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

Abstract

The present invention provides a culture medium for primary ovarian cancer cells, an in vitro culture method, and applications thereof. The culture medium comprises an MST1 / 2 kinase inhibitor; at least one Rho protein kinase inhibitor selected from Y27632, fasudil, and H-1152; an insulin-transferrin-selenium supplement; insulin; prostaglandin E2; epidermal growth factor; gastrin; insulin-like growth factor-1; cholera toxin; amphiregulin; N2; and B27. Compared to existing culture methods, in vitro culture using the culture medium of the present invention has higher expansion efficiency. Culturing primary ovarian cancer cells using this culture medium can maintain the morphological structure and pathological characteristics of the primary tissue, thereby improving the success rate and survival rate of primary ovarian cancer cell culture.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a culture medium and application thereof, and more specifically to a culture medium, a culture method and application of primary ovarian cancer cells. Background Art

[0002] Ovarian cancer refers to a malignant tumor that develops in the ovaries. It is one of the most common malignant tumors of the female reproductive organs, second only to cervical cancer and uterine corpus cancer in incidence. Epithelial carcinomas are the most common type of ovarian cancer, followed by malignant germ cell tumors. Among these, epithelial ovarian cancer leads the mortality rate among all gynecological cancers, posing a serious threat to women's lives. While ovarian cancer is often asymptomatic in its early stages, advanced stages may present with digestive symptoms such as lower abdominal discomfort, bloating, and decreased appetite. Treatment options include surgical resection, medications, and radiation therapy, with an overall poor prognosis.

[0003] Chemotherapy is one of the main treatments for ovarian tumors. Although a wide range of chemotherapy drugs are currently available, the efficacy rate for ovarian tumors is only approximately 25%. This is primarily due to the fact that current chemotherapy regimens are often based on clinicians' experience, without considering individual patient differences. This approach, which involves trial-evaluation-drug switching and re-evaluation, not only fails to improve drug efficacy but also misses the optimal treatment window, leading to advanced tumor progression. Furthermore, throughout treatment, patients endure the burden of drug side effects and extremely high medical costs.

[0004] Therefore, establishing primary tumor models in vitro and utilizing them for efficient drug screening experiments is a promising approach. Currently, the primary method for establishing primary ovarian tumor models in vitro is the patient-derived tumor xenograft (PDX) model. This involves transplanting patient tumor cells into nude mice and then evaluating the therapeutic effects of non-anti-tumor drugs. However, the PDX approach has several drawbacks, including species differences between humans and mice; long testing cycles (over 4 weeks); high costs (over 200,000 yuan); and false positives and false negatives. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a culture medium for primary ovarian cancer cells and an in vitro culture method and application.

[0006] One aspect of the present invention is to provide a culture medium for primary ovarian cancer cells, comprising an MST1 / 2 kinase inhibitor; a Rho protein kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152; an insulin-transferrin-selenium supplement; insulin; prostaglandin E2; epidermal growth factor; gastrin; insulin-like growth factor-1; cholera toxin; amphiregulin; N2; and B27.

[0007] Wherein, the MST1 / 2 kinase inhibitor includes a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,

[0008]

[0009] in,

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

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

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

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

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

[0015]

[0016] in,

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

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

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

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

[0021]

[0022]

[0023]

[0024]

[0025]

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

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

[0028] (1) The content of the MST1 / 2 kinase inhibitor in the culture medium is 2.5 to 20 μM;

[0029] (2) the content of the Rho protein kinase inhibitor in the culture medium is 2.5 to 20 μM;

[0030] (3) the volume ratio of the insulin-transferrin-selenium supplement to the culture medium is 1:800 to 1:50;

[0031] (4) The content of insulin in the culture medium is 1 to 27 μg / mL;

[0032] (5) The content of prostaglandin E2 in the culture medium is 2.5 to 10 μM;

[0033] (6) The content of the epidermal growth factor in the culture medium is 2.5 to 40 ng / mL;

[0034] (7) The content of gastrin in the culture medium is 1 to 9 nM;

[0035] (8) The content of insulin-like growth factor-1 in the culture medium is 25 to 100 ng / mL;

[0036] (9) The content of cholera toxin in the culture medium is 0.05-0.8 μg / mL;

[0037] (10) The content of amphiregulin in the culture medium is 1 to 81 ng / mL;

[0038] (11) The volume ratio of the B27 additive to the culture medium is preferably in the range of 1:25 to 1:400;

[0039] (12) The volume ratio of the N2 additive to the culture medium is preferably in the range of 1:50 to 1:400.

[0040] In an embodiment of the present invention, the culture medium further contains 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.

[0041] In a preferred embodiment, when the antibiotic is selected from streptomycin / penicillin, the concentration of streptomycin is in the range of 25 to 400 μg / mL, and the concentration of penicillin is in the range of 25 to 400 U / mL. When the antibiotic is selected from amphotericin B, the concentration range is 0.25 to 4 μg / mL. When the antibiotic is selected from Primocin, the concentration range is 25 to 400 μg / mL.

[0042] According to a second aspect, the present invention further provides an in vitro culture method for primary ovarian cancer cells. In the in vitro culture method for primary ovarian cancer cells of the present invention, the primary ovarian cancer cells are cultured in vitro using the primary ovarian cancer cell culture medium of the present invention.

