Culture medium for primary ovarian cancer cells, method for culturing primary ovarian cancer cells and use thereof

Abstract

A culture medium for primary ovarian cancer cells, a method and use of in vitro culture of primary ovarian cancer cells. The culture medium contains an MST1 / 2 kinase inhibitor, a Rho protein kinase inhibitor selected from at least one of Y27632, fasudil and H-1152, an insulin-transferrin-sodium selenite supplement, insulin, prostaglandin E2, epidermal growth factor, gastrin, insulin-like growth factor-1, cholera toxin, amphiregulin, N2 and B27. Compared with existing culture methods, in vitro culture using the culture medium has a higher amplification efficiency. The use of the culture medium in the culture of primary ovarian cancer cells can maintain the morphological structure and pathological characteristics of the primary tissue, and improve the success rate of culture of primary ovarian cancer cells and the survival rate of the cultured primary ovarian cancer cells.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedical technology, in particular to culture media and uses thereof, more particularly to culture media for primary ovarian cancer cells, and methods and uses for culturing primary ovarian cancer cells. [Background technology]

[0002] Ovarian cancer refers to a malignant tumor disease that develops in the ovaries. It is one of the common malignant tumors of the female reproductive system and is the disease with the third highest incidence rate after cervical cancer and uterine cancer. Epithelial ovarian cancer is the most common type of ovarian cancer, followed by malignant ovarian germ cell tumors. Epithelial ovarian cancer has the highest mortality rate among all types of gynecological tumors and can pose a serious threat to women's lives. Ovarian cancer is often asymptomatic in the early stages, but in later stages, gastrointestinal symptoms such as lower abdominal discomfort, bloating, and loss of appetite may appear. The main treatment options for ovarian cancer include surgical resection, chemotherapy, and radiation therapy, and the overall prognosis is poor.

[0003] Chemotherapy is one of the main treatments for ovarian tumors. Although numerous chemotherapy drugs are currently available for clinical use, the clinical efficacy rate for ovarian tumors is only approximately 25%. The main reason for this is that most chemotherapy regimens used by patients are based on the clinician's experience, and dosage changes and re-evaluations are conducted based on clinical trial evaluations without taking into account individual patient differences. This not only fails to improve the therapeutic efficacy of the drugs, but also results in missed optimal treatment periods, leading to tumor progression. Furthermore, patients endure drug side effects and extremely high medical costs throughout the treatment process.

[0004] Therefore, establishing an in vitro primary tumor model and using it to conduct efficient drug screening experiments is a promising solution. Currently, the main method for establishing an in vitro culture model of primary ovarian tumors is the patient-derived tumor xenograft (PDX) model, which involves transplanting tumor cells from patients into nude mice and testing the therapeutic effects of various antitumor drugs. However, the PDX method has several drawbacks, such as differences between human and mouse species, a long testing cycle (more than 4 weeks), high costs (more than 200,000), and false positives and false negatives. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides a culture medium for primary ovarian cancer cells, and its in vitro culture method and use.

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

[0007] wherein the MST1 / 2 kinase inhibitor is represented by formula (I): [ka] (In the formula, R1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl optionally substituted with 1-2 independent R6 (e.g., phenyl and naphthyl, etc.), arylC1-C6 alkyl optionally substituted with 1-2 independent R6 (e.g., phenylmethyl, etc.), and heteroaryl optionally substituted with 1-2 independent R6 (e.g., thienyl, etc.); R2 and R3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl; R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, hydroxylC1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, and C3-C6 heterocyclylC1-C6 alkyl (heterocyclyl is, for example, selected from piperidyl, tetrahydropyranyl, etc.); R6 includes compounds selected from halogen (preferably fluoro and chloro, more preferably fluoro), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoromethyl), or a pharmaceutically acceptable salt or solvate thereof.

[0008] In a preferred embodiment, the MST1 / 2 kinase inhibitor has formula (Ia): [ka] (In the formula, R1 is selected from C1-C6 alkyl, phenyl optionally substituted with 1-2 independent R6, thienyl optionally substituted with 1-2 independent R6, and phenylmethyl optionally substituted with 1-2 independent R6, more preferably R1 is phenyl optionally substituted with 1-2 independent R6; R5 is selected from hydrogen, C1-C6 alkyl and C3-C6 cycloalkyl, more preferably R5 is hydrogen; R6 is independently selected from halogen, C1-C6 alkyl and C1-C6 haloalkyl; more preferably, R6 is fluoro, methyl or trifluoromethyl), or a pharmaceutically acceptable salt or solvate thereof.

[0009] Preferably, the MST1 / 2 kinase inhibitor is at least one selected from the following compounds or a pharmaceutically acceptable salt or solvate thereof:

[0010] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0011] Most preferably, the MST1 / 2 kinase inhibitor of the present invention is Compound 1.

[0012] In an embodiment of the present invention, the amounts of ingredients in the culture medium of the present invention are determined according to the following conditions: (1) The amount of MST1 / 2 kinase inhibitor in the culture medium is 2.5 μM to 20 μM; (2) the amount of Rho kinase inhibitor in the culture medium is 2.5 μM to 20 μM; (3) the volume ratio of insulin-transferrin-sodium selenite supplement to culture medium is 1:800 to 1:50; (4) The amount of insulin in the culture medium is 1 μg / mL to 27 μg / mL; (5) The amount of prostaglandin E2 in the culture medium is 2.5 μM to 10 μM; (6) The amount of epidermal growth factor in the culture medium is 2.5 ng / mL to 40 ng / mL; (7) The amount of gastrin in the culture medium is 1 nM to 9 nM; (8) The amount of insulin-like growth factor-1 in the culture medium is 25 ng / mL to 100 ng / mL; (9) The amount of cholera toxin in the culture medium is 0.05 μg / mL to 0.8 μg / mL; (10) The amount of amphiregulin in the culture medium is 1 ng / mL to 81 ng / mL; (11) The volume ratio of B27 additive to culture medium is preferably 1:25 to 1:400; (12) The volume ratio of N2 additive to culture medium is preferably 1:50 to 1:400; Satisfy one or more or all of the following.

[0013] In an embodiment of the invention, the culture medium further comprises an initial medium selected from the group consisting of DMEM / F12, DMEM, F12 or RPMI-1640, and one or more antibiotics selected from the group consisting of streptomycin / penicillin, amphotericin B and Primocin.

