CN115975932A - Culture medium, culture method and application of ovarian cancer primary cells - Google Patents

Abstract

The invention provides a culture medium and a culture method of primary ovarian cancer cells. The medium comprises an MST1/2 kinase inhibitor; sodium pyruvate; forskolin; an epidermal growth factor; a gastrin; fibroblast growth factor 7; nicotinamide; SB431542; and fetal bovine serum. Compared with the existing culture mode, the in vitro culture using the culture medium has higher amplification efficiency; the culture medium is used for primary ovarian cancer culture, the morphological structure and pathological features of primary tissues can be maintained, and the success rate and survival rate of primary ovarian cancer cell culture are improved.

Description

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

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a culture medium and application thereof, in particular to a culture medium of ovarian cancer primary cells, a culture method and application thereof.

Background

Ovarian cancer refers to a malignant tumor disease occurring in the ovary, is one of the common malignant tumors of female reproductive organs, and has the incidence rate second to cervical cancer and uterine corpus cancer. Ovarian cancer is the most common of epithelial cancers, and is followed by malignant germ cell tumors, wherein the death rate of ovarian epithelial cancers accounts for the first part of various gynecological tumors, and can cause serious threat to the life of women. Ovarian cancer is asymptomatic in early stage, digestive tract symptoms such as lower abdominal discomfort, abdominal distension and appetite reduction can appear in later stage, the main treatment modes comprise surgical resection, drug treatment and radiation treatment, and the overall prognosis is poor.

Chemotherapy is one of the major methods of treating ovarian tumors. Although a plurality of chemotherapy drugs are available in clinic at present, the clinical treatment effective rate of ovarian tumor is only about 25%. The main reason is that most of the current chemotherapeutic drug regimens used by patients are based on the experience of clinicians, and on the premise of not considering individual differences of patients, the method of trial-evaluation-dressing change trial-reevaluation not only fails to improve the treatment effect of the drugs, but also misses the optimal treatment period, thereby leading the tumor to enter the late stage. In addition, patients are burdened with drug side effects and extremely high medical costs throughout the treatment process.

Therefore, establishing a primary tumor model in vitro and using the model to perform efficient drug screening experiments is a scheme with good prospect. At present, a main method for establishing an in-vitro culture model of a primary ovarian tumor is a human-Derived tumor xenotransplantation model (PDX), wherein tumor cells of a Patient are transplanted into a nude mouse, and then the treatment effect of the nude mouse without using anti-tumor drugs is investigated. However, the PDX method also has some disadvantages, such as: species differences between humans and mice; the test period is long (more than 4 weeks); the cost is high (more than 20 ten thousand); false positive and false negative, etc.

Disclosure of Invention

In order to solve the technical problems, the invention provides a culture medium and a culture method for quickly amplifying ovarian cancer primary cells in vitro.

One aspect of the invention provides a culture medium of ovarian cancer primary cells, the culture medium comprising an MST1/2 kinase inhibitor; sodium pyruvate; forskolin; an epidermal growth factor; a 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,

wherein the content of the first and second substances,

R ₁ selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R ₆ Substituted aryl (e.g., phenyl, naphthyl, and the like), arylC 1-C6 alkyl (e.g., benzyl, and the like), and heteroaryl (e.g., thienyl, and the like);

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

R ₄ and R ₅ Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, and C3-C6 heterocycloC 1-C6 alkyl (said heterocyclyl is selected from, for example, piperidinyl, tetrahydropyranyl, and the like);

R ₆ selected from the group consisting of halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoromethyl).

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

wherein the content of the first and second substances,

R ₁ selected from C1-C6 alkyl, optionally substituted with 1-2 independent R ₆ Substituted phenyl, optionally substituted with 1-2 independent R ₆ Substituted thienyl, and optionally substituted with 1-2 independent R ₆ Substituted benzyl, R ₁ More preferably optionally substituted with 1-2 independent R ₆ Substituted phenyl;

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

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

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

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

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

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

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

(3) The content of the forskolin in the culture medium is 2.5-10 mu M;

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

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

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

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

(8) The content of the SB431542 in the culture medium is 3.75-30 mu M;

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

In an embodiment of the invention, said medium further comprises a starting 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.

In a preferred embodiment, the concentration of streptomycin ranges from 25 to 400. Mu.g/mL when the antibiotic is selected from streptomycin/penicillin, the concentration of penicillin ranges from 25 to 400U/mL, the concentration ranges from 0.25 to 4. Mu.g/mL when the antibiotic is selected from amphotericin B, and the concentration ranges from 25 to 400. Mu.g/mL when the antibiotic is selected from Primocin.

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

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

1. isolation of Primary ovarian cancer cells

(1) Separating ovarian cancer tissue samples, adding a basic culture medium and tissue digestive juice (the adding amount of the tissue digestive juice is about 5-10mL for 1g of tumor tissues) according to the volume ratio of 1:3, and placing the mixture in a constant temperature shaking table for digestion, wherein the digestion temperature is 4-37 ℃, and the digestion rotating speed is 200-350 rpm;

(2) The digestion can be stopped until no obvious tissue block is found, and the digestion time is 3 to 6 hours;

(3) And removing supernatant after centrifugation, wherein the centrifugation rotating speed is 1200-1600 rpm, the centrifugation time is 2-6 minutes, and a basic culture medium is added for re-suspension for later use.

