Culture medium for culturing hepatocellular carcinoma organoids, method for culturing hepatocellular carcinoma organoids, and uses thereof

Abstract

A culture medium for hepatocellular carcinoma organoid culture, comprising an MST1 / 2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, an ITS cell culture additive, Y27632, dexamethasone, neuregulin-1, insulin, epidermal growth factor, GlutaMAX, and non-essential amino acids. The present invention further relates to a method for culturing hepatocellular carcinoma organoids and their use. By using the hepatocellular carcinoma organoid culture medium, effective and rapid expansion of hepatocellular carcinoma organoids can be achieved, and the organoids obtained by such expansion can maintain the pathological characteristics of the patient, improve the success rate of culturing and the expansion rate of hepatocellular carcinoma organoids, and provide a research basis for personalized treatment of patients.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, in particular to a culture medium for culturing hepatocellular carcinoma organoids, a method for culturing hepatocellular carcinoma organoids using the culture medium, and its use in evaluating and screening the efficacy of drugs. [Background technology]

[0002] In recent years, postoperative adjuvant chemotherapy for liver cancer, including postoperative TACE therapy and oral drug therapy, has gradually attracted the attention of clinicians and gained recognition as a new type of adjuvant treatment. However, due to the lack of standardized chemotherapy regimens, traditional empirical chemotherapy often ignores individual differences and has blind spots, resulting in unsatisfactory efficacy, with monotherapy or combination therapy achieving an efficacy rate of less than 20% (Non-Patent Document 1). Although emerging targeted drugs have somewhat reduced toxic side effects, the drug dose is too low, treatment costs are high, and efficacy varies from patient to patient, making it difficult to meet the treatment needs of most patients. The lack of an effective liver cancer drug sensitivity testing system makes precise chemotherapy impossible. Therefore, matching in vitro drug sensitivity results with clinical in vivo responses for liver cancer is key to treatment.

[0003] Traditional clinical drug sensitivity testing often uses two-dimensional (2D) cell cultures. However, 2D cultured cells can only simulate physiological tissue conditions to a limited extent and lack the actual in vivo tissue structure, leading to low levels of differentiation and loss of physiological cell function. This makes it difficult to predict actual clinical outcomes from experimental results. Organoids are a type of three-dimensional (3D) cell culture derived primarily from human embryonic stem cells, induced pluripotent stem cells, and adult stem cells, which have differentiation potential. Endogenous tissue stem cells play an important role in maintaining the functional morphology of each organ and are present in various tissues and organs. These stem cells can self-organize under specific in vitro induction conditions to form miniature structures only a few millimeters in diameter. Tumor organoids are miniature 3D tumor cell models cultured in the laboratory from primary tumors harvested from patients. Tumor organoids can highly simulate the characteristics of original tumor tissue while maintaining tumor heterogeneity between individuals, making them useful for functional testing such as high-throughput drug screening and personalized precision therapy.

[0004] Currently, hepatocellular carcinoma organoid culture methods often use basal culture medium (DMEM or DMEM / F12), R-spondin-1, noggin, and several expensive protein factors, resulting in high organoid culture costs. Furthermore, the complex operation and difficulty of this technique limit its large-scale commercial application. Therefore, the development of a low-cost, simple, and highly successful organoid culture method and culture medium is needed. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Jindal A, Thadi A, Shailubhai K. Hepatocellular Carcinoma: Etiology and Current and Future Drugs [J]. J Clin Exp Hepatol, 2019, 9(2): 221-232 Summary of the Invention

[0006] To solve the above-mentioned technical problems, the present invention provides a culture medium and a culture method for rapidly expanding and growing hepatocellular carcinoma organoids in vitro.

[0007] One aspect of the present invention is to provide a culture medium for hepatocellular carcinoma organoids, comprising an MST1 / 2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, ITS cell culture additive, Y27632, dexamethasone, neuregulin-1, insulin, epidermal growth factor, GlutaMAX, and a non-essential amino acid, wherein the MST1 / 2 kinase inhibitor is represented by formula (I): [ka] (In the formula, R1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl optionally substituted with 1-2 independent R6 (e.g., phenyl and naphthyl, etc.), arylC1-C6 alkyl optionally substituted with 1-2 independent R6 (e.g., phenylmethyl, etc.), and heteroaryl optionally substituted with 1-2 independent R6 (e.g., thienyl, etc.); R2 and R3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl; R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, hydroxylC1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, and C3-C6 heterocyclylC1-C6 alkyl (heterocyclyl is, for example, selected from piperidyl, tetrahydropyranyl, etc.); R6 includes compounds selected from halogen (preferably fluoro and chloro, more preferably fluoro), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoromethyl), or a pharmaceutically acceptable salt or solvate thereof.

