CN117736992A - Culture medium, culture method and application of primary cells of neuroblastoma - Google Patents

Abstract

The invention provides a culture medium for primary cells of neuroblastoma, which comprises the following components: initial medium, MST1/2 kinase inhibitor, fetal bovine serum, Y-27632, insulin-transferrin-selenium supplement, insulin-like growth factor-1, fibroblast growth factor 7, gastrin, hepatocyte growth factor. The invention also provides a culture method for culturing the primary cell of the neuroblastoma by using the culture medium. Meanwhile, the invention also provides application of the culture medium in-vitro rapid amplification of primary cells of neuroblastoma. The method does not need xenograft, and only needs to seed the primary cells of the neuroblastoma into a culture medium when in use; the culture medium and the culture method can avoid species difference of xenograft, and have the advantages of high amplification efficiency, controllable culture cost, high success rate and no false positive and false negative.

Description

Culture medium, culture method and application of primary cells of neuroblastoma

Technical Field

The invention relates to the technical field of biological medicine, in particular to a culture medium of primary cells of neuroblastoma, a culture method and application.

Background

Neuroblastoma is the third tumor in childhood and is the primary malignancy that threatens childhood life. 90% of patients are diagnosed less than 5 years old and the average age of onset is 19 months. The early diagnosis of neuroblastoma is difficult, the malignancy degree is high, the transfer speed is high, and the 5-year survival rate of the conventional treatment method is only 10-20%. Almost 15% of all childhood cancer deaths. Thus, neuroblastoma is referred to as "childhood tumor king".

Neuroblastoma can be usually confirmed by urinary catecholamine and imaging examinations. However, in order to establish biology (i.e., cytogenetics, pathology) and risk stratification, biopsies of the primary site (or bone marrow for staging) are required. Treatment is primarily dependent on risk stratification, and varies from observation of only certain low-risk patients to intensive multimode therapy for high-risk patients. For patients with low risk disease, the prognosis is good, while for patients with high risk disease, the prognosis is poor. Recurrent or refractory (high-risk) diseases are difficult to cure and survival rates are extremely low.

Therefore, it is a promising approach to build a primary tumor model in vitro and use it for efficient drug screening experiments. At present, a main method for establishing an in-vitro culture model of primary neuroblastoma is a humanized tumor xenograft model (patent-Derived tumor Xenograft, PDX), and the method is to transplant tumor cells of a Patient into a nude mouse body and then examine the treatment effect of the tumor cells without an anti-tumor drug. However, PDX methods also have some drawbacks, such as: species differences between human and murine; the test period is long (more than 4 weeks); the cost is high (more than 20 ten thousand); false positives and false negatives are liable to occur.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a culture medium for rapidly expanding primary cells of neuroblastoma in vitro, a culture method and application, and the method does not need xenograft, and only needs to seed the primary cells of the neuroblastoma into the culture medium when in use; the culture medium and the culture method can avoid species difference of xenograft, and have the advantages of high amplification efficiency, controllable culture cost, high success rate and no false positive and false negative.

The invention adopts the following technical scheme to solve the technical problems:

a culture medium for primary cells of neuroblastoma, comprising the following components: initial medium, MST1/2 kinase inhibitor, fetal bovine serum, Y-27632, insulin-transferrin-selenium supplement, insulin-like growth factor-1, fibroblast growth factor 7, gastrin, hepatocyte growth factor;

the initial culture medium is selected from DMEM/F12, DMEM, F12 or RPMI-1640;

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

the volume percentage content of the fetal bovine serum relative to the culture medium is 2.5-40%;

The content of the Y-27632 in the culture medium is 2.5-20 mu M;

the volume ratio content of the insulin-transferrin-selenium supplement relative to the culture medium is 1:25 to 1:400;

the content of the insulin in the culture medium is 2.5-40 mug/mL;

the content of the insulin-like growth factor-1 in the culture medium is 12.5-200 ng/mL;

the content of the fibroblast growth factor 7 in the culture medium is 2.5-40 ng/mL;

the content of the gastrin in the culture medium is 1-81 nM;

the content of the hepatocyte growth factor in the culture medium is 1-81 ng/mL.

As one of the preferred modes of the present invention, the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof;

wherein R is ₁ Selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally R, independently of 1 to 2 ₆ Substituted aryl (e.g., phenyl, naphthyl, etc.), aryl C1-C6 alkyl (e.g., benzyl, etc.), and heteroaryl (e.g., thienyl, etc.);

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

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

R ₆ Selected from halogen, C1-C6 alkyl, C1-C6 alkoxy and C1-C6 haloalkyl.

Specifically, among the compounds of formula (I):

R ₁ selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally R, independently of 1 to 2 ₆ Substituted phenyl, naphthyl, benzyl, and thienyl;

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

R ₄ and R is ₅ Each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylamino C1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, piperidinyl C1-C6 alkyl and tetrahydropyranyl C1-C6 alkyl;

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

As one of the preferred modes of the present invention, the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof;

wherein R is ₁ Selected from C1-C6 alkyl, optionally substituted with 1-2 independently R ₆ Substituted phenyl, optionally substituted with 1 to 2 groups R ₆ Substituted thienyl, and optionally substituted with 1-2 independently R ₆ A substituted benzyl group;

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

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

Specifically, among the compounds of formula (Ia):

R ₁ is optionally substituted with 1 to 2 independent R ₆ A substituted phenyl group;

R ₅ is hydrogen;

R ₆ is fluorine, methyl or trifluoromethyl.

As one of the preferred modes of the present invention, the MST1/2 kinase inhibitor is selected from at least one of the compounds in table 1 or pharmaceutically acceptable salts, or solvates thereof.

TABLE 1 list of MST1/2 kinase inhibitor compounds of the invention

Wherein, compound 1 is the optimal MST1/2 kinase inhibitor.

As one of the preferred modes of the present invention, there is also included an N2 cell culture additive; the volume ratio content of the N2 cell culture additive in the culture medium is 1:25 to 1:400.

as one of preferred modes of the present invention, antibiotics are also included; the antibiotics are selected from one or more of streptomycin, penicillin, amphotericin B and Primocin; when the antibiotics select streptomycin, the adding concentration is 25-400 mug/mL; when penicillin is selected as the antibiotic, the concentration of the antibiotic is 25-400U/mL; when the antibiotics select amphotericin B, the concentration is 0.25-4 mug/mL; when Primocin is selected as the antibiotic, the addition concentration is 25-400 mug/mL.

