WO2023060677A1 - Culture medium, culture method and use of primary ovarian cancer cells - Google Patents

Abstract

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

Description

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

The invention belongs to the technical field of biomedicine, specifically relates to a culture medium and its application, and more specifically relates to a culture medium, a culture method and an application of ovarian cancer primary cells.

Background technique

Ovarian cancer refers to a malignant tumor disease that occurs in the ovary. It is one of the common malignant tumors of female reproductive organs, and its incidence rate is second only to cervical cancer and uterine body cancer. Ovarian cancer is the most common epithelial cancer, followed by malignant germ cell tumors. Among them, the mortality rate of ovarian epithelial cancer accounts for the first of all kinds of gynecological tumors, which can pose a serious threat to women's lives. Ovarian cancer is mostly asymptomatic in the early stage, but gastrointestinal symptoms such as lower abdominal discomfort, abdominal distension, and loss of appetite may appear in the late stage. The main treatment methods include surgical resection, drug therapy, and radiation therapy, and the overall prognosis is poor.

Chemotherapy is one of the main methods for treating ovarian tumors. Although there are many chemotherapeutic drugs that can be used clinically, the effective rate of clinical treatment of ovarian tumors is only about 25%. The main reason is that most of the chemotherapeutic drug regimens currently used by patients are based on the experience of clinicians. Without considering the individual differences of patients, through the method of trial-assessment-change drug trial-reassessment, not only failed Improve the therapeutic effect of drugs, and at the same time miss the best treatment period, so that the tumor will enter the advanced stage. In addition, patients suffer from drug side effects and high medical costs throughout the course of treatment.

Therefore, establishing a primary tumor model in vitro and using it for efficient drug screening experiments is a promising solution. At present, the main method for establishing the in vitro culture model of primary ovarian tumors is patient-derived tumor xenograft model (Patient-Derived Tumor Xenograft, PDX). its therapeutic effect. However, the PDX method also has some shortcomings, such as: species differences between humans and mice; long test period (more than 4 weeks); high cost (more than 200,000); false positives and false negatives.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention provides a culture medium and a culture method for rapid expansion of ovarian cancer primary cells in vitro.

One aspect of the present invention is to provide a culture medium for ovarian cancer primary cells, said culture medium comprising MST1/2 kinase inhibitors; sodium pyruvate; forskolin; epidermal growth factor; gastrin; fibroblast growth Factor 7; Nicotinamide; SB431542; and Fetal Bovine Serum.

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

in,

R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by _1-2 independent R (such as phenyl and naphthyl, etc.), aryl C1-C6 alkyl (such as benzyl, etc.) and heteroaryl (such as thienyl, etc.);

R ₂ and R ₃ are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

R ₄ and R ₅ are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyran base, etc.);

_R 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 trifluoro methyl).

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

in,

_R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently _R6 , thienyl optionally substituted by 1-2 independently _R6 , and optionally substituted by 1 -2 independently _R6 substituted benzyl, _R1 is more preferably optionally 1-2 independently _R6 substituted phenyl;

R ₅ is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R ₅ is more preferably hydrogen;

R ₆ is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R ₆ is more preferably fluorine, methyl or trifluoromethyl.

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

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

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

(1) The content of the MST1/2 kinase inhibitor in the medium is 2.5-10 μM;

(2) The content of the sodium pyruvate in the culture medium is 0.25～1mM;

(3) The content of the forskolin in the medium is 2.5-10 μM;

(4) The content of the epidermal growth factor in the medium is 5-80 ng/mL;

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

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

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

(8) The content of the SB431542 in the medium is 3.75-30 μM;

(9) The volume ratio of the fetal bovine serum relative to the culture medium is 2.5% (v/v)-40% (v/v).

In an embodiment of the present invention, the medium also contains an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one selected from streptomycin/penicillin, amphotericin B and Primocin one or more antibiotics.

In a preferred embodiment, when the antibiotic is selected from streptomycin/penicillin, the streptomycin concentration ranges from 25 to 400 μg/mL, and the penicillin concentration ranges from 25 to 400 U/mL; when the antibiotic is selected from amphotericin B, The concentration range is 0.25-4 μg/mL, and when the antibiotic is selected from Primocin, the concentration range is 25-400 μg/mL.

According to the second aspect, the present invention also provides a method for culturing primary ovarian cancer cells in vitro. In the method for culturing primary ovarian cancer cells in vitro of the present invention, the primary ovarian cancer cell culture medium of the present invention is used to culture primary ovarian cancer cells in vitro.