2. Culturing Using the ovarian cancer Primary cell culture Medium of the present invention

Resuspending and counting the ovarian cancer primary cells obtained in the step 1 by using the ovarian cancer primary cell culture medium of the invention according to the cell density of 1-10 multiplied by 10 ⁴ Per cm ² Planting the mixture into a culture dish and simultaneously performing cell density 2-3 multiplied by 10 ⁴ Per cm ² And adding the feeder cells until the culture dish is full of more than 90% of the cells for digestion and passage.

Wherein the basic medium formulation in step 1 comprises a starting 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. The formula of the tissue digestive fluid comprises 1640 culture medium, collagenase II (1-2 mg/mL), collagenase IV (1-2 mg/mL), DNA enzyme (50-100U/mL), hyaluronidase (0.5-1 mg/mL), calcium chloride (1-5 mM) and bovine serum albumin BSA (5-10 mg/mL). The trophoblasts in the step 2 can be irradiated NIH-3T3 cells, the irradiation source is X rays or gamma rays, preferably gamma rays, and the irradiation dose is 20-50 Gy, preferably 30Gy.

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

(1) Culturing the ovarian cancer primary cells by using the culture method of the ovarian cancer primary cells for drug screening;

(2) Selecting a medicine to be detected and diluting according to a required concentration gradient;

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

(4) Cell viability assays were performed.

The technical scheme of the invention can achieve the following technical effects:

(1) The success rate of ovarian cancer primary cell culture is improved, and tumor tissues from multiple sample sources such as epithelial cancer, malignant germ cell tumor, interstitial tumor, metastatic tumor and the like can be cultured, and the success rate reaches over 80 percent;

(2) The ovarian cancer primary cells subjected to in vitro primary culture can keep the pathological characteristics of a patient;

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

(4) High amplification efficiency, as long as 10 ⁵ The cell number of the grade can be successfully amplified to 10 in about one week ⁶ The expanded ovarian cancer primary cells can also be continuously passaged;

(5) The culture cost is controllable: expensive factors such as Wnt agonist, R-spondin family protein, BMP inhibitor, FGF10 and the like do not need to be added into the culture medium;

(6) The ovarian cancer primary cells obtained by the culture of the technology have large quantity and high homogenization degree, and are suitable for screening new candidate compounds at high flux and providing high-flux medicine in-vitro sensitivity function tests for patients.

Drawings

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

FIGS. 2A-2I are graphs showing the effect of different concentrations of an ovarian cancer primary cell culture medium with added factors on the growth of ovarian cancer primary cells.

FIGS. 3A-3J are photographs of observing, with a microscope, ovarian cancer primary cells cultured using the ovarian cancer primary cell culture medium of the present invention.

FIGS. 4A-4G show immunohistochemical results for primary ovarian cancer tissue cells.

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

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

FIGS. 7A-7E are graphs showing the results of different generations of ovarian cancer cells cultured using the ovarian cancer primary cell culture medium of the present invention for drug screening.

Detailed Description

For a better understanding of the present invention, the present invention will be further described with reference to the following examples, which are set forth to illustrate, but are not to be construed as the limit of the present invention.

[ preparation example of MST1/2 kinase inhibitor ]

In the present specification, an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly down-regulates MST1/2 signaling. In general, MST1/2 kinase inhibitors, for example, bind to and reduce the activity of MST1/2 kinase. Due to the structural similarity of MST1 and MST2, MST1/2 kinase inhibitors may also be compounds that bind to and reduce the activity of MST1 or MST2, for example.

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) benzenesulfonamide 1

Methyl 2-amino-2- (2,6-difluorophenyl) acetate (A2): after 2-amino-2- (2,6-difluorophenyl) acetic acid (2.0 g) was added to the round bottom flask, methanol (30 ml) was added followed by thionyl chloride (1.2 ml) dropwise under ice-bath. The reaction system was reacted at 85 ℃ overnight. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure to obtain a white solid, which was used in the next step.

Methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2,6-difluorophenyl) acetate (A3): to a round bottom flask was added methyl 2-amino-2- (2,6-difluorophenyl) acetate (2 g), followed by acetone (30 ml) and potassium carbonate (2.2 g), then the system was cooled to-10 ℃ with an ice salt bath, followed by the slow addition of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction was stirred at room temperature overnight. After the reaction, the reaction mixture was filtered, the solvent was removed from the filtrate under reduced pressure, and the residue was purified by pressure silica gel column chromatography to obtain Compound A3.LC/MS: m + H359.0.

2-chloro-7- (2,6-difluorophenyl) -7,8-dihydropteridin-6 (5H) -one (A4): to a round bottom flask was added methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2,6-difluorophenyl) acetate (2.5 g) followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction was stirred at 60 ℃ for two hours. After the reaction was completed, the solvent was evaporated under reduced pressure, and the obtained product was neutralized to be alkaline with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. Filtering the organic phase, and evaporating to dryness under reduced pressure to obtain a crude product. Washing the crude product with diethyl ether to obtain a compound A4.LC/MS: m + H297.0.

