Culture medium, culture method and application of ovarian cancer organoids

Abstract

The present invention provides a culture medium and culture method for ovarian cancer organoids. The culture medium contains an MST1 / 2 kinase inhibitor; a B27 additive; an N2 additive; an insulin-transferrin-selenium supplement; insulin; epidermal growth factor; fetal bovine serum; basic fibroblast growth factor; fibroblast growth factor 7; and a Rho protease inhibitor. Compared with existing culture media and culture methods, in vitro culture using the culture medium of the present invention has higher expansion efficiency. Cultivating ovarian cancer organoids using this culture medium can maintain the morphological structure and pathological characteristics of the primary tissue, improve the success rate and survival rate of ovarian cancer organoid culture, and provide a research basis for personalized treatment of patients.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a culture medium for culturing ovarian cancer organoids, a method for culturing ovarian cancer organoids using the culture medium, and an application of the culture medium in evaluating and screening drug efficacy. Background Art

[0002] Ovarian cancer, a malignant tumor that develops in the ovaries, 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, digestive symptoms such as lower abdominal discomfort, bloating, and decreased appetite may develop in its later stages. Treatment options include surgical resection, medications, and radiation therapy, with an overall poor prognosis.

[0003] Traditional clinical drug sensitivity testing mostly uses two-dimensional cell culture. However, these cells only simulate tissue physiological conditions to a limited extent and lack authentic in vivo tissue structure, which can easily lead to low differentiation levels and loss of cellular physiological function. This, in turn, makes the experimental results difficult to predict actual clinical outcomes. Organoids, a three-dimensional (3D) cell culture, are primarily derived from human embryonic stem cells, induced pluripotent stem cells, and adult stem cells with the ability to differentiate. Endogenous tissue stem cells exist in various tissues and organs and play a vital role in maintaining the functional morphology of each organ. Under certain inductive conditions in vitro, these stem cells can self-organize into miniature structures measuring only a few millimeters in diameter. Tumor organoids are miniature 3D tumor cell models cultured in the laboratory using primary tumors taken from patients. Tumor organoids closely mimic the characteristics of the original tumor tissue, preserving the heterogeneity between individual tumors. They can be used for functional testing, such as high-throughput drug screening and personalized precision medicine.

[0004] Currently, ovarian cancer organoid culture methods often use R-spondin-1, Noggin, and other expensive protein factors, resulting in high costs. Furthermore, the technology is complex and technically challenging, limiting its large-scale commercial application. Therefore, there is a need to develop a low-cost, simple, and highly successful organoid culture method and culture medium. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a culture medium and a culture method for rapidly expanding ovarian cancer organoids in vitro.

[0006] One aspect of the present invention is to provide a culture medium for ovarian cancer organoids, comprising an MST1 / 2 kinase inhibitor; a B27 supplement; an N2 supplement; an insulin-transferrin-selenium supplement; insulin; epidermal growth factor; fetal bovine serum; basic fibroblast growth factor; fibroblast growth factor 7; and at least one Rho protease inhibitor selected from Y27632, fasudil, and H-1152. The MST1 / 2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.

[0007]

[0008] in,

[0009] 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.);

[0010] R2 and R3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

[0011] 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.);

[0012] 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).

[0013] In a preferred embodiment, the MST1 / 2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,

[0014]

[0015] in,

[0016] 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;

[0017] R5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R5 is more preferably hydrogen;

[0018] R6 are each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R6 is more preferably fluorine, methyl or trifluoromethyl.

[0019] Preferably, the MST1 / 2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.

[0020]

[0021]

[0022]

[0023]

[0024]

[0025] Most preferably, the MST1 / 2 kinase inhibitor of the present invention is Compound 1.

[0026] In addition, preferably, the Rho protein kinase inhibitor of the present invention is Y27632.