[0014] In a preferred embodiment, when streptomycin / penicillin is used as the antibiotic, the concentration range of streptomycin is 25 μg / mL to 400 μg / mL, and the concentration range of penicillin is 25 U / mL to 400 U / mL; when amphotericin B is used as the antibiotic, the concentration range is 0.25 μg / mL to 4 μg / mL; and when Primocin is used as the antibiotic, the concentration range is 25 μg / mL to 400 μg / mL.

[0015] According to a second aspect, the present invention also provides a method for culturing primary ovarian cancer cells in vitro. In the method of the present invention for culturing primary ovarian cancer cells in vitro, the primary ovarian cancer cells are cultured in vitro using a culture medium for primary ovarian cancer cells of the present invention.

[0016] The method of the present invention for culturing primary ovarian cancer cells in vitro comprises the following steps.

[0017] 1. Primary Ovarian Cancer Cell Isolation (1) Isolate an ovarian cancer tissue sample, add basal medium and tissue digestion solution (Note: The amount of tissue digestion solution to be added is approximately 5 mL to 10 mL per 1 g of tumor tissue) at a ratio of 1:3, place in a thermostatic shaker, and perform digestion at a digestion temperature of 4°C to 37°C and a speed of 200 rpm to 350 rpm. (2) Digestion may be stopped when digestion is complete, i.e., when no obvious tissue debris is observed and the digestion time is 3 to 6 hours. (3) After centrifugation at a centrifugation speed of 1200 rpm to 1600 rpm for 2 to 6 minutes, discard the supernatant and add basal medium to resuspend the cells for later use.

[0018] Here, the basal medium formulation includes an initial medium selected from the group consisting of DMEM / F12, DMEM, F12, or RPMI-1640, and one or more antibiotics selected from the group consisting of streptomycin / penicillin, amphotericin B, and Primocin. The tissue digestion solution formulation includes 1640 medium, collagenase II (1 mg / mL to 2 mg / mL), collagenase IV (1 mg / mL to 2 mg / mL), DNase (50 U / mL to 100 U / mL), hyaluronidase (0.5 mg / mL to 1 mg / mL), calcium chloride (1 mM to 5 mM), and bovine serum albumin (BSA) (5 mg / mL to 10 mg / mL).

[0019] 2. Culturing cells using the culture medium for primary ovarian cancer cells of the present invention The basal medium is mixed with Matrigel (Corning™, 356231) at a ratio of 25:1 to 100:1 on ice. After plating the cells, the plate is incubated in a 37°C, 5% CO2 incubator for 25 to 35 minutes, and the supernatant is discarded. The primary ovarian cancer cells obtained in step 1 above are resuspended in the culture medium for primary ovarian cancer cells of the present invention, and the cells are counted. The cell density is adjusted to 2 x 10 4 cells / cm 2 ~8×10 4 cells / cm 2 The culture medium for the primary ovarian cancer cells of the present invention is added, and the cells are cultured in an incubator.

[0020] In yet another aspect, the present invention provides a method for producing a method for manufacturing a semiconductor device, comprising the steps of: (1) culturing primary ovarian cancer cells using the method for culturing primary ovarian cancer cells of the present invention; (2) selecting a test drug and diluting the drug to a desired concentration gradient; (3) adding a drug diluted to various concentration gradients to the cells cultured in step (1); (4) detecting cell viability; Also provided is a drug screening method for ovarian cancer disease, comprising:

[0021] The technical solution of the present invention can achieve the following technical effects: (1) The success rate of culturing primary ovarian cancer cells has been improved, and the present invention makes it possible to culture tumor tissues from various sources, such as epithelial carcinomas, malignant germ cell tumors, stromal tumors, and metastatic tumors, with a success rate of over 80%; (2) primary ovarian cancer cells cultured in vitro reliably maintain the pathological characteristics of patients; (3) cultured primary ovarian cancer cells are not interfered with by stromal cells such as fibroblasts and adipocytes; (4) The expansion efficiency is high, and primary ovarian cancer cells can be successfully cultured in approximately one week. The expanded primary ovarian cancer cells have the ability to be continuously passaged; (5) The culture medium does not require expensive factors such as Wnt agonists, which reduces culture costs; (6) The culture medium is serum-free, which can avoid co-culture of primary cells with stromal cells and also solves a series of problems existing in conventional culture protocols, such as the inclusion of serum and co-culture with stromal cells; (7) Primary ovarian cancer cells cultured by the above techniques are large in number and highly homogenized, making them suitable for high-throughput screening of new candidate compounds and providing high-throughput drug sensitivity in vitro for patients. [Brief explanation of the drawings]

[0022] [Figure 1] 1 is a graph showing the effect of various combinations of supplemental factors in culture media for primary ovarian cancer cells on the growth of primary ovarian cancer cells. [Figure 2-1] 2A to 2F are graphs showing the effect of various concentrations of each factor added to the culture medium for primary ovarian cancer cells on the growth of the primary ovarian cancer cells. [Figure 2-2] 2G to 2L are graphs showing the effect of various concentrations of each factor added to the culture medium for primary ovarian cancer cells on the growth of primary ovarian cancer cells. [Figure 3] 3A to 3F are photographs taken under a microscope of primary ovarian cancer cells cultured using the culture medium for primary ovarian cancer cells of the present invention. [Figure 4-1] 4A to 4F show the immunohistochemical results of the original ovarian cancer tissue cells. [Figure 4-2] FIG. 4G shows the immunohistochemistry results of the original ovarian cancer tissue cells. [Figure 5-1] 5A to 5D show the immunohistochemical results of primary ovarian cancer cells obtained by culturing original ovarian cancer tissue cells up to the fourth passage using the culture medium for primary ovarian cancer cells of the present invention. [Figure 5-2] 5E to 5G show the immunohistochemical results of primary ovarian cancer cells obtained by culturing original ovarian cancer tissue cells up to the fourth passage using the culture medium for primary ovarian cancer cells of the present invention. [Figure 6] 6A to 6C show cell growth curves of primary ovarian cancer cells cultured using the culture medium for primary ovarian cancer cells of the present invention, a culture medium of the prior art, and a commercially available culture medium, respectively. [Figure 7] 7A to 7F show the results of drug screening of primary ovarian cancer cells cultured for various passages using the culture medium for primary ovarian cancer cells of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0023] For a better understanding of the present invention, the following further description is provided in conjunction with embodiments and drawings. The following examples are presented for illustrative purposes only, and are not intended to limit the present invention.