A method for culturing primary neuroblastoma cells comprises culturing primary neuroblastoma cells with the medium.

As one of preferred embodiments of the present invention, the method comprises the steps of:

(1) Preparing a culture medium of the primary neuroblastoma cells;

(2) The primary cell of neuroblastoma is processed according to the cell density of 1 to 10 multiplied by 10 ⁴ Individual/cm ² Seed into a petri dish and then culture using the medium of the primary neuroblastoma cells.

The application of the culture medium of the primary neuroblastoma cells in the in-vitro rapid amplification of the primary neuroblastoma cells is that the primary neuroblastoma cells are subjected to in-vitro rapid amplification culture.

Compared with the prior art, the invention has the advantages that:

(1) The in vitro rapid expansion culture of the primary neuroblastoma cells can be realized by only planting the primary neuroblastoma cells into a culture medium when the primary neuroblastoma cells are used without the need of xenograft, and the species difference of the xenograft can be avoided;

(2) The amplification efficiency is high: as long as there is 10 ⁵ The cell number of the grade can be amplified into 10 successfully within about 1 to 2 weeks ⁶ The primary cell of the neuroblastoma of the order of magnitude can be continuously passaged;

(3) The success rate is high: the method can culture tumor tissues with multiple sample sources such as neuroblastoma (the most common type), gangliocytoneuroblastoma, gangliocytoma and the like, and the success rate reaches more than 80 percent;

(4) The culture cost is controllable: the culture medium does not need to be added with factors such as expensive Wnt agonists, R-spondin family proteins, BMP inhibitors, FGF10 and the like;

(5) No false positives and false negatives are present: the primary neuroblastoma cells cultured in vitro can keep the pathological characteristics of patients, and the cultured primary neuroblastoma cells are not interfered by interstitial cells such as fibroblasts, adipocytes and the like;

(6) The primary cells of the neuroblastoma obtained by the culture of the technology have large quantity and high uniformity, and are suitable for high-flux screening of new candidate compounds and in-vitro sensitivity functional test of high-flux drugs for patients.

Drawings

FIG. 1 is a graph showing the effect of combinations of different additive factors in the primary cell culture medium of neuroblastoma on the growth of primary neuroblastoma cells in test example 2;

FIG. 2 is a graph showing the effect of different concentrations of additive factors on primary neuroblastoma cell growth in test example 3 (A, B, C, D, E, F, G graph shows the effect of different concentrations of compound 1, hepatocyte growth factor, insulin, N2, Y-27632, insulin-transferrin-selenium supplement, gastrin, insulin-like growth factor-1, fibroblast growth factor 7, fetal bovine serum on primary neuroblastoma cell growth, respectively);

FIG. 3 is a photograph of primary neuroblastoma cells obtained by culturing the primary neuroblastoma cells of the present invention in test example 4 using a microscope (in the figure, A, B, C, D, E, F corresponds to the photographs of samples 7, 8, 9, 10, 11, and 12, respectively);

FIG. 4 is a graph showing the result of immunohistochemistry of primary neuroblastoma tissue in test example 4 (in the figures, the A graph is a graph of labeled CK antibody, the B graph is a graph of labeled Syn antibody, the C graph is a graph of labeled NF antibody, the D graph is a graph of labeled S-100 antibody, and the E graph is a graph of labeled Ki-67 antibody);

FIG. 5 is a diagram showing the result of immunohistochemistry of primary neuroblastoma cells obtained by culturing primary neuroblastoma tissue cells of the present invention in test example 4 to the seventh generation using the primary neuroblastoma cell culture medium (in the figures, panel A shows a picture of labeled CK antibody, panel B shows a picture of labeled Syn antibody, panel C shows a picture of labeled NF antibody, panel D shows a picture of labeled S-100 antibody, and panel E shows a picture of labeled Ki-67 antibody);

FIG. 6 is a photograph of a gastric cancer organoid obtained by culturing a gastric cancer organoid using comparative medium 1 and primary medium NB-1 of the present invention in test example 5 (in the figures, A is a photograph of sample 13 cultured using comparative medium 1; B is a photograph of sample 13 cultured using primary medium NB-1 of the present invention; C is a photograph of sample 14 cultured using comparative medium 1; D is a photograph of sample 14 cultured using primary medium NB-1 of the present invention);

FIG. 7 is a photograph of primary cervical cancer cells obtained by culturing primary cervical cancer cells in test example 5 using comparative medium 2 and primary culture medium NB-1 of the present invention (in the drawing, A is a photograph obtained by culturing sample 15 in comparative medium 2; B is a photograph obtained by culturing sample 15 in primary culture medium NB-1 of the present invention);

FIG. 8 is a photograph of a primary cell culture of a neuroblastoma obtained by culturing a primary cell of the neuroblastoma with comparative medium 2, comparative medium 1 and primary medium NB-1 of the present invention in sample 16 of test example 5 (in the drawing, A is a photograph obtained by culturing sample 16 with comparative medium 2; B is a photograph obtained by culturing sample 16 with comparative medium 1; C is a photograph obtained by culturing sample 16 with primary medium NB-1 of the present invention);

FIG. 9 is a photograph of a primary cell culture of a neuroblastoma obtained by culturing a primary cell of a neuroblastoma in test example 5 with comparative medium 2, comparative medium 1 and primary medium NB-1 of the present invention (in the drawing, A is a photograph obtained by culturing sample 17 with comparative medium 2; B is a photograph obtained by culturing sample 17 with comparative medium 1; C is a photograph obtained by culturing sample 17 with primary medium NB-1 of the present invention).

Detailed Description

The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples. Meanwhile, the reagent products and experimental methods used in the following examples are not specifically described, and are conventional reagents or methods in the art, and will not be described in detail.

[ preparation example of MST1/2 kinase inhibitor ]

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

1. Preparation of MST1/2 kinase inhibitor compound 1 (compound 1 in table 1):

the preparation route and process of the compound 1, namely 4- ((7- (2, 6-difluorophenyl) -5, 8-dimethyl-6-oxo-5, 6,7, 8-tetrahydropteridin-2-yl) amino) benzenesulfonamide 1, are as follows:

(1) Preparation of methyl 2-amino-2- (2, 6-difluorophenyl) acetate (A2): after adding 2-amino-2- (2, 6-difluorophenyl) acetic acid (A1, 2.0 g) to the round bottom flask, methanol (30 ml) was added followed by dropwise addition of thionyl chloride (1.2 ml) under ice-bath. The reaction was allowed to react overnight at 85 ℃. After the reaction was completed, the solvent was evaporated under reduced pressure to give a white solid which was used directly in the next step.