[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 concentration of the MST1 / 2 kinase inhibitor is generally in the range of 2.5 to 40 μM, more preferably 5 to 20 μM;

[0029] (2) The volume ratio of the B27 additive to the culture medium is generally in the range of 1:25 to 1:400, more preferably 1:50 to 1:100;

[0030] (3) The volume ratio of the N2 additive to the culture medium is generally in the range of 1:100 to 1:800, more preferably 1:200 to 1:800;

[0031] (4) The concentration range of the epidermal cytokine is usually 2.5 to 10 ng / mL;

[0032] (5) The concentration of insulin is generally in the range of 0.25 to 4 μg / mL, more preferably 0.5 to 1 μg / mL;

[0033] (6) The volume ratio of the insulin-transferrin-selenium supplement to the culture medium is generally in the range of 1:50 to 1:400, more preferably 1:50 to 1:200;

[0034] (7) The concentration range of the Rho protein kinase inhibitor is generally 2.5 to 40 μM, more preferably 5 to 10 μM;

[0035] (8) The volume ratio of the fetal bovine serum to the culture medium is generally in the range of 1.25% (v / v) to 20% (v / v), more preferably 5% (v / v) to 20% (v / v);

[0036] (9) The concentration of the basic fibroblast growth factor is generally in the range of 2.5 to 20 ng / mL, more preferably 2.5 to 10 ng / mL;

[0037] (10) The concentration of the fibroblast growth factor 7 is generally in the range of 2.5 to 20 ng / mL, more preferably 5 to 20 ng / mL.

[0038] 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.

[0039] 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.

[0040] According to a second aspect, the present invention further provides an in vitro culture method for ovarian cancer organoids. In the in vitro culture method for ovarian cancer organoids of the present invention, primary ovarian cancer cells are cultured in vitro using the ovarian cancer organoid culture medium of the present invention.

[0041] The ovarian cancer organoid culture method of the present invention comprises the following steps:

[0042] 1. Isolate samples from ovarian cancer solid tumor tissue to obtain primary ovarian cancer cells. The process includes the following steps.

[0043] (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 about 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 range of 4-37°C and a rotation speed range of 200 rpm-350 rpm.

[0044] (2) Digestion can be terminated when no obvious tissue blocks are observed. The digestion time is 3 to 6 hours.

[0045] (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.

[0046] 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).

[0047] 2. Cultivation using the ovarian cancer organoid culture medium of the present invention

[0048] The primary ovarian cancer cells obtained in step 1 above were resuspended in basal medium and counted, and mixed evenly with matrigel on ice at a volume ratio of 1:1. The final cell density was 1 to 8 × 10 5 / mL, take the matrix gel and cell suspension to form solidified droplets in the culture plate, let the culture plate stand at 4-37°C for 10-60 minutes to wait for the matrix gel to completely solidify, add the ovarian cancer organoid culture medium of the present invention, and culture in an incubator.

[0049] The present invention also provides a method for evaluating or screening drugs for treating ovarian cancer, comprising the following steps:

[0050] (1) Cultivating ovarian cancer organoids using the ovarian cancer organoid culture method of the present invention;

[0051] (2) Select the drug to be tested and dilute it according to the required concentration gradient;

[0052] (3) adding the diluted drug to the organoids cultured in (1);

[0053] (4) Conduct organoid size or organoid viability tests.

[0054] The technical solution of the present invention can achieve the following technical effects:

[0055] (1) Improve the success rate of ovarian cancer organoid culture to over 90%;

[0056] (2) ensuring that primary cultured ovarian cancer organoids in vitro can maintain the patient's pathological characteristics;

[0057] (3) High amplification efficiency, capable of rapidly culturing ovarian cancer organoids, and the amplified ovarian cancer organoids can be continuously passaged;

[0058] (4) The culture cost is controllable, and the culture medium does not need to add expensive Wnt agonists, R-spondin family proteins, Noggin proteins, BMP inhibitors, fibroblast growth factor 10 (FGF10) and other factors;

[0059] (5) The technology described herein can produce a large number of ovarian cancer organoids, which are suitable for high-throughput screening of candidate compounds and providing high-throughput in vitro drug sensitivity functional testing for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 A graph showing the effects of different combinations of added factors in ovarian cancer organoid culture medium on the growth of ovarian cancer organoids.