[0024] (Preparation example of MST1 / 2 kinase inhibitor) 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 reduces the activity of MST1 / 2 kinase, for example, by binding to MST1 / 2 kinase. Because MST1 and MST2 have similar structures, an MST1 / 2 kinase inhibitor may be, for example, a compound that binds to MST1 or MST2 and reduces its activity.

[0025] 1. Preparation of MST1 / 2 kinase inhibitor compound 1 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzsulfamide 1 [ka]

[0026] Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): 2-Amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) and methanol (30 mL) were added to a round-bottom flask, followed by the dropwise addition of thionyl chloride (1.2 mL) in an ice bath. The reaction mixture was allowed to react overnight at 85 °C. After completion of the reaction, the mixture was evaporated under reduced pressure to dry the solvent, and the resulting white solid was used directly in the next step.

[0027] Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (A3): Methyl 2-amino-2-(2,6-difluorophenyl)acetate (2 g) was added to a round-bottom flask, followed by acetone (30 mL) and potassium carbonate (2.2 g). The mixture was then cooled to -10°C in an ice-salt bath, and a solution of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone was slowly added. The reaction mixture was stirred overnight at room temperature. After completion of the reaction, the reaction 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 give compound A3. LC / MS: M+H 359.0.

[0028] 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): To a round-bottom flask was added methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g), followed by acetic acid (50 mL) and iron powder (3.9 g). The reaction mixture was stirred at 60°C for 2 hours. After completion of the reaction, the reaction mixture was evaporated under reduced pressure to dryness. The resulting mixture was neutralized to alkaline with saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to give the crude product. The crude product was washed with diethyl ether to give compound A4. LC / MS: M+H 297.0.

[0029] 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 and cooled to -35 °C. Iodomethane (0.9 mL) and sodium hydride (615 mg) were then added, and the reaction mixture was stirred for 2 hours. After completion of the reaction, the reaction mixture was quenched with water and extracted with ethyl acetate. 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 give the crude product. The crude product was washed with diethyl ether to give compound A5. LC / MS: M+H 325.0.

[0030] 4-((7-(2,6-Difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzsulfamide (1): To a round-bottom flask was added 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (100 mg), sulfanilamide (53 mg), p-toluenesulfonic acid (53 mg), and sec-butanol (5 mL). The reaction was stirred at 120° C. overnight. After completion of the reaction, the reaction mixture was filtered and washed with methanol and diethyl ether to give compound 1. LC / MS: M+H 461.1.

[0031] 2. Preparation of Other MST1 / 2 Inhibitor Compounds of the Invention Other MST1 / 2 inhibitor compounds of the present invention were synthesized in a similar manner to compound 1, and their structures and mass spectral data are shown in the table below.

[0032] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0033] Example 1. Effect of various factors added to culture medium for primary ovarian cancer cells on the growth of primary ovarian cancer cells (1) Preparation of culture medium for primary ovarian cancer cells First, a basal medium containing an initial medium was prepared. The initial medium can be selected from the group consisting of DMEM / F12, DMEM, F12, or RPMI-1640, which are commonly used in the art. In this example, the formulation of the basal medium was DMEM / F12 medium (purchased from Corning) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (volume / volume), commercially available concentration is 50 mg / mL). Various types of additives (see Table 1) were added to the basal medium to prepare culture media for primary ovarian cancer cells containing various components.

[0034] (2) Isolation and processing of primary ovarian cancer cells 1. Sample Selection Ovarian cancer solid tumor tissue samples (intraoperative) were obtained from patients by specialized medical personnel at specialized medical institutions, and all patients signed informed consent. 3 intraoperative specimen, 0.025cm in size 3 Endoscopic samples were stored and transported using a commercially available tissue preservation solution (manufacturer: Miltenyi Biotec).

[0035] 2. Material Preparation After surface sterilization, 15 mL sterilized centrifuge tubes, pipettors, 10 mL pipettes, sterilized pipette tips, etc. were placed on an ultraclean work bench and irradiated with UV light for 30 minutes. The basal medium was removed from a 4°C refrigerator 30 minutes before the sterilization, and the tissue digestion solution was removed from a -20°C refrigerator 30 minutes before the sterilization.

[0036] Tissue digestion solution: 1640 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), BSA (10 mg / mL).

[0037] The above-mentioned collagenase II, collagenase IV, DNase, and hyaluronidase were all purchased from Sigma Corporation, calcium chloride was purchased from Sangon Biotech (Shanghai) Co., Ltd., and BSA was purchased from Biofroxx Corporation.

[0038] 3. Primary Ovarian Cancer Cell Isolation 3.1. The tissue sample was transferred from the ultraclean work bench to a culture dish, and blood-contaminated tissue was removed. The tissue sample was washed twice with basal medium, then transferred to another culture dish and cut into 1 × 1 × 1 mm pieces with a sterile scalpel. 3 The tissue was mechanically cut into tissue blocks of about 1 / 4 inch in size.

[0039] 3.2. The cut intraoperative tissue was aspirated into a 15 mL centrifuge tube, to which 5 mL of basal medium was added, mixed thoroughly, and then centrifuged at 1500 rpm for 4 minutes.

[0040] 3.3. The supernatant was discarded, and a 1:3 mixture of basal medium and tissue digestion solution (approximately 10 mL of tissue digestion solution was added to 1 g of tumor tissue). The samples were labeled and numbered, sealed with sealing film, and then digested at 37°C and 300 rpm in a shaker (Zhichu Instrument ZQLY-180N). Completion of digestion was determined based on the presence or absence of visible particles, observed every 30 minutes. The digestion time was 4 hours.

[0041] 3.4. After digestion was complete, the undigested tissue mass was filtered through a 100 μM filter mesh. The tissue mass on the filter mesh was washed into a centrifuge tube with basal medium to reduce cell loss. The resulting mixture was centrifuged at 25°C, 1500 rpm, for 4 minutes.

[0042] 3.5. The supernatant was discarded and the resulting material was observed to determine the presence of blood cells. If blood cells were present, 8 mL of blood cell lysis solution (purchased from Sigma) was added to the resulting material, mixed thoroughly, and allowed to lyse at 4°C for 20 minutes, with one inversion during this process. The resulting material was centrifuged at 25°C, 1500 rpm, and 4 minutes.