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, cooled to-35 deg.C, iodomethane (0.9 mL) was added followed by sodium hydride (615 mg) and the reaction was stirred for an additional two hours. After the reaction, water was added to quench, ethyl acetate was used for extraction, and the organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. Filtering the organic phase, and evaporating to dryness under reduced pressure to obtain a crude product. Washing the crude product with diethyl ether to obtain a compound A5.LC/MS: m + H325.0.

4- ((7- (2,6-difluorophenyl) -5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl) amino) benzenesulfonamide (1): into a round bottom flask was added 2-chloro-7- (2,6-difluorophenyl) -5,8-dimethyl-7,8-dihydropteridin-6 (5H) -one (100 mg), sulfanilamide (53 mg), p-toluenesulfonic acid (53 mg), and sec-butanol (5 ml). The reaction was stirred at 120 ℃ overnight. After the reaction is finished, filtering, and washing by methanol and ether to obtain the compound 1.LC/MS: m + H461.1.

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

Other MST1/2 inhibitor compounds of the invention were synthesized in analogy to compound 1 and their structural and mass spectral data are shown in the table below.

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Example 1 Effect of factors added to ovarian cancer Primary cell Medium on proliferation of ovarian cancer Primary cells

(1) Preparation of ovarian cancer primary cell culture medium

First, a basal medium containing an initial medium is prepared. The starting medium may be selected from DMEM/F12, DMEM, F12 or RPMI-1640, commonly used in the art. In this example, the formulation of the basal medium is: DMEM/F12 medium (from Corning) + 100. Mu.g/mL Primocin (from InvivoGen, 0.2% (v/v), commercial product concentration 50 mg/mL). Different additives (see table 1) are added into the basic culture medium respectively to prepare the 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 (intraoperatively) were obtained by medical professionals at a specialized medical facility from patients who signed informed consent. Intraoperative sample 0.25cm ³ Endoscope sample 0.025cm ³ (ii) a The tissue preservation solution (manufacturer: miltenyi Biotec) is commercially available for storage and transportation.

2 Material preparation

And (3) sterilizing the surfaces of a 15mL sterile centrifuge tube, a pipette, a 10mL pipette, a sterile gun head and the like, and then placing the sterilized surfaces into a super clean bench for ultraviolet irradiation for 30 minutes. The wash medium was removed from the 4 ℃ freezer 30 minutes earlier and the tissue digest was removed from the-20 ℃ freezer 30 minutes earlier.

Basic culture medium: DMEM/F12 medium contained 100. Mu.g/mL Primocin (from Invivogen, 0.2% (v/v), a commercial product concentration of 50 mg/mL).

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

Collagenase ii, collagenase iv, dnase, and hyaluronidase mentioned above were all purchased from Sigma company; calcium chloride was purchased from bio-engineering (shanghai) incorporated; BSA was purchased from Biofrox.

3. Isolation of Primary ovarian cancer cells

3.1 taking tissue sample in a culture dish in a super clean bench, removing the tissue with blood, washing with a basic culture medium for 2 times, transferring the tissue to another culture dish, mechanically separating with a sterile scalpel, and dividing the tissue block into 1 × 1 × 1mm ³ Size;

3.2 sucking the cut intraoperative tissue into a 15mL centrifuge tube, adding 5mL of basal medium, uniformly mixing, and centrifuging at 1500rpm for 4 minutes;

3.3, discarding the supernatant, adding a basal medium and a tissue digestive juice according to a proportion of 1:3 (note: the adding amount of the tissue digestive juice is about 10mL for 1g of tumor tissue), marking the name and the number of a sample, sealing by a sealing film, digesting in a shaker (ZQLY-180N) at 300rpm at 37 ℃, observing whether the digestion is completed every 30 minutes, judging whether particles visible to naked eyes exist according to the judgment, and the digestion time is 4 hours;

3.4 after the digestion is finished, filtering out undigested tissue agglomerates by a 100-micron filter screen, flushing the tissue agglomerates on the filter screen into a centrifuge tube by using a basic culture medium to reduce cell loss, and centrifuging at 1500rpm for 4 minutes at 25 ℃;

3.5 discarding the supernatant, observing whether there are blood cells, if there are blood cells, adding 8mL of blood cell lysate (purchased from Sigma), mixing, lysing for 20 minutes at 4 ℃, reversing and mixing once, and centrifuging at 1500rpm at 25 ℃ for 4 minutes;

3.6 discard the supernatant, add 2mL of basal medium to resuspend the cells for use.

4 cell counting and processing

4.1 Observation under a lens: a small amount of the resuspended cells are removed and spread in a culture dish, and the density and morphology of the cancer cells are observed under a microscope (CNOPTEC, BDS 400);

4.2 viable cell count: after 12. Mu.L of the resuspended cell suspension and 12. Mu.L of trypan blue stain (Biotechnology (Shanghai) Co., ltd.) were mixed well, 20. Mu.L of the mixture was added to a cell counting plate (Countstar, specification: 50 plates/cassette), and the percentage of viable large cells (cell diameter >10 μm) calculated by a cell counter (Countstar, IC 1000) was = viable cell count/total cell count × 100%.