[0008] In a preferred embodiment, the MST1 / 2 kinase inhibitor has formula (Ia): [ka] (In the formula, R1 is selected from C1-C6 alkyl, phenyl optionally substituted with 1-2 independent R6, thienyl optionally substituted with 1-2 independent R6, and phenylmethyl optionally substituted with 1-2 independent R6, more preferably R1 is phenyl optionally substituted with 1-2 independent R6; R5 is selected from hydrogen, C1-C6 alkyl and C3-C6 cycloalkyl, more preferably R5 is hydrogen; R6 is independently selected from halogen, C1-C6 alkyl and C1-C6 haloalkyl; more preferably, R6 is fluoro, methyl or trifluoromethyl), or a pharmaceutically acceptable salt or solvate thereof.

[0009] Preferably, the MST1 / 2 kinase inhibitor is at least one selected from the following compounds or a pharmaceutically acceptable salt or solvate thereof:

[0010] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0011] Most preferably, the MST1 / 2 kinase inhibitor of the present invention is Compound 1.

[0012] In an embodiment of the invention, the amounts of components in the culture media of the invention satisfy one or more or all of the following conditions: (1) The concentration of the MST1 / 2 kinase inhibitor is 2.5 μM to 10 μM; (2) the volume ratio of B27 or N2 cell culture additive in the culture medium is 1:25 to 1:100; (3) the concentration of hepatocyte growth factor is 1 ng / mL to 25 ng / mL; (4) The volume ratio of ITS cell culture additives in the culture medium is 1:30 to 1:300; (5) The concentration of Y27632 is 3 μM to 30 μM; (6) The concentration of dexamethasone is 0.1 μM to 1 μM; (7) The concentration of neuregulin-1 is 1 ng / mL to 25 ng / mL; (8) Insulin concentration is 1 μg / mL to 10 μg / mL; (9) The concentration of epidermal growth factor is 2 ng / mL to 18 ng / mL; (10) The volume ratio of GlutaMAX in the culture medium is 1:30 to 1:300; (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 50 μM to 200 μM.

[0013] In an embodiment of the invention, the culture medium further comprises an initial medium selected from the group consisting of DMEM / F12, DMEM, F12 or RPMI-1640, and one or more antibiotics selected from the group consisting of streptomycin / penicillin, amphotericin B and Primocin.

[0014] In a preferred embodiment, when streptomycin / penicillin is used as the antibiotic, the concentration range of streptomycin is 25 μg / mL to 400 μg / mL, and the concentration range of penicillin is 25 U / mL to 400 U / mL; when amphotericin B is used as the antibiotic, the concentration range is 0.25 μg / mL to 4 μg / mL; and when primocin is used as the antibiotic, the concentration range is 25 μg / mL to 400 μg / mL.

[0015] The present invention also provides a method for culturing hepatocellular carcinoma organoids, in which the hepatocellular carcinoma organoids are cultured using a culture medium for hepatocellular carcinoma organoids of the present invention.

[0016] The method for culturing hepatocellular carcinoma organoids of the present invention comprises the following steps.

[0017] 1. Isolate a sample from solid liver cancer tissue to obtain primary liver cancer cells. This process includes the following steps: (1) Isolate liver cancer tissue samples, add basal medium and tissue digestion solution (approximately 10 mL of tissue digestion solution per 1 g of tumor tissue) in a ratio of 1:3, place in a thermostatic shaker, and digest for 3 to 6 hours at a digestion temperature of 4°C to 37°C and a rotation speed of 200 rpm to 350 rpm. (2) After digestion, the mixture is centrifuged at a speed of 1200 rpm to 1600 rpm for 2 to 6 minutes, and the supernatant is then discarded.

[0018] Here, the basal medium formulation includes an initial medium selected from the group consisting of DMEM / F12, DMEM, F12, or RPMI-1640, and one or more antibiotics selected from the group consisting of streptomycin / penicillin, amphotericin B, and Primocin. The tissue digestion solution formulation includes 1640 medium, collagenase II (1 mg / mL to 2 mg / mL), collagenase IV (1 mg / mL to 2 mg / mL), DNase (50 U / mL to 100 U / mL), hyaluronidase (0.5 mg / mL to 1 mg / mL), calcium chloride (1 mM to 5 mM), and bovine serum albumin (BSA) (5 mg / mL to 10 mg / mL).