[0043] 3.6. The supernatant was discarded, 2 mL of basal medium was added, and the cells were resuspended and stored.

[0044] 4. Cell Counting and Processing 4.1. Microscopic observation: A small amount of resuspended cells was plated on a culture dish, and the density and morphology of cancer cells were observed under a microscope (CNOPTEC, BDS400).

[0045] 4.2. Counting viable cells: 12 μL of the resuspended cell suspension was thoroughly mixed with 12 μL of trypan blue staining solution (Sangon Biotech (Shanghai) Co., Ltd.), and 20 μL of the mixture was added to a cell counting plate (Countstar, specifications: 50 cells / box). Using a cell counter (Countstar, IC1000), the percentage of viable large cells (cells larger than 10 μm) was calculated as follows: Number of viable cells / Total number of cells × 100%.

[0046] (3) Culture of primary ovarian cancer cells Primary ovarian cancer cells isolated from two ovarian cancer tissues (codes L40 and LQQ) according to step (2) above were resuspended in the culture medium listed in Table 1, and the cell numbers were counted. The basal medium was mixed with Matrigel (Corning™, 356231) at a ratio of 50:1 on ice. 300 μL of the mixture was plated and incubated for 25 to 35 minutes, preferably 30 minutes, in a 5% CO2 incubator at 37°C. The supernatant was discarded. The culture medium containing the various components listed in Table 1, containing primary ovarian cancer cells, was added to each well at a concentration of 4 × 10 cells / well. 4 Cells were added to a 48-well plate to a total volume of 500 μL per well. Basal medium without any additives was used as a control. After 7–10 days of culture, when the cells reached 85% growth, the culture medium was discarded and the cells were rinsed once with 100 μL of 0.05% trypsin (purchased from Gibco) per well. After aspirating the trypsin, 200 μL of 0.05% trypsin was added to each well. The plate was then placed in an incubator at 37°C and 5% CO2 for 10 minutes. Complete digestion was confirmed by observation under a microscope (CNOPTEC, BDS400). Next, 300 μL of DMEM / F12 medium containing 10% serum (Excell Bio, FND500) was added to stop the digestion, and 20 μL of the resulting solution was added to a cell counting plate (Countstar, size: 50 cells / box). The total number of cells was counted using a cell counter (Countstar, IC1000). The experimental results are shown in Table 1.

[0047] [Table 3]

[0048] Here, "+" indicates that, compared to the basal medium, the culture medium supplemented with the additive(s) has the effect of promoting the growth of two cases of primary ovarian cancer cells isolated from ovarian cancer tissue; "-" indicates that the culture medium supplemented with the additive(s) has the effect of promoting the growth of one case of primary ovarian cancer cells isolated from ovarian cancer tissue; and "◯" indicates that the culture medium supplemented with the additive(s) has no significant effect on the growth of at least two cases of primary ovarian cancer cells isolated from ovarian cancer tissue.

[0049] Based on the above results, factors such as Compound 1, prostaglandin E2, B27, N2, epidermal growth factor, insulin, insulin-transferrin-sodium selenite supplement, amphiregulin, Y-27632, insulin-like growth factor-1, gastrin, and cholera toxin were selected for further culture experiments.

[0050] Example 2: Effect of various combinations of factors added to culture medium for primary ovarian cancer cells on the growth of primary ovarian cancer cells Culture media for primary ovarian cancer cells containing various combinations of supplementary factors were prepared according to the ingredients in Table 2, and the growth-promoting effects of the various combinations of supplementary factors on primary ovarian cancer cells were investigated.

[0051] [Table 4]

[0052] Primary ovarian cancer cells were obtained from ovarian cancer tissues (designated L46, L55, and L56) according to the process of step (2)-3 in Example 1. The resulting cell suspension was divided into 14 equal parts and then centrifuged at 1500 rpm for 4 minutes. After centrifugation, the cells were resuspended in 200 μL of BM and culture medium numbers 1 to 13, respectively. The basal medium was mixed with Matrigel (Corning™, 356231) at a ratio of 50:1 on ice. 300 μL of the mixture was plated into a 48-well plate and incubated for 25 to 35 minutes, preferably 30 minutes, in a 5% CO2 incubator at 37°C. The supernatant was discarded. Primary ovarian cancer cells were cultured in a 48-well plate at a viable cell density of 4×10 4 Cells were seeded at 40,000 cells per well. Each well of a 48-well plate was supplemented with the corresponding culture medium to a volume of 1 mL, and the resulting mixture was mixed thoroughly. After surface sterilization, the plate was placed in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific) and cultured.

[0053] Once the cells had grown to over 85% in the 48-well plate, the culture medium was discarded. The cells were rinsed once with 100 μL of 0.05% trypsin (purchased from Gibco) per well. After aspirating the trypsin, 200 μL of 0.05% trypsin was added to each well. The plate was then placed in an incubator at 37°C and 5% CO2 for 10 minutes. Cell digestion was monitored under a microscope (CNOPTEC, BDS400). 300 μL of DMEM medium containing 10% fetal bovine serum was added to stop the digestion, and 20 μL of the resulting solution was added to a cell counting plate (Countstar, specification: 50 cells / box). The total cell number was counted using a cell counter (Countstar, IC1000). The results obtained from primary ovarian cancer cells isolated from L46, L55, and L56 (intraoperative) are shown in Figure 1.

[0054] According to the results in Figure 1, compared with the basal medium, the above culture media Nos. 1 to 13 can promote the proliferation of primary ovarian cancer cells to varying degrees. Therefore, primary ovarian cancer cells can be better proliferated when cultured in a culture medium containing additives such as Compound 1, prostaglandin E2, B27, N2, epidermal growth factor, insulin, insulin-transferrin-sodium selenite supplement, amphiregulin, Y-27632, insulin-like growth factor-1, gastrin, and cholera toxin.

[0055] Example 3: Effect of various concentrations of additives contained in the culture medium for primary ovarian cancer cells on the proliferation of primary ovarian cancer cells Primary ovarian cancer cells were obtained from tissue samples (codes A26083, L56, L62) according to step (2)-3 of Example 1. Culture was performed using a culture medium containing the effective factor combination described in Example 2. The basal medium was mixed with Matrigel (Corning™, 356231) at a ratio of 50:1 on ice. 4 mL of the mixture was plated in a T12.5 culture flask and incubated for 25 to 35 minutes, preferably 30 minutes, in a 5% CO2 incubator at 37°C. The supernatant was discarded.