[0061] Figures 2A-2J A graph showing the effects of different concentrations of added factors to ovarian cancer organoid culture medium on the growth of ovarian cancer organoids.

[0062] Figures 3A-3F These are photographs of ovarian cancer organoids cultured using the ovarian cancer organoid culture medium of the present invention observed under a microscope.

[0063] Figures 4A-4G These are the immunohistochemical results of primitive ovarian cancer tissue cells.

[0064] Figures 5A-5G The immunohistochemical results of ovarian cancer organoids obtained by culturing primary ovarian cancer tissue cells to the third passage using the ovarian cancer organoid culture medium of the present invention.

[0065] Figure 6 These are microscopic photographs of ovarian cancer organoids obtained by culturing primary ovarian cancer cells using the ovarian cancer organoid culture medium of the present invention and the culture medium from the literature, respectively.

[0066] Figure 7A and 7B The results of drug sensitivity testing of ovarian cancer organoids obtained by culturing the ovarian cancer organoid culture medium of the present invention are shown, wherein Figure 7A Photos showing organoid growth without drug treatment and after 3 days of drug treatment; Figure 7B A plot showing drug sensitivity of ovarian cancer organoids grown at different concentrations of test drugs. DETAILED DESCRIPTION

[0067] 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.

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

[0069] 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.

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

[0071] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide 1

[0072]

[0073] 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). Thionyl chloride (1.2 mL) was then added dropwise under an ice bath. The reaction was allowed to react at 85°C overnight. After completion of the reaction, the solvent was evaporated under reduced pressure to yield a white solid, which was used directly in the next step.

[0074] 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 mixture 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.

[0075] 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.

[0076] 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.

[0077] 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.

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

[0079] 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.

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Example 1 Effects of Various Added Factors in Ovarian Cancer Organoid Culture Medium on the Growth of Ovarian Cancer Organoids

[0087] (1) Preparation of ovarian cancer organoid culture medium

[0088] First, a basal culture medium containing an initial culture medium is prepared. The initial culture medium can be selected from DMEM / F12, DMEM, F12, or RPMI-1640 commonly used in the art. In this embodiment, the formula of the basal culture medium is: DMEM / F12 culture medium (purchased from Corning) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available product concentration 50 mg / ml). Different types of additives (see Table 1) are added to the basal culture medium to prepare ovarian cancer organoid culture media containing different additives.

[0089] (2) Isolation and processing of primary ovarian cancer cells

[0090] 1. Sample selection

[0091] Ovarian cancer solid tumor tissue samples (intraoperative / endoscopic) 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.

[0092] 2. Material Preparation

[0093] After surface disinfection, sterile 15mL centrifuge tubes, pipettes, 10mL pipettes, and sterile pipette tips should be placed in a clean bench and exposed to 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 (recipe below) from the -20°C refrigerator 30 minutes in advance.

[0094] 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).

[0095] 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.

[0096] 3. Isolation of Primary Ovarian Cancer Cells

[0097] 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;

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

[0099] 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 sealing film, 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 approximately 4 hours.

[0100] 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.

[0101] 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.

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

[0103] 4. Cell Counting and Processing

[0104] 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).

[0105] 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.), mixed thoroughly, and 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%.

[0106] (3) Culture of ovarian cancer organoids

[0107] The primary ovarian cancer cells isolated from two ovarian cancer tissues (numbered L55 and L56) according to the above step (2) were resuspended in basal culture medium and counted, and then mixed with matrigel ( 356231) were mixed evenly on ice, and the final cell density was 5×10 5 / mL, take 7μL of matrix gel and cell suspension to form a solidified droplet in the center of each well of a 96-well culture plate, and let the culture plate stand at 37°C for 30 minutes to wait for the matrix gel to completely solidify. The culture medium with different components in Table 1 was added to the 96-well plate at a volume of 100μL / well. Among them, as an experimental control, a basic culture medium without any added components was used. After culturing for 7 to 10 days, 50μL CellTiter-Glo (purchased from Promega) luminescent reagent was added to each well, allowed to stand for 10 minutes and then mixed, and detected using a multifunctional microplate reader (PerkinElmer Envision). The relative cell viability (%) was calculated according to the formula = chemiluminescence value of the experimental well / chemiluminescence value of the control well × 100%, and the promoting effect of different added components on the growth of ovarian cancer organoids was inferred. The experimental results are shown in Table 1.