The ovarian cancer primary cell culture method of the present invention comprises the following steps:

1. Isolation of Ovarian Cancer Primary Cells

(1) Separate ovarian cancer tissue samples, add basal medium and tissue digestion solution at a volume ratio of 1:3 (note: the amount of tissue digestion solution is about 5-10mL tissue digestion solution for 1g of tumor tissue), and place at constant temperature Carry out digestion in a shaker, the digestion temperature is 4-37°C, and the digestion speed is 200rpm-350rpm;

(2) Digestion can be terminated until no obvious tissue blocks are seen, and the digestion time is 3 to 6 hours;

(3) Discard the supernatant after centrifugation, the centrifugation speed is 1200-1600 rpm, the centrifugation time is 2-6 minutes, add the basal medium and resuspend for use.

2. use ovarian cancer primary cell culture medium of the present invention to cultivate

The ovarian cancer primary cells obtained in the above step 1 were resuspended and counted with the ovarian cancer primary cell culture medium of the present invention, planted into a culture dish according to the cell density of 1-10× ¹⁰⁴ cells/ ^cm2 , and at the same time according to the cell density The density is 2-3×10 ⁴ cells/cm ² and trophoblasts are added until the cells in the culture dish are over 90% full and can be digested and passaged.

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

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

(1) using the method for culturing primary ovarian cancer cells of the present invention to cultivate primary ovarian cancer cells for drug screening;

(2) Select the drug to be detected and dilute it according to the required concentration gradient;

(3) Adding the drugs in various concentration gradients to the cells cultured in (1);

(4) Carry out cell activity test.

The technical solution of the present invention can obtain the following technical effects:

(1) Improve the success rate of ovarian cancer primary cell culture, and be able to culture tumor tissues derived from multiple sources such as epithelial cancer, malignant germ cell tumor, mesenchymal tumor and metastatic tumor, with a success rate of more than 80%;

(2) The ovarian cancer primary cells cultured in vitro can maintain the pathological characteristics of the patient;

(3) The cultured primary ovarian cancer cells are not disturbed by mesenchymal cells such as fibroblasts and adipocytes;

(4) The amplification efficiency is high. As long as there are 10 ⁵ cells, primary ovarian cancer cells of the order of 10 ⁶ can be successfully amplified within a week, and the amplified primary ovarian cancer cells can also be continuously passaged ;

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

(6) The number of ovarian cancer primary cells cultured by the described technique is large and highly homogeneous, which is suitable for high-throughput screening of new candidate compounds and providing patients with high-throughput drug sensitivity functional tests in vitro.

Description of drawings

Fig. 1 is a graph showing the effects of different combinations of factors added in primary ovarian cancer cell culture medium on the growth of primary ovarian cancer cells.

2A-2I are graphs showing the effects of different concentrations of factors added to primary ovarian cancer cell culture medium on the growth of primary ovarian cancer cells.

3A-3J are photographs of ovarian cancer primary cells cultured using the primary ovarian cancer cell culture medium of the present invention observed under a microscope.

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

5A-5G are the immunohistochemical results of primary ovarian cancer cells obtained by culturing primary ovarian cancer tissue cells to the fourth generation using the ovarian cancer primary cell culture medium of the present invention.

6A-6D are the cell growth curves of primary ovarian cancer cells cultured using the primary ovarian cancer cell culture medium of the present invention, literature culture medium and commercial medium respectively.

7A-7E are results of drug screening of ovarian cancer cells of different passages cultured using the ovarian cancer primary cell culture medium of the present invention.

Detailed ways

In order to better understand the present invention, the present invention will be further described below in conjunction with the embodiments and accompanying drawings, and the following embodiments are only to illustrate the present invention rather than limit it.

[Preparation Example of MST1/2 Kinase Inhibitor]

In the present specification, an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signal transduction. In general, MST1/2 kinase inhibitors, for example, bind to MST1/2 kinase and reduce its activity. Due to the similarity in the structures of MST1 and MST2, MST1/2 kinase inhibitors may also be, for example, compounds that bind to MST1 or MST2 and reduce their activity.

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)benzene Sulfonamide 1

2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): In a round bottom flask was added 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) Methanol (30 mL) was then added, followed by the dropwise addition of thionyl chloride (1.2 mL) under ice-cooling. The reaction system was reacted overnight at 85°C. After the reaction, the system was evaporated to dryness under reduced pressure to obtain a white solid, which was directly used in the next step.

Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (A3): Add 2-amino- After adding acetone (30 ml) and potassium carbonate (2.2 g) to 2-(2,6-difluorophenyl) methyl acetate (2 g), the system was cooled to -10 ° C with an ice-salt bath, and then slowly added 2,4-Dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction was stirred overnight at room temperature. After the reaction was completed, it was filtered, and the solvent was removed from the filtrate under reduced pressure, and the residue was purified by pressurized silica gel column chromatography to obtain compound A3. LC/MS: M+H 359.0.

2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): add 2-((2-chloro- Methyl 5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g) was added followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction system was stirred at 60°C for two hours. After the reaction, the system was evaporated to dryness under reduced pressure, and the resultant was neutralized to alkalinity with saturated sodium bicarbonate. 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 obtain a crude product. The crude product was washed with ether to obtain compound A4. LC/MS: M+H 297.0.