(3) Culture of Primary ovarian cancer cells

The different components of the medium in table 1 were added to 48 well plates in 1 mL/well volume. Two ovarian cancer tissues (numbered L40, LQQ) were isolated according to step (2) above

The obtained ovarian cancer primary cells are cultured at 4X10 ⁴ Cell density per well was seeded in 48-well plates and 2X 10 cells per well were added ⁴ NIH-3T3 cells (purchased from ATCC, resuspended using basal medium) irradiated with gamma rays (irradiation dose 30 Gy)/well at 37 ℃ with 5% CO ₂ The culture was performed under the condition of concentration. After 7 to 10 days of culture, the cells were grown to 85%, the medium was discarded, the cells were rinsed 1 time with 100. Mu.L of 0.05% trypsin (purchased from Gibco) per well, and 200. Mu.L of 0.05% trypsin was added to each well after aspiration. At 37 ℃ C, 5% CO ₂ The reaction in the incubator is 10 minutesAfter the cells were completely digested under a microscope (CNOPTEC, BDS 400), 300. Mu.L of DMEM/F12 medium containing 10% serum (Excell Bio, FND 500) was added to stop the digestion, 20. Mu.L of the medium was added to a cell counter (Countstar, standard: 50 plates/cassette), and the total number of cells was counted by a cell counter (Countstar, IC 1000). Among them, as an experimental control, a basal medium without any additive was used, and the experimental results are shown in table 1.

TABLE 1 additional ingredients in culture Medium and cell proliferation promoting effects

Wherein "+" indicates that the medium added with the additive has proliferation promoting effect on at least two cases of ovarian cancer primary cells separated from ovarian cancer tissues compared with the basic medium; "-" indicates that the medium to which the additive was added showed an effect of promoting proliferation of one example of ovarian cancer primary cells isolated from ovarian cancer tissue; ". O" indicates that the medium to which the additive was added had no significant effect on the proliferation of at least two cases of ovarian cancer primary cells isolated from ovarian cancer tissue.

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

Example 2 Effect of combinations of different additional factors in ovarian cancer Primary cell culture Medium on ovarian cancer Primary cell proliferation

Ovarian cancer primary cell culture media with different additive factor combinations are prepared according to the components in the table 2, and the proliferation promoting effect of the different additive factor combinations on ovarian cancer primary cells is examined.

TABLE 2 preparation of the media of different compositions (final concentration)

Ovarian cancer primary cells were obtained from ovarian cancer tissues (numbered L37, L40, L43, L44) according to the method of 3 of step (2) of example 1, and the obtained cell suspension was divided into 18 portions on average and centrifuged at 1500rpm for 4 minutes. After centrifugation, 200. Mu.L of BM and No.1 to 17 medium were used for resuspension, respectively, at a viable cell density of 4X10 ⁴ Per cm ² Seeded in 48-well plates (4 ten thousand cells per well) and subsequently at a cell density of 2X 10 ⁴ Per cm ² NIH-3T3 cells (purchased from ATCC and resuspended by using a Basal Medium (BM)) irradiated by gamma rays (irradiation dose is 30 Gy) are added, and finally, the pore volume of each 48-well plate is filled to 1mL by using a corresponding medium, and the mixture is fully mixed. Surface sterilized, then treated at 37 deg.C and 5% CO ₂ Incubators (purchased from Saimeri fly) for culture.

Cells grown to over 85% in 48-well plates were removed from the medium, rinsed 1 time with 100. Mu.L of 0.05% trypsin (from Gibco), aspirated, and 200. Mu.L of 0.05% trypsin was added to each well. At 37 ℃ C, 5% CO ₂ After 10 minutes of reaction in the incubator, the cells were observed under a microscope (CNOPTEC, BDS 400) to be completely digested, and the digestion was terminated by adding 300. Mu.L of DMEM/F12 medium containing 10% serum (Excell Bio, FND 500), and 20. Mu.L of the medium was added to a cell counter (Countstar, standard: 50 plates/box), and the total number of cells was counted by a cell counter (Countstar, IC 1000). The results obtained from ovarian cancer primary cells isolated from intraoperative samples L37, L40, L43, L44 are shown in fig. 1.

From the results shown in FIG. 1, it is seen that the growth effect is the best when ovarian cancer primary cells are cultured using a medium containing additional components such as sodium pyruvate, forskolin, epidermal growth factor, gastrin, fibroblast growth factor 7, nicotinamide, SB431542, compound 1, and fetal bovine serum, as compared with the Basal Medium (BM) in the cases of the above-mentioned media Nos. 1 to 17.