[0019] 2. Prepare a culture medium for the hepatocellular carcinoma organoids of the present invention and culture the primary hepatocellular carcinoma cells obtained in the above steps. The primary hepatoma cells obtained in step 1 above are resuspended in the culture medium for hepatocellular carcinoma organoids of the present invention and counted. 6 cells / mL~10×10 6 Dilute the cell suspension to 100 cells / mL, add an equal volume of Matrigel matrix gel to the diluted cell suspension, and mix thoroughly. Next, seed the mixture into a multi-well plate and place the plate in a culture incubator for 30-60 minutes until the Matrigel completely solidifies. Then, add culture medium for hepatocellular carcinoma organoids for expansion and culture.

[0020] The present invention also provides a method for evaluating or screening a therapeutic agent for liver cancer, comprising the steps of: (1) culturing hepatocellular carcinoma organoids using the hepatocellular carcinoma organoid culture method of the present invention; (2) selecting a test drug and diluting it to the required concentration gradient; (3) adding the diluted drug to the organoids obtained in step (1); (4) Detecting the size or viability of the organoids.

[0021] Beneficial effects of the present invention include the following: (1) The success rate of hepatocellular carcinoma organoid culture is improved with a success rate of over 90%; (2) Ensure that primary in vitro cultured hepatocellular carcinoma organoids maintain the pathological characteristics of patients; (3) HCC organoids can be rapidly cultured with high expansion efficiency, and the expanded HCC organoids have the ability to be continuously passaged; (4) The culture medium does not require expensive factors such as Wnt agonists, R-spondin family proteins, Noggin proteins, BMP inhibitors, and fibroblast growth factor 10 (FGF10), reducing the cost of culture; (5) This technology allows for the mass cultivation and provision of hepatocellular carcinoma organoids, making it suitable for high-throughput screening of candidate compounds and for in vitro high-throughput drug sensitivity functional testing in patients. [Brief explanation of the drawings]

[0022] [Figure 1-1] 1A to 1H are diagrams showing the effects of different concentrations of factors added to the culture medium for hepatocellular carcinoma organoids of the present invention on the growth of hepatocellular carcinoma organoids. [Figure 1-2] 1I to 1K are diagrams showing the effects of different concentrations of factors added to the culture medium for hepatocellular carcinoma organoids of the present invention on the growth of hepatocellular carcinoma organoids. [Figure 2]Figures 2A to 2D are microscopic photographs of hepatocellular carcinoma organoids cultured using the culture medium for hepatocellular carcinoma organoids of the present invention. Figure 2A shows a photograph of organoids cultured for 10 days from sample GL-003. Figure 2B shows a photograph of organoids cultured for 10 days from sample GL-006. Figure 2C shows a photograph of organoids cultured for 12 days from sample GL-008. Figure 2D shows a photograph of organoids cultured for 15 days from sample GL-013. [Figure 3] Figure 3A shows the results of pathological and immunohistochemical characterization of hepatocellular carcinoma organoids cultured from sample GL-006 using the culture medium for hepatocellular carcinoma organoids of the present invention. Figure 3B shows the results of pathological and immunohistochemical characterization of tissue sample GL-006. [Figure 4] Figure 4 shows the comparison results of hepatocellular carcinoma organoid culture between the culture medium for hepatocellular carcinoma organoid of the present invention and existing culture medium. Figure 4A shows the photograph of the organoid cultured for 25 days using the HC-3 culture medium of the present invention. Figure 4B shows the photograph of the organoid cultured for 25 days using Laura culture medium. Figure 4C shows the bar graph comparing the relative size of the organoid cultured by HC-3 culture medium and Laura culture medium. [Figure 5] Figure 5A shows the results of different drug sensitivity tests for hepatocellular carcinoma organoids that are cultured using the culture medium for hepatocellular carcinoma organoids of the present invention.Figure 5A shows the photo of organoid growth without drug treatment and the photo of organoid growth after 5 days of drug treatment.Figure 5B shows the bar graph of the inhibition rate of the growth of hepatocellular carcinoma organoids that different concentrations of test drugs cause. DETAILED DESCRIPTION OF THE INVENTION

[0023] For a better understanding of the present invention, the following further description is provided in conjunction with embodiments and drawings. The following examples are presented for illustrative purposes only, and are not intended to limit the present invention.