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

4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide (1): Add 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridine-6(5H) to a round bottom flask - Ketone (100 mg), sulfonamide (53 mg), p-toluenesulfonic acid (53 mg) and sec-butanol (5 ml). The reaction system was stirred overnight at 120°C. After the reaction, filter and wash with methanol and ether to obtain compound 1. LC/MS: M+H 461.1.

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

Other MST1/2 inhibitor compounds of the present invention were synthesized according to a method similar to compound 1, and their structures and mass spectrometry data are shown in the table below.

Example 1 Effects of various added factors in primary ovarian cancer cell culture medium on the proliferation of ovarian cancer primary cells

(1) Preparation of ovarian cancer primary cell culture medium

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

(2) Isolation and processing of ovarian cancer primary cells

1 Sample Selection

Ovarian cancer solid tumor tissue samples (intraoperative) were obtained from patients by professional medical staff of professional medical institutions, and all patients signed informed consent. The intraoperative sample is 0.25cm ³ , and the endoscopic sample is 0.025cm ³ ; commercialized tissue preservation solution (manufacturer: Miltenyi Biotec) is used for storage and transportation.

2 Material preparation

After surface disinfection of 15mL sterile centrifuge tubes, pipette guns, 10mL pipettes, sterile pipette tips, etc., put them in the ultra-clean workbench and irradiate them with ultraviolet light for 30 minutes. Take out the washing medium from the 4°C refrigerator 30 minutes in advance, and take out the tissue digestion solution from the -20°C refrigerator 30 minutes in advance.

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

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

Collagenase II, collagenase IV, DNase, and hyaluronidase mentioned above were all purchased from Sigma Company; calcium chloride was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; BSA was purchased from Biofroxx Company.

3. Isolation of Ovarian Cancer Primary Cells

3.1 Take the tissue samples in the culture dish in the ultra-clean bench, remove the tissue with blood, wash it twice with the basal medium, transfer the tissue to another culture dish and use a sterile scalpel for mechanical separation, and divide the tissue block into 1 ×1×1mm ³ size;

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

3.3 Discard the supernatant, add basal medium and tissue digestion solution at a ratio of 1:3 (note: the amount of tissue digestion solution added is about 10 mL of tissue digestion solution for 1 g of tumor tissue), mark the sample name and number, and seal it with a parafilm. Digestion was carried out at 37°C in a 300rpm shaker (ZQLY-180N), during which the digestion was observed every 30 minutes to see if there were no particles visible to the naked eye, and the digestion time was 4 hours;

3.4 After the digestion is completed, filter the undigested tissue mass through a 100μm filter, wash the tissue mass on the filter with the basal medium into the centrifuge tube to reduce cell loss, and centrifuge at 1500rpm at 25°C for 4 minutes;

3.5 Discard the supernatant and observe whether there are blood cells. If there are blood cells, add 8mL blood cell lysate (purchased from Sigma Company), mix well, and lyse at 4°C for 20 minutes.

3.6 Discard the supernatant, add 2mL basal medium to resuspend the cells, and set aside.

4 Cell Counting and Processing

4.1 Observation under the microscope: transfer a small amount of resuspended cells and spread them on a culture dish, and observe the density and shape of cancer cells under a microscope (CNOPTEC, BDS400);

4.2 Viable cell counting: Take 12 μL of the resuspended cell suspension and 12 μL trypan blue staining solution (Sangon Bioengineering (Shanghai) Co., Ltd.) after mixing thoroughly, take 20 μL and add it to a cell counting plate (Countstar, specification: 50 pieces/ box), the percentage of living large cells (cell size > 10 μm) was calculated by a cell counter (Countstar, IC1000) = number of viable cells/number of total cells × 100%.

(3) Culture of ovarian cancer primary cells

Add the medium with different components in Table 1 into the 48-well plate at a volume of 1 mL/well. The ovarian cancer primary cells isolated from two cases of ovarian cancer tissues (numbered L40 and LQQ) according to the above step (2) were seeded in a 48-well culture plate at a cell density of ⁴ ×10 cells/well, and each well was Add 2×10 ⁴ /well of NIH-3T3 cells (purchased from ATCC, resuspended using basal medium) irradiated by γ-rays (irradiation dose is 30Gy), under the conditions of 37°C and 5% CO ₂ To cultivate. After culturing for 7-10 days, the cells grew to 85%, the medium was discarded, rinsed once with 100 μL 0.05% trypsin (purchased from Gibco) per well, and then 200 μL 0.05% trypsin was added to each well. Place it in a 37°C, 5% _CO2 incubator for 10 minutes, observe under a microscope (CNOPTEC, BDS400) that the cells have been completely digested, and add 300 μL of DMEM/F12 medium containing 10% serum (Excell Bio, FND500) to terminate the digestion. 20 μL was added to a cell counting plate (Countstar, specification: 50 pieces/box), and the cell counting instrument (Countstar, IC1000) counted the total number of cells. Among them, as an experimental control, a basal medium without any additives was used, and the experimental results are shown in Table 1.