[0043] The in vitro culture method of primary ovarian cancer cells of the present invention comprises the following steps:

[0044] 1. Isolation of Primary Ovarian Cancer Cells

[0045] (1) Ovarian cancer tissue samples were separated and basal culture medium and tissue digestion solution were added at a volume ratio of 1:3 (Note: the amount of tissue digestion solution added is approximately 5-10 mL of tissue digestion solution for 1 g of tumor tissue). The samples were placed in a constant temperature shaker for digestion at a temperature of 4-37°C and a speed of 200-350 rpm.

[0046] (2) Digestion can be terminated when no obvious tissue blocks are observed. The digestion time is 3 to 6 hours.

[0047] (3) After centrifugation, discard the supernatant at a speed of 1200-1600 rpm for 2-6 minutes, add basal culture medium and resuspend for later use.

[0048] The basal culture medium 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 includes 1640 culture medium, collagenase II (1-2 mg / mL), collagenase IV (1-2 mg / mL), DNase (50-100 U / mL), hyaluronidase (0.5-1 mg / mL), calcium chloride (1-5 mM), and bovine serum albumin (BSA) (5-10 mg / mL).

[0049] 2. Cultivation using the ovarian cancer primary cell culture medium of the present invention

[0050] Use basal culture medium and matrigel at a ratio of 50:1 356231) were mixed evenly on ice, plated, and incubated in a 37°C, 5% CO2 incubator for 25-35 minutes. The supernatant was discarded. The primary ovarian cancer cells obtained in step 1 above were resuspended in the ovarian cancer primary cell culture medium of the present invention and counted. The cell density was adjusted to 2-8×10 4 cells / cm 2 , add the primary ovarian cancer cell culture medium of the present invention, and culture in an incubator.

[0051] In yet another aspect, the present invention further provides a method for screening drugs for ovarian cancer, comprising the following steps:

[0052] (1) Cultivating primary ovarian cancer cells using the culturing method of primary ovarian cancer cells of the present invention;

[0053] (2) Select the drug to be tested and dilute it according to the required concentration gradient;

[0054] (3) adding the drug at various concentration gradients to the cells cultured in (1);

[0055] (4) Conduct cell activity test.

[0056] The technical solution of the present invention can achieve the following technical effects:

[0057] (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%;

[0058] (2) Primary ovarian cancer cells cultured in vitro can maintain the patient's pathological characteristics;

[0059] (3) The cultured primary ovarian cancer cells are not interfered with by stromal cells such as fibroblasts and adipocytes;

[0060] (4) High amplification efficiency, primary ovarian cancer cells can be successfully cultured within about a week, and the amplified primary ovarian cancer cells can be continuously passaged;

[0061] (5) The culture cost is controllable, and the culture medium does not need to be added with expensive Wnt agonists and other factors;

[0062] (6) The culture medium does not contain serum and can avoid the co-culture of primary cells and stromal cells, which also solves a series of problems existing in traditional culture schemes such as serum-containing and co-culture with stromal cells;

[0063] (7) The primary ovarian cancer cells cultured by the technology are large in number and highly homogenized, which are suitable for high-throughput screening of new candidate compounds and providing high-throughput in vitro drug sensitivity for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The figure shows the effects of different combinations of added factors in the culture medium of primary ovarian cancer cells on the growth of primary ovarian cancer cells.

[0065] Figures 2A-2L The figure shows the effects of different concentrations of added factors to the culture medium of primary ovarian cancer cells on the growth of primary ovarian cancer cells.

[0066] Figures 3A-3F These are photographs of ovarian cancer primary cells cultured using the ovarian cancer primary cell culture medium of the present invention observed under a microscope.

[0067] Figures 4A-4G These are the immunohistochemical results of primitive ovarian cancer tissue cells.

[0068] Figures 5A-5G The present invention provides immunohistochemical results of primary ovarian cancer cells obtained by culturing original ovarian cancer tissue cells to the fourth passage using the primary ovarian cancer cell culture medium of the present invention.

[0069] Figures 6A-6C The cell growth curves of primary ovarian cancer cells cultured using the primary ovarian cancer cell culture medium of the present invention, a culture medium from literature, and a commercial culture medium are shown.

[0070] Figures 7A-7F The results of drug screening using primary ovarian cancer cells cultured to different passages using the ovarian cancer primary cell culture medium of the present invention are shown. DETAILED DESCRIPTION

[0071] For a better understanding of the present invention, the present invention is further described below in conjunction with embodiments and drawings. The following embodiments are merely illustrative of the present invention and are not intended to limit the present invention.

[0072] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0073] As used herein, an MST1 / 2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1 / 2 signaling. Generally, an MST1 / 2 kinase inhibitor, for example, binds to and reduces the activity of MST1 / 2 kinases. Due to the structural similarities between MST1 and MST2, an MST1 / 2 kinase inhibitor may also be a compound that binds to and reduces the activity of either MST1 or MST2.

[0074] 1. Preparation of MST1 / 2 kinase inhibitor compound 1

[0075] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)

[0076] Benzenesulfonamide 1

[0077]

[0078] Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): Add 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) to a round-bottom flask, followed by methanol (30 mL). Then, add thionyl chloride (1.2 mL) dropwise under an ice bath. The reaction mixture is allowed to react at 85°C overnight. After completion of the reaction, the solvent is evaporated under reduced pressure to yield a white solid, which is used directly in the next step.

