Culture medium and culture method of liver cancer suspension organoids

Abstract

The present application relates to a culture medium for liver cancer suspension organoid culture, comprising MST1 / 2 kinase inhibitor, Y27632, at least one cell culture additive selected from N2 and B27, SB202190, insulin, fibroblast growth factor 10, cholera toxin, ITS cell culture additive, hepatocyte growth factor, GlutaMAX, and non-essential amino acids. The present application also relates to a culture method of liver cancer suspension organoid and its use. By using the liver cancer suspension organoid culture medium of the present application, effective and rapid expansion of liver cancer cells can be achieved, the liver cancer cells obtained by expansion retain the pathological characteristics of the patient, the culture success rate and expansion rate of the liver cancer cells are improved, and research basis can be provided for personalized treatment of the patient.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a culture medium for liver cancer suspension organoids, a method for culturing liver cancer suspension organoids using the culture medium, and application thereof in efficacy evaluation and screening of drugs. BACKGROUND

[0002] Liver cancer is the third most common malignant tumor with a mortality rate second only to stomach cancer and esophageal cancer. In 2020, more than 900,000 people worldwide were newly diagnosed with liver cancer, and more than 830,000 people died of liver cancer, close to the number of new cases.

[0003] In recent years, postoperative adjuvant chemotherapy for liver cancer as a new type of adjuvant therapy has gradually been valued and recognized by clinicians, including postoperative TACE treatment, oral drug treatment, etc. However, due to the lack of standardized chemotherapy regimens, conventional chemotherapy is based on experience, ignores individual differences, and is blind to a certain extent, so the effect has not been good, and the effective rate of single drug and combined drug is less than 20% (Jindal A, Thadi A, Shailubhai K. Hepatocellular Carcinoma: Etiology and Current and Future Drugs [J]. J Clin Exp Hepatol, 2019, 9(2): 221-232). Although the emerging targeted drugs have reduced the side effects to a certain extent, the number is too small, the treatment cost is high, and the effective rate varies with individual differences, making it difficult to meet the treatment needs of most patients. Due to the lack of an effective liver cancer drug sensitivity test system, it is difficult to achieve precision chemotherapy, so it is crucial to correspond the in vitro drug sensitivity results of liver cancer to the in vivo response.

[0004] Traditional clinical drug sensitivity detection mostly uses two-dimensional cell culture. However, two-dimensional cultured cells only simulate the physiological conditions of tissues to a limited extent, lack the real tissue structure in vivo, are prone to cause low differentiation level and loss of cell physiological function, and thus the experimental results obtained are difficult to predict the actual clinical results. Organoids are mainly derived from human embryonic stem cells, induced pluripotent stem cells and adult stem cells with differentiation ability. Endogenous tissue stem cells exist in different organs, and play an important role in maintaining the functional morphology of each organ. These stem cells can self-organize into a mini structure with a diameter of only a few millimeters under certain induction conditions in vitro. Tumor organoids are some micro 3D tumor cell models cultured in the laboratory from primary tumors taken from patients. Tumor organoids highly simulate the characteristics of the source tumor tissue, retain the tumor heterogeneity between individuals, and can be used for functional tests, such as high-throughput drug screening and individualized precision treatment.

[0005] Currently, the liver cancer organoid culture method mostly uses R-spondin-1, Noggin and some expensive protein factors, resulting in high cost of organoid culture; and the technology needs to be coated with Matrigel in advance, and the culture and subculture operation is complex and difficult in technology; due to the coating of Matrigel, the large-scale culture cost is high, the standardization is difficult, and the large-scale commercial application is limited. Therefore, it is necessary to develop a low-cost, simple and high-success-rate organoid culture method and culture medium. SUMMARY

[0006] In order to solve the above technical problems, the present application provides a culture medium and culture method for rapidly expanding liver cancer suspension organoids in vitro.

[0007] One aspect of the present application is to provide a culture medium for liver cancer suspension organoids, which comprises an MST1 / 2 kinase inhibitor, Y27632, at least one cell culture additive selected from N2 and B27, SB202190, insulin, fibroblast growth factor 10, cholera toxin, ITS cell culture additive, hepatocyte growth factor, GlutaMAX, and non-essential amino acids. Wherein, the MST1 / 2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,

[0008]

[0009] wherein,

[0010] R1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl (e.g., phenyl and naphthyl, etc.) optionally substituted with 1-2 independent R6, aryl C1-C6 alkyl (e.g., benzyl, etc.) and heteroaryl (e.g., thienyl, etc.);

[0011] R2 and R3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

[0012] R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyranyl, etc.);

[0013] R6 is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoromethyl).

[0014] In preferred embodiments, the MST1 / 2 kinase inhibitor comprises a compound of Formula (Ia), or a pharmaceutically acceptable salt, or solvate thereof,

[0015]

[0016] wherein,

[0017] R1is selected from the group consisting of C1-C6alkyl, phenyl optionally substituted with 1-2 independent R6, thienyl optionally substituted with 1-2 independent R6, and benzyl optionally substituted with 1-2 independent R6, R1is more preferably phenyl optionally substituted with 1-2 independent R6;

[0018] R5is selected from the group consisting of hydrogen, C1-C6alkyl, and C3-C6cycloalkyl, R5is more preferably hydrogen;

[0019] each R6is independently selected from the group consisting of halogen, C1-C6alkyl, and C1-C6haloalkyl, R6is more preferably fluorine, methyl or trifluoromethyl.