(2) Preparation of methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2, 6-difluorophenyl) acetate (A3): after adding methyl 2-amino-2- (2, 6-difluorophenyl) acetate (2 g) to a round bottom flask, acetone (30 ml) and potassium carbonate (2.2 g) were added, then the system was cooled to-10 ℃ with an ice salt bath, followed by slow addition of a solution of 2, 4-dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction was stirred at room temperature overnight. After the reaction, filtering, removing the solvent from the filtrate under reduced pressure, and purifying the residue by pressurized silica gel column chromatography to obtain the compound A3.LC/MS: M+H 359.0.

(3) Preparation of 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 system was stirred at 60℃for two hours. After the reaction was completed, the solvent was evaporated under reduced pressure, and the resultant was neutralized with saturated sodium hydrogencarbonate to be alkaline. The mixture was extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, and dried over anhydrous sodium sulfate. The organic phase is filtered and evaporated to dryness under reduced pressure to obtain a crude product. The crude product is washed by diethyl ether to obtain a compound A4.LC/MS: m+h 297.0.

(4) Preparation of 2-chloro-7- (2, 6-difluorophenyl) -5, 8-dimethyl-7, 8-dihydropteridin-6 (5H) -one (A5): 2-chloro-7- (2, 6-difluorophenyl) -7, 8-dihydro-pteridin-6 (5H) -one (2 g) and N, N-dimethylacetamide (10 ml) were added to a round bottom flask, cooled to-35℃and methyl iodide (0.9 ml) was added followed by sodium hydride (615 mg) and the reaction stirred for an additional two hours. After the completion of the reaction, the mixture was quenched with water, extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate. The organic phase is filtered and evaporated to dryness under reduced pressure to obtain a crude product. The crude product is washed by diethyl ether to obtain a compound A5.LC/MS: m+h325.0.

(5) Preparation of 4- ((7- (2, 6-difluorophenyl) -5, 8-dimethyl-6-oxo-5, 6,7, 8-tetrahydropteridin-2-yl) amino) benzenesulfonamide (1): 2-chloro-7- (2, 6-difluorophenyl) -5, 8-dimethyl-7, 8-dihydro-pteridin-6 (5H) -one (100 mg), sulfanilamide (53 mg), p-toluenesulfonic acid (53 mg) and sec-butanol (5 ml) were added to the round bottom flask. The reaction was stirred at 120℃overnight. After the reaction, filtering, and washing with methanol and diethyl ether to obtain the compound 1.LC/MS: m+h461.1.

2. Preparation of other MST1/2 kinase inhibitor Compounds of the invention (other Compounds in Table 1)

Other MST1/2 kinase inhibitor compounds of the present invention were synthesized in a similar manner to compound 1, with the structure and mass spectral data shown in Table 2 below.

TABLE 2 Structure and Mass Spectrometry data for MST1/2 kinase inhibitor Compounds of the invention

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Example 1

The culture medium for primary cells of neuroblastoma of the embodiment comprises the following components: initial medium, MST1/2 kinase inhibitor, fetal bovine serum, Y-27632, insulin-transferrin-selenium supplement, insulin-like growth factor-1, fibroblast growth factor 7, gastrin, hepatocyte growth factor, N2 cell culture additive, antibiotics.

Wherein, the initial culture medium is selected from DMEM culture medium; MST1/2 kinase inhibitor the aforementioned MST1/2 kinase inhibitor compound 1 (i.e., compound 1 of Table 1) was selected at a level of 2.5. Mu.M in the medium; the volume percentage content of the fetal bovine serum relative to the culture medium is 2.5 percent; y-27632 is present in the medium in an amount of 5. Mu.M; the volume ratio content of insulin-transferrin-selenium supplement to the culture medium is 1:25, a step of selecting a specific type of material; insulin was contained in the medium at 2.5. Mu.g/mL; the content of the insulin-like growth factor-1 in the culture medium is 12.5ng/mL; the content of the fibroblast growth factor 7 in the culture medium is 2.5ng/mL; the content of gastrin in the culture medium is 1nM; the content of the hepatocyte growth factor in the culture medium is 1ng/mL; the volume ratio content of the N2 cell culture additive in the culture medium is 1:25, a step of selecting a specific type of material; the antibiotic was streptomycin, added at a concentration of 200. Mu.g/mL.

The preparation method comprises the following steps: according to the above composition, MST1/2 kinase inhibitor, fetal bovine serum, Y-27632, insulin-transferrin-selenium supplement, insulin-like growth factor-1, fibroblast growth factor 7, gastrin, hepatocyte growth factor, N2 cell culture additive, antibiotic were added to the prepared DMEM medium.

Example 2

The culture medium for primary cells of neuroblastoma of the embodiment comprises the following components: initial medium, MST1/2 kinase inhibitor, fetal bovine serum, Y-27632, insulin-transferrin-selenium supplement, insulin-like growth factor-1, fibroblast growth factor 7, gastrin, hepatocyte growth factor, N2 cell culture additive, antibiotics.

Wherein the initial medium is selected from F12 medium; MST1/2 kinase inhibitor the aforementioned MST1/2 kinase inhibitor compound 1 (i.e., compound 1 of Table 1) was selected to be present in the medium at a concentration of 10. Mu.M; the volume percentage content of the fetal bovine serum relative to the culture medium is 10 percent; y-27632 is present in the medium in an amount of 2.5. Mu.M; the volume ratio content of insulin-transferrin-selenium supplement to the culture medium is 1:50; insulin was contained in the medium at 9. Mu.g/mL; the content of the insulin-like growth factor-1 in the culture medium is 50ng/mL; the content of the fibroblast growth factor 7 in the culture medium is 10ng/mL; the content of gastrin in the culture medium is 9nM; the content of the hepatocyte growth factor in the culture medium is 9ng/mL; the volume ratio content of the N2 cell culture additive in the culture medium is 1:100; primocin is selected as the antibiotic, and the adding concentration is 100 mug/mL.