EXAMPLE 3 proliferation of ovarian cancer Primary cells by different concentrations of factors added to ovarian cancer Medium

Ovarian cancer primary cells were obtained from tissue samples (nos. L53, L55, L56) according to the method of 3 of step (2) of example 1. The obtained ovarian cancer primary cells have a viable cell density of 3 × 10 ⁴ Per cm ² Seeded in 6-well plates (30 ten thousand cells per well) at a cell density of 2X 10 ⁴ Per cm ² Adding NIH-3T3 cells irradiated by gamma ray (irradiation dose 30 Gy), and mixing. Surface sterilizing, adding into 37 deg.C and 5% CO ₂ Incubators (purchased from Saimeri fly) for culture. And expanded on the basis of a combined medium containing the effective factors identified in example 2 (basal medium BM, 1mM sodium pyruvate, 10. Mu.M forskolin, 20ng/mL epidermal growth factor, 27nM gastrin, 5ng/mL fibroblast growth factor 7, 4mM nicotinamide, 15. Mu.M SB431542, 10. Mu.M Compound 1, 10% (v/v) fetal bovine serum). Until the cells grew to 85% or more, 500. Mu.L of 0.05% trypsin (from Gibco) was added for rinsing for 1 minute, and after aspiration, 1mL of 0.05% trypsin was added to each well, and the mixture was left at 37 ℃ and 5% CO ₂ The reaction was carried out in an incubator for 2-10 minutes until the cells had been completely digested, and digestion was stopped by adding 1mL of DMEM/F12 medium containing 10% serum (Excell Bio, FND 500). After centrifugation at 1500rpm for 4 minutes, the supernatant was discarded. DMEM/F12 resuspends the cell pellet. mu.L of the suspension was added to a cell counting plate (manufacturer: countstar, specification: 50 plates/box), and the total number of cells was counted by a cell counter (Countstar, IC 1000). The resulting cells were used in the following culture experiments.

Next, the following 9 media formulations were prepared for the experiments:

formula 1: the components of the primary ovarian cancer cell culture medium do not contain sodium pyruvate;

and (2) formula: the components of the primary ovarian cancer cell culture medium do not contain forskolin;

and (3) formula: the components of the primary ovarian cancer cell culture medium do not contain epidermal growth factors;

and (4) formula: the components of the primary ovarian cancer cell culture medium do not contain gastrin;

and (5) formula: the components of the primary ovarian cancer cell culture medium do not contain fibroblast growth factor 7;

and (6) formula: the components of the primary ovarian cancer cell culture medium do not contain nicotinamide;

and (3) formula 7: the components of the primary ovarian cancer cell culture medium do not contain SB431542;

and (4) formula 8: the components of the primary ovarian cancer cell culture medium do not contain the compound 1;

formula 9: the components of the primary ovarian cancer cell culture medium do not contain fetal calf serum.

Add 20. Mu.l of 4X10 to each well ⁴ The cell suspension was diluted with 1mL of the medium of the above formulations 1 to 9.

When the culture medium of formula 1 is used, 1mL of prepared sodium pyruvate is added to each well of a 48-well plate inoculated with primary cells, and the final concentrations of the sodium pyruvate are 0.25mM, 0.5mM, 1mM, 2mM and 4mM, respectively; and control wells (BC) were set using medium of formula 1.

When the culture medium of formula 2 is used, 1mL of the prepared forskolin is added into 48-well plates inoculated with primary cells respectively, and the final concentrations of the forskolin are 2.5 muM, 5 muM, 10 muM, 20 muM and 40 muM respectively; and control wells (BC) were set using medium of formula 2.

When the culture medium of the formula 3 is used, 1mL of the prepared epidermal growth factor is added into a 48-well plate inoculated with primary cells respectively, and the final concentrations of the epidermal growth factor are 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL and 80ng/mL respectively; and control wells (BC) were set using medium of formula 3.

When the culture medium of formula 4 is used, 1mL of the prepared gastrin is added into 48-well plates inoculated with primary cells respectively, and the final concentrations of the gastrin are 1nM, 3nM, 9nM, 27nM and 81nM respectively; and control wells (BC) were set using medium of formula 4.

When the culture medium of formula 5 is used, 1mL of prepared fibroblast growth factor 7 is added into a 48-well plate inoculated with primary cells, and the final concentrations of the fibroblast growth factor 7 are respectively 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 40ng/mL; and control wells (BC) were set using medium of formulation 5.

When the culture medium of formula 6 is used, 1mL of prepared nicotinamide is added to each well of 48-well plates inoculated with primary cells, and the final concentrations of the nicotinamide are 1mM, 2mM, 4mM, 8mM and 16mM respectively; and control wells (BC) were set using medium of formula 6.

When the culture medium of formula 7 was used, prepared SB431542 was added to each well of 1mL in 48-well plates inoculated with primary cells, and the final concentrations of SB431542 were 3.75. Mu.M, 7.5. Mu.M, 15. Mu.M, 30. Mu.M, and 60. Mu.M, respectively; and control wells (BC) were set using medium of formula 7.

When the culture medium of formula 8 was used, 1mL of the prepared compound 1 was added to each well of 48-well plates inoculated with primary cells, and the final concentrations of the compound 1 were 2.5 μ M, 5 μ M, 10 μ M, 20 μ M, and 40 μ M, respectively; and control wells (BC) were set using medium of formula 8.