[0108] Table 1 Additives in culture medium and their effects on promoting cell proliferation

[0109]

[0110]

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

[0112] Based on the above results, compound 1, B27, N2, epidermal cell factor, insulin, insulin-transferrin-selenium supplement, Y-27632, fetal bovine serum, basic fibroblast growth factor, Forsklin, fibroblast growth factor 7, cholera toxin and other added ingredients were selected for further culture experiments.

[0113] Example 2 Effects of different combinations of added factors in ovarian cancer organoid culture medium on the growth of ovarian cancer organoids

[0114] Ovarian cancer organoid culture media with different combinations of added factors were prepared according to the components in Table 2 to investigate the growth-promoting effects of different combinations of added factors on ovarian cancer organoids.

[0115] Table 2 Preparation of different components of culture medium (concentration is final concentration)

[0116]

[0117]

[0118] According to the method of step (2) of Example 1, primary ovarian cancer cells were obtained from ovarian cancer tissues (numbered L53, L57, and L58), resuspended in basal culture medium and counted, and then mixed with matrix gel ( 356231) were mixed evenly on ice, and the final cell density was 5×10 5 / mL, take 20μL of matrix gel and cell suspension to form a solidified droplet in the center of each well of a 48-well culture plate, and let the culture plate stand at 37°C for 30 minutes to wait for the matrix gel to completely solidify. Finally, add 1mL of BM culture medium and culture medium corresponding to No. 1 to No. 13 to the 48-well plate respectively. After 7 to 10 days of culture, when the organoids are obviously formed, add 20μL of CellTiter-Glo (purchased from Promega) luminescent reagent to each well, let it stand for 10 minutes and then mix it, and use a multifunctional microplate reader (PerkinElmer Envision) to detect. According to the formula, the relative viability of the cells is calculated (%) = chemiluminescence value of the experimental well / chemiluminescence value of the control well × 100%, and the effect of different component culture media on the growth promotion of ovarian cancer organoids is obtained. The experimental results are shown in Figure 1 .

[0119] according to Figure 1The results show that, compared with the basal culture medium, when using the above-mentioned No. 1 to No. 13 culture media, the proliferation of primary ovarian cancer cells can be promoted to varying degrees. When the added factors Forsklin (No. 11) and cholera toxin (No. 13) are omitted, the proliferation-promoting effect of the culture medium formula is more obvious. Therefore, in subsequent examples, factors such as compound 1, B27, N2, epidermal cell factor, insulin, insulin-transferrin-selenium supplement, Y-27632, fetal bovine serum, basic fibroblast growth factor, and fibroblast growth factor 7 are used as culture medium formulas for culturing ovarian cancer organoids for further study.

[0120] Example 3 Effects of Different Concentrations of Factors Added to Ovarian Cancer Organoid Culture Medium on the Proliferation of Ovarian Cancer Organoids

[0121] According to the method of step (2) of Example 1, primary ovarian cancer cells were obtained from ovarian cancer tissue (numbered L74), resuspended in basal culture medium and counted, and then mixed with matrix gel ( 356231) were mixed evenly on ice, and the final cell density was 5×10 5 7 μL of matrix gel and cell suspension were taken to form a solidified droplet in the center of each well of a 96-well culture plate. The culture plate was placed at 37°C for 30 minutes to allow the matrix gel to completely solidify for the following culture experiments.

[0122] Next, ovarian cancer organoid culture medium containing basal culture medium BM, 20 μM compound 1, 1:50 (v / v) B27, 1:100 (v / v) N2, 10 ng / mL epidermal cell factor, 1 μg / mL insulin, 1:100 (v / v) insulin-transferrin-selenium supplement, 10 μM Y-27632, 10% (v / v) fetal bovine serum, 10 ng / mL basic fibroblast growth factor, 5 ng / mL fibroblast growth factor 7 and the following 10 formula culture media were prepared for experiments.