The preparation method comprises the following steps: MST1/2 kinase inhibitor, fetal bovine serum, Y-27632, insulin-transferrin-selenium supplement, insulin-like growth factor-1, fibroblast growth factor 7, gastrin, hepatocyte growth factor, N2 cell culture additive, antibiotics were added to the formulated F12 medium according to the above composition.

Example 3

The culture medium for primary cells of neuroblastoma of the embodiment comprises the following components: initial medium, MST1/2 kinase inhibitor, fetal bovine serum, Y-27632, insulin-transferrin-selenium supplement, insulin-like growth factor-1, fibroblast growth factor 7, gastrin, hepatocyte growth factor, N2 cell culture additive, antibiotics.

Wherein the initial medium is selected from DMEM/F12 medium; MST1/2 kinase inhibitor the aforementioned MST1/2 kinase inhibitor compound 1 (i.e., compound 1 of Table 1) was selected at a level of 2.5. Mu.M in the medium; the volume percentage content of the fetal bovine serum relative to the culture medium is 5%; y-27632 is present in the medium at 10. Mu.M; the volume ratio content of insulin-transferrin-selenium supplement to the culture medium is 1:200; insulin was present in the medium at 10. Mu.g/mL; the content of the insulin-like growth factor-1 in the culture medium is 50ng/mL; the content of the fibroblast growth factor 7 in the culture medium is 5ng/mL; the content of gastrin in the culture medium is 9nM; the content of the hepatocyte growth factor in the culture medium is 9ng/mL; the volume ratio content of the N2 cell culture additive in the culture medium is 1:200; primocin is selected as the antibiotic, and the adding concentration is 100 mug/mL.

The preparation method comprises the following steps: MST1/2 kinase inhibitor, fetal bovine serum, Y-27632, insulin-transferrin-selenium supplement, insulin-like growth factor-1, fibroblast growth factor 7, gastrin, hepatocyte growth factor, N2 cell culture additive, antibiotics were added to the prepared DMEM/F12 medium according to the above composition.

Example 4

The culture medium for primary cells of neuroblastoma of the embodiment comprises the following components: initial medium, MST1/2 kinase inhibitor, fetal bovine serum, Y-27632, insulin-transferrin-selenium supplement, insulin-like growth factor-1, fibroblast growth factor 7, gastrin, hepatocyte growth factor, N2 cell culture additive, antibiotics.

Wherein, the initial culture medium is RPMI-1640 culture medium; MST1/2 kinase inhibitor the aforementioned MST1/2 kinase inhibitor compound 1 (i.e., compound 1 of Table 1) was selected at a level of 5. Mu.M in the medium; the volume percentage content of the fetal bovine serum relative to the culture medium is 40%; y-27632 is present in the medium at 20. Mu.M; the volume ratio content of insulin-transferrin-selenium supplement to the culture medium is 1:400; the content of insulin in the culture medium is 40 mug/mL; the content of the insulin-like growth factor-1 in the culture medium is 200ng/mL; the content of the fibroblast growth factor 7 in the culture medium is 40ng/mL; the content of gastrin in the culture medium is 81nM; the content of hepatocyte growth factor in the culture medium was 81ng/mL. The volume ratio content of the N2 cell culture additive in the culture medium is 1:400; the antibiotic selected amphotericin B was added at a concentration of 2. Mu.g/mL.

The preparation method comprises the following steps: MST1/2 kinase inhibitor, fetal bovine serum, Y-27632, insulin-transferrin-selenium supplement, insulin-like growth factor-1, fibroblast growth factor 7, gastrin, hepatocyte growth factor, N2 cell culture additive, antibiotics were added to the formulated RPMI-1640 medium according to the above composition.

Test example 1

This test example was used to verify the effect of each additive factor in the primary neuroblastoma cell culture medium on the proliferation of primary neuroblastoma cells.

1. Preparation of primary cell culture medium for neuroblastoma

First, a basal medium containing the initial medium was prepared. Wherein the initial culture medium is selected from DMEM/F12, DMEM, F12 or RPMI-1640 commonly used in the art. In this test example, the formulation of the basal medium was: DMEM/F12 medium (available from Corning company) +100. Mu.g/mL Primocin (available from InvivoGen company). Different kinds of additives (see table 3) were added to the basal medium, respectively, to prepare a primary neuroblastoma cell culture medium containing different additive components.

2. Isolation and treatment of primary neuroblastoma cells

(1) Sample selection

The neuroblastoma solid tumor tissue sample (intra-operative) is obtained from the patient by a medical professional in a medical professional, who has signed an informed consent. Samples 0.25cm during surgery ³ The method comprises the steps of carrying out a first treatment on the surface of the Commercial tissue preservation solutions (manufacturer: miltenyi Biotec) were used for storage and transport.

(2) The surfaces of a 15mL sterile centrifuge tube, a pipette, a 10mL pipette, a sterile gun head and the like are sterilized and then put into an ultra-clean workbench for ultraviolet irradiation for 30min. Taking out the cleaning culture medium from the refrigerator at 4 ℃ 30min in advance, and taking out the tissue digestion solution from the refrigerator at-20 ℃ 30min in advance.

Basal medium: DMEM/F12 medium+100 μg/mL Primocin (available from InvivoGen Co.).

Tissue digestion solution: 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; calcium chloride was purchased from the division of bioengineering (Shanghai); BSA was purchased from Biofroxx corporation.

(3) Isolation of primary cells of neuroblastoma

Tumor tissue samples are taken from an ultra clean bench and placed in a culture dish, the tissue with blood is removed, the tissue is washed 2 times by a basic culture medium, the tissue was transferred to another petri dish and mechanically separated with a sterile scalpel, dividing tissue blocks into 1X 1mm ³ Size of the product.

The excised intraoperative tissue was aspirated into a 15mL centrifuge tube, 5mL of basal medium was added, mixed well, and centrifuged at 1500rpm for 4min.

The supernatant was discarded, and the basal medium and the tissue digestion solution were added in a ratio of 1:3 (note: the amount of tissue digestion solution added was about 10mL of tissue digestion solution was used for 1g of tumor tissue), the sample name and number were labeled, the sample was sealed with a sealing film, and digestion was performed in a shaker (know Chu instrument ZQLY-180N) at 37℃at 300rpm, and whether the digestion was completed was observed every 30 minutes, and the basis was no macroscopic particulate matter, and the digestion time was 4 hours.