When the medium of formula 9 was used, 1mL of prepared fetal bovine serum was added to each well of 48-well plates inoculated with primary cells at a ratio of 2.5% (v/v), 5% (v/v), 10% (v/v), 20% (v/v), or 40% (v/v); and control wells (BC) were set using medium of formula 9.

After the cells were expanded to about 85% of the 48 wells and digested and counted, the number of cells in the control well (BC) was compared to calculate the proliferation fold, and the results are shown in FIGS. 2A to 2I, respectively. In FIGS. 2A to 2I, the ratio is the ratio of the number of cells obtained by one-pass culture using each medium to the number of cells obtained by one-pass culture using the corresponding control well. The ratio is more than 1, which indicates that the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is better than that of a control Kong Peiyang medium; if the ratio is less than 1, the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is weaker than that of the culture medium of the control hole.

According to the results of FIGS. 2A to 2I, the content of sodium pyruvate is preferably 0.25 to 1mM, and the cell proliferation effect is most significant at a concentration of 0.5 mM; the content of forskolin is preferably 2.5-10 μ M, and the cell proliferation effect is most obvious when the concentration is 2.5 μ M; the content of the epidermal growth factor is preferably 5-80 ng/mL, more preferably 5-20 ng/mL, and the cell proliferation effect is most obvious when the concentration is 10 ng/mL; the content of gastrin is preferably 3-81 nM, more preferably 9-81 nM, and the cell proliferation effect is most obvious when the concentration is 27 nM; the content of the fibroblast growth factor 7 is preferably 5-40 ng/ml, more preferably 5-20 ng/ml, and the cell proliferation effect is most obvious when the concentration is 10 ng/ml; the content of nicotinamide in the culture medium is preferably 1-16 mM, more preferably 1-4 mM, and the cell proliferation effect is most obvious when the concentration is 1mM; the content of SB431542 is preferably 3.75-30 μ M, more preferably 3.75-15 μ M, and the cell proliferation effect is most obvious when the concentration is 7.5 μ M; the content of the compound 1 is preferably 2.5-10 mu M, more preferably 2.5-5 mu M, and the cell proliferation effect is most obvious when the concentration is 5 mu M; the fetal bovine serum preferably has a volume content of 2.5 to 40% (v/v), more preferably 5 to 20% (v/v), and the cell proliferation effect is most remarkable at a concentration of 10% (v/v).

The most preferred concentrations of each additive factor in the above media were used as the ovarian cancer primary cell culture medium of the present invention used in the following examples, which contained: basal medium BM, 0.5mM sodium pyruvate, 2.5. Mu.M forskolin, 10ng/mL epidermal growth factor, 27nM gastrin, 10ng/mL fibroblast growth factor 7, 1mM nicotinamide, 7.5. Mu.MSB 431542, 5. Mu.M 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 3 of step (2) of example 1, and cultured using the OC-1 medium of example 3, and the obtained ovarian cancer primary cells were cultured at a viable cell density of 3X 10 ⁴ Per cm ² Seeded in 6-well plates (30 ten thousand cells per well) at a cell density of 2X 10 ⁴ Per cm ² Adding NIH-3T3 cells irradiated by gamma rays (irradiation dose is 30 Gy), and mixing evenly. Surface sterilized, then treated at 37 deg.C and 5% CO ₂ Incubators (purchased from Saimeri fly) for culture.

The cultured ovarian cancer primary cells were observed using a microscope (EVOS M500, invitrogen), and FIGS. 3A to 3J are photographs taken under a 10-fold objective lens, in which the cells were closely arranged under the lens and were slightly irregular in morphology.

About 0.25cm was taken from the intraoperative tissue (sample No. L62) of one ovarian cancer patient ³ The size of the cancer tissue was fixed by immersion in 1mL of 4% paraformaldehyde. The sample L62 was continuously cultured up to passage 4 using the method of example 3 using the medium OC-1 of the present invention. The 4% paraformaldehyde fixed tissue or cells were paraffin embedded and sliced into 4 μm thick tissue sections with a microtome. Conventional immunohistochemical detection was then performed (see, for specific steps, li et al, nature Communication, (2018) 9. The primary antibodies used were ER, PR, P53, napsinA, pax-8, WT-1, ki-67 (all available from CST).

FIGS. 4A-4G and 5A-5G are graphs comparing immunohistochemical results for primary tissue cells and ovarian cancer primary cells obtained by culturing the cells in the ovarian cancer primary culture medium OC-1 of the present invention, respectively. FIGS. 4A and 5A are respectively a picture of a labeled ER antibody of an ovarian cancer tissue and cultured ovarian cancer primary cells, FIGS. 4B and 5B are respectively a picture of a labeled PR antibody of the ovarian cancer tissue and cultured ovarian cancer primary cells, FIGS. 4C and 5C are respectively a picture of a labeled P53 antibody of the ovarian cancer tissue and cultured ovarian cancer primary cells, FIGS. 4D and 5D are respectively a picture of a labeled NapsinA antibody of the ovarian cancer tissue and cultured ovarian cancer primary cells, FIGS. 4E and 5E are respectively a picture of a labeled Pax-8 antibody of the ovarian cancer tissue and cultured ovarian cancer primary cells, FIGS. 4F and 5F are respectively a picture of a labeled WT-1 antibody of the ovarian cancer tissue and cultured ovarian cancer primary cells, and FIGS. 4G and 5G are respectively a picture of a labeled Ki-67 antibody of the ovarian cancer tissue and cultured ovarian cancer primary cells. From this, it was confirmed that, when the ovarian cancer primary cells cultured by the technique of the present invention were cultured up to the 4 th generation, the expression of the ovarian cancer-associated biomarker on the ovarian cancer primary cells was substantially identical to the expression of the marker in the original tissue section from which the ovarian cancer primary cells were derived. This demonstrates that the primary ovarian cancer cells cultured by the present technology retain the original pathological characteristics of the cancer tissue of ovarian cancer patients.