[0079] Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (A3): To a round-bottom flask, add methyl 2-amino-2-(2,6-difluorophenyl)acetate (2 g), followed by acetone (30 ml) and potassium carbonate (2.2 g). The mixture was then cooled to -10°C in an ice-salt bath. An acetone solution of 2,4-dichloro-5-nitropyrimidine (3.1 g) was then slowly added. The reaction system was stirred at room temperature overnight. After completion of the reaction, the mixture was filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by pressurized silica gel column chromatography to yield compound A3. LC / MS: M+H 359.0.

[0080] 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): To a round-bottom flask, add methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g), followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction system was stirred at 60°C for two hours. After completion of the reaction, the solvent was evaporated under reduced pressure, and the resulting product was neutralized with saturated sodium bicarbonate until alkaline. Extraction was performed with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain compound A4. LC / MS: M+H 297.0.

[0081] 2-Chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (A5): 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (2 g) and N,N-dimethylacetamide (10 mL) were added to a round-bottom flask. The mixture was cooled to -35°C, and iodomethane (0.9 mL) was added, followed by sodium hydride (615 mg). The reaction system was stirred for two hours. After completion of the reaction, the mixture was quenched with water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, then dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain compound A5. LC / MS: M+H 325.0.

[0082] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzenesulfonamide (1): 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (100 mg), sulfonamide (53 mg), p-toluenesulfonic acid (53 mg), and sec-butanol (5 mL) were added to a round-bottom flask. The reaction system was stirred at 120°C overnight. After completion of the reaction, the mixture was filtered and washed with methanol and diethyl ether to obtain compound 1. LC / MS: M+H 461.1.

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

[0084] Other MST1 / 2 inhibitor compounds of the present invention were synthesized in a similar manner to compound 1, and their structures and mass spectrometry data are shown in the following table.

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] Example 1 Effects of Various Added Factors in Ovarian Cancer Primary Cell Culture Medium on the Growth of Ovarian Cancer Primary Cells

[0092] (1) Preparation of culture medium for primary ovarian cancer cells

[0093] First, a basal medium containing an initial culture medium was prepared. The initial culture medium can be selected from DMEM / F12, DMEM / F12, or RPMI-1640, commonly used in the art. In this example, the basal medium formulation was: DMEM / F12 (purchased from Corning) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available at a concentration of 50 mg / ml). Culture media containing different additives (see Table 1) were then added to the basal medium to prepare primary ovarian cancer cell culture media containing various additives.

[0094] (2) Isolation and processing of primary ovarian cancer cells

[0095] 1. Sample selection

[0096] Ovarian cancer solid tumor tissue samples (intraoperative) were obtained from patients by professional medical staff of professional medical institutions, and all patients signed informed consent. 3 , endoscopic sample 0.025cm 3 Commercial tissue preservation solution (manufacturer: Miltenyi Biotec) was used for storage and transportation.

[0097] 2. Material Preparation

[0098] After disinfecting the surfaces of sterile 15mL centrifuge tubes, pipettes, 10mL pipettes, and sterile pipette tips, place them in a clean bench and irradiate with UV light for 30 minutes. Remove the basal culture medium from the 4°C refrigerator 30 minutes in advance, and remove the tissue digestion solution from the -20°C refrigerator 30 minutes in advance.

[0099] Tissue digestion solution: 1640 culture medium (Corning, 10-040-CVR), collagenase II (2 mg / mL), collagenase IV (2 mg / mL), DNase (50 U / mL), hyaluronidase (0.75 mg / mL), calcium chloride (3.3 mM), and BSA (10 mg / mL).

[0100] The above-mentioned collagenase II, collagenase IV, DNA enzyme, and hyaluronidase were purchased from Sigma; calcium chloride was purchased from Shanghai Shenggong Biotechnology Co., Ltd.; and BSA was purchased from Biofroxx.

[0101] 3. Isolation of Primary Ovarian Cancer Cells

[0102] 3.1 Take the tissue sample in the clean bench and place it in a culture dish. Remove the blood-stained tissue and rinse it twice with basal culture medium. Transfer the tissue to another culture dish and perform mechanical separation with a sterile scalpel. Cut the tissue into 1×1×1 mm 3 size;

[0103] 3.2 Pipette the cut tissue into a 15 mL centrifuge tube, add 5 mL of basal culture medium, mix well, and centrifuge at 1500 rpm for 4 minutes;

[0104] 3.3 Discard the supernatant and add basal culture medium and tissue digestion solution in a 1:3 ratio (Note: the amount of tissue digestion solution added is approximately 10 mL for 1 g of tumor tissue). Label the sample name and number, seal with parafilm, and digest at 37°C in a shaker (Zhichu Instrument ZQLY-180N) at 300 rpm. Observe the digestion completion every 30 minutes, judging by the absence of visible particulate matter. The digestion time is 4 hours.

[0105] 3.4 After digestion is complete, filter the undigested tissue clumps through a 100 μm filter. Rinse the tissue clumps on the filter with basal culture medium into a centrifuge tube to reduce cell loss and centrifuge at 1500 rpm for 4 minutes at 25°C.

[0106] 3.5 Discard the supernatant and observe whether there are blood cells. If there are blood cells, add 8 mL of blood cell lysis buffer (purchased from Sigma), mix well, and lyse at 4°C for 20 minutes, inverting once during the process. Centrifuge at 1500 rpm at 25°C for 4 minutes.

[0107] 3.6 Discard the supernatant and add 2 mL of basal culture medium to resuspend the cells for later use.

[0108] 4. Cell Counting and Processing

[0109] 4.1 Observation under microscope: Pipette a small amount of resuspended cells and spread them flatly in a culture dish. Observe the density and morphology of cancer cells under a microscope (CNOPTEC, BDS400).