[0056] The obtained primary ovarian cancer cells were cultured in a T12.5-well plate (300,000 cells per well) at a viable cell density of 2.4 × 10 4 cells / cm 2The plates were then surface sterilized and placed in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific). The cells were cultured and expanded in a culture medium containing the effective factor combination determined in Example 2 (basal medium (BM), 20 μM Compound 1, 5 μM prostaglandin E2, 1:100 (vol / vol) B27, 1:100 (vol / vol) N2, 10 ng / mL epidermal growth factor, 3 μg / mL insulin, 1:50 (vol / vol) insulin-transferrin-sodium selenite 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) until the cells reached 85% or greater growth. 500 μL of 0.05% trypsin (purchased from Gibco Scientific) was added and the cells were rinsed for 1 minute. After aspirating the trypsin, 500 μL of 0.05% trypsin was added to each well. The plate was placed in a 37°C, 5% CO2 incubator and incubated for 2 to 10 minutes. Digestion was stopped when the cells were completely digested. After centrifugation at 1500 rpm for 4 minutes, the supernatant was discarded. The cell pellet was resuspended in DMEM / F12, and 20 μL of the suspension was added to a cell counting plate (manufacturer: Countstar, specifications: 50 cells / box), and the total cell number was counted using a cell counter (Countstar, IC1000). The resulting cells were used in the following culture experiments.

[0057] The following 12 culture medium formulations were prepared for the experiment:

[0058] Formulation 1: the culture medium for primary ovarian cancer cells described above without Compound 1; Formulation 2: the above-mentioned culture medium for primary ovarian cancer cells without prostaglandin E2; Formulation 3: Culture medium for primary ovarian cancer cells as described above without B27; Formulation 4: the above-mentioned culture medium for primary ovarian cancer cells without N2; Formulation 5: Culture medium for primary ovarian cancer cells as described above, without epidermal growth factor; Formulation 6: Culture medium for primary ovarian cancer cells as described above without insulin; Formulation 7: Culture medium for primary ovarian cancer cells as described above without insulin-transferrin-sodium selenite supplement; Formulation 8: Culture medium for primary ovarian cancer cells as described above, which does not contain amphiregulin; Formulation 9: the above-mentioned culture medium for primary ovarian cancer cells without Y-27632; Formulation 10: Culture medium for primary ovarian cancer cells as described above, without insulin-like growth factor-1; Formulation 11: Culture medium for primary ovarian cancer cells as described above without gastrin; Formulation 12: Culture medium for primary ovarian cancer cells as described above without cholera toxin.

[0059] 4×10 4 20 μl of the cell resuspension containing the cells was added to each well, and then diluted with 1 mL of the culture medium of each of the above formulations 1 to 12.

[0060] When using the culture medium of Formulation 1, 1 mL of the prepared Compound 1 was added to each well of a 48-well plate seeded with primary cells so that the final concentrations of Compound 1 were 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively. Blank control (BC) wells were set up using the culture medium of Formulation 1.

[0061] When using the culture medium of Formulation 2, 1 mL of the prepared prostaglandin E2 was added to each well of a 48-well plate seeded with primary cells to achieve final prostaglandin E2 concentrations of 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively. Blank control (BC) wells were also set up using the culture medium of Formulation 2.

[0062] When using the culture medium of Formulation 3, 1 mL of the prepared B27 was added to each well of a 48-well plate seeded with primary cells to achieve final B27 concentrations of 1:400 (volume / volume), 1:200 (volume / volume), 1:100 (volume / volume), 1:50 (volume / volume), and 1:25 (volume / volume), respectively. Blank control (BC) wells were set up using the culture medium of Formulation 3.

[0063] When using the culture medium of Formula 4, 1 mL of the prepared N2 was added to each well of a 48-well plate seeded with primary cells to achieve final N2 concentrations of 1:400 (volume / volume), 1:200 (volume / volume), 1:100 (volume / volume), 1:50 (volume / volume), and 1:25 (volume / volume), respectively. Blank control (BC) wells were set up using the culture medium of Formula 4.

[0064] When using the culture medium of Formulation 5, 1 mL of the prepared epidermal growth factor was added to each well of a 48-well plate seeded with primary cells to achieve final epidermal growth factor concentrations of 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively. Blank control (BC) wells were also set up using the culture medium of Formulation 5.

[0065] When using the culture medium of Formulation 6, 1 mL of prepared insulin was added to each well of a 48-well plate seeded with primary cells to achieve final insulin concentrations of 1 μg / mL, 3 μg / mL, 9 μg / mL, 27 μg / mL, and 81 μg / mL, respectively. Blank control (BC) wells were also set up using the culture medium of Formulation 6.

[0066] When using Formulation 7 culture medium, 1 mL of the prepared insulin-transferrin-sodium selenite supplement was added to each well of a 48-well plate seeded with primary cells to achieve final concentrations of insulin-transferrin-sodium selenite supplement of 1:800 (v / v), 1:400 (v / v), 1:200 (v / v), 1:100 (v / v), and 1:50 (v / v), respectively. Blank control (BC) wells were also set up using Formulation 7 culture medium.

[0067] When using the culture medium of Formulation 8, 1 mL of the prepared amphiregulin was added to each well of a 48-well plate seeded with primary cells to achieve final amphiregulin concentrations of 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, and 81 ng / mL, respectively. Blank control (BC) wells were also set up using the culture medium of Formulation 8.

[0068] When using the culture medium of Formula 9, 1 mL of the prepared Y-27632 was added to each well of a 48-well plate seeded with primary cells to achieve final concentrations of Y-27632 of 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively. Blank control (BC) wells were also set up using the culture medium of Formula 9.

[0069] When using the formulation 10 culture medium, 1 mL of the prepared insulin-like growth factor-1 was added to each well of a 48-well plate seeded with primary cells to achieve final insulin-like growth factor-1 concentrations of 12.5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL, respectively. Blank control (BC) wells were also set up using the formulation 10 culture medium.