After digestion was completed, undigested tissue pellet was filtered through a 100 μm screen, and the tissue pellet on the screen was rinsed into a centrifuge tube with basal medium to reduce cell loss and centrifuged at 1500rpm for 4min at 25 ℃.

The supernatant was discarded, and the presence of blood cells was observed, and if any, 8mL of blood cell lysate (purchased from Sigma Co.) was added,

mixing, cracking at 4deg.C for 20min, mixing at 25deg.C for 4min, and centrifuging at 1500 rpm.

The supernatant was discarded and the cells were resuspended in 2mL of basal medium for further use.

(4) Cell counting and processing

And (5) observing under a mirror: a small amount of resuspended cells were plated in a petri dish and observed for cancer cell density and morphology under a microscope (cnopotec, BDS 400).

Viable cell count: after 12. Mu.L of the resuspended cell suspension and 12. Mu.L of trypan blue dye (available from Shanghai Biotechnology Co., ltd.) were thoroughly mixed, 20. Mu.L was added to a cell counting plate (Countstar, gauge: 50 pieces/box) and the percentage of viable large cells (cell size >10 μm) was calculated by a cell counter (Countstar, IC 1000) =viable cell number/total cell number×100%.

3. Culture of primary neuroblastoma cells

The medium of the different compositions in Table 3 was added to a 48-well plate at 1 mL/well volume. The neuroblastoma tissues (numbered sample 1, sample 2) were isolated from two examples according to step 2 above.

The obtained primary neuroblastoma cells were used in 5×10 ⁴ Cell density of individual/well cells were seeded in 48-well plates at 37℃with 5% CO ₂ The culture was performed under the condition of concentration. After 5-10 days of culture, the cells grew to 85%, the medium was discarded, and 100. Mu.L of 0.05% trypsin (from Gibco) per well was used for rinsing 1 time, and 200. Mu.L of 0.05% trypsin per well was added after blotting. Placing at 37deg.C and 5% CO ₂ The reaction was carried out in an incubator for 10min, and after complete digestion of the cells was observed under a microscope (CNOPTEC, BDS 400), digestion was stopped by adding 300. Mu.L of DMEM/F12 medium containing 10% serum (vivacell, C04002-500), 20. Mu.L was added to a cell counting plate (Countstar, specification: 50 pieces/box), and the total number of cells was counted by a cell counter (Countstar, IC 1000). Among them, as experimental controls, a basal medium without any additives was used, and experimental results are shown in table 3.

TABLE 3 additive components in the culture medium and cell proliferation promoting effect

In Table 3, "+" indicates that the medium to which the additive was added has an effect of promoting proliferation of at least two of the primary neuroblastoma cells isolated from the neuroblastoma tissue, as compared to the basal medium; "-" indicates that the medium to which the additive is added exhibits an effect of inhibiting proliferation on at least one of the primary neuroblastoma cells isolated from the neuroblastoma tissue; "" indicates that the medium to which the additive was added had no significant effect on proliferation of at least two of the primary neuroblastoma cells isolated from the neuroblastoma tissue.

Based on the above results, further culture experiments were performed with factors selected from hepatocyte growth factor, compound 1, N2, insulin-transferrin-selenium supplement, neuregulin-1, amphiregulin, Y-27632, fetal bovine serum, insulin-like growth factor-1, forskolin, gastrin, fibroblast growth factor 7, and the like.

Test example 2

This test example was used to verify the effect of a combination of different additive factors in the primary neuroblastoma cell culture medium on the proliferation of primary neuroblastoma cells.

A neuroblastoma primary cell culture medium was prepared from the components shown in table 4, and the proliferation promoting effect of the different additive factor combinations on the neuroblastoma primary cells was examined.

TABLE 4 preparation of different component Medium (final concentration)

The primary neuroblastoma cells were obtained from the neuroblastoma tissues (sample 3, sample 4) according to the "step 2 (3)" method of test example 1, and the obtained cell suspension was equally divided into 15 parts and centrifuged at 1500rpm for 4min. After centrifugation, 200. Mu.L of BM and No.1 to No. 14 medium were used to resuspend each, respectively, according to a viable cell density of 5X 10 ⁴ Individual/cm ² Inoculating into 48-well plate (5 ten thousand cells per well), filling each well volume in 48-well plate to 1mL with corresponding culture medium, and mixing thoroughly. Sterilizing the surface, placing at 37deg.C and 5% CO ₂ Incubator (purchased from zemoeid) culture.

Cells in the 48-well plate grew to over 85%, the medium was discarded, rinsed 1-time with 100. Mu.L of 0.05% trypsin (available from Gibco corporation), and 200. Mu.L of 0.05% trypsin was added to each well after blotting. Placing at 37deg.C and 5% CO ₂ The reaction was carried out in an incubator for 10min, and cells were observed to have been completely digested under a microscope (CNOPTEC, BDS 400), and the digestion was stopped by adding 300. Mu.L of DMEM/F12 medium containing 10% serum (vivacell, C04002-500), and 20. Mu.L was added to a cell counting plate (Countstar, specification: 50 pieces/cassette),the results obtained for the primary neuroblastoma cells of the cell counts of samples 3 and 4 are shown in FIG. 1.

From the results of fig. 1, it can be seen that: when the above-mentioned culture media No.1 to No.14 are used, the proliferation effect is best when the primary neuroblastoma cells are cultured in a medium containing "hepatocyte growth factor, compound 1, N2, insulin-transferrin-selenium supplement, Y-27632, insulin-like growth factor-1, gastrin, fibroblast growth factor 7, fetal bovine serum" and other additives, as compared with the Basal Medium (BM).

Test example 3

This test example was used to verify the proliferation of primary neuroblastoma cells at different concentrations of factors added to the neuroblastoma medium.