[0110] 4.2 Viable Cell Counting: 12 μL of the resuspended cell suspension was added to 12 μL of trypan blue dye (Shanghai Sangon Biotech Co., Ltd.). After thorough mixing, 20 μL was added to a cell counting plate (Countstar, specification: 50 plates / box). The percentage of viable large cells (cell size >10 μm) was calculated using a cell counter (Countstar, IC1000) as follows: number of viable cells / total number of cells × 100%.

[0111] (3) Culture of primary ovarian cancer cells

[0112] The primary ovarian cancer cells isolated from two ovarian cancer tissues (numbered L40 and LQQ) according to the above step (2) were resuspended in basal medium and counted. The basal medium was mixed with matrigel ( 356231) were mixed on ice, 300 μL was taken and plated, incubated in a 37°C, 5% CO2 incubator for 25 to 35 minutes, preferably 30 minutes, and the supernatant was discarded. The culture medium containing the different components in Table 1 containing primary ovarian cancer cells was added at a rate of 4×10 4 Cells were plated in a 48-well plate at a volume of 500 μL / well. As a control, basal medium without any additives was used. 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 (Gibco) per well. The cells were then aspirated and 200 μL of 0.05% trypsin was added to each well. The plates were incubated at 37°C and 5% CO₂ 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 (ExcellBio, FND500). 20 μL of the culture medium was added to a cell counting plate (Countstar, 50 plates / box) and the total number of cells was counted using a cell counter (Countstar, IC1000). The results are shown in Table 1.

[0113] Table 1 Additives in culture medium and their effects on promoting cell proliferation

[0114]

[0115]

[0116] Among them, "+" indicates that compared with the basal medium, the culture medium to which the additive is added has a proliferation-promoting effect on 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.

[0117] Based on the above results, factors such as compound 1, prostaglandin E2, B27, N2, epidermal growth factor, insulin, insulin-transferrin-selenium supplement, amphiregulin, Y-27632, insulin-like growth factor-1, gastrin, and cholera toxin were selected for further culture experiments.

[0118] 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

[0119] According to the components in Table 2, culture medium for primary ovarian cancer cells with different combinations of added factors were prepared to investigate the proliferation-promoting effects of different combinations of added factors on primary ovarian cancer cells.

[0120] Table 2 Preparation of different components of culture medium (concentration is final concentration)

[0121]

[0122]

[0123] According to the method of step (2) 3 of Example 1, primary ovarian cancer cells were obtained from ovarian cancer tissues (numbered L46, L55, and L56). The obtained cell suspension was divided into 14 equal parts and centrifuged at 1500 rpm for 4 minutes. After centrifugation, 200 μL of BM and No. 1 to 13 culture medium were used to resuspend the cells. The basal culture medium was mixed with matrigel ( 356231) were mixed on ice, 300 μL was plated on a 48-well plate, and incubated in a 37°C, 5% CO2 incubator for 25 to 35 minutes, preferably 30 minutes, and the supernatant was discarded. 4 pieces / cm 2 Cells were seeded into a 48-well plate (40,000 cells per well), and the volume of each well was filled to 1 mL with the corresponding culture medium. The cells were mixed thoroughly. After surface disinfection, the cells were cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific).

[0124] When the cells in the 48-well plate grow to more than 85%, the culture medium is discarded and 100 μL of 0.05% trypsin (purchased from Gibco) is used to rinse once. After aspiration, 200 μL of 0.05% trypsin is added to each well. The cells are placed in a 37°C, 5% CO2 incubator for 10 minutes. The cells are observed under a microscope (CNOPTEC, BDS400) to ensure that they have been completely digested. 300 μL of DMEM culture medium containing 10% fetal bovine serum is added to terminate the digestion. 20 μL is taken and added to a cell counting plate (Countstar, specification: 50 plates / box). The total number of cells is counted using a cell counter (Countstar, IC1000). The results obtained from the primary ovarian cancer cells isolated from L46, L55, and L56 during surgery are shown in Figure 2. Figure 1 .

[0125] according to Figure 1 The results show that, compared to the basal medium, the aforementioned media Nos. 1 to 13 all promoted the proliferation of primary ovarian cancer cells to varying degrees. Therefore, when cultured with media containing supplemental ingredients such as Compound 1, prostaglandin E2, B27, N2, epidermal growth factor, insulin, insulin-transferrin-selenium supplement, amphiregulin, Y-27632, insulin-like growth factor-1, gastrin, and cholera toxin, primary ovarian cancer cells showed a better proliferation effect.

[0126] Example 3 Effects of Different Concentrations of Additive Factors in Ovarian Cancer Primary Cell Culture Medium on the Proliferation of Ovarian Cancer Primary Cells

[0127] Obtain primary ovarian cancer cells from tissue samples (numbered A26083, L56, and L62) according to step (2) of Example 1. Culture them using the combined culture medium of the effective factors in Example 2. Use basal culture medium and matrigel ( 356231) on ice, mix well, take 4 mL and plate in a T12.5 culture flask, incubate in a 37°C, 5% CO2 incubator for 25 to 35 minutes, preferably 30 minutes, and discard the supernatant.