[0070] When using the culture medium of Formula 11, 1 mL of the prepared gastrin was added to each well of a 48-well plate seeded with primary cells to achieve final gastrin concentrations of 1 nM, 3 nM, 9 nM, 27 nM, and 81 nM, respectively. Blank control (BC) wells were also set up using the culture medium of Formula 11.

[0071] When using the culture medium of Formula 12, 1 mL of the prepared cholera toxin was added to each well of a 48-well plate seeded with primary cells to achieve final concentrations of cholera toxin of 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL, and 0.8 μg / mL, respectively. Blank control (BC) wells were also set up using the culture medium of Formula 12.

[0072] When the cells expanded to approximately 85% of the 48 wells, they were digested and counted. The proliferation fold was calculated by comparing the cell count in the blank control (BC) wells. The results are shown in Figures 2A-2L. In Figures 2A-2L, the ratios represent the ratio of the number of first-passage cells cultured using each culture medium to the number of first-passage cells cultured in the corresponding blank control wells. A ratio greater than 1 indicates that the proliferation-promoting effect of the prepared culture medium containing various concentrations of factors or small molecule compounds is more favorable than that of the culture medium in the blank control wells. A ratio less than 1 indicates that the proliferation-promoting effect of the prepared culture medium containing various concentrations of factors or small molecule compounds is less favorable than that of the culture medium in the blank control wells.

[0073] The results in Figures 2A-2L show that Compound 1, prostaglandin E2, B27, N2, epidermal growth factor, insulin, insulin-transferrin-sodium selenite supplement, amphiregulin, Y-27632, insulin-like growth factor-1, gastrin, and cholera toxin have significant proliferation-promoting effects on primary ovarian cancer cells. According to the results of this example, the amount of Compound 1 is preferably 2.5 μM to 20 μM, more preferably 2.5 μM to 10 μM, with the most significant cell proliferation effect occurring at a concentration of 5 μM. The amount of prostaglandin E2 is preferably 2.5 μM to 10 μM, with the most significant cell proliferation effect occurring at a concentration of 2.5 μM. The amount of B27 is preferably 1:25 (volume / volume) to 1:400 (volume / volume), more preferably 1:50 (volume / volume) to 1:200 (volume / volume), and the cell proliferation effect is most pronounced when the concentration is 1:100 (volume / volume). The amount of N2 is preferably 1:50 (volume / volume) to 1:400 (volume / volume), and the cell proliferation effect is most pronounced when the concentration is 1:400 (volume / volume). The amount of epidermal growth factor is preferably 2.5 ng / mL to 40 ng / mL, more preferably 5 ng / mL to 20 ng / mL, and the cell proliferation effect is most pronounced when the concentration is 10 ng / mL. The amount of insulin is preferably 1 μg / mL to 27 μg / mL, more preferably 3 μg / mL to 9 μg / mL, and the cell proliferation effect is most pronounced when the concentration is 9 μg / mL. The amount of insulin-transferrin-sodium selenite supplement is preferably 1:50 (volume / volume) to 1:800 (volume / volume), more preferably 1:400 (volume / volume) to 1:100 (volume / volume), with the most pronounced cell proliferation effect at a concentration of 1:200 (volume / volume). The amount of amphiregulin is preferably 1 ng / mL to 81 ng / mL, more preferably 1 ng / mL to 27 ng / mL, with the most pronounced cell proliferation effect at a concentration of 3 ng / mL. The amount of Y-27632 is preferably 2.5 μM to 20 μM, with the most pronounced cell proliferation effect at a concentration of 5 μM.The amount of insulin-like growth factor-1 is preferably 25 ng / mL to 100 ng / mL, and the cell proliferation effect is most pronounced when the concentration is 50 ng / mL. The amount of gastrin is preferably 1 nM to 9 nM, and the cell proliferation effect is most pronounced when the concentration is 3 nM. The amount 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 pronounced when the concentration is 0.1 μg / mL.

[0074] Using the most preferred concentrations of each factor added to the culture medium described above, the culture medium for primary ovarian cancer cells of the present invention used in the following examples contains basal medium (BM), 5 μM Compound 1, 2.5 μM prostaglandin E2, 1:100 (vol / vol) B27, 1:400 (vol / vol) N2, 10 ng / mL epidermal growth factor, 9 μg / mL insulin, 1:200 (vol / vol) insulin-transferrin-sodium selenite 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 culture medium "OC-2").

[0075] Example 4 Cultivation of primary ovarian cancer cells Primary ovarian cancer cells were obtained from six intraoperative tissue samples (codes A26083, L65, L66, L72, L74, and L84) according to step (2)-3 of Example 1 and cultured using culture medium OC-2. The basal medium was mixed with Matrigel (Corning™, 356231) at a ratio of 50:1 on ice. 2 mL of the mixture was plated into a 6-well plate and incubated at 37°C in a 5% CO2 incubator for 25 to 35 minutes, preferably 30 minutes, and the supernatant was discarded. The obtained primary ovarian cancer cells were plated into a 6-well plate (250,000 cells per well) at a viable cell density of 2 x 10 4 cells / cm 2 After surface sterilization, the plate was placed in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific) and cultured.

[0076] Cultured primary ovarian cancer cells were observed under a microscope (EVOS M500, Invitrogen). Figures 3A-3F show photographs of primary ovarian cancer cells obtained from samples A26083, L65, L66, L72, L74, and L84, respectively, after 4-6 days of culture, taken with a 10x objective lens. When observed under the microscope, the cells were densely arranged and slightly irregular in shape.

[0077] Example 5 Histochemical Characterization of Primary Ovarian Cancer Cell Cultures Approximately 0.25 cm 3 Cancer tissue (sample L74) was removed from an ovarian cancer patient during surgery and fixed by immersion in 1 mL of 4% paraformaldehyde. Using the method described in Example 4, sample L74 was cultured up to the fourth passage using the OC-2 culture medium for primary ovarian cancer cells of the present invention. The 4% paraformaldehyde-fixed primary ovarian cancer cells were embedded in paraffin and cut into 4 μm-thick tissue sections using a microtome. Routine immunohistochemical detection was then performed (see Li et al., Nature Communication, (2018) 9: 2983 for specific procedures). The primary antibodies used were ER, PR, P53, NapsinA, Pax-8, WT-1, and Ki-67 (all purchased from CST).