[0020] Preferably, the MST1 / 2 inhibitor is at least one selected from the following compounds, or a pharmaceutically acceptable salt, or solvate thereof.

[0021]

[0022]

[0023]

[0024]

[0025]

[0026] Most preferably, the MST1 / 2 kinase inhibitor of the present application is Compound 1.

[0027] In embodiments of the present application, the content of each component in the culture medium of the present application satisfies any one or more or all of the following:

[0028] (1) the concentration of the MST1 / 2 kinase inhibitor is preferably 2.5-40 μM, more preferably 2.5-20 μM;

[0029] (2) the volume ratio of B27 or N2 cell culture additives to the culture medium is 1:25-1:200, more preferably 1:25-1:100;

[0030] (3) the concentration of hepatocyte growth factor is preferably 1.25-20 ng / mL, more preferably 1.25-10 ng / mL;

[0031] (4) The volume ratio of the ITS cell culture supplement to the medium is preferably 1:12.5 to 1:200, more preferably 1:50 to 1:200;

[0032] (5) The concentration of SB202190 is preferably 5 to 40 nM, more preferably 5 to 20 nM;

[0033] (6) The concentration of fibroblast growth factor 10 is preferably 2.5 to 20 ng / mL, more preferably 2.5 to 10 ng / mL;

[0034] (7) The concentration of cholera toxin is preferably 1.25 to 20 ng / mL, more preferably 2.5 to 10 ng / mL;

[0035] (8) The concentration of insulin is preferably 2.5 to 40 μg / mL, more preferably 2.5 to 20 μg / mL;

[0036] (9) The concentration of Y27632 is preferably 2.5 to 40 μM, more preferably 5 to 20 μM;

[0037] (10) The volume ratio of GlutaMAX to the medium is preferably 1:12.5 to 1:200;

[0038] (11) The non-essential amino acid is one or more selected from the group consisting of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine, and the concentration of the non-essential amino acid is preferably 12.5 to 200 μM, more preferably 100 to 200 μM.

[0039] In the embodiment of the present application, the medium further contains an initial medium selected from the group consisting of DMEM / F12, DMEM, F12, and RPMI-1640; and an antibiotic selected from the group consisting of streptomycin / penicillin, amphotericin B, and Primocin.

[0040] In the preferred embodiment, when the antibiotic is selected from streptomycin / penicillin, the concentration of streptomycin ranges from 25 to 400 μg / mL, and the concentration of penicillin ranges from 25 to 400 U / mL, when the antibiotic is selected from amphotericin B, the concentration ranges from 0.25 to 4 μg / mL, and when the antibiotic is selected from Primocin, the concentration ranges from 25 to 400 μg / mL.

[0041] The present application also provides a culture method of a liver cancer suspension organoid. In the culture method of the liver cancer suspension organoid of the present application, liver cancer primary cells are subjected to suspension culture using the liver cancer suspension organoid medium of the present application. "Suspension culture" refers to a tissue culture system in which single cells and small cell clusters are cultured in a liquid medium subjected to constant agitation or shaking or in a low-adsorption culture dish, and is a culture method of non-adhesion-dependent cells.

[0042] The liver cancer suspension organoid culture method of the present application comprises the following steps.

[0043] 1. Isolate samples from liver cancer solid tumor tissues to obtain liver cancer primary cells. The process comprises the following steps:

[0044] (1) Isolate liver cancer tissue samples, add 1:3 ratio of basic culture medium and tissue digestion solution (the amount of tissue digestion solution added is about 10 mL per 1 g of tumor tissue), and place in a constant temperature shaker for digestion. The digestion temperature is 4-37℃, the shaker speed is 200-350 rpm, and the digestion time is 3-6 hours.

[0045] (2) After digestion is complete, centrifuge and discard the supernatant. The centrifugation speed is 1200-1600 rpm, and the centrifugation time is 2-6 minutes.

[0046] The basic culture medium formula includes an initial culture medium selected from DMEM / F12, DMEM, F12, or RPMI-1640; and one or more antibiotics selected from streptomycin / penicillin, amphotericin B, and Primocin. The tissue digestion solution formula includes 1640 culture medium, collagenase II (1-2 mg / mL), collagenase IV (1-2 mg / mL), DNAase (50-100 U / mL), hyaluronidase (0.5-1 mg / mL), calcium chloride (1-5 mM), and bovine serum albumin BSA (5-10 mg / mL).

[0047] 2. Prepare the liver cancer suspension organoid culture medium of the present application, and culture the liver cancer primary cells obtained in the above step.

[0048] Resuspend the liver cancer primary cells obtained in step 1 above with the liver cancer suspension organoid culture medium of the present application and count them. Seed the cells in a low-adsorption culture plate at a density of 1-5 x 10 5 per well, and add the corresponding volume of liver cancer suspension organoid culture medium for expansion culture.