Table 1 Added components in the medium and their effect on promoting cell proliferation

Among them, "+" means that compared with the basal medium, the medium added with this additive has the effect of promoting the proliferation of at least two cases of ovarian cancer primary cells isolated from ovarian cancer tissue; "-" means that the additive is added One case of the primary ovarian cancer cells isolated from ovarian cancer tissue showed that the culture medium of Proliferation was not significantly affected in at least two cases.

According to the above results, SB202190, compound 1, hydrocortisone, SB431542, epidermal growth factor, insulin, insulin-transferrin-selenium supplement, nicotinamide, non-essential amino acid, Y-27632, fetal bovine serum, hair Throat, sodium pyruvate, gastrin, fibroblast growth factor 7, cholera toxin and other factors were further cultured.

Example 2 Effect of the combination of different added factors in primary ovarian cancer cell culture medium on the proliferation of ovarian cancer primary cells

According to the ingredients in Table 2, ovarian cancer primary cell culture media with different additive factor combinations were prepared, and the proliferation-promoting effects of different additive factor combinations on ovarian cancer primary cells were investigated.

Table 2 Preparation of medium with different components (concentration is the final concentration)

Obtain ovarian cancer primary cells from ovarian cancer tissues (numbered L37, L40, L43, L44) according to the method of step (2) of Example 1, divide the obtained cell suspension into 18 parts, and centrifuge at 1500rpm for 4 minute. After centrifugation, use 200 μL BM and No.1-17 medium to resuspend, inoculate in a 48-well plate according to the viable cell density of 4× ¹⁰⁴ / ^cm2 (40,000 cells per well), and then according to the cell density NIH-3T3 cells (purchased from ATCC and resuspended using basal medium (BM)) irradiated by γ-rays (irradiation dose of 30Gy) were added at 2× ¹⁰ cells/cm ² , and finally the corresponding medium was used Make up the volume of each well in the 48-well plate to 1 mL, and mix well. After surface disinfection, they were cultured in a 37°C, 5% CO ₂ incubator (purchased from Thermo Fisher).

When the cells in the 48-well plate grew to more than 85%, the culture medium was discarded, rinsed once with 100 μL 0.05% trypsin (purchased from Gibco), and then 200 μL 0.05% trypsin was added to each well. Place it in a 37°C, 5% _CO2 incubator for 10 minutes, observe under a microscope (CNOPTEC, BDS400) that the cells have been completely digested, and add 300 μL of DMEM/F12 medium containing 10% serum (Excell Bio, FND500) to terminate the digestion. 20 μL was added to a cell counting plate (Countstar, specification: 50 pieces/box), and the cell counting instrument (Countstar, IC1000) counted the total number of cells. The results obtained from primary ovarian cancer cells isolated from intraoperative samples L37, L40, L43, L44 are shown in FIG. 1 .

According to the results in Figure 1, compared with the basal medium (BM), when using the above-mentioned No.1-No.17 medium, when using the medium containing sodium pyruvate, forskolin, epidermal growth factor, gastrin, Primary ovarian cancer cells have the best proliferation effect when cultured in the medium with added components such as fibroblast growth factor 7, nicotinamide, SB431542, compound 1, and fetal bovine serum.

Example 3 Effects of Different Concentrations of Factors Added to the Ovarian Cancer Medium on the Proliferation of Ovarian Cancer Primary Cells

According to the method of step (2)-3 of Example 1, primary ovarian cancer cells were obtained from tissue samples (numbered L53, L55, and L56). The primary ovarian cancer cells obtained were seeded in 6-well plates at a living cell density of ³ ×10 ⁴ cells/cm ² (300,000 cells per well), ^and added with γ- The NIH-3T3 cells irradiated with radiation (irradiation dose 30Gy) were mixed evenly. After surface disinfection, they were cultured in a 37°C, 5% CO ₂ incubator (purchased from Thermo Fisher). And use the combined culture medium of the effective factor determined in embodiment 2 (containing basal medium BM, 1mM sodium pyruvate, 10 μ M forskolin, 20ng/mL epidermal growth factor, 27nM gastrin, 5ng/mL fibroblast Growth factor 7, 4 mM nicotinamide, 15 μM SB431542, 10 μM compound 1, 10% (v/v) fetal calf serum) were cultured and expanded. When the cells grow to more than 85%, add 500 μL 0.05% trypsin (purchased from Gibco) to rinse for 1 minute, then add 1 mL 0.05% trypsin to each well, and place in a 37°C, 5% _CO2 incubator React for 2-10 minutes, until the cells have been digested completely, add 1 mL of DMEM/F12 medium containing 10% serum (Excell Bio, FND500) to terminate the digestion. After centrifugation at 1500 rpm for 4 minutes, the supernatant was discarded. Resuspend the cell pellet in DMEM/F12. Take 20 μL and add it to a cell counting plate (manufacturer: Countstar, specifications: 50 pieces/box), and a cell counter (Countstar, IC1000) to count the total number of cells. The obtained cells were used for the following culture experiments.

