It can be confirmed from Figures 6A-6D that the cell expansion rate of the ovarian cancer primary cells cultured in the medium OC-1 of the present application remains basically unchanged when the cells are continuously cultured and expanded for at least 60 days, and the cells still have the ability to continue to expand; the expansion rate of the ovarian cancer primary cells cultured using the literature medium and the commercial medium is significantly lower than that of the OC-1 medium, and the cells stop proliferating after at most 2 passages. In summary, compared with the literature medium and the commercial culture, the ovarian cancer primary cell culture medium of the present application has a significantly better proliferation efficiency in the in vitro culture of ovarian cancer cells.

[0157] Example 6: Use of the ovarian cancer primary cells expanded using the medium of the present application for drug screening and efficacy evaluation

[0158] 1. Cell culture and plating

[0159] The ovarian cancer primary cells (numbered as L62) were isolated according to the method of step (2) of Example 1, and were used as the first generation and cultured using the ovarian cancer primary cell culture medium OC-1 of the present application. When the cells were expanded to 85%, they were passaged. The cells were counted according to step (2) of Example 1, and the cells were plated at a viable cell density of 1 x 10 5 After the sample wells were mixed thoroughly, the cells were cultured in 384-well non-transparent white cell culture plates (purchased from Corning) at a volume of 50 μL per well, and the number of cells was 5000 cells per well. The ovarian cancer primary cell culture medium of the present application was added to the edge of the well plate to seal the plate, and the plate was labeled with the sample name, the drug addition time and the CellTiter-Glo (purchased from Promega) detection time. The surface was sterilized with 75% alcohol (purchased from Lijian Kang), and the plate was cultured in a 37°C, 5% CO2 incubator. The drug was added after 24 hours. The first, second, third, fourth and fifth generations of cells were obtained for drug screening, and the drug sensitivity of the ovarian cancer primary cells cultured using the medium of the present application was tested.

[0160] 2. Preparation of screening drugs

[0161] Five drugs (Cytarabine, Doxorubicin, Pracinostat, Azacitidine, Homoharringtonine; all purchased from MCE) were prepared in six concentration gradients according to the following table, 30 μL was added to each well of the 384-well drug plate (purchased from Thermo), and stored for use.

[0162] Table 3 Drug action concentration setting

[0163]

[0164] 3. High-throughput drug addition

[0165] The prepared drug plate was taken out and placed at room temperature, and then centrifuged at 1000 rpm for 1 minute in a centrifuge (Beckman) at room temperature. High-throughput drug addition was performed using a high-throughput automated sample addition system (Perkin Elmer JANUS). 0.1 μL of the corresponding concentration of the screening drug was added to each well of the 384-well plate containing the primary ovarian cancer cells. After drug addition, the 384-well plate was sterilized and then moved to the incubator, and the cell activity was measured after 72 hours.

[0166] 4. Cell activity test

[0167] The CellTiter-Glo luminescent reagent (purchased from Promega) was taken out from the 4°C refrigerator, and 10 mL of the reagent was placed in a sample addition slot. The 384-well plate to be tested was taken out from the incubator, 10 μL of the CellTiter-Glo luminescent reagent was added to each well, and after standing for 10 minutes, the mixture was mixed well and detected using a multifunctional enzyme label instrument (Perkin Elmer Envision).

[0168] 5. Data processing

[0169] The cell inhibition rate after the action of different drugs on the cells was calculated according to the formula: cell inhibition rate (%) = 100% - (chemiluminescence value of the drug addition well) / (chemiluminescence value of the control well) x 100%, and the half maximal inhibitory concentration (IC50) of the drug on the cells was calculated using graphpad prism software. The results are shown in Figures 7A-7E .

[0170] From Figures 7A-7E It can be confirmed that the primary ovarian cancer cells cultured using the ovarian cancer primary cell culture medium OC-1 of the present application for drug screening have basically consistent inhibition effects (consistent inhibition curves) on the same drug for cells of different generations. The sensitivity of the cells of the same patient to different drugs at the maximum blood drug concentration in the human body is different. According to the results, the effectiveness of the ovarian cancer patient in the clinical use of the drug can be judged, and it can be shown that the sensitivity of the tumor cells of different generations obtained by the culture method of the present application to the drug is stable.

[0171] Industrial applicability

[0172] The application provides a culture medium and a culture method for culturing ovarian cancer primary cells in vitro, and the cells obtained through the culture can be applied to drug efficacy evaluation and screening.

[0173] Although the application has been described in detail, it should be understood that various modifications can be made to the application without departing from the spirit and scope thereof, and it is therefore contemplated to fall within the scope of the application.

Claims

1. A culture medium for primary ovarian cancer cells, characterized in that Made with the following ingredients: MST1 / 2 kinase inhibitor; sodium pyruvate; forskolin; epidermal growth factor; gastrin; fibroblast growth factor 7; nicotinamide; SB431542; fetal bovine serum; An initial culture medium selected from DMEM / F12, DMEM, F12, or RPMI-1640; and antibiotics; Wherein, the MST1 / 2 kinase inhibitor is compound 1 or a pharmaceutically acceptable salt or solvate thereof, in, (1) the content of the MST1 / 2 kinase inhibitor in the culture medium is 2.5 to 10 μM; (2) the content of sodium pyruvate in the culture medium is 0.25 to 1 mM; (3) the content of forskolin in the culture medium is 2.5 to 10 μM; (4) the content of the epidermal growth factor in the culture medium is 5 to 80 ng / mL; (5) the content of gastrin in the culture medium is 3 to 81 nM; (6) the content of the fibroblast growth factor 7 in the culture medium is 5 to 40 ng / mL; (7) The content of nicotinamide in the culture medium is 1 to 16 mM; (8) the content of SB431542 in the culture medium is 3.75-30 μM; (9) The volume concentration of the fetal bovine serum relative to the culture medium is 2.5% (v / v) to 40% (v / v).

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

3. A method for culturing primary ovarian cancer cells, characterized in that: (1) preparing a culture medium for primary ovarian cancer cells according to claim 1 or 2; (2) Obtain primary ovarian cancer cells and culture at a cell density of 1 to 10 × 10 4 pieces / cm 2 Plant them in culture dishes and keep the cell density at 2-3×10 4 pieces / cm 2 Feeder cells are added, and then culture is performed using the culture medium of the primary ovarian cancer cells obtained in step (1).

4. The method for culturing primary ovarian cancer cells according to claim 3, wherein: The trophoblast cells are irradiated NIH-3T3 cells, the irradiation source is X-rays or gamma rays, and the irradiation dose is 20 to 50 Gy.

5. A method for screening drugs for treating ovarian cancer, characterized in that: The following steps are involved: (1) Culturing primary ovarian cancer cells using the culture method according to claim 3 or 4; (2) Select the drug to be tested and dilute it according to the required concentration gradient; (3) adding the drug at various concentration gradients to the cells cultured in (1); (4) Conduct cell activity test.

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