[0123] Formulation 1: The above ovarian cancer organoid culture medium components do not contain compound 1;

[0124] Recipe 2: The above ovarian cancer organoid culture medium components do not contain B27;

[0125] Recipe 3: The above ovarian cancer organoid culture medium components do not contain N2;

[0126] Recipe 4: The above ovarian cancer organoid culture medium components do not contain epidermal cytokine;

[0127] Recipe 5: The above ovarian cancer organoid culture medium components do not contain insulin;

[0128] Recipe 6: The above ovarian cancer organoid culture medium components do not contain insulin-transferrin-selenium supplement;

[0129] Recipe 7: The above ovarian cancer organoid culture medium components do not contain Y-27632;

[0130] Recipe 8: The above-mentioned ovarian cancer organoid culture medium components do not contain fetal bovine serum;

[0131] Recipe 9: The above-mentioned ovarian cancer organoid culture medium components do not contain basic fibroblast growth factor;

[0132] Recipe 10: The above-mentioned ovarian cancer organoid culture medium components do not contain fibroblast growth factor 7;

[0133] When using the culture medium of Formula 1, 100 μL of the prepared compound 1 was added to each well of a 96-well plate seeded with primary cells, and the final concentrations of compound 1 were 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.

[0134] When using the culture medium of Formula 2, 100 μL of the prepared B27 was added to each well of a 96-well plate seeded with primary cells. The volume ratios of B27 to the culture medium were 1:400 (v / v), 1:200 (v / v), 1:100 (v / v), 1:50 (v / v), and 1:25 (v / v), respectively; and control wells (BC) were set using the culture medium of Formula 2.

[0135] When using the culture medium of Formula 3, 100 μL of the prepared N2 was added to each well of a 96-well plate seeded with primary cells. The volume ratios of N2 to the culture medium 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 3.

[0136] When using the culture medium of Formula 4, 100 μL of the prepared epidermal cytokine was added to each well of a 96-well plate seeded with primary cells. The final concentrations of the epidermal cytokine 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 4.

[0137] When using the culture medium of Formula 5, 100 μL of prepared insulin was added to each well of a 96-well plate seeded with primary cells. The final concentrations of insulin were 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 5.

[0138] When using the culture medium of Formula 6, 100 μL of the prepared insulin-transferrin-selenium supplement was added to each well of a 96-well plate seeded with primary cells. The volume ratios of the insulin-transferrin-selenium supplement to the culture medium were 1:400 (v / v), 1:200 (v / v), 1:100 (v / v), 1:50 (v / v), and 1:25 (v / v), respectively; and control wells (BC) were set using the culture medium of Formula 6.

[0139] When using the culture medium of Formula 7, 100 μL of the prepared Y-27632 was added to each well of a 96-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 using the culture medium of Formula 7.

[0140] When using the culture medium of Formula 8, 100 μL of prepared fetal bovine serum was added to each well of a 96-well plate seeded with primary cells. The volume ratios of fetal bovine serum to culture medium were 1.25% (v / v), 2.5% (v / v), 5% (v / v), 10% (v / v), and 20% (v / v), respectively; and control wells (BC) were set using the culture medium of Formula 8.

[0141] When using the culture medium of Formula 9, 100 μL of the prepared basic fibroblast growth factor was added to each well of a 96-well plate seeded with primary cells. The final concentrations of basic fibroblast 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 9.

[0142] When using the culture medium of Formula 10, 100 μL of the prepared fibroblast growth factor 7 was added to each well of a 96-well plate seeded with primary cells. The final concentrations of fibroblast growth factor 7 were 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively. Control wells (BC) were set using the culture medium of Formula 10.