[0128] The obtained primary ovarian cancer cells were cultured at a viable cell density of 2.4×10 4 pieces / cm 2The cells were seeded into T12.5 well plates (300,000 cells per well), and after surface disinfection, they were cultured in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher Scientific). Cells were cultured and expanded in a combination of the effective factors identified in Example 2 (containing basal medium BM, 20 μM Compound 1, 5 μM prostaglandin E2, 1:100 (v / v) B27, 1:100 (v / v) N2, 10 ng / mL epidermal growth factor, 3 μg / mL insulin, 1:50 (v / v) insulin-transferrin-selenium supplement, 27 ng / mL amphiregulin, 5 μM Y-27632, 50 ng / mL insulin-like growth factor-1, 27 nM gastrin, and 0.1 μg / mL cholera toxin). Cells were grown to more than 85% of their original size and then rinsed with 500 μL of 0.05% trypsin (purchased from Gibco) for 1 minute. After aspiration, 500 μL of 0.05% trypsin was added to each well. The cells were then incubated at 37°C, 5% CO2 for 2-10 minutes, or until the cells were completely digested. After centrifugation at 1500 rpm for 4 minutes, discard the supernatant. Resuspend the cell pellet in DMEM / F12. Transfer 20 μL of the pellet to a cell counting plate (Countstar, 50 plates / box) and count the total number of cells using a cell counter (Countstar, IC1000). The resulting cells were used in the following culture experiments.

[0129] The following 12 culture media were prepared for the experiment.

[0130] Formulation 1: The above-mentioned ovarian cancer primary cell culture medium components do not contain compound 1;

[0131] Formula 2: The above-mentioned ovarian cancer primary cell culture medium components do not contain prostaglandin E2;

[0132] Recipe 3: The above-mentioned ovarian cancer primary cell culture medium components do not contain B27;

[0133] Recipe 4: The above-mentioned ovarian cancer primary cell culture medium components do not contain N2;

[0134] Recipe 5: The above-mentioned ovarian cancer primary cell culture medium components do not contain epidermal growth factor;

[0135] Recipe 6: The above-mentioned ovarian cancer primary cell culture medium components do not contain insulin;

[0136] Formula 7: The above-mentioned ovarian cancer primary cell culture medium components do not contain insulin-transferrin-selenium supplement;

[0137] Recipe 8: The above-mentioned primary ovarian cancer cell culture medium components do not contain amphiregulin;

[0138] Recipe 9: The above-mentioned ovarian cancer primary cell culture medium components do not contain Y-27632;

[0139] Formula 10: The above-mentioned primary ovarian cancer cell culture medium components do not contain insulin-like growth factor-1;

[0140] Formulation 11: The above-mentioned primary ovarian cancer cell culture medium components do not contain gastrin;

[0141] Formula 12: The above-mentioned primary ovarian cancer cell culture medium components do not contain cholera toxin;

[0142] Add 20 μl of 4x10 4 For each cell resuspension, 1 mL of the culture medium of formulas 1 to 12 was used to dilute the cell suspension.

[0143] When using the culture medium of Formula 1, 1 mL of prepared Compound 1 was added to each well of a 48-well plate seeded with primary cells, with final concentrations of Compound 1 of 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively; and control wells (BC) were set using the culture medium of Formula 1.

[0144] When using the culture medium of Formula 2, 1 mL of prepared prostaglandin E2 was added to each well of a 48-well plate seeded with primary cells, with final concentrations of prostaglandin E2 of 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively; and control wells (BC) were set up using the culture medium of Formula 2.

[0145] When using the culture medium of Formula 3, 1 mL of prepared B27 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of B27 were 1:400 (V / V), 1:200 (V / V), 1:100 (V / V), 1:50 (V / V), and 1:25 (V / V), respectively. Control wells (BC) were set up using the culture medium of Formula 3.

[0146] When using the culture medium of Formula 4, 1 mL of prepared N2 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of N2 were 1:400 (V / V), 1:200 (V / V), 1:100 (V / V), 1:50 (V / V), and 1:25 (V / V), respectively. Control wells (BC) were set up using the culture medium of Formula 4.

[0147] When using the culture medium of Formula 5, 1 mL of prepared epidermal growth factor was added to each well of a 48-well plate seeded with primary cells. The final concentrations of epidermal growth factor were 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 5.

[0148] When using the culture medium of Formula 6, 1 mL of prepared insulin was added to each well of a 48-well plate seeded with primary cells. The final concentrations of insulin were 1 μg / mL, 3 μg / mL, 9 μg / mL, 27 μg / mL, and 81 μg / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 6.

[0149] When using the culture medium of Formula 7, 1 mL of the prepared insulin-transferrin-selenium supplement was added to each well of a 48-well plate seeded with primary cells. The final concentrations of the insulin-transferrin-selenium supplement were 1:800 (V / V), 1:400 (V / V), 1:200 (V / V), 1:100 (V / V), and 1:50 (V / V), respectively; and control wells (BC) were set using the culture medium of Formula 7.

[0150] When using the medium of Formula 8, 1 mL of prepared amphiregulin was added to each well of a 48-well plate seeded with primary cells. The final concentrations of amphiregulin were 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, and 81 ng / mL, respectively. Control wells (BC) were set up using the medium of Formula 8.

[0151] When using the medium of Formula 9, 1 mL of prepared Y-27632 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of Y-27632 were 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively. Control wells (BC) were set up using the medium of Formula 9.

[0152] When using the culture medium of Formula 10, 1 mL of prepared insulin-like growth factor-1 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of insulin-like growth factor-1 were 12.5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 10.

[0153] When using the culture medium of Formula 11, 1 mL of prepared gastrin was added to each well of a 48-well plate seeded with primary cells, with final concentrations of gastrin of 1 nM, 3 nM, 9 nM, 27 nM, and 81 nM, respectively; and control wells (BC) were set using the culture medium of Formula 11.