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

[0050] (1) Culture liver cancer organoids using the liver cancer suspension organoid culture method of the present application;

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

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

[0053] (4) Perform organoid size or organoid viability test.

[0054] The beneficial effects of the present application include:

[0055] (1) Improve the success rate of liver cancer organoid culture, the success rate reaches more than 90%;

[0056] (2) Ensure that the liver cancer organoids cultured in vitro can maintain the pathological characteristics of the patient;

[0057] (3) High expansion efficiency, liver cancer organoids can be quickly cultured, and the expanded liver cancer organoids can also be continuously passaged;

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

[0059] (5) The culture method does not need to use matrix glue, the culture and passage operation is simple, and is suitable for large-scale culture;

[0060] (6) The liver cancer organoids obtained by the technology are large in number, suitable for high-throughput screening of candidate compounds and providing high-throughput drug in vitro sensitivity function test for patients. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figures 1A-1K A graph showing the effect of different concentrations of factors added to the liver cancer suspension organoid culture medium of the present application on the proliferation of liver cancer organoids.

[0062] Figures 2A-2D A photo of liver cancer suspension organoids cultured using the liver cancer suspension organoid culture medium of the present application, wherein Figure 2A A photo showing the organoids cultured from sample GL-003 after 7 days; Figure 2B A photo showing the organoids cultured from sample GL-006 after 7 days; Figure 2C A photo showing the organoids cultured from sample GL-008 after 7 days; Figure 2D A photo showing the organoids cultured from sample GL-013 after 7 days.

[0063] Figure 3A A view of pathological and immunohistochemical identification of liver cancer suspension organoids of sample GL-006 cultured using the liver cancer suspension organoid culture medium of the present application; Figure 3B A view of pathological and immunohistochemical identification of the original tissue of sample GL-006.

[0064] Figures 4A-4CA comparison result chart of liver cancer organoids cultured using the liver cancer suspension organoid medium of the present application and existing media, wherein Figure 4A A photograph showing liver cancer suspension organoids obtained after 7 days of culture using the HC-XF medium of the present application; Figure 4B A photograph showing liver cancer suspension organoids obtained after 7 days of culture using the Laura medium; Figure 4C A comparison bar chart showing the relative size of liver cancer suspension organoids obtained by culture using the HC-XF medium and the Laura medium.

[0065] Figure 5A and 5B A result chart showing different drug sensitivity tests of liver cancer organoids cultured using the liver cancer suspension organoid medium of the present application, wherein Figure 5A A photograph showing the growth of organoids after 5 days of treatment with different concentrations of test drugs; Figure 5B A bar chart showing the inhibition rate of liver cancer organoid growth by different concentrations of test drugs. DETAILED DESCRIPTION

[0066] In order to better understand the present application, the present application will be further described below in conjunction with examples and drawings. The following examples are merely illustrative of the present application and are not intended to limit it.

[0067] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0068] In the present specification, the MST1 / 2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1 / 2 signaling. Generally, the MST1 / 2 kinase inhibitor, for example, binds to MST1 / 2 kinase and reduces its activity. Since MST1 and MST2 have structural similarity, the MST1 / 2 kinase inhibitor can also be, for example, a compound that binds to MST1 or MST2 and reduces its activity.

[0069] 1. Preparation of MST1 / 2 Kinase Inhibitor Compound 1

[0070] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydrophtalidine-2- yl)amino)benzene Sulfonamide 1

[0071]

[0072] 2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): After adding 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) to a round-bottom flask, add methanol (30 mL), and then add dichlorosulfoxide (1.2 mL) dropwise under ice bath. The reaction system is reacted at 85°C overnight. After the reaction is completed, the system is evaporated under reduced pressure to remove the solvent, and the obtained white solid is directly used for the next step.

[0073] 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetic acid methyl ester (A3): In a round bottom flask, 2-amino-2-(2,6-difluorophenyl)acetic acid methyl ester (2 g) was added followed by acetone (30 mL) and potassium carbonate (2.2 g), then the system was cooled to -10 °C with ice-salt bath, followed by the slow addition of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction system was stirred at room temperature overnight. After the reaction was completed, it was filtered, the filtrate was removed under reduced pressure, and the residue was purified by column chromatography on pressurized silica gel to obtain compound A3. LC / MS: M+H 359.0.

[0074] 2-chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): In a round bottom flask, 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetic acid methyl ester (2.5 g) was added followed by acetic acid (50 mL) and iron powder (3.9 g). The reaction system was stirred at 60 °C for two hours. After the reaction was completed, the system was evaporated under reduced pressure to remove the solvent, and the obtained product was neutralized to basic with saturated sodium bicarbonate. Ethyl acetate was extracted, and the organic phase was washed with water, saturated brine, and dried with anhydrous sodium sulfate. The organic phase was filtered and evaporated under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain compound A4. LC / MS: M+H 297.0.

[0075] 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (A5): In a round bottom flask, 2-chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (2 g) and N,N-dimethylacetamide (10 mL) were added and cooled to -35 °C, followed by the addition of iodomethane (0.9 mL), and then sodium hydride (615 mg). The reaction system was continuously stirred for two hours. After the reaction was completed, it was quenched with water, extracted with ethyl acetate, and the organic phase was washed with water, saturated brine, and dried with anhydrous sodium sulfate. The organic phase was filtered and evaporated under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain compound A5. LC / MS: M+H 325.0.