[0024] (Preparation example of MST1 / 2 kinase inhibitor) As used herein, an MST1 / 2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1 / 2 signaling. Generally, an MST1 / 2 kinase inhibitor reduces the activity of MST1 / 2 kinase, for example, by binding to MST1 / 2 kinase. Because MST1 and MST2 have similar structures, an MST1 / 2 kinase inhibitor may be, for example, a compound that binds to MST1 or MST2 and reduces its activity.

[0025] 1. Preparation of MST1 / 2 kinase inhibitor compound 1 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzsulfamide 1 [ka]

[0026] Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): 2-Amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) and methanol (30 ml) were added to a round-bottom flask, followed by the dropwise addition of thionyl chloride (1.2 ml) in an ice bath. The reaction mixture was allowed to react overnight at 85°C. After completion of the reaction, the mixture was evaporated under reduced pressure to dry the solvent, and the resulting white solid was used directly in the next step.

[0027] Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (A3): Methyl 2-amino-2-(2,6-difluorophenyl)acetate (2 g) was added to a round-bottom flask, followed by acetone (30 ml) and potassium carbonate (2.2 g). The mixture was then cooled to -10°C in an ice-salt bath, and a solution of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone was slowly added. The reaction mixture was stirred overnight at room temperature. After completion of the reaction, the reaction mixture was filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by pressurized silica gel column chromatography to give compound A3. LC / MS: M+H 359.0.

[0028] 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 mixture was stirred at 60°C for 2 hours. After completion of the reaction, the reaction mixture was evaporated under reduced pressure to dryness. The resulting mixture was neutralized to alkaline with saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to give the crude product. The crude product was washed with diethyl ether to give compound A4. LC / MS: M+H 297.0.

[0029] 2-Chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (A5): A round-bottom flask was charged with 2-chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (2 g) and N,N-dimethylacetamide (10 ml) and cooled to -35°C. Iodomethane (0.9 ml) and sodium hydride (615 mg) were then added, and the reaction mixture was stirred for 2 hours. After completion of the reaction, the reaction mixture was quenched with water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to give the crude product. The crude product was washed with diethyl ether to give compound A5. LC / MS: M+H 325.0.

[0030] 4-((7-(2,6-Difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzsulfamide (1): To a round-bottom flask was added 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (100 mg), sulfanilamide (53 mg), p-toluenesulfonic acid (53 mg), and sec-butanol (5 ml). The reaction was stirred at 120° C. overnight. After completion of the reaction, the reaction mixture was filtered and washed with methanol and diethyl ether to give compound 1. LC / MS: M+H 461.1.

[0031] 2. Preparation of Other MST1 / 2 Inhibitor Compounds of the Invention Other MST1 / 2 inhibitor compounds of the present invention were synthesized in a similar manner to compound 1, and their structures and mass spectral data are shown in the table below.

[0032] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0033] Example 1. Effect of various factors added to culture medium for hepatocellular carcinoma organoids on the growth of hepatocellular carcinoma organoids (1) Preparation of culture medium for hepatocellular carcinoma organoids First, a basal medium containing an initial medium was prepared. The initial medium can be selected from the commonly used DMEM / F12, DMEM, F12, or RPMI-1640. In this embodiment, the formulation of the basal medium is DMEM / F12 medium (purchased from Corning) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (volume / volume), commercially available concentration is 50 mg / mL).

[0034] Different types of additives (see Table 1) were added to the basal medium to prepare culture media for hepatocellular carcinoma organoids containing different components.

[0035] (2) Isolation and processing of primary hepatoma cells 1. Sample Selection Tissue samples of solid tumors from liver cancer (intraoperatively) were collected from patients by specialized medical personnel at specialized medical institutions, and all patients signed informed consent. 3 The intraoperative samples were stored and transported using a commercially available tissue preservation solution (manufacturer: Miltenyi Biotec).

[0036] 2. Material Preparation After surface sterilization, 15 mL sterilized centrifuge tubes, pipettors, 10 mL pipettes, and sterilized pipette tips were placed on an ultra-clean workbench and irradiated with UV light for 30 minutes. The basal medium was removed from a 4°C refrigerator 30 minutes before use, and the tissue digestion solution was removed from a -20°C refrigerator 30 minutes before use.

[0037] Tissue digestion solution: 1640 medium (Corning, 10-040-CVR), collagenase II (2 mg / mL), collagenase IV (2 mg / mL), DNase (50 U / mL), hyaluronidase (0.75 mg / mL), calcium chloride (3.3 mM), BSA (10 mg / mL).

[0038] The above-mentioned collagenase II, collagenase IV, DNase and hyaluronidase were all purchased from Sigma Corporation, calcium chloride was purchased from Sangon Biotech (Shanghai) Co., Ltd., and BSA was purchased from Biofroxx Corporation.