Primary neuroblastoma cells were obtained from the tissue samples (numbered sample 5, sample 6) according to the "step 2 (3)" method of test example 1. The obtained primary neuroblastoma cells have a viable cell density of 1×10 ⁵ Individual/cm ² Inoculating into 6-well plate (50 ten thousand cells per well), sterilizing, placing at 37deg.C, and 5% CO ₂ Incubator (purchased from zemoeid) culture. And amplified by culture using the combination medium of the identified effective factors in test example 2 (containing basal medium BM, 9ng/mL hepatocyte growth factor, 10. Mu.M Compound 1, 1:100 (v/v) N2, 9. Mu.g/mL insulin, 1:50 (v/v) insulin-transferrin-selenium supplement, 2.5. Mu. M Y-27632, 50ng/mL insulin-like growth factor-1, 9nM gastrin, 10ng/mL fibroblast growth factor 7, 10% (v/v) fetal bovine serum). Adding 500 μL of 0.05% trypsin (available from Gibco company) to soak for 1min until the cells grow to above 85%, sucking, adding 1mL of 0.05% trypsin into each well, and standing at 37deg.C and 5% CO ₂ The reaction was carried out in the incubator for 2 to 10 minutes until the cells had been digested, and the digestion was stopped by adding 1mL of DMEM/F12 medium containing 10% serum (excel Bio, FND 500). After centrifugation at 1500rpm for 4min, the supernatant was discarded. The cell pellet was resuspended in DMEM/F12. mu.L was added to a cell counting plate (manufacturer: countstar, specification: 50 pieces/cassette) and the total number of cells was counted by a cell counter (Countstar, IC 1000). The cells obtained were used in the following And (5) culturing experiments.

Next, experiments were performed by preparing the following 10 formulation media:

formula 1: the primary cell culture medium component of the neuroblastoma does not contain hepatocyte growth factor;

formula 2: the primary cell culture medium component of the neuroblastoma does not contain a compound 1;

formula 3: the primary cell culture medium component of the neuroblastoma does not contain N2;

formula 4: the primary cell culture medium component of the neuroblastoma does not contain insulin;

formula 5: the primary cell culture medium component of the neuroblastoma does not contain insulin-transferrin-selenium supplement;

formula 6: the primary cell culture medium component of the neuroblastoma does not contain Y-27632;

formula 7: the primary cell culture medium component of the neuroblastoma does not contain insulin-like growth factor-1;

formula 8: the primary cell culture medium component of the neuroblastoma does not contain gastrin;

formula 9: the primary cell culture medium component of the neuroblastoma does not contain fibroblast growth factor 7;

formula 10: the primary cell culture medium component of the neuroblastoma does not contain fetal bovine serum;

mu.L of a solution containing 5X 10 is added to each well ⁴ Cell suspensions of individual cells were diluted with 1mL of the medium of the above formulations 1 to 10, respectively.

When using the medium of formula 1: 1mL of prepared hepatocyte growth factor per hole is respectively added into a 48-hole plate inoculated with primary cells, and the final concentration of the hepatocyte growth factor is respectively 1ng/mL, 3ng/mL, 9ng/mL, 27ng/mL and 81ng/mL; and control wells (BC) were set using the medium of formula 1.

When using the medium of formula 2: 1mL of compound 1 prepared was added to each well of a 48-well plate inoculated with primary cells, and the final concentration of compound 1 was 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M, respectively; and control wells (BC) were set using the medium of formula 2.

When using the medium of formula 3: 1mL of N2 per well prepared was added to a 48-well plate inoculated with primary cells at a final concentration of 1:25 (v/v), 1:50 (v/v), 1:100 (v/v), 1:200 (v/v), 1:400 (v/v), respectively; and control wells (BC) were set using the medium of formula 3.

When using the medium of formula 4: 1mL of prepared insulin is added into a 48-well plate inoculated with primary cells, wherein the final concentration of the insulin is 2.5 mug/mL, 5 mug/mL, 10 mug/mL, 20 mug/mL and 40 mug/mL respectively; and control wells (BC) were set using the medium of formula 4.

When using the medium of formula 5: 1mL of the prepared insulin-transferrin-selenium supplement is added into a 48-well plate inoculated with primary cells, wherein the final concentration of the insulin-transferrin-selenium supplement is 1:25 (v/v), 1:50 (v/v), 1:100 (v/v), 1:200 (v/v) and 1:400 (v/v); and control wells (BC) were set using the medium of formula 5.

When using the medium of formula 6: 1mL of prepared Y-27632 per well is added to a 48-well plate inoculated with primary cells, and the final concentration of Y-27632 is 2.5 mu M, 5 mu M, 10 mu M, 20 mu M and 40 mu M respectively; and control wells (BC) were set using the medium of formula 6.

When using the medium of formula 7: 1mL of the prepared insulin-like growth factor-1 is respectively added into a 48-hole plate inoculated with primary cells, and the final concentration of the insulin-like growth factor-1 is respectively 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 40ng/mL; and control wells (BC) were set using the medium of formula 7.

When using the medium of formula 8: 1mL of prepared gastrin is added into a 48-well plate inoculated with primary cells, and the final concentration of the gastrin is 1nM, 3nM, 9nM, 27nM and 81nM respectively; and control wells (BC) were set using the medium of formula 8.

When using the medium of formula 9: 1mL of prepared fibroblast growth factor 7 is respectively added into a 48-hole plate inoculated with primary cells, and the final concentration of the fibroblast growth factor 7 is respectively 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 40ng/mL; and control wells (BC) were set using the medium of formula 9.

When using the medium of formula 10: 1mL of prepared fetal bovine serum is respectively added into a 48-well plate inoculated with primary cells, wherein the adding proportion of the fetal bovine serum is 2.5% (v/v), 5% (v/v), 10% (v/v), 20% (v/v) and 40% (v/v); and control wells (BC) were set using the medium of formulation 10.

The proliferation fold was calculated by comparing the cell number of control wells (BC) to the cell number of 48 wells when the cells were expanded to about 85% of the digestion count, and the results are shown in FIGS. 2A to 2J, respectively. In FIGS. 2A to 2J, the ratio is the ratio of the number of cells obtained by culturing the first generation using each medium to the number of cells obtained by culturing the first generation in the corresponding control well; the ratio of more than 1 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 base; the ratio is smaller than 1, which indicates that 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 with the control holes.