[0076] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzenesulfonamide (1): In a round bottom flask, 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) were added. The reaction system was stirred at 120 °C overnight. After the reaction was completed, it was filtered and washed with methanol and diethyl ether to obtain compound 1. LC / MS: M+H 461.1.

[0077] 2. Preparation of other MST1 / 2 inhibitor compounds of the present application

[0078] Other MST1 / 2 inhibitor compounds of the present application were synthesized according to the similar method as compound 1, and their structures and mass spectrometry data are shown in the following table.

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Effects of various additives in the hepatocarcinoma suspension organoid culture medium on the proliferation of hepatocarcinoma suspension organoids in Example 1

[0086] (1) Preparation of hepatocarcinoma suspension organoid culture medium

[0087] First, the base medium containing the initial medium was prepared. The initial medium can be selected from DMEM / F12, DMEM, F12 or RPMI-1640 commonly used in the art. In this example, the formula of the base medium is: DMEM / F12 medium (purchased from Corning Company) + 100 μg / mL Primocin (purchased from InvivoGen Company, 0.2% (v / v), commercially available product concentration 50 mg / ml).

[0088] Different types of additives were added to the base medium respectively (see Table 1) to prepare hepatocarcinoma suspension organoid culture media containing different additives.

[0089] (2) Isolation and treatment of hepatocarcinoma primary cells

[0090] 1 Sample selection

[0091] The hepatocarcinoma solid tumor tissue samples (intraoperative) were obtained from patients by professional medical personnel of professional medical institutions, and the patients all signed the informed consent form. The intraoperative sample was 0.25 cm 3 stored and transported using a commercial tissue preservation solution (manufacturer: Miltenyi Biotec).

[0092] 2 Material preparation

[0093] 15 mL sterile centrifuge tube, pipette gun, 10 mL pipette, surface sterilization of sterile gun head and put into ultraclean workbench after ultraviolet irradiation for 30 minutes. Take the basic medium from 4℃ refrigerator 30 minutes in advance, and take the tissue digestion solution from -20℃ refrigerator 30 minutes in advance.

[0094] Tissue digestion solution formula: 1640 culture medium (Corning, 10-040-CVR), collagenase II (2 mg / mL), collagenase IV (2 mg / mL), DNAase (50 U / mL), hyaluronidase (0.75 mg / mL), calcium chloride (3.3 mM), bovine serum albumin BSA (10 mg / mL).

[0095] The above-mentioned collagenase II, collagenase IV, DNAase, hyaluronidase are purchased from Sigma Company; calcium chloride is purchased from Shengong Bioengineering (Shanghai) Co., Ltd.; BSA is purchased from Biofroxx Company.

[0096] 3 sample separation

[0097] 3.1 In the ultraclean bench, take the tissue sample in a culture dish, remove the tissue with blood, rinse twice with the basic medium, transfer the tissue to another culture dish and mechanically separate it with a sterile scalpel, and divide the tissue block into 1*1*1 mm 3 size;

[0098] 3.2 After cutting, the intraoperative tissue is sucked into a 15 mL centrifuge tube, 5 mL of basic medium is added, mixed, and centrifuged at 1500 rpm for 4 minutes;

[0099] 3.3 Discard the supernatant, add 1:3 ratio of basic medium and tissue digestion solution (Note: the amount of tissue digestion solution added is about 10 mL for 1 g of tumor tissue), label the sample name and number, seal with sealing film, and digest in a 37℃ shaking bed (ZQLY-180N of Zhi Chu Instrument) at 300 rpm. Observe whether the digestion is complete every 30 minutes during the period, and the judgment basis is no visible particulate matter;

[0100] 3.4 After digestion is completed, filter out the undigested tissue mass through a 100 μm filter screen, and rinse the tissue mass on the filter screen into the centrifuge tube with the basic medium to reduce cell loss, and centrifuge at 1500 rpm for 4 minutes at 25℃;

[0101] 3.5 Discard the supernatant, observe whether there are blood cells, if there are blood cells, add 8 mL of blood cell lysis solution (purchased from Sigma Company), mix well, lyse at 4℃ for 20 minutes, and mix well once during the period, and centrifuge at 1500 rpm for 4 minutes at 25℃;

[0102] 3.6 Discard the supernatant, add 2 mL of basic medium to resuspend the cells, and reserve for use.

[0103] 4 Cell counting and processing

[0104] 4.1 Microscopic observation: A small amount of resuspended cells was taken and spread on a culture dish, and the cancer cell density and morphology were observed under a microscope (CNOPTEC, BDS400).

[0105] 4.2 Live cell counting: 12 μL of resuspended cell suspension was mixed with 12 μL of trypan blue dye (manufacturer: Shenguo Bioengineering (Shanghai) Co., Ltd.), and then 20 μL was added to a cell counting plate (manufacturer: Countstar, specifications: 50 pieces / box). The percentage of live large cells (cell size > 10 μm) was calculated by a cell counter (Countstar, IC1000) as follows: live cell number / total cell number*100%.