[0154] When using the culture medium of Formula 12, 1 mL of prepared cholera toxin was added to each well of a 48-well plate seeded with primary cells. The final concentrations of cholera toxin were 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL, and 0.8 μg / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 12.

[0155] When the cells have expanded to about 85% of the 48 wells, digest and count them, and calculate the proliferation times by referring to the number of cells in the control wells (BC). The results are shown in Figures 2A to 2L . Figures 2A to 2L The ratio is the ratio of the number of cells cultured in each culture medium for one generation to the number of cells cultured in the corresponding control well for one generation. A ratio greater than 1 indicates that the culture medium containing the factor or small molecule compound at different concentrations promotes cell proliferation more effectively than the culture medium in the control well. A ratio less than 1 indicates that the culture medium containing the factor or small molecule compound at different concentrations promotes cell proliferation less effectively than the culture medium in the control well.

[0156] according to Figures 2A to 2LThe results showed that compound 1, prostaglandin E2, B27, N2, epidermal growth factor, insulin, insulin-transferrin-selenium supplement, amphiregulin, Y-27632, insulin-like growth factor-1, gastrin, and cholera toxin had a significant proliferative effect on primary ovarian cancer cells. According to the results of this example, the content of compound 1 is preferably 2.5 μM to The cell proliferation effect is most obvious when the concentration is 5 μM; the content of prostaglandin E2 is preferably 2.5 μM to 10 μM, and the cell proliferation effect is most obvious when the concentration is 2.5 μM; the content of B27 is preferably 1:25 (V / V) to 1:400 (V / V), more preferably 1:50 (V / V) to 1:200 (V / V), and the cell proliferation effect is most obvious when the concentration is 1:100 (V / V); the content of N2 is preferably 1:50 (V / V) to 1:400 (V / V), and the cell proliferation effect is most obvious when the concentration is 1 :400 (V / V) when the cell proliferation effect is most obvious; the content of epidermal growth factor is preferably 2.5ng / ml to 40ng / ml, more preferably 5ng / ml to 20ng / ml, and the cell proliferation effect is most obvious when the concentration is 10ng / mL; the content of insulin is preferably 1μg / ml to 27μg / ml, more preferably 3μg / ml to 9μg / ml, and the cell proliferation effect is most obvious when the concentration is 9μg / mL; the content of insulin-transferrin-selenium supplement is preferably 1:50 (V / V) to 1:800 (V / V), The concentration of amphiregulin is preferably 1:400 (V / V) to 1:100 (V / V), and the cell proliferation effect is most obvious at a concentration of 1:200 (V / V); the content of amphiregulin is preferably 1 ng / ml to 81 ng / ml, more preferably 1 ng / ml to 27 ng / ml, and the cell proliferation effect is most obvious at a concentration of 3 ng / mL; the content of Y-27632 is preferably 2.5 μM to 20 μM, and the cell proliferation effect is most obvious at a concentration of 5 μM; the content of insulin-like growth factor-1 is preferably 25 ng / ml to 100 ng / ml, and the cell proliferation effect is most obvious at a concentration of 50 ng / mL; the content of gastrin is preferably 1 nM to 9 nM, and the cell proliferation effect is most obvious at a concentration of 3 nM; the content of cholera toxin is preferably 0.05 μg / ml to 0.8 μg / ml, more preferably 0.05 μg / ml to 0.2 μg / ml, and the cell proliferation effect is most obvious at a concentration of 0.1 μg / mL.

[0157] The optimal concentrations of each added factor in the above-mentioned culture medium were used as the culture medium for primary ovarian cancer cells of the present invention used in the following examples, which contained: basal culture medium BM, 5 μM compound 1, 2.5 μM prostaglandin E2, 1:100 (V / V) B27, 1:400 (V / V) N2, 10 ng / mL epidermal growth factor, 9 μg / mL insulin, 1:200 (V / V) insulin-transferrin-selenium supplement, 3 ng / mL amphiregulin, 5 μM Y-27632, 50 ng / mL insulin-like growth factor-1, 3 nM gastrin, and 0.1 μg / mL cholera toxin (hereinafter referred to as "OC-2" culture medium).

[0158] Example 4 Culture of primary ovarian cancer cells

[0159] According to the method of step (2) 3 of Example 1, primary ovarian cancer cells were obtained from 6 intraoperative tissue samples (numbered A26083, L65, L66, L72, L74, and L84) and cultured in OC-2 medium. The basal medium was mixed with matrigel ( 356231) were mixed on ice, 2 mL was plated on a 6-well culture plate, and incubated in a 37°C, 5% CO2 incubator for 25 to 35 minutes, preferably 30 minutes, and the supernatant was discarded. The obtained primary ovarian cancer cells were cultured at a viable cell density of 2×10 4 pieces / cm 2 Cells were seeded into 6-well plates (250,000 cells per well), and 4 mL of OC-2 medium was added. After surface disinfection, the cells were cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific).

[0160] The cultured primary ovarian cancer cells were observed using a microscope (Invitrogen EVOS M500). Figures 3A to 3F This is a photo taken under a 10x objective lens. The cells are closely arranged under the microscope and have slightly irregular shapes.