[0143] After 7-10 days of organoid culture, the relative proliferation times were calculated by comparing the cell numbers in the control wells (BC) and the results were shown in Figures 2A to 2J . Figures 2A to 2JThe relative proliferation fold is the ratio of the proliferation fold of ovarian cancer organoids cultured in each medium for 7–10 days to the proliferation fold of ovarian cancer organoids cultured in the corresponding control wells for 7–10 days. A ratio greater than 1 indicates that the culture medium containing the factor or small molecule compound at different concentrations is more effective in promoting proliferation than the culture medium in the control wells. A ratio less than 1 indicates that the culture medium containing the factor or small molecule compound at different concentrations is less effective in promoting proliferation than the culture medium in the control wells.

[0144] according to Figures 2A to 2J As a result, the concentration range of compound 1 is preferably 2.5-40 μM, more preferably 5-20 μM; the volume ratio of B27 additive to culture medium is preferably 1:25 (v / v)-1:400 (v / v), more preferably 1:50 (v / v)-1:100 (v / v); the volume ratio of N2 additive to culture medium is preferably 1:100-1:800, more preferably 1:200-1:800; the concentration range of epidermal cytokine is preferably 2.5-10 ng / mL; the content of insulin is preferably 0.25-4 μg / ml, more preferably 0.25-1 μg / ml; the content of insulin-transferrin- The volume ratio range of selenium supplement to culture medium is preferably 1:50 (v / v) to 1:400 (v / v), more preferably 1:50 (v / v) to 1:200 (v / v); the content of Y-27632 is preferably 2.5 to 40 μM, more preferably 5 to 10 μM; the volume content of fetal bovine serum is preferably 1.25 to 20% (v / v), more preferably 5 to 20% (v / v); the content of basic fibroblast growth factor is preferably 2.5 to 20 ng / ml, more preferably 2.5 to 10 ng / ml; the content of fibroblast growth factor 7 is preferably 2.5 to 20 ng / ml, more preferably 5 to 20 ng / ml.

[0145] The ovarian cancer organoid culture medium of the present invention used in the following examples contains: basal medium BM, 10 μM compound 1, 1:100 (v / v) B27, 1:200 (v / v) N2, 5 ng / mL epidermal cell factor, 0.5 μg / mL insulin, 1:200 (v / v) insulin-transferrin-selenium supplement, 10 μM Y-27632, 10% (v / v) fetal bovine serum, 5 ng / mL basic fibroblast growth factor, and 5 ng / mL fibroblast growth factor 7 (hereinafter referred to as "OC-3" medium).

[0146] Example 4 Ovarian Cancer Organoid Culture

[0147] According to the method described in Example 1 (2), primary ovarian cancer cells (L65, L66, L70, L74, L82, L84) were obtained, resuspended in basal culture medium and counted, and mixed with matrigel ( 356231) were mixed evenly on ice, and the final cell density was 5×10 5 / mL, take 50 μL of matrix gel and cell suspension to form a solidified droplet in the center of each well of a 24-well culture plate, let the culture plate stand at 37°C for 30 minutes to allow the matrix gel to completely solidify, and use a pipette to gently add 1 mL of the ovarian cancer organoid culture medium OC-3 of the present invention at room temperature to each well along the side wall of the well. After the surface of the 24-well culture plate is disinfected, it is placed in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific) for culture. After culturing for 4 to 10 days, the cultured ovarian cancer organoids are observed using a microscope (Invitrogen, EVOS M500). Figures 3A-3F This photo shows ovarian cancer organoids taken with a 10x objective lens. Under the microscope, the ovarian cancer organoids appear spherical with a smooth surface.

[0148] Example 5 Histochemical Identification of Cultured Ovarian Cancer Organoids

[0149] A 0.25 cm spherical ... 3 The size of the cancer tissue was immersed in 1 mL of 4% paraformaldehyde for fixation. Sample L74 was continuously cultured to the third generation using the ovarian cancer organoid culture medium of the present invention using the method of Example 3. The ovarian cancer organoids fixed with 4% paraformaldehyde were embedded in paraffin 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).