Example 5 comparison of culture Effect with existing Medium and calculation of culture cycle and cell count of Primary ovarian cancer cells and dosage Doubling (PD) value

Literature medium (Xuefeng Liu et al, nat. Protoc.,12 (2): 439-451, 2017)) formulated as DMEM/F12 medium +250ng/ml amphotericin B (from Selleck company) + 10. Mu.g/ml gentamycin (from MCE company) +0.1nM cholera toxin +0.125ng/ml EGF +25ng/ml hydrocortisone + 10. Mu. M Y27632+10 FBS.

Commercial culture medium: defined K-SFM, keratinocyte Serum Medium (from gibco, 10744-019)

Ovarian cancer primary cells were obtained from 4 sample ovarian cancer tissue samples (accession numbers BNYT1083, L57, L58, L60) according to the method of 3 of step (2) of example 1. The ovarian cancer primary cells obtained were cultured in a literature medium, a commercial medium, and OC-1 medium in example 3 at a viable cell density of 3X 10 ⁴ Per cm ² The cells were seeded in a 6-well plate and cultured, digested and counted after the cells were expanded to 95%, and the number of days of culture until digestion was recorded as one culture cycle. Culturing under the experimental condition, amplifying the cells obtained by amplification in different generations, digesting each generation, counting and recording the corresponding culture period, calculating the PD according to the formula of amplification dosing (PD) =3.32 log10 (total number of cells digested/number of cells initially seeded), the formula is shown in Chapman et al, stem Cell Research&Therapy 2014,5:60。

FIGS. 6A-6D show growth curves of 4 primary cells cultured using literature media, commercial media and the ovarian cancer primary cell culture medium OC-1 of the present invention, plotted using Graphpad Prism software, with the abscissa representing the number of days in cell culture, and the ordinate representing the cumulative fold of cell proliferation, representing the fold of cells expanded during the culture period, and the larger the number, the greater the number of cells expanded within a given period, i.e., the greater the number of cells obtained, and the slope representing the rate of cell expansion.

From FIGS. 6A-6D, it can be confirmed that the ovarian cancer primary cells cultured by the culture medium OC-1 of the present invention have the ability to continue to expand at a substantially constant cell expansion rate for at least 60 days; ovarian cancer primary cells cultured using literature and commercial media expand at a significantly lower rate than OC-1 media and cease proliferation up to passage 2. In conclusion, the ovarian cancer cells cultured in the primary ovarian cancer cell culture medium of the invention have significantly better proliferation efficiency than the culture medium of the literature and commercial culture.

Example 6 ovarian cancer Primary cells expanded using the culture Medium of the present invention for drug screening and efficacy assessment

1. Cell culture and plating

Ovarian cancer primary cells (numbered L62) were isolated as a generation according to the method of 3 of step (2) of example 1, and cultured using the ovarian cancer primary cell culture medium OC-1 of the present invention, and passaging was performed until the cells were expanded to 85%. Cells were subcultuted according to 4 of step (2) in example 1, and cells were subcultuted according to the viable cell density of 1X 10 ⁵ After being well mixed in a sample addition well (purchased from corning), each/mL of cells were cultured in 384-well opaque white cell culture plates (purchased from corning), each of which had a volume of 50 μ L and a cell number of 5000 cells/well. Ovarian cancer primary cell culture medium sealing plates of the invention were added from the edge of the well plate, and the plate was labeled with the sample name, the dosing time, and the CellTiter-Glo (purchased from Promega corporation) detection time. Surface sterilized with 75% alcohol (ex Lierkang) and incubated at 37 deg.C, 5% CO ₂ Culturing in incubator, and adding medicine after 24 hr. Cells of 1 st generation, 2 nd generation, 3 rd generation, 4 th generation and 5 th generation are obtained and cultured respectively to carry out drug screening, and the drug sensitivity of the ovarian cancer primary cells cultured by the culture medium of the invention through continuous passage is tested.

2. Screening drug formulation

5 drugs (cytarabine, doxorubicin, panobinostat, azacitidine, homoharringtonine; all from MCE) were prepared in 6 concentration gradients according to the following table, and 30. Mu.L of each drug was added to each well of a 384-well plate (from Saimer Fei) and stored for later use.