[0161] Example 5 Histochemical Identification of Primary Ovarian Cancer Cell Culture

[0162] A 0.25 cm spherical ... 3Cancer tissues of different sizes were immersed in 1 mL of 4% paraformaldehyde for fixation. Sample L74 was cultured to the fourth generation using the ovarian cancer primary cell culture medium OC-2 of the present invention using the method of Example 4. Ovarian cancer primary cells fixed with 4% paraformaldehyde were paraffin-embedded and cut into 4 μm thick tissue sections using a microtome. Conventional immunohistochemistry was then performed (for specific steps, see Li et al., Nature Communication, (2018) 9: 2983). The primary antibodies used were ER, PR, P53, Napsin A, Pax-8, WT-1, and Ki-67 (all purchased from CST).

[0163] Figures 4A-4G 5A-5G are comparative diagrams of the immunohistochemical results of the original tissue cells and the primary ovarian cancer cells obtained by culturing the cells with the ovarian cancer primary culture medium OC-2 of the present invention. Figure 4A and Figure 5A These are pictures of ovarian cancer tissue and cultured primary ovarian cancer cells labeled with ER antibodies. Figure 4B and Figure 5B These are pictures of ovarian cancer tissue and cultured primary ovarian cancer cells labeled with PR antibodies. Figure 4C and Figure 5C These are pictures of ovarian cancer tissue and cultured primary ovarian cancer cells labeled with P53 antibodies. Figure 4D and Figure 5D These are pictures of ovarian cancer tissue and cultured primary ovarian cancer cells labeled with NapsinA antibodies. Figure 4E and Figure 5E These are pictures of ovarian cancer tissue and cultured primary ovarian cancer cells labeled with Pax-8 antibodies. Figure 4F and Figure 5F These are pictures of ovarian cancer tissue and cultured primary ovarian cancer cells labeled with WT-1 antibody. Figure 4G and Figure 5G The images show ovarian cancer tissue and cultured primary ovarian cancer cells labeled with Ki-67 antibodies. This confirms that the expression of ovarian cancer-related biomarkers in primary ovarian cancer cells cultured using the technology of the present invention at passage 4 is essentially consistent with that in the original tissue sections from which they were derived. This demonstrates that primary ovarian cancer cells cultured using the technology of the present invention retain the original pathological characteristics of ovarian cancer tissue from patients.

[0164] Example 6 Comparison of culture effects with existing culture media and statistics of culture cycle and cell number of primary ovarian cancer cells and calculation of Population Doubling (PD) value

[0165] The culture medium was prepared from the literature (Xuefeng Liu et al., Nat. Protoc., 12(2):439-451, 2017), which was composed of DMEM / F12 medium + 250 ng / ml amphotericin B (purchased from Selleck) + 10 μg / ml gentamicin (purchased from MCE) + 0.1 nM cholera toxin + 0.125 ng / ml EGF + 25 ng / ml hydrocortisone + 10 μM Y27632 + 10% FBS.

[0166] Commercial culture medium: Defined K-SFM, Keratinocyte Serum Medium (purchased from Gibco, 10744-019)

[0167] According to the method of step (2) 3 of Example 1, primary ovarian cancer cells were obtained from three ovarian cancer tissue samples (numbered L65, L66, and A22083). The obtained primary ovarian cancer cells were cultured using literature culture medium, commercial culture medium, and OC-2 culture medium in Example 3, respectively, at a viable cell density of 3×10 4 pieces / cm 2 Cells were seeded in 6-well plates and cultured. After cell proliferation reached 95%, the cells were digested and counted. The number of days in culture until digestion was recorded, and the number of days in culture until digestion was considered a culture cycle. Continued culture under these experimental conditions resulted in the cells being expanded for different generations. After each generation, the cells were digested and counted, and the corresponding culture cycle was recorded. PD was calculated using the formula: Population Doubling (PD) = 3.32 * log10 (total number of cells after digestion / initial seeded cell number). The formula is described in Chapman et al., Stem Cell Research & Therapy 2014, 5: 60.

[0168] Figures 6A-6C The graph shows the growth curves of three primary cells cultured using a culture medium from the literature, a commercial culture medium, and the OC-2 culture medium for primary ovarian cancer cells of the present invention, drawn using Graphpad Prism software. The horizontal axis represents the number of days of cell culture, and the vertical axis represents the cumulative cell proliferation multiple, which represents the multiple of cell expansion during the culture cycle. A larger value indicates a greater number of cell expansions within a certain cycle, i.e., a greater number of cells are obtained by expansion. The slope represents the rate of cell expansion. Figures 6A-6C It can be confirmed that when the primary ovarian cancer cells cultured in the medium OC-2 of the present invention are continuously cultured and expanded for at least 30 days, the cell expansion rate remains substantially unchanged and still has the ability to continue to expand; Figures 6A-6CThe results show that the proliferation rate of primary ovarian cancer cells cultured using literature culture medium and commercial culture medium is significantly lower than that of OC-2 culture medium. In summary, compared with literature culture medium and commercial culture medium, the ovarian cancer primary cell culture medium of the present invention has a significantly better proliferation efficiency of ovarian cancer cells in vitro.