[0106] (3) Culture of liver cancer suspension organoids

[0107] The primary liver cancer cells obtained in the above step were resuspended and counted with pre-cooled DMEM / F12, and the cells were inoculated into a 24-well low-adsorption culture plate at a density of 1-5 x 10 5 per well, and then the culture medium shown in Table 1 was added to each well after being restored to room temperature. The medium was replaced every three days for expansion culture. After 7 days, the cultured organoids were photographed, and the diameters of the organoids were measured and counted to compare the promoting effects of various factors on the proliferation of liver cancer suspension organoids. As a control, the basic medium without any additives was used, and the experimental results are shown in Table 1.

[0108] Table 1: Additive components in the culture medium and the effect of promoting organoid proliferation

[0109] Serial Number Culture Medium Additive Category Supplier Final Concentration Proliferation Promoting Degree Classification 1 N2 Gibco 1:50 + 2 Epidermal Cell Growth Factor EGF R&D 5 ng / mL ○ 3 R-spondin 1 R&D 20 ng / mL ○ 4 Prostaglandin E2 Tocris 0.5 μΜ ○ 5 Insulin Peprotech 10 μg / mL + 6 B27 Gibco 1:50 + 7 A8301 MCE 100 nM - 8 SB202190 MCE 10 nM + 9 Basic Fibroblast Growth Factor bFGF R&D 10 ng / mL - 10 Hydrocortisone Sigma 10 ng / mL ○ 11 Noggin R&D 30 ng / mL ○ 12 Fetal Bovine Serum FBS Excell 5％ + 13 Insulin-like Growth Factor-1 IGF-1 R&D 45 ng / mL ○ 14 Keratinocyte Cell Growth Factor KGF R&D 5 ng / mL ○ 15 GlutaMAX Gibco 1:100 + 16 Non-essential Amino Acids Corning 100 μΜ + 17 Dexamethasone MCE 0.1 μΜ ○ 18 Neuregulin 1 NRG1 Sino Biological 5 ng / mL ○ 19 Y27632 MCE 10 μΜ + 20 ITS Cell Culture Additives Gibco 1:100 + 21 Compound 1 Preparation Example 5 μΜ + 22 CHIR99021 MCE 2.5 μΜ - 23 Hepatocyte Growth Factor HGF R&D 5 ng / mL + 24 Cholera Toxin CTX MCE 5 ng / mL + 25 Fibroblast Growth Factor 10 R&D 5 ng / mL +

[0110] Among them, "+" means that the culture medium with the additive has a promoting effect on the proliferation of at least two cases of liver cancer suspension organoids isolated from liver cancer tissues compared with the basic medium; "-" means that the culture medium with the additive shows an inhibitory effect on the proliferation of at least one case of liver cancer suspension organoids isolated from liver cancer tissues; and "O" means that the culture medium with the additive has no obvious effect on the proliferation of at least two cases of liver cancer suspension organoids isolated from liver cancer tissues.

[0111] According to the above results, B27, hepatocyte growth factor (HGF), ITS cell culture additive, Y27632, SB202190, fibroblast growth factor 10 (FGF10), insulin, cholera toxin (CTX), GlutaMAX, compound 1, non-essential amino acids, etc. were selected for further culture experiments.

[0112] Effect of different concentrations of medium additive factors on the proliferation of liver cancer suspension organoids

[0113] Primary liver cancer cells were obtained from intraoperative tissue samples (Nos. GL-003, GL-004) according to the method of (2) of Example 1, and organoid culture was performed using the medium formulations in Table 2 below.

[0114] Table 2 Medium formulations (concentrations are final concentrations)

[0115]

[0116] When using the medium of Formula 1, 200 μL of prepared B27 was added to each well of the 96-well plate inoculated with the suspension organoids on the basis of Formula 1, and the final concentration of B27 was 1:200, 1:100, 1:50, 1:25, 1:12.5, respectively; and a control well (BC) was set using the medium of Formula 1. The final concentrations of other additive factors in this series of media were the same as those in the HC-XF medium. The following experiments of Formulas 1-11 were also performed in the same manner, and are not described in detail.

[0117] When using the medium of Formula 2, 200 μL of prepared HGF was added to each well of the 96-well plate inoculated with the suspension organoids on the basis of Formula 2, and the final concentration of HGF was 1.25 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, respectively; and a control well (BC) was set using the medium of Formula 2.

[0118] When using the medium of Formula 3, 200 μL of prepared ITS cell culture additive was added to each well of the 96-well plate inoculated with the organoids on the basis of Formula 3, and the final concentration of ITS cell culture additive was 1:200, 1:100, 1:50, 1:25, 1:12.5, respectively; and a control well (BC) was set using the medium of Formula 3.

[0119] When using the medium of Formula 4, 200 μL of prepared Y27632 was added to each well of the 96-well plate inoculated with the organoids on the basis of Formula 4, and the final concentration of Y27632 was 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, respectively; and a control well (BC) was set using the medium of Formula 4.