Next, prepare the following 9 kinds of formula media for experimentation:

Formula 1: Sodium pyruvate is not included in the above-mentioned primary ovarian cancer cell culture medium components;

Formula 2: Forskolin is not included in the above-mentioned primary ovarian cancer cell culture medium components;

Formula 3: the above-mentioned ovarian cancer primary cell culture medium components do not contain epidermal growth factor;

Formula 4: Gastrin is not contained in the above-mentioned primary ovarian cancer cell culture medium components;

Formula 5: the above-mentioned ovarian cancer primary cell culture medium components do not contain fibroblast growth factor 7;

Formula 6: Nicotinamide is not included in the above-mentioned primary ovarian cancer cell culture medium components;

Formula 7: SB431542 is not included in the above-mentioned primary ovarian cancer cell culture medium components;

Formula 8: Compound 1 is not contained in the above-mentioned primary ovarian cancer cell culture medium components;

Recipe 9: Fetal bovine serum is not included in the above-mentioned components of the ovarian cancer primary cell culture medium.

Add 20 μl of cell suspension containing 4 x 10 ⁴ cells to each well, and use 1 mL of the above-mentioned medium of recipes 1 to 9 to dilute the cell suspension.

When using the medium of Formula 1, add 1 mL of prepared sodium pyruvate to each well of the 48-well plate inoculated with primary cells, and the final concentrations of sodium pyruvate are 0.25 mM, 0.5 mM, 1 mM, 2 mM, 4 mM; and set up control wells (BC) using the medium of Formulation 1.

When using the medium of formula 2, add 1 mL of prepared forskolin to each well of the 48-well plate inoculated with primary cells, and the final concentrations of forskolin are 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively. ; and set up control wells (BC) using the medium of recipe 2.

When the medium of formula 3 was used, 1 mL of prepared epidermal growth factor was added to each well of the 48-well plate inoculated with primary cells, and the final concentrations of epidermal growth factor were 5 ng/mL, 10 ng/mL, 20 ng/mL, 40ng/mL, 80ng/mL; and use the medium of formula 3 to set up control wells (BC).

When the medium of Formula 4 was used, 1 mL of prepared gastrin was added to each well of the 48-well plate inoculated with primary cells, and the final concentrations of gastrin were 1 nM, 3 nM, 9 nM, 27 nM, and 81 nM; And set up control wells (BC) using the medium of recipe 4.

When using the medium of Formula 5, add 1 mL of prepared fibroblast growth factor 7 to each well of the 48-well plate inoculated with primary cells, and the final concentration of fibroblast growth factor 7 is 2.5 ng/mL , 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL; and set up control wells (BC) using the medium of formula 5.

When using the medium of formula 6, add 1 mL of prepared nicotinamide to each well of the 48-well plate inoculated with primary cells, and the final concentrations of nicotinamide are 1 mM, 2 mM, 4 mM, 8 mM, and 16 mM respectively; and use The media of formulation 6 set up control wells (BC).

When using the medium of Formula 7, add 1 mL of prepared SB431542 to each well of the 48-well plate inoculated with primary cells, and the final concentrations of SB431542 are 3.75 μM, 7.5 μM, 15 μM, 30 μM, and 60 μM; and use The media of formulation 7 set up control wells (BC).

When the medium of Formula 8 was used, 1 mL of prepared compound 1 was added to each well of the 48-well plate inoculated 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 up using formulation 8 medium.

When using the medium of Formula 9, add 1 mL of prepared fetal bovine serum to each well of the 48-well plate inoculated with primary cells, and the addition ratio of fetal bovine serum is 2.5% (v/v) and 5% respectively. (v/v), 10% (v/v), 20% (v/v), 40% (v/v); and set up control wells (BC) using the medium of Formulation 9.

After the cells were expanded to about 85% of the 48 wells, they were digested and counted, and the proliferation multiples were calculated with reference to the number of cells in the control well (BC), and the results were shown in Figures 2A-2I. In FIGS. 2A-2I , the ratio is the ratio of the number of cells obtained by using each medium for one generation of culture to the number of cells obtained by the corresponding control well for one generation of culture. A ratio greater than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds has a better effect on promoting proliferation than the culture medium of the control well; a ratio less than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds promotes proliferation The effect was weaker than that of the culture medium in the control well.