[0078] Figures 4A-4G and 5A-5G are photographs comparing immunohistochemical results between the original tissue cells and primary ovarian cancer cells obtained by culturing the original tissue cells in the OC-2 culture medium for primary ovarian cancer cells of the present invention. Figures 4A and 5A are photographs of ER antibody labeling in ovarian cancer tissue and cultured primary ovarian cancer cells, respectively. Figures 4B and 5B are photographs of PR antibody labeling in ovarian cancer tissue and cultured primary ovarian cancer cells, respectively. Figures 4C and 5C are photographs of P53 antibody labeling in ovarian cancer tissue and cultured primary ovarian cancer cells, respectively. Figures 4D and 5D are photographs of NapsinA antibody labeling in ovarian cancer tissue and cultured primary ovarian cancer cells, respectively. Figures 4E and 5E are photographs of Pax-8 antibody labeling in ovarian cancer tissue and cultured primary ovarian cancer cells, respectively. Figures 4F and 5F are photographs of WT-1 antibody labeling in ovarian cancer tissue and cultured primary ovarian cancer cells, respectively. Figures 4G and 5G are photographs of Ki-67 antibody labeling in ovarian cancer tissue and cultured primary ovarian cancer cells. This confirms that when primary ovarian cancer cells cultured using the technology of the present invention are cultured up to the fourth passage, the expression of ovarian cancer-related biomarkers on the primary ovarian cancer cells is consistent with the expression in the original tissue section from which the primary ovarian cancer cells were derived. This demonstrates that primary ovarian cancer cells cultured using the technology of the present invention maintain the original pathological characteristics of the cancer tissue of ovarian cancer patients.

[0079] Example 6 Comparison of culture effects with existing culture media, statistics of culture cycles and cell numbers of primary ovarian cancer cells, and calculation of population doublings (PD) A conventional culture medium (Xuefeng Liu et al., Nat. Protoc., 12(2): 439-451, (2017)) had the following formulation: 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.

[0080] Commercially available culture medium: Keratinocyte Serum Medium (K-SFM), a standard medium (purchased from Gibco, 10744-019)

[0081] Primary ovarian cancer cells were obtained from three ovarian cancer tissue samples (codes L65, L66, and A22083) according to the process of step (2)-3 in Example 1. The obtained primary ovarian cancer cells were cultured using a conventional culture medium, a commercially available culture medium, and the culture medium OC-2 in Example 3, respectively, and the cells were plated in a 6-well plate at a viable cell density of 3 × 10 4 cells / cm 2 After the cells expanded to 95%, they were digested and counted, and the number of days of culture until digestion was recorded as the culture cycle. Under this experimental condition, the expanded cells were expanded for different passages. The cells of each passage were digested and counted, and the corresponding culture cycle was recorded. The PD number was calculated according to the following formula: Population doubling (PD) = 3.32 × log 10 (Total cell number after digestion / Initial number of cells inoculated). For the formula, see Chapman et al., Stem Cell Research & Therapy 2014, 5: 60.

[0082] Figures 6A-6C show growth curves of three primary cells cultured using a conventional culture medium, a commercially available culture medium, and the OC-2 culture medium for primary ovarian cancer cells of the present invention, plotted using Graphpad Prism software. The horizontal axis represents the number of days of cell culture, and the vertical axis represents the cumulative fold increase in cell growth, i.e., the fold increase in cell growth per culture cycle. The higher the value, the more times the cells expand within a given cycle, i.e., the more cells expand. The slope represents the cell expansion rate. Figures 6A-6C show that when primary ovarian cancer cells cultured in the OC-2 culture medium of the present invention are continuously cultured and expanded for at least 30 days, the cell expansion rate remains essentially unchanged, indicating that the cells still have the ability to continue expanding. The results in Figures 6A-6C indicate that the expansion rates of primary ovarian cancer cells cultured in the conventional culture medium and the commercially available medium are significantly lower than those in OC-2 medium. In summary, the growth efficiency of ovarian cancer cells cultured in vitro using the culture medium for primary ovarian cancer cells of the present invention is significantly better than that of prior art culture media and commercially available media.

[0083] Example 7 Use of primary ovarian cancer cells expanded using the culture medium of the present invention for drug screening and efficacy evaluation 1. Cell Culture and Plating Primary ovarian cancer cells (code A22083) were isolated and used for one generation according to the process in step (2)-3 of Example 1. They were cultured in the culture medium OC-2 for primary ovarian cancer cells of the present invention until the cells expanded to 85% and were then passaged. The cells were passaged and counted according to step (2)-4 of Example 1. The cells were loaded into a loading slot (purchased from Corning) at a viable cell density of 1 x 10 5After thorough mixing, the cells were placed into a 384-well opaque white cell culture plate (purchased from Corning) at a volume of 50 μL per well and a cell count of 5,000 cells / well. The culture medium for the primary ovarian cancer cells of the present invention was added from the edge of the plate to seal it, and the sample name, administration time, and CellTiter-Glo (Promega) test time were marked on the plate. The surface of the plate was disinfected with 75% alcohol (LIRCON), and the plate was cultured in a 5% CO2 incubator at 37°C, followed by administration 24 hours later. For drug screening, cells from the first, second, third, fourth, and fifth passages of culture were obtained, respectively, to test the drug sensitivity of the primary ovarian cancer cells cultured using the culture medium of the present invention for continuous passage.

[0084] 2. Preparation of Candidate Drugs Six concentration gradients of six drugs (cytarabine, doxorubicin, bortezomib, panobinostat, azacitidine, and homoharringtonine, all purchased from MCE) were prepared according to the table below, and these were added to a 384-well plate (Thermo Fisher) at a volume of 30 μL per well and stored until use.

[0085] [Table 5]

[0086] 3. High-throughput Dosing The prepared drug-containing plates were removed and kept at room temperature. The plates were centrifuged at room temperature in a centrifuge (Beckman) at 1000 rpm for 1 minute and then removed from the centrifuge. A high-throughput automated workstation (JANUS, Perkin Elmer) was used for high-throughput administration. 0.1 μL of the corresponding concentration of candidate drug was added to each well of a 384-well plate containing cultured primary ovarian cancer cells. After administration, the surface of the 384-well plate was disinfected and moved to an incubator. Cell viability was measured 72 hours later.