According to the results of FIGS. 2A to 2J, the content of hepatocyte growth factor is preferably 1 to 81ng/mL, and the cell proliferation effect is most obvious when the concentration is 9 ng/mL; the content of the compound 1 is preferably 2.5-10 mu M, and the cell proliferation effect is most obvious when the concentration is 2.5 mu M; the content of N2 is preferably 1:25-1:400 (v/v), and the cell proliferation effect is most obvious when the concentration is 1:200 (v/v); the content of insulin is preferably 2.5-40 mug/mL, and the cell proliferation effect is most obvious when the concentration is 10 mug/mL; the content of the insulin-transferrin-selenium supplement is preferably 1:25-1:400 (v/v), and the cell proliferation effect is most obvious when the concentration is 1:200 (v/v); the content of Y-27632 in the culture medium is preferably 2.5-20 mu M, and the cell proliferation effect is most obvious when the concentration is 10 mu M; the content of the insulin-like growth factor-1 is preferably 12.5-200 ng/mL, and the cell proliferation effect is most obvious when the concentration is 50 ng/mL; the content of the gastrin is preferably 1-81 nM, and the cell proliferation effect is most obvious when the concentration is 9 nM; the content of the fibroblast growth factor 7 is preferably 2.5-40 ng/mL, and the cell proliferation effect is most obvious when the concentration is 5 ng/mL; the preferred volume content of the fetal bovine serum is 2.5-40% (v/v), and the cell proliferation effect is most obvious when the concentration is 5% (v/v).

The most preferable concentration of each additive factor in the above-mentioned medium was used as the primary cell culture medium (i.e., the optimal medium formulation of the present invention) for neuroblastoma of the present invention used in the following test examples, which contains: basal medium BM, 9ng/mL hepatocyte growth factor, 2.5. Mu.M Compound 1, 1:200 (v/v) N2, 10. Mu.g/mL insulin, 1:200 (v/v) insulin-transferrin-selenium supplement, 10. Mu. M Y-27632, 50ng/mL insulin-like growth factor-1, 9nM gastrin, 5ng/mL fibroblast growth factor 7, 5% (v/v) fetal bovine serum (hereinafter "NB-1" medium).

Test example 4

The test example is used for verifying the primary cell culture and identification results of neuroblastoma.

1. Primary cell culture of neuroblastoma

Primary neuroblastoma cells were obtained from 6 tissue samples (numbered sample 7, sample 8, sample 9, sample 10, sample 11, sample 12) according to the "step 2 (3)" method of test example 1, and cultured using NB-1 medium in test example 3; the obtained primary neuroblastoma cells have a viable cell density of 1×10 ⁵ Individual/cm ² Inoculating into 6-well plate (50 ten thousand cells per well), and mixing. Sterilizing the surface, placing at 37deg.C and 5% CO ₂ Incubator (purchased from zemoeid) culture.

The cultured primary neuroblastoma cells were observed using a microscope (EVOS M500 from Invitrogen company), and fig. 3A to 3F are photographs taken under a 10-fold objective lens, and the cells were closely arranged under the lens with a slightly irregular morphology like neurons.

2. Neuroblastoma cell immunohistochemical identification after neuroblastoma tissue subculture

About 0.25cm of the tissue (sample No. 13) was removed from the operation of one patient with neuroblastoma ³ Cancer tissues of the size were fixed by immersing in 1mL of 4% paraformaldehyde. Sample 13 was continuously cultured to passage 7 using the method of test example 3 using the culture medium NB-1 of the present invention. The 4% paraformaldehyde fixed tissue or cells were paraffin embedded and cut into 4 μm thick tissue sections using a microtome.Conventional immunohistochemical assays were then performed. The primary antibodies used were CK, syn, NF, S-100, ki-67 (all available from CST).

FIGS. 4A-4E and 5A-5E are comparative diagrams of immunohistochemical results of primary cells of neuroblastoma obtained by culturing primary cells of neuroblastoma of the present invention in NB-1, respectively. Fig. 4A and 5A are pictures of labeled CK antibody of neuroblastoma tissue and cultured neuroblastoma primary cells, respectively, fig. 4B and 5B are pictures of labeled Syn antibody of neuroblastoma tissue and cultured neuroblastoma primary cells, respectively, fig. 4C and 5C are pictures of labeled NF antibody of neuroblastoma tissue and cultured neuroblastoma primary cells, respectively, fig. 4D and 5D are pictures of labeled S-100 antibody of neuroblastoma tissue and cultured neuroblastoma primary cells, respectively, and fig. 4E and 5E are pictures of labeled Ki-67 antibody of neuroblastoma tissue and cultured neuroblastoma primary cells, respectively, whereby it can be confirmed that the expression of a biomarker associated with neuroblastoma on the neuroblastoma primary cells is substantially identical to the expression of a marker of a primary tissue derived from neuroblastoma primary cells when the neuroblastoma primary cells were cultured to the 7 th generation. The primary cell of the neuroblastoma cultured by the technology of the invention maintains the original pathological characteristics of the cancer tissue of the patient with the neuroblastoma.

Test example 5

This test example was used to verify the uniqueness of the inventive culture medium to the primary cell culture of neuroblastoma.

The present inventors have studied a gastric cancer organoid medium (hereinafter abbreviated as "comparative medium 1", product model "Columbrellas pre, # PRS-GCM-3D") and a cervical cancer primary cell medium (hereinafter abbreviated as "comparative medium 2", product model "Columbrellas pre, # PRS-CCM-2D"). Wherein, although the two comparison media are partially identical with the culture media of the invention in terms of component species selection, the comparison medium 1 is only suitable for gastric cancer organoids, and the comparison medium 2 is only suitable for cervical cancer primary cells.

1. Cultivation of gastric cancer organoids

A single primary gastric cancer cell was obtained from 2 gastric cancer samples (sample 13 and sample 14) according to the method of test example 1 of the present invention. The gastric cancer primary cells obtained by separation are resuspended and counted by a basic culture medium, and the gastric cancer primary cells are mixed with matrigel according to the volume ratio of 1:1356231 Mixing on ice to obtain a final cell density of 5×10 ⁵ At each mL, 50 mu L of matrigel and cell suspension are taken to form coagulated liquid drops at the center of each hole of a 24-hole culture plate, and the culture plate is kept stand at 37 ℃ for 30min until the matrigel is completely coagulated. Culture medium 1 (gastric carcinoma organoid culture) and primary culture medium NB-1 of the present invention (neuroblastoma cells) were cultured separately. / >

FIGS. 6A and 6B are photographs of gastric cancer organoids obtained by culturing sample 13 with comparative medium 1 (gastric cancer organoid culture) and primary medium NB-1 (neuroblastoma cells) of the present invention, respectively; FIGS. 6C and 6D are photographs of gastric cancer organoids obtained by culturing sample 14 with comparative medium 1 (gastric cancer organoid culture) and primary medium NB-1 (neuroblastoma cells) of the present invention, respectively. From this, it was confirmed that the gastric cancer organoids cultured using the medium of the present invention did not proliferate significantly. It was demonstrated that the culture results were completely different based on the difference between the comparative medium 1 and the medium composition of the present invention; the culture medium based on the composition of the invention cannot be used for the culture of gastric cancer organoids.