According to the result of Fig. 2A～2I, the content of sodium pyruvate is preferably 0.25～1mM, and the cell proliferation effect is the most obvious when the concentration is 0.5mM; The content of forskolin is preferably 2.5～10μM, and the cell proliferation effect is the best when the concentration is 2.5μM Obviously; the content of epidermal growth factor is preferably 5-80 ng/mL, more preferably 5-20 ng/mL, and the cell proliferation effect is the most obvious when the concentration is 10 ng/mL; the content of gastrin is preferably 3-81 nM, more preferably 9-81nM, the cell proliferation effect is the most obvious when the concentration is 27nM; the content of fibroblast growth factor 7 is preferably 5-40ng/ml, more preferably 5-20ng/ml, and the cell proliferation effect is the most obvious when the concentration is 10ng/ml The content of nicotinamide in the medium is preferably 1-16mM, more preferably 1-4mM, and the cell proliferation effect is the most obvious when the concentration is 1mM; the content of SB431542 is preferably 3.75-30μM, more preferably 3.75-15μM, and the concentration is 7.5 The cell proliferation effect is the most obvious when μM; the content of Compound 1 is preferably 2.5～10 μM, more preferably 2.5～5 μM, and the cell proliferation effect is the most obvious when the concentration is 5 μM; the volume content of fetal bovine serum is preferably 2.5～40% (v/ v), more preferably 5-20% (v/v), and the cell proliferation effect is most obvious when the concentration is 10% (v/v).

The most preferred concentration of each added factor in the above-mentioned medium is used as the ovarian cancer primary cell culture medium of the present invention used in the following examples, which contains: basal medium BM, 0.5mM sodium pyruvate, 2.5 μM forskolin , 10ng/mL epidermal growth factor, 27nM gastrin, 10ng/ml fibroblast growth factor 7, 1mM nicotinamide, 7.5μM SB431542, 5μM compound 1, 10% (v/v) fetal bovine serum (hereinafter referred to as " OC-1" medium).

Example 4 Ovarian cancer primary cell culture and identification

Ovarian cancer primary cells were obtained from 10 tissue samples (numbered L53, L54, L56, L58, L60, L62, L65, L66, L69, L74) according to the method of step (2) of Example 1, and the obtained The primary ovarian cancer cells were seeded in a 6-well plate (300,000 cells per well) according to the viable cell density of 3×10 ⁴ cells/cm ² , and added in the γ-ray irradiated cells according to the cell density of 2×10 ⁴ cells/cm ² The NIH-3T3 cells irradiated (irradiation dose 30Gy) were mixed, and the OC-1 medium in Example 3 was added. After surface disinfection, they were cultured in a 37°C, 5% CO ₂ incubator (purchased from Thermo Fisher).

The cultured primary ovarian cancer cells were observed using a microscope (EVOS M500 from Invitrogen Company). Figures 3A-3J are photos taken under a 10x objective lens.

A cancer tissue with a size of about 0.25 cm ³ was taken from the intraoperative tissue of a patient with ovarian cancer (sample number L62), soaked in 1 mL of 4% paraformaldehyde and fixed. Using the method of Example 3, the sample L62 was continuously cultured to the fourth generation using the medium OC-1 of the present invention. Tissues or cells fixed with 4% paraformaldehyde were embedded in paraffin and cut into 4 μm thick tissue sections with a microtome. Then routine immunohistochemical detection was performed (see Li et al., Nature Communication, (2018) 9:2983 for specific steps). The primary antibodies used were ER, PR, P53, NapsinA, Pax-8, WT-1, Ki-67 (all purchased from CST).

Figures 4A-4G and 5A-5G are comparisons of the immunohistochemical results of primitive tissue cells and ovarian cancer primary cells obtained by culturing the cells with the primary ovarian cancer medium OC-1 of the present invention, respectively. Figure 4A and Figure 5A are pictures of labeled ER antibody on ovarian cancer tissue and primary ovarian cancer cells after culture, respectively, and Figure 4B and Figure 5B are pictures of labeled PR antibody on ovarian cancer tissue and primary ovarian cancer cells after culture, respectively Picture, Figure 4C and Figure 5C are the pictures of labeled P53 antibody of ovarian cancer tissue and primary ovarian cancer cells after culture, respectively, and Figure 4D and Figure 5D are the marker NapsinA of ovarian cancer tissue and primary ovarian cancer cells after culture, respectively The pictures of the antibody, Figure 4E and Figure 5E are the pictures of the labeled Pax-8 antibody on the ovarian cancer tissue and the primary ovarian cancer cells after culture, respectively, and Figure 4F and Figure 5F are the ovarian cancer tissue and the primary ovarian cancer cells after culture, respectively The pictures of cells labeled with WT-1 antibody, Figure 4G and Figure 5G are pictures of ovarian cancer tissue and cultured ovarian cancer primary cells labeled with Ki-67 antibody, respectively. It can thus be confirmed that when the ovarian cancer primary cells cultured by the technology of the present invention are cultured to the fourth generation, the expression of ovarian cancer-related biomarkers on the ovarian cancer primary cells is the same as that of the original tissue from which the ovarian cancer primary cells originate. The expression of markers in slices was basically the same. This shows that the ovarian cancer primary cells cultured by the technology of the present invention maintain the original pathological characteristics of the ovarian cancer patient's cancer tissue.