TABLE 3 drug action concentration settings

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3. High flux dosing

The prepared drug plate is taken out, placed at room temperature, centrifuged in a centrifuge (Beckman) at room temperature of 1000rpm for 1 minute and then taken out. High-throughput dosing was performed using a high-throughput automated loading system (JANUS, perkin Elmer). To 384 well plates cultured with ovarian cancer primary cells, 0.1. Mu.L of the screening drug was added per well at the corresponding concentration. After the dosing is finished, the surface of the 384-hole plate is disinfected and then is moved into an incubator, and the cell activity is measured after 72 hours.

4. Cell viability assay

CellTiter-Glo luminescence reagent (purchased from Promega corporation) was taken out from a4 ℃ freezer, and 10mL of the reagent was put into a sample addition well. The 384-well plate to be detected was taken out from the incubator, 10. Mu.L of CellTiter-Glo luminescence reagent was added to each well, and the mixture was left to stand for 10 minutes and mixed, followed by detection using a multifunctional microplate reader (Envision, perkin Elmer Co., ltd.).

5. Data processing

According to the formula of cell inhibition rate (%) =100% -chemical luminescence value of dosing hole/chemical luminescence value of control hole x 100%, calculating the cell inhibition rate after different drugs act on the cell, and calculating the half inhibition rate (IC 50) of the drugs act on the cell by using the graphpad prism software. The results are shown in FIGS. 7A to 7E.

From FIGS. 7A to 7E, it was confirmed that when primary ovarian cancer cells cultured on the ovarian cancer primary cell culture medium OC-1 of the present invention were subjected to drug screening, the inhibitory effects of the same drug on different generations of cultured cells were substantially consistent (the inhibitory curves were substantially consistent). The sensitivity of cells of the same patient to the maximum blood concentration of different drugs in the human body varies. According to the results, the effectiveness of the ovarian cancer patients in clinical use of the medicine can be judged, and meanwhile, the sensitivity of tumor cells with different generations obtained by the culture method of the invention to the medicine can be proved to be stable.

Industrial applicability

The invention provides a culture medium and a culture method for culturing ovarian cancer primary cells in vitro, which can be used for evaluating and screening the curative effect of a medicament by using the cultured cells. 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 based on the principle of the present invention, and therefore, various modifications made in accordance with the principle of the present invention should be construed as falling within the scope of the present invention.

Claims (10)

1. A culture medium of primary ovarian cancer cells, comprising:

an MST1/2 kinase inhibitor; sodium pyruvate; forskolin; an epidermal growth factor; a 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,

wherein the content of the first and second substances,

R ₁ selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R ₆ Substituted aryl, arylC 1-C6 alkyl, and heteroaryl;

R ₂ and R ₃ Each independently selected from C1-C6 alkyl;

R ₄ and R ₅ Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, and C3-C6 heterocycloC 1-C6 alkyl;

R ₆ selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.

2. The culture medium of claim 1, wherein

R ₁ Selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R ₆ Substituted phenyl, naphthyl, benzyl and thienyl;

R ₂ and R ₃ Each independently selected from C1-C3 alkyl;

R ₄ and R ₅ Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, piperidinylC 1-C6 alkyl, and tetrahydropyranyl C1-C6 alkyl;

R ₆ selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.

3. The culture medium of claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,

wherein the content of the first and second substances,

R ₁ selected from C1-C6 alkyl, optionally substituted by 1-2 independent R ₆ Substituted phenyl, optionally substituted with 1-2 independent R ₆ Substituted thienyl, and optionally substituted with 1-2 independent R ₆ A substituted benzyl group;

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

R ₆ each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl.

4. The culture medium of claim 3, wherein

R ₁ Is optionally substituted by 1-2 independent R ₆ A substituted phenyl group;

R ₅ is hydrogen;

R ₆ preferably fluorine, methyl or trifluoromethyl.

5. The culture medium of claim 1, wherein the MST1/2 kinase inhibitor is selected from at least one of the following compounds or a pharmaceutically acceptable salt thereof:

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6. a culture medium according to any one of claims 1 to 5, wherein 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 culture medium is 2.5-10 mu M;

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

(3) The content of the forskolin in the culture medium is 2.5-10 mu M;

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

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

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

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

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

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

7. The culture medium according to any one of claims 1 to 5, further comprising:

a starting 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 primary ovarian cancer cells, which is characterized by comprising the following steps:

(1) Preparing a culture medium of the ovarian cancer primary cells of any one of claims 1 to 7;

(2) Obtaining primary ovarian cancer cells according to the cell density of 1-10 multiplied by 10 ⁴ Per cm ² Planting into culture dish according to cell density 2-3X 10 ⁴ Per cm ² Adding the trophoblasts, and then culturing by using the culture medium of the ovarian cancer primary cells obtained in the step (1).

9. The method for culturing primary ovarian cancer cells according to claim 8, wherein the feeder cells are irradiated NIH-3T3 cells, the irradiation source is X-rays or gamma-rays, and the irradiation dose is 20-50 Gy.

10. A method of screening or evaluating a drug for treating ovarian cancer, comprising the steps of:

(1) Culturing ovarian cancer primary cells according to the culture method of claim 8 or 9;

(2) Selecting a medicine to be detected and diluting according to a required concentration gradient;

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

(4) Cell viability assays were performed.

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