[0169] Example 7: Primary ovarian cancer cells amplified using the culture medium of the present invention are used for drug screening and efficacy evaluation

[0170] 1. Cell Culture and Plating

[0171] Ovarian cancer primary cells (numbered A22083) were isolated and cultured using the OC-2 culture medium for primary ovarian cancer cells of the present invention according to step (2) of Example 1. After the cells expanded to 85%, they were passaged. The cell passages were counted according to step (2) of Example 1, and the cells were plated at a viable cell density of 1×10 5 Cells were placed in a sample loading well (from Corning) and thoroughly mixed. The cells were then cultured in a 384-well opaque white cell culture plate (from Corning) with a volume of 50 μL per well and 5,000 cells per well. The ovarian cancer primary cell culture medium of the present invention was added to the edge of the plate to seal the plate. The plate was labeled with the sample name, drug addition time, and CellTiter-Glo (from Promega) detection time. The surface was disinfected with 75% alcohol (from Lierkang) and incubated in a 37°C, 5% CO2 incubator. Drugs were added after 24 hours. Cells were obtained at the first, second, third, fourth, and fifth passages for drug screening. The drug sensitivity of the ovarian cancer primary cells cultured in the culture medium of the present invention was tested.

[0172] 2. Screening drug preparation

[0173] Five drugs (cytarabine, doxorubicin, bortezomib, panobinostat, azacitidine, homoharringtonine; all purchased from MCE) were prepared in six concentration gradients according to the table below. 30 μL of the drugs were added to each well of a 384-well drug plate (purchased from Thermo Fisher Scientific) and stored for later use.

[0174] Table 3 Drug action concentration settings

[0175]

[0176] 3. High-throughput dosing

[0177] The prepared drug plate was removed, placed at room temperature, and centrifuged at 1000 rpm for 1 minute in a Beckman centrifuge. High-throughput drug addition was performed using a high-throughput automated sample delivery system (JANUS, Perkin Elmer). 0.1 μL of the selected drug at the corresponding concentration was added to each well of a 384-well plate containing primary ovarian cancer cells. After drug addition, the 384-well plate was surface disinfected and moved to an incubator. Cell viability was measured 72 hours later.

[0178] 4. Cell activity test

[0179] Remove CellTiter-Glo luminescent reagent (purchased from Promega) from a 4°C refrigerator and place 10 mL of the reagent in a sample reservoir. Remove the 384-well plate to be tested from the incubator and add 10 μL of CellTiter-Glo luminescent reagent to each well. Let it stand for 10 minutes, then mix thoroughly and analyze using a multi-function microplate reader (Perkin Elmer Envision).

[0180] 5. Data processing

[0181] The cell inhibition rate after different drugs acted on cells was calculated according to the formula: cell inhibition rate (%) = 100% - chemiluminescence value of drug-treated wells / chemiluminescence value of control wells × 100%. The half inhibition rate (IC) of drug action on cells was calculated using GraphPad Prism software. 50 The results are shown in Figures 7A-7F .

[0182] Depend on Figures 7A-7E It has been confirmed that drug screening using primary ovarian cancer cells cultured in the OC-2 culture medium of the present invention shows essentially consistent inhibitory effects of the same drug on cells of different passages (inhibition curves remain essentially the same). Cells from the same patient exhibit varying sensitivities to different drugs at their maximum blood concentrations in the human body. These results can be used to assess the clinical effectiveness of this drug in ovarian cancer patients and demonstrate the stable drug sensitivity of tumor cells of different passages cultured using the present method.

[0183] Industrial Applicability

[0184] The present invention provides a primary cell culture medium and a culture method for culturing primary ovarian cancer cells in vitro. The cultured cells can be used for evaluating and screening drug efficacy. Therefore, the present invention is suitable for industrial application.

[0185] Although the present invention is described in detail herein, the present invention is not limited thereto. Those skilled in the art may make modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the scope of protection of the present invention.

Claims

1. A culture medium for primary ovarian cancer cells, characterized in that Made with the following ingredients: MST1 / 2 kinase inhibitor; Y27632; insulin-transferrin-selenium supplement; insulin; prostaglandin E2; epidermal growth factor; gastrin; insulin-like growth factor-1; cholera toxin; amphiregulin; N2 supplement; B27 supplement; starting 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, Compound 1 in, (1) The content of the MST1 / 2 kinase inhibitor is 2.5 to 20 μM; (2) The content of Y27632 is 2.5-20 μM; (3) The volume ratio of the insulin-transferrin-selenium supplement to the culture medium of the primary ovarian cancer cells is 1:800 to 1:50; (4) The insulin content is 1 to 27 μg / mL; (5) The content of prostaglandin E2 is 2.5 to 10 μM; (6) The content of the epidermal growth factor is 2.5 to 40 ng / mL; (7) The gastrin content is 1 to 9 nM; (8) The content of insulin-like growth factor-1 is 25 to 100 ng / mL; (9) The cholera toxin content is 0.05 to 0.8 μg / mL; (10) The amphiregulin content is 1 to 81 ng / mL; (11) The volume ratio of the B27 additive to the culture medium of the primary ovarian cancer cells is in the range of 1:25 to 1:400; (12) The volume ratio of the N2 additive to the culture medium of the primary ovarian cancer cells is in the range of 1:50 to 1:

400.

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: The following steps are involved: (1) preparing a culture medium for primary ovarian cancer cells according to claim 1 or 2; (2) Obtaining primary ovarian cancer cells from ovarian cancer tissue samples; (3) Adding the culture medium of the primary ovarian cancer cells obtained in step (1) to the primary ovarian cancer cells obtained in step (2) for culturing.

4. A method for screening a drug for treating ovarian cancer, comprising the following steps: (1) Cultivating primary ovarian cancer cells using the culture method according to claim 3; (2) Select the drug to be tested and dilute it according to the required concentration gradient; (3) adding the drug in various concentration gradients to the primary ovarian cancer cells cultured in (1); (4) Conduct cell activity test.

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