[0150] Figures 4A-4G 5A-5G are comparative diagrams of the immunohistochemical results of the original tissue and the ovarian cancer organoids obtained by culturing the original tissue cells using the ovarian cancer organoid culture medium OC-3 of the present invention. Figure 4A and Figure 5A These are pictures of ovarian cancer tissue and cultured ovarian cancer organoids labeled with ER antibodies. Figure 4B and Figure 5B These are pictures of ovarian cancer tissue and cultured ovarian cancer organoids labeled with PR antibodies. Figure 4C and Figure 5C These are pictures of ovarian cancer tissue and cultured ovarian cancer organoids labeled with P53 antibodies. Figure 4D and Figure 5D These are pictures of NapsinA antibodies labeled on ovarian cancer tissue and cultured ovarian cancer organoids. Figure 4E and Figure 5E These are pictures of Pax-8 antibodies labeled for ovarian cancer tissue and cultured ovarian cancer organoids. Figure 4F and Figure 5F These are pictures of ovarian cancer tissue and cultured ovarian cancer organoids labeled with WT-1 antibodies. Figure 4G and Figure 5G The images show ovarian cancer tissue and cultured ovarian cancer organoids, each labeled with the Ki-67 antibody. This confirms that, at the third passage, the expression of ovarian cancer-related biomarkers in ovarian cancer organoids cultured using the technology of this invention is essentially consistent with that in the original tissue sections from which they were derived. This demonstrates that ovarian cancer organoids cultured using the technology of this invention retain the original pathological characteristics of ovarian cancer tissue from patients.

[0151] Comparison of culture effects of Example 5 with literature culture medium

[0152] (1) Preparation of culture medium

[0153] Literature culture medium (Oded Kopper et al., Nature medicine, 2019, 838-849): DMEM / F12+25% (v / v) Wnt3A (purchased from Invitrogen) +1% penicillin / streptomycin +1:100 (v / v) glutamine supplement (purchased from thermo) +1% (v / v) HEPES (purchased from Gibco) +1:100 (v / v) N2 (purchased from Gibco) +10ng / mL epidermal cytokine (purchased from R&D) +100ng / mL Noggin (purchased from R&D) +100ng / mL FGF10 (purchased from R&D) +1mM nicotinamide (purchased from MCE) +9μM Y-27632 (purchased from MCE) +0.5μM SB431542 (purchased from MCE).

[0154] (2) Obtaining primary ovarian cancer cells and culturing ovarian cancer organoids

[0155] According to the method of step (2) of Example 1, primary ovarian cancer cells were obtained from the intraoperative tissue sample (numbered L68), resuspended in basal culture medium and counted, and then mixed with matrix gel ( 356231) were mixed evenly on ice, and the final cell density was 5×10 5 / mL, take 50μL of matrix gel and cell suspension to form a solidified droplet in the center of each well of a 24-well culture plate, let the culture plate stand at 37°C for 30 minutes to wait for the matrix gel to completely solidify, and use a pipette to gently add 1mL of the ovarian cancer organoid culture medium OC-3 of the present invention and the culture medium from the literature at room temperature to each well along the side wall of the well. After the surface of the 24-well culture plate is disinfected, it is placed in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific) for culture. After 7 days of culture, the cultured primary ovarian cancer cells were observed using a microscope (Invitrogen EVOS M500). Figure 6 This is an ovarian cancer organoid photographed and recorded under a 10x objective lens.

[0156] according to Figure 6 The results show that compared with the culture medium in the literature, the ovarian cancer organoid culture medium of the present invention has a large number of organoids and a complete organoid structure when culturing ovarian cancer organoids in vitro, and its effect is significantly better than the culture medium in the literature.

[0157] Example 6: Ovarian cancer organoids expanded using the culture medium of the present invention for drug screening

[0158] (1) Ovarian cancer organoid culture

[0159] Primary ovarian cancer cells were isolated from an intraoperative ovarian cancer sample (L74) according to the method of Example 1 (2), and organoids were cultured using OC-3 medium. Drug screening was performed when the diameter of the ovarian cancer organoids exceeded 50 μm.

[0160] (2) Screening drug preparation

[0161] Prepare 5 concentration gradients of 2 drugs (carboplatin and gemcitabine; both purchased from MCE) according to the table below and store for later use.