[0120] When using the medium of Formula 5, 200 μL of prepared SB202190 was added to each well of the 96-well plate inoculated with the organoids on the basis of Formula 5, and the final concentration of SB202190 was 2.5 nM, 5 nM, 10 nM, 20 nM, 40 nM, respectively; and a control well (BC) was set using the medium of Formula 5.

[0121] When using the medium of Formula 6, 200 μL of prepared FGF10 was added to each well of the 96-well plate inoculated with the organoids on the basis of Formula 6, and the final concentration of FGF10 was 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, respectively; and a control well (BC) was set using the medium of Formula 6.

[0122] When using the medium of Formula 7, 200 μL of prepared insulin was added to each well of the 96-well plate inoculated with the organoids on the basis of Formula 7, and the final concentration of insulin was 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL, respectively; and a control well (BC) was set using the medium of Formula 7.

[0123] When using the medium of Formula 8, 200 μL of prepared CTX was added to each well of the 96-well plate inoculated with the organoids on the basis of Formula 8, and the final concentration of CTX was 1.25 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL, respectively; and a control well (BC) was set using the medium of Formula 8.

[0124] When using the medium of Formula 9, 200 μL of prepared GlutaMAX was added to each well of the 96-well plate inoculated with the organoids on the basis of Formula 9, and the final concentration of GlutaMAX was 1:200, 1:100, 1:50, 1:25, and 1:12.5, respectively; and a control well (BC) was set using the medium of Formula 9.

[0125] When using the medium of Formula 10, 200 μL of prepared Compound 1 was added to each well of the 96-well plate inoculated with the organoids on the basis of Formula 10, and the final concentration of Compound 1 was 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively; and a control well (BC) was set using the medium of Formula 10.

[0126] When using the medium of Formula 11, 200 μL of prepared non-essential amino acids was added to each well of the 96-well plate inoculated with the organoids on the basis of Formula 11, and the final concentration of non-essential amino acids was 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively; and a control well (BC) was set using the medium of Formula 11.

[0127] After 14 days, the cultured organoids were photographed, and the diameters of the organoids were measured and counted to compare the promoting effects of various concentrations of factors on the proliferation of liver cancer organoids. The data collected from two samples are shown in Table 1. Figures 1A-1K . Figures 1A-1KThe ratio is the ratio of the organoid diameter obtained by culturing with each medium for 14 days to the diameter of the organoid obtained by culturing with the corresponding BC control well for 14 days. A ratio greater than 1 indicates that the medium containing the factor or small molecule at the different concentrations promotes proliferation more effectively than the medium in the control well. A ratio less than 1 indicates that the medium containing the factor or small molecule at the different concentrations promotes proliferation less effectively than the medium in the control well.

[0128] according to Figures 1A-1K The results show that the volume concentration of B27 is preferably 1:25 to 1:200, more preferably 1:25 to 1:100; the content of hepatocyte growth factor is preferably 1.25 to 20 ng / mL, more preferably 1.25 to 10 ng / mL; the volume concentration of ITS cell culture additive is preferably 1:12.5 to 1:200, more preferably 1:50 to 1:200; the content of Y27632 is preferably 2.5 to 40 μM, more preferably 5 to 20 μM; the content of SB202190 is preferably 5 to 40 nM, more preferably 5 to 20 nM; the content of fibroblast growth factor 10 is preferably 2 .5~20ng / mL, more preferably 2.5~10ng / mL; the content of insulin is preferably 2.5~40μg / mL, more preferably 2.5~20μg / mL; the content of cholera toxin is preferably 1.25~20ng / mL, more preferably 2.5~10ng / mL; the volume concentration of GlutaMAX is preferably 1:12.5~1:200; the content of MST1 / 2 kinase inhibitor compound 1 is preferably 2.5~40μM, more preferably 2.5~20μM; the content of non-essential amino acids is preferably 12.5~200μM, more preferably 100~200μM.

[0129] Example 3 Liver cancer suspension organoid culture and identification

[0130] The primary liver cancer cells (GL-003, GL-006, GL-008, and GL-013) obtained according to the method described in Example 1 (2) were resuspended in the liver cancer suspension organoid culture medium HC-XF of the present invention and counted. The cells were cultured at a rate of 5×10 5 The cells were seeded at a density of / well in a 24-well low-adhesion culture plate, 1 mL of liver cancer suspension organoid culture medium was added, and the culture was expanded by replacing the culture medium every three days.

[0131] On day 7, the cultured hepatoma suspension organoids were observed using a microscope (Invitrogen EVOS M500). Figures 2A-2D The images above are taken with a 10x objective lens. The liver cancer suspension organoids, obtained after seven days of culture, were obtained from samples GL-003, GL-006, GL-008, and GL-013. Under the microscope, the liver cancer suspension organoids appear spherical with a smooth surface.

[0132] The cultured liver cancer suspension organoids were subjected to pathological and immunohistochemical identification, and the corresponding tissue samples were subjected to pathological and immunohistochemical identification, and the consistency of the results of the organoids and the original tissues was compared.