Example 5 Comparison with existing medium culture effects and ovarian cancer primary cell culture cycle and cell number statistics and Population Doubling (PD) value calculation

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

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

The ovarian cancer primary cells were obtained from 4 samples of ovarian cancer tissue samples (numbered BNYT1083, L57, L58, and L60) according to the method of step (2)-3 of Example 1. For the primary ovarian cancer cells obtained, culture medium, commercial medium and OC-1 medium in Example 3 were used to culture, and the cells were seeded in 6 wells according to the viable cell density of 3×10 ⁴ cells/cm ² After the cells were expanded to 95%, they were digested and counted. At the same time, the number of days of culture until digestion was recorded, and the number of days of culture until digestion was regarded as a culture cycle. Continue to cultivate under the experimental conditions, expand the cells obtained by different generations, count and record the corresponding culture period after each generation is digested, according to the formula Population Doubling (PD)=3.32*log10 (total number of cells after digestion/ Initial seeded cell number) to calculate PD, see Chapman et al., Stem Cell Research & Therapy 2014, 5:60 for the formula.

Figure 6A-6D shows the growth curves of 4 cases of primary cells cultured by Graphpad Prism software using literature medium, commercial medium and ovarian cancer primary cell culture medium OC-1 of the present invention, and the abscissa indicates the cells The number of days of culture, the ordinate is the cumulative cell proliferation multiple, indicating the multiple of cell expansion during the culture period, the larger the value, the more times the cells are expanded within a certain period, that is, the greater the number of expanded cells Many, the slope represents the rate of cell expansion.

It can be confirmed from Figures 6A-6D that when the ovarian cancer primary cells cultured in the medium OC-1 of the present invention are continuously cultured and expanded for at least 60 days, the cell expansion rate remains basically unchanged and still has the ability to continue to expand; The proliferation rate of ovarian cancer primary cells cultured in literature medium and commercial medium was significantly lower than that in OC-1 medium, and the proliferation stopped after passage at most 2 passages. To sum up, compared with literature culture medium and commercial culture, the ovarian cancer primary cell culture medium of the present invention has significantly better proliferation efficiency of ovarian cancer cell culture in vitro.

Example 6 The ovarian cancer primary cells expanded using the culture medium of the present invention are used for drug screening and curative effect evaluation

1. Cell culture and plating

According to the method of step (2) of Example 1, the primary ovarian cancer cells (numbered as L62) were isolated and used as a first generation, and cultured using the ovarian cancer primary cell culture medium OC-1 of the present invention, and the cells were expanded. Increased to 85%, for subculture. According to step (2)-4 of Example 1, the cell subculture was counted, and the cells were placed in the sample tank (purchased from Corning Corporation) according to the viable cell density of 1× ¹⁰⁵ cells/mL and mixed thoroughly, and then placed in the 384-well The cells were cultured in an opaque white cell culture plate (purchased from Corning Corporation), with a volume of 50 μL per well, and the number of cells was 5000 per well. Add the ovarian cancer primary cell culture medium of the present invention from the edge of the hole plate to seal the plate, and label the sample name, drug addition time and CellTiter-Glo (purchased from Promega Company) detection time on the plate. The surface was sterilized with 75% alcohol (purchased from Lierkang), placed in a 37°C, 5% CO ₂ incubator for cultivation, and the drug was added after 24 hours. The cultured first generation, second generation, third generation, fourth generation and fifth generation cells were respectively obtained for drug screening, and the drug sensitivity of the primary ovarian cancer cells cultured in the medium of the present invention for continuous passage was tested.

2. Screen drug preparation

According to the table below, 5 drugs (cytarabine, doxorubicin, panobinostat, azacitidine, homoharringtonine; all purchased from MCE Company) with 6 concentration gradients were prepared. Add 30 μL to each well of the plate (purchased from Thermo Fisher), and save it for later use.

Table 3 Drug action concentration settings

3. High-throughput dosing

Take out the prepared medicine plate, place it at room temperature, centrifuge at 1000 rpm in a centrifuge (Beckman) for 1 minute at room temperature, and then take it out. A high-throughput automated sample addition system (Perkin Elmer JANUS) was used for high-throughput drug addition. Add 0.1 μL of corresponding concentration of screening drugs to each well of the 384-well plate cultured with ovarian cancer primary cells. After the drug addition, the surface of the 384-well plate was sterilized and moved to the incubator, and the cell viability was measured 72 hours later.

4. Cell Viability Test

The CellTiter-Glo luminescent reagent (purchased from Promega) was taken out from the refrigerator at 4°C, and 10 mL of the reagent was placed in the sampling tank. Take out the 384-well plate to be tested in the incubator, add 10 μL CellTiter-Glo luminescent reagent to each well, let it stand for 10 minutes, mix well, and use a multifunctional microplate reader (Envision of Perkin Elmer Company) to detect.