[0162] Table 3 Drug action concentration settings

[0163]

[0164] (3) Dosing

[0165] Remove the prepared drug and place it at room temperature. Prepare the drug with OC-3 medium and set aside. Remove the organoids cultured according to step (1) from the incubator, remove the medium in the culture wells, and slowly pour 400 μL of the medium containing the drug into the culture wells along the well walls. After the addition of the drug, disinfect the surface of the 48-well plate and move it to the incubator for further culture. After 3 days, measure the viability of the organoids.

[0166] (4) Organoid Viability Test

[0167] The CellTiter-Glo luminescent reagent (purchased from Promega) was taken out of the 4°C refrigerator, and the 48-well plate to be tested was taken out of the incubator. 50 μL of CellTiter-Glo luminescent reagent was added to each well. After standing for 30 minutes, the plate was mixed and detected using a multifunctional microplate reader (Perkin Elmer Envision).

[0168] (5) Data processing

[0169] The relative activity of different drugs was calculated according to the formula: drug relative activity (%) = (chemiluminescence value of the drug-treated group on the third day of culture well / chemiluminescence value of the drug-treated group on the zero day of culture well) * 100%. The results are shown in Figure 7A and 7B . Figure 7A The photos of organoid growth taken under a 4x objective microscope (Invitrogen EVOS M500) were taken before drug treatment and after drug treatment for 3 days. Figure 7B The figure shows the drug sensitivity curve of ovarian cancer organoids grown with different concentrations of test drugs.

[0170] Depend on Figure 7A and 7B It can be confirmed that ovarian cancer organoids cultured using the organoid culture medium OC-3 of the present invention can be used for drug screening, and the results can be used to determine the effectiveness of the drug in clinical use in ovarian cancer patients.

[0171] Industrial Applicability

[0172] The present invention provides a culture medium and method for culturing ovarian cancer organoids. The cultured organoids can be used for evaluating and screening drug efficacy. Therefore, the present invention is suitable for industrial applications.

[0173] 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 ovarian cancer organoids, characterized in that Made with the following ingredients: MST1 / 2 kinase inhibitor; B27 supplement; N2 supplement; insulin-transferrin-selenium supplement; insulin; epidermal growth factor; fetal bovine serum; basic fibroblast growth factor; fibroblast growth factor 7; Y27632; An initial culture medium selected from DMEM / F12, DMEM, F12, or RPMI-1640; and antibiotics; Wherein, the MST1 / 2 kinase inhibitor is compound 1 or a pharmaceutically acceptable salt or solvate thereof, in, (1) The concentration of the MST1 / 2 kinase inhibitor is 2.5 to 40 μM; (2) The volume ratio of the B27 additive to the culture medium is 1:25 to 1:400; (3) The volume ratio of the N2 additive to the culture medium is 1:100 to 1:800; (4) the concentration of the epidermal cytokine is 2.5 to 10 ng / mL; (5) The concentration of the insulin is 0.25 to 4 μg / mL; (6) The volume ratio of the insulin-transferrin-selenium supplement to the culture medium is 1:400 to 1:50; (7) The concentration of Y27632 is 2.5 to 40 μM; (8) The volume concentration of the fetal bovine serum relative to the culture medium is 1.25% (v / v) to 20% (v / v); (9) The concentration of the basic fibroblast growth factor is 2.5 to 20 ng / mL; (10) The concentration of the fibroblast growth factor 7 is 2.5 to 20 ng / mL.

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 ovarian cancer organoids, characterized in that The following steps are involved: (1) preparing a culture medium for the ovarian cancer organoid according to claim 1 or 2; (2) obtaining primary ovarian cancer cells from ovarian cancer tissue samples, and mixing the obtained primary ovarian cancer cell suspension with matrigel; (3) Adding the culture medium of the ovarian cancer organoids obtained in step (1) to the mixture of the primary ovarian cancer cells and matrigel obtained in step (2) for culturing.

4. A method for screening drugs for treating ovarian cancer, characterized in that: The following steps are involved: (1) Cultivating ovarian cancer organoids using the ovarian cancer organoid 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 diluted drug to the organoids cultured in (1); (4) Conduct organoid size or organoid viability testing.

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