[0087] 4. Measuring Cell Viability CellTiter-Glo Luminescent Reagent (Promega) was removed from a 4°C refrigerator, and 10 mL of the reagent was added to the loading slot. The 384-well plate to be tested was removed from the incubator, and 10 μL of CellTiter-Glo Luminescent Reagent was added to each well. After allowing to stand for 10 minutes, the assay was performed using a multifunction microplate reader (Envision, Perkin Elmer).

[0088] 5. Data Processing The cell inhibition rate of cells treated with various drugs was calculated according to the formula: cell inhibition rate (%) = 100% - chemiluminescence value of drug-treated wells / chemiluminescence value of control wells × 100%, and the half-maximal inhibition rate (IC) of the drug on the cells was calculated. 50 ) was calculated using GraphPad Prism software. The results are shown in Figures 7A to 7F.

[0089] Figures 7A-7F show that when primary ovarian cancer cells cultured from the OC-2 culture medium for primary ovarian cancer cells of the present invention are used for drug screening, the inhibitory effect of the same drug on cultured cells of different passages is substantially consistent (the inhibition curves are substantially consistent). Cells from the same patient have different sensitivities to different drugs at the maximum blood concentration in the human body. Based on these results, the effectiveness of drugs in clinical use in ovarian cancer patients can be determined. At the same time, the results also show that the drug sensitivity of tumor cells of different passages obtained by the culture method of the present invention is stable. [Industrial Applicability]

[0090] The present invention provides a primary cell culture medium and a culture method for culturing primary ovarian cancer cells in vitro, and the cultured cells can be used for drug efficacy evaluation and screening. Therefore, the present invention is suitable for industrial application.

[0091] Although the present invention has been described in detail in this specification, the present invention is not limited thereto, and those skilled in the art can make modifications according to the principles of the present invention. Therefore, it should be understood that any modifications made according to the principles of the present invention fall within the protection scope of the present invention.

Claims

1. 1. A culture medium for primary ovarian cancer cells, comprising: The present invention is characterized by comprising an MST1 / 2 kinase inhibitor, at least one Rho kinase inhibitor selected from the group consisting of Y27632, fasudil, and H-1152, an insulin-transferrin-sodium selenite supplement, insulin, prostaglandin E2, epidermal growth factor, gastrin, insulin-like growth factor-1, cholera toxin, amphiregulin, N2, and B27; The MST1 / 2 kinase inhibitor is represented by formula (I): 【Chemistry 1】 (In the formula, R １ is C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and one to two independent R ６ aryl optionally substituted with one to two independent R ６ aryl C1-C6 alkyl optionally substituted with, and one to two independent R ６ heteroaryl optionally substituted with R ２ and R ３ are each independently selected from C1-C6 alkyl; R ４ and R ５ are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, hydroxylC1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, and C3-C6 heterocyclylC1-C6 alkyl; R ６ is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl, or a pharmaceutically acceptable salt or solvate thereof.

2. R １ is C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and one to two independent R ６ phenyl optionally substituted with one to two independent R ６ naphthyl optionally substituted with one to two independent R ６ phenylmethyl optionally substituted with, and one to two independent R ６ thienyl optionally substituted with R ２ and R ３ are each independently selected from C1-C3 alkyl; R ４ and R ５ are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, hydroxylC1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, piperidylC1-C6 alkyl, and tetrahydropyranylC1-C6 alkyl; R ６ is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl; The culture medium of claim 1 .

3. The MST1 / 2 kinase inhibitor is represented by formula (Ia): 【Chemistry 2】 (In the formula, R １ is one to two independent R ６ phenyl optionally substituted with one to two independent R ６ thienyl optionally substituted with, and one to two independent R ６ phenylmethyl optionally substituted with R ５ is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; R ６ is independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl), or a pharmaceutically acceptable salt or solvate thereof.

4. R １ There are one to two independent R ６ is phenyl optionally substituted with R ５ is hydrogen, R ６ The culture medium of claim 3 , wherein is fluoro, methyl, or trifluoromethyl.

5. The culture medium according to claim 1, wherein the MST1 / 2 kinase inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts thereof: Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5

6. The amounts of components in the culture medium are as follows: (1) the amount of the MST1 / 2 kinase inhibitor in the culture medium is 2.5 μM to 20 μM; (2) the amount of the Rho kinase inhibitor in the culture medium is 2.5 μM to 20 μM; (3) the volume ratio of the insulin-transferrin-sodium selenite supplement to the culture medium is 1:800 to 1:50; (4) the amount of insulin in the culture medium is 1 μg / mL to 27 μg / mL; (5) the amount of prostaglandin E2 in the culture medium is 2.5 μM to 10 μM; (6) The amount of the epidermal growth factor in the culture medium is 2.5 ng / mL to 40 ng / mL; (7) the amount of gastrin in the culture medium is 1 nM to 9 nM; (8) The amount of insulin-like growth factor-1 in the culture medium is 25 ng / mL to 100 ng / mL; (9) The amount of the cholera toxin in the culture medium is 0.05 μg / mL to 0.8 μg / mL; (10) The amount of amphiregulin in the culture medium is 1 ng / mL to 81 ng / mL; (11) The volume ratio of the B27 additive to the culture medium is 1:25 to 1:400; (12) The volume ratio of the N2 additive to the culture medium is 1:50 to 1:400; The culture medium according to any one of claims 1 to 5, characterized in that it satisfies any one or more or all of the following requirements.

7. The culture medium according to any one of claims 1 to 6, further comprising an initial medium selected from the group consisting of DMEM / F12, DMEM, F12 or RPMI-1640, and one or more antibiotics selected from the group consisting of streptomycin / penicillin, amphotericin B and primocin.

8. 1. A method of culturing primary ovarian cancer cells, comprising: (1) preparing a culture medium for primary ovarian cancer cells according to any one of claims 1 to 7; (2) obtaining primary ovarian cancer cells from the ovarian cancer tissue sample; (3) adding the culture medium for the primary ovarian cancer cells obtained in step (1) to the primary ovarian cancer cells obtained in step (2) and culturing them; A method comprising:

9. 1. A method for evaluating or screening a drug for treating ovarian cancer, comprising: (1) culturing primary ovarian cancer cells using the method for culturing primary ovarian cancer cells according to claim 8; (2) selecting a test drug and diluting the drug to a desired concentration gradient; (3) adding the drug diluted to various concentration gradients to the primary ovarian cancer cells cultured in step (1); (4) detecting cell viability; A method comprising:

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