5. Data processing

According to the formula cell inhibition rate (%)=100%-chemiluminescence value of drug-dosing well/chemiluminescence value of control well×100%, calculate the cell inhibition rate after different drugs act on cells, and use graphpad prism software to calculate the effect of drugs on cells Half inhibition rate (IC50). The results are shown in Figures 7A-7E.

It can be confirmed from Figures 7A-7E that the ovarian cancer primary cells cultured using the ovarian cancer primary cell culture medium OC-1 of the present invention are used for drug screening, and the same drug has basically the same inhibitory effect on cultured cells of different generations (inhibition The curves are basically the same). Cells from the same patient differ in their sensitivity to different drugs at their maximum blood concentration in the human body. According to the results, the effectiveness of the clinical use of the drug in ovarian cancer patients can be judged, and at the same time, it can be explained that the sensitivity of the tumor cells of different generations obtained according to the culture method of the present invention is stable to the drug.

Industrial Applicability

The invention provides a culture medium and a culture method for culturing ovarian cancer primary cells in vitro, and the cultured cells can be applied to the curative effect evaluation and screening of drugs. Thus, the present invention is suitable for industrial applications.

Although the present invention has been described in detail herein, the present invention is not limited thereto, and those skilled in the art can make modifications according to the principle of the present invention. Therefore, all modifications made according to the principle of the present invention should be understood as falling within protection scope of the present invention.

Claims (10)

1. A culture medium for ovarian cancer primary cells, characterized in that it comprises:

   MST1/2 kinase inhibitors; sodium pyruvate; forskolin; epidermal growth factor; gastrin; fibroblast growth factor 7; nicotinamide; SB431542; and fetal bovine serum;

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

   

   in,

   R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by _1-2 independent R Base, aryl C1-C6 alkyl and heteroaryl;

   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, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl;

   R ₆ is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
2. culture medium as claimed in claim 1, wherein

   _R is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and benzene optionally substituted by _1-2 independently R phenyl, naphthyl, benzyl and thienyl;

   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, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, piperidinyl C1-C6 alkyl, and tetrahydropyranyl C1-C6 alkyl;

   R ₆ is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
3. The culture medium according to claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt thereof, or a solvate,

   

   in,

   _R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently _R6 , thienyl optionally substituted by 1-2 independently _R6 , and optionally substituted by 1 -2 independently _R6 substituted benzyl groups;

   R _is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl;

   Each R ₆ is independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl.
4. culture medium as claimed in claim 3, wherein

   R ₁ is phenyl optionally substituted by 1-2 independently R ₆ ;

   _R is hydrogen;

   _R6 is preferably fluoro, methyl or trifluoromethyl.
5. The culture medium according to claim 1, wherein the MST1/2 kinase inhibitor is selected from at least one of the following compounds or pharmaceutically acceptable salts thereof:

   

   

   

   

   

   
6. The culture medium according to any one of claims 1 to 5, characterized in that the content of each component in the culture medium satisfies any one or more or all of the following:

   (1) The content of the MST1/2 kinase inhibitor in the medium is 2.5-10 μM;

   (2) The content of the sodium pyruvate in the culture medium is 0.25～1mM;

   (3) The content of the forskolin in the medium is 2.5-10 μM;

   (4) The content of the epidermal growth factor in the medium is 5-80 ng/mL;

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

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

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

   (8) The content of the SB431542 in the medium is 3.75-30 μM;

   (9) The volume ratio of the fetal bovine serum relative to the culture medium is 2.5% (v/v)-40% (v/v).
7. The culture medium according to any one of claims 1 to 6, further comprising:

   An initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and an antibiotic selected from one or more of streptomycin/penicillin, amphotericin B and Primocin.
8. A method for culturing ovarian cancer primary cells, characterized in that:

   (1) preparing the culture medium of ovarian cancer primary cells as described in any one of claims 1 to 7;

   (2) Obtain primary ovarian cancer cells, seed them into culture dishes according to the cell density of 1-10×10 ⁴ cells/cm ² , and add trophoblast cells according to the cell density of 2-3×10 ⁴ cells/cm ² , and then use the steps (1) The culture medium of the obtained ovarian cancer primary cells was cultured.
9. The culture method of ovarian cancer primary cells according to claim 8, wherein the trophoblast cells are irradiated NIH-3T3 cells, the radiation source is X-ray or γ-ray, and the radiation dose is 20- 50Gy.
10. A method for screening or evaluating drugs for the treatment of ovarian cancer, comprising the following steps:

    (1) cultivating ovarian cancer primary cells according to the culture method described in claim 8 or 9;

    (2) Select the drug to be detected and dilute it according to the required concentration gradient;

    (3) Adding the drugs in various concentration gradients to the cells cultured in (1);

    (4) Carry out cell activity test.

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