2. Culture of primary cells for cervical cancer

Individual cervical cancer primary cells were obtained from 1 cervical cancer sample (sample 15) according to the method of test example 1. The separated primary cervical cancer cells are resuspended and counted by using a basic culture medium, 50 ten thousand cells per well are inoculated into 6-well culture plates, and a contrast medium 2 (primary cervical cancer cells) and a primary culture medium NB-1 (neuroblastoma cells) of the invention are respectively added for culture.

FIGS. 7A and 7B are photographs of primary cell cultures of cervical cancer obtained by culturing sample 15 with comparative medium 2 (cervical cancer primary cells) and primary medium NB-1 of the present invention (neuroblastoma cells), respectively. From this, it was confirmed that the primary cells of cervical cancer cultured by the medium of the present invention were not significantly proliferated and were in an extremely poor state. It was demonstrated that the culture results were completely different based on the difference between the comparative medium 2 and the medium composition of the present invention; the culture medium based on the composition of the present invention cannot be used for culturing primary cells of cervical cancer.

3. Culture medium (neuroblastoma cells) uniqueness verification of the invention

Single neuroblastoma primary cells were obtained from 2 neuroblastoma samples (sample 16, sample 17) according to the method of test example 1. The primary neuroblastoma cells obtained by separation are resuspended and counted by using a basic culture medium, 50 ten thousand cells per well are inoculated into 6-well culture plates, and a comparison culture medium 1 (gastric cancer organoid culture), a comparison culture medium 2 (cervical cancer primary cells) and the primary culture medium NB-1 (neuroblastoma cells) are respectively added for culture.

FIGS. 8A-8C are photographs of primary neuroblastoma cell cultures obtained by culturing sample 16 with comparative medium 2 (cervical cancer primary cells), comparative medium 1 (gastric cancer organoid culture), and primary medium NB-1 (neuroblastoma cells) of the present invention, respectively. FIGS. 9A-9C are photographs of primary neuroblastoma cell cultures obtained by culturing sample 17 with comparative medium 2 (cervical cancer primary cells), comparative medium 1 (gastric cancer organoid culture) and primary medium NB-1 (neuroblastoma cells) of the present invention, respectively. From this, it was confirmed that the primary neuroblastoma cells cultured in the document comparative medium 2 (cervical cancer primary cell) and comparative medium 1 (gastric cancer organoid culture) did not proliferate significantly. It is stated that only media based on the components of the invention are most suitable for culturing neuroblastoma primary cells.

Application example

The invention relates to application of a culture medium of primary cells of neuroblastoma.

1. First, the above-mentioned culture medium for primary neuroblastoma cells was used for in vitro rapid expansion culture of primary neuroblastoma cells:

(1) Preparing a culture medium of the primary neuroblastoma cells;

(2) The primary cell of neuroblastoma is processed according to the cell density of 1 to 10 multiplied by 10 ⁴ Individual/cm ² Seed into a petri dish and then culture using the medium of the primary neuroblastoma cells.

2. The cells obtained by culture are applied to the curative effect evaluation and screening of medicines.

The invention is suitable for industrial application.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (7)

1. A medium for primary cells of neuroblastoma, comprising the following components: initial medium, MST1/2 kinase inhibitor, fetal bovine serum, Y-27632, insulin-transferrin-selenium supplement, insulin-like growth factor-1, fibroblast growth factor 7, gastrin, hepatocyte growth factor;

The initial culture medium is selected from DMEM/F12, DMEM, F12 or RPMI-1640;

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

the volume percentage content of the fetal bovine serum relative to the culture medium is 2.5-40%;

the content of the Y-27632 in the culture medium is 2.5-20 mu M;

the volume ratio content of the insulin-transferrin-selenium supplement relative to the culture medium is 1:25 to 1:400;

the content of the insulin in the culture medium is 2.5-40 mug/mL;

the content of the insulin-like growth factor-1 in the culture medium is 12.5-200 ng/mL;

the content of the fibroblast growth factor 7 in the culture medium is 2.5-40 ng/mL;

the content of the gastrin in the culture medium is 1-81 nM;

the content of the hepatocyte growth factor in the culture medium is 1-81 ng/mL.

2. The medium of neuroblastoma primary cells according to claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof;

wherein R is ₁ Selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally R, independently of 1 to 2 ₆ Substituted aryl, aryl C1-C6 alkyl and heteroaryl;

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

R ₄ and R is ₅ Each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylamino C1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl and C3-C6 heterocyclylC 1-C6 alkyl;

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

3. The medium of neuroblastoma primary cells of claim 1, further comprising an N2 cell culture additive; the volume ratio content of the N2 cell culture additive in the culture medium is 1:25 to 1:400.

4. the medium of neuroblastoma primary cells of claim 1, further comprising an antibiotic; the antibiotics are selected from one or more of streptomycin, penicillin, amphotericin B and Primocin; when the antibiotics select streptomycin, the adding concentration is 25-400 mug/mL; when penicillin is selected as the antibiotic, the concentration of the antibiotic is 25-400U/mL; when the antibiotics select amphotericin B, the concentration is 0.25-4 mug/mL; when Primocin is selected as the antibiotic, the addition concentration is 25-400 mug/mL.

5. A method for culturing primary neuroblastoma cells, comprising culturing primary neuroblastoma cells in the medium according to any one of claims 1 to 4.

6. The method for primary cell culture of neuroblastoma according to claim 5, comprising the steps of:

(1) Preparing a medium of the primary neuroblastoma cells according to any one of claims 1 to 4;

(2) The primary cell of neuroblastoma is processed according to the cell density of 1 to 10 multiplied by 10 ⁴ Individual/cm ² Seed into a petri dish and then culture using the medium of the primary neuroblastoma cells.

7. Use of a medium of primary neuroblastoma cells according to any one of claims 1-4 for rapid expansion of primary neuroblastoma cells in vitro.