[0133] Figure 3A The results of pathological and immunohistochemical identification of liver cancer suspension organoids obtained by in vitro culture of sample GL-006 are shown in the following figures, which are pictures taken under a 20x objective. As shown in the following figures, the HE results show that the structure of the organoids is in the form of cancer tissue; CK19, Heppar-1, and Ki67 expression suggest that the sample is liver cancer. Figure 3A The pathological and immunohistochemical results of the corresponding tissue of GL-006 before culture are shown in the following figures. The results show that the diagnosis results of the liver cancer organoids cultured using the culture medium HC-XF of the present application are consistent with those of the liver cancer tissue before culture. Figure 3B The pathological and immunohistochemical results of the corresponding tissue of GL-006 before culture are shown in the following figures. The results show that the diagnosis results of the liver cancer organoids cultured using the culture medium HC-XF of the present application are consistent with those of the liver cancer tissue before culture.

[0134] Comparison of the culture effects of Example 4 and existing organoid culture medium

[0135] (1) Preparation of control medium

[0136] The medium used in the literature (Laura et al., Nat Med. 2017, 23(12): 1424-1435) was prepared, and the formula was Advanced DMEM / F12 medium (purchased from Corning Company) + 1:100 Penicillin / Streptomycin (purchased from Corning Company) + 1:100 GlutaMAX (purchased from Corning Company) + 10 mM HEPES (purchased from Thermo Fisher Company) + 1:50 B27 (purchased from Gibco Company) + 1:100 N2 (purchased from Gibco Company) + 1.25 mmol / L N-acetyl cysteine (purchased from MCE Company) + 10 mmol / L nicotinamide (purchased from MCE Company) + 10 nM gastrin (purchased from MCE Company) + 50 ng / ml epidermal growth factor (purchased from R&D Company) + 100 ng / ml fibroblast growth factor 10 (purchased from sino biological company) + 25 ng / ml hepatocyte growth factor (purchased from R&D Company) + 10 μmol / L Forskolin (purchased from MCE Company) + 5 μmol / L A8301 (purchased from MCE Company) + 10 μmol / L Y27632 (purchased from MCE Company) + 3 nmol / L dexamethasone (purchased from MCE Company). Hereinafter referred to as Laura medium.

[0137] (2) Culture of liver cancer suspension organoids

[0138] Primary hepatocarcinoma cells were obtained from the intraoperative tissue sample GL-018 according to the method of (2) of Example 1, and were cultured in suspension organoids using the HC-XF medium and the Laura medium, respectively, according to the method of Example 3.

[0139] On the 7th day of culture, the liver cancer suspension organoids obtained by culture were observed using a microscope (Invitrogen EVOS M500). Figure 4A and 4B are photos of the organoids obtained by culture using the HC-XF medium and the Laura medium, respectively, taken under a 4-fold objective lens, Figure 4C is a comparative column chart of the relative sizes of the organoids obtained by culture using the two media.

[0140] According to the results, Figures 4A-4C it can be seen that, compared with the Laura medium, the HC-XF medium can significantly promote the expansion and culture of liver cancer organoids.

[0141] Example 5: Use of liver cancer suspension organoids obtained by expansion using the medium of the application for drug screening

[0142] (1) Liver cancer suspension organoid culture

[0143] Primary hepatocarcinoma cells were isolated from the intraoperative sample of liver cancer (GL-006) according to the method of (2) of Example 1, and were cultured in suspension organoids using the HC-XF medium, and drug screening was performed when the liver cancer suspension organoids had a diameter of more than 50 μm.

[0144] (2) Preparation of screening drugs

[0145] Three drugs (epirubicin, daunorubicin, doxorubicin; all purchased from MCE) were prepared in two concentration gradients according to the following table, and were stored for use.

[0146] Table 3 Preparation of drug addition solutions of epirubicin, doxorubicin, and daunorubicin

[0147] Epirubicin Formulated Concentration (mM) 2 0.2 Doxorubicin Formulated Concentration (mM) 2 0.2 Daunorubicin Formulated Concentration (mM) 2 0.2

[0148] (3) Drug addition

[0149] The prepared drugs were taken out and placed at room temperature, and the drugs were diluted 1000-fold with the HC-XF medium and stored for use. The organoids obtained by culture according to step (1) were taken out from the incubator, and were transferred to a 15-ml centrifuge tube and centrifuged at 1500 rpm for 5 minutes. The organoids were resuspended with the HC-XF medium, counted, and the concentration of the organoids was adjusted to 50 per 50 μL. The adjusted organoid suspension was added to 50 μL in a 96-well plate, and 50 μL of the diluted drug was added. The surface of the 96-well plate was sterilized and transferred to the incubator for continued culture, and the viability of the organoids was determined after 5 days.

[0150] (4) Organoid viability test

[0151] CellTiter-Glo Luminescent Reagent (purchased from Promega Corporation) was taken out from a 4℃ refrigerator, 10 milliliters of the reagent were taken to a sample slot, 20 μL of CellTiter-Glo Luminescent Reagent was added to each well of a 96-well plate taken out from an incubator, and after standing for 10 minutes, the mixture was mixed, and a multifunctional enzyme label instrument (Envision of Perkin Elmer Corporation) was used for detection.

[0152] (5) Data processing

[0153] According to the formula drug inhibition rate (%) = 100% - (fifth day culture hole chemiluminescence value 药物处理组 / zero day culture hole chemiluminescence value 药物处理组 ) / (fifth day culture hole chemiluminescence value DMSO / zero day culture hole chemiluminescence value DMSO )*100%, the inhibition rate of different drugs was calculated, and the results are shown in Figure 5A 5B . Figure 5A Fig. 1 is a photograph of organoid growth after 5 days of drug treatment taken by a 4x objective microscope (EVOS M500 of Invitrogen Corporation), Figure 5B Fig. 2 is a column chart of inhibition rates of different concentrations of test drugs for inhibiting the growth of liver cancer organoids.

[0154] Fig. 3 is a column chart of inhibition rates of three different concentrations of test drugs for inhibiting the growth of liver cancer organoids. Figure 5A It can be confirmed that the organoids cultured using the liver cancer suspension organoid culture medium of the present application are obviously inhibited in growth after high-concentration drug treatment. Figure 5B Fig. 4 is a column chart of inhibition rates of three different concentrations of test drugs for inhibiting the growth of liver cancer organoids. Figure 5B It can be seen that the data error values of the drug treatment groups are very small, indicating that the data of repeated wells of the same drug basically remain consistent when the system is used for drug screening. Among the three antitumor drugs, high concentrations have a strong effect of inhibiting the growth of organoids, and low concentrations have a large difference in the inhibiting effect on the growth of organoids, which indicates that the organoids of the same patient have different effectiveness and sensitivity to different drugs. According to the results, the effectiveness and effective amount of the drug can be judged when the drug is used clinically for liver cancer patients.

[0155] Industrial Applicability

[0156] The present application provides a culture medium and a culture method for liver cancer suspension organoid culture, and the organoids obtained by culture can be applied to drug efficacy evaluation and screening. Thus, the present application is suitable for industrial application.

[0157] ​Although the present application has been described in detail, but the present application is not limited thereto, those skilled in the art can make modifications according to the principles of the present application, therefore, all kinds of modifications made according to the principles of the present application should be understood as falling within the scope of the present application.

Claims

1. A culture medium for liver cancer suspension organoids, characterized in that Made with the following ingredients: MST1 / 2 kinase inhibitor; Y27632; B27 supplement; SB202190; insulin; fibroblast growth factor 10; cholera toxin; ITS cell culture supplement; hepatocyte growth factor; GlutaMAX; non-essential amino acids; initial culture medium selected from DMEM / F12, DMEM, F12 or RPMI-1640; and antibiotics, Wherein, the MST1 / 2 kinase inhibitor is compound 1 or a pharmaceutically acceptable salt or solvate thereof, in, The concentration of the MST1 / 2 kinase inhibitor is 2.5 to 40 μM; The volume ratio of the B27 additive to the culture medium is 1:25 to 1:200; The concentration of the hepatocyte growth factor is 1.25-20 ng / mL; The volume ratio of the ITS cell culture additive to the culture medium is 1:12.5 to 1:200; The concentration of Y27632 is 2.5-40 μM; The concentration of SB202190 is 5-40 nM; The concentration of the fibroblast growth factor 10 is 2.5 to 20 ng / mL; The concentration of the insulin is 2.5 to 40 μg / mL; The concentration of the cholera toxin is 1.25 to 20 ng / mL; The volume ratio of the GlutaMAX to the culture medium is 1:12.5 to 1:200; The non-essential amino acids are one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the concentration thereof is 12.5 to 200 μM.

2. The culture medium according to claim 1, wherein The content of each component in the culture medium satisfies any one, multiple or all of the following: The concentration of the MST1 / 2 kinase inhibitor is 2.5 to 20 μM; The volume ratio of the B27 additive to the culture medium is 1:25 to 1:100; The concentration of the hepatocyte growth factor is 1.25-10 ng / mL; The volume ratio of the ITS cell culture additive to the culture medium is 1:50 to 1:200; The concentration of Y27632 is 5 to 20 μM; The concentration of SB202190 is 5-20 nM; The concentration of the fibroblast growth factor 10 is 2.5 to 10 ng / mL; The concentration of the insulin is 2.5 to 20 μg / mL; The concentration of the cholera toxin is 2.5 to 10 ng / mL; The concentration of the non-essential amino acids is 100-200 μM.

3. The culture medium according to claim 1 or 2, wherein: The antibiotic is selected from one or more of streptomycin / penicillin, amphotericin B and primocin.

4. A method for culturing liver cancer suspension organoids, characterized in that The following steps are involved: (1) Isolate samples from solid liver cancer tissue to obtain primary liver cancer cells; (2) preparing a culture medium for liver cancer suspension organoids according to any one of claims 1 to 3, and performing suspension culture on the primary liver cancer cells obtained in step (1).

5. A method for screening drugs for treating liver cancer, characterized in that: The following steps are involved: (1) culturing liver cancer suspension organoids using the liver cancer suspension organoid culture method according to claim 4; (2) Select the drug to be tested and dilute it according to the required concentration gradient; (3) adding the diluted drug to the suspended organoids cultured in (1); (4) Conduct organoid size or organoid viability testing.

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