[0039] 3. Sample Isolation 3.1. The tissue sample was transferred from the ultraclean workbench to a culture dish, and blood-contaminated tissue was removed. The tissue sample was washed twice with basal medium, then transferred to another culture dish and cut into 1 × 1 × 1 mm pieces with a sterile scalpel. 3 The tissue was mechanically cut into tissue blocks of about 10 mm in size.

[0040] 3.2. The cut intraoperative tissue was aspirated into a 15 mL centrifuge tube, to which 5 mL of basal medium was added, mixed thoroughly, and then centrifuged at 1500 rpm for 4 minutes.

[0041] 3.3. The supernatant was discarded, and a 1:3 mixture of basal medium and tissue digestion solution (approximately 10 mL of tissue digestion solution was added to 1 g of tumor tissue). The samples were labeled and numbered, sealed with sealing film, and then digested in a shaker (Zhichu Instrument ZQLY-180N) at 37°C and 300 rpm. Completion of digestion was determined by observing every 30 minutes whether or not visible particles were present.

[0042] 3.4. After digestion was complete, the undigested tissue mass was filtered through a 100 μM filter mesh. The tissue mass on the filter mesh was washed into a centrifuge tube with basal medium to reduce cell loss. The resulting mixture was centrifuged at 25°C, 1500 rpm, for 4 minutes.

[0043] 3.5. The supernatant was discarded and the resulting material was observed to determine the presence of blood cells. If blood cells were present, 8 mL of blood cell lysis solution (purchased from Sigma) was added to the resulting material, mixed thoroughly, and allowed to lyse at 4°C for 20 minutes, with one inversion during this process. The resulting material was centrifuged at 25°C, 1500 rpm, and 4 minutes.

[0044] 3.6. The supernatant was discarded, 2 mL of basal medium was added, and the cells were resuspended and stored.

[0045] 4. Cell Counting and Processing 4.1 Microscopic observation: A small amount of resuspended cells was plated on a culture dish, and the density and morphology of cancer cells were observed under a microscope (CNOPTEC, BDS400).

[0046] 4.2 Counting viable cells: 12 μL of the resuspended cell suspension was thoroughly mixed with 12 μL of trypan blue staining solution (manufacturer: Sangon Biotech (Shanghai) Co., Ltd.), and 20 μL of the mixture was added to a cell counting plate (manufacturer: Countstar, specification: 50 cells / box). Using a cell counter (Countstar, IC1000), the percentage of viable large cells (cells larger than 10 μm) was calculated as follows: viable cell number / total cell number × 100%.

[0047] (3) Cultivation of hepatocellular carcinoma organoids The primary hepatoma cells obtained in the above steps were resuspended in pre-chilled DMEM / F12 and counted. 6 cells / mL~10×10 6The cells were diluted to 1000 cells / mL, and an equal volume of Matrigel matrix gel (Corning) was added to 400 μL of the diluted cell suspension and mixed gently. The mixture was then seeded into a 96-well plate at 5 μL / well. The plate was placed in an incubator for 30 minutes until the Matrigel solidified completely. The culture medium listed in Table 1, which had been warmed to room temperature, was then added. The culture medium was changed every 3 days for expansion and culture. After 7 days, the cultured organoids were photographed, and their diameters were measured and counted to compare the promoting effects of various factors on the growth of hepatocellular carcinoma organoids. A basal medium containing no additives was used as an experimental control. The experimental results are shown in Table 1.

[0048] [Table 3]

[0049] Here, "+" indicates that, compared to the basal medium, the medium supplemented with the additive(s) has the effect of promoting the growth of hepatocellular carcinoma organoids isolated from liver cancer tissue in at least two cases; "-" indicates that the medium supplemented with the additive(s) has the effect of inhibiting the growth of hepatocellular carcinoma organoids isolated from liver cancer tissue in at least one case; and "○" indicates that the medium supplemented with the additive(s) has no significant effect on the growth of hepatocellular carcinoma organoids isolated from liver cancer tissue in at least two cases.

[0050] According to the above results, factors including B27, hepatocyte growth factor (HGF), ITS cell culture additive, Y27632, dexamethasone, neuregulin-1 (NRG1), insulin, epidermal growth factor (EGF), GlutaMAX, Compound 1, and non-essential amino acids were selected for further culture experiments.

[0051] Example 2. Effect of different concentrations of factors added to the culture medium on the growth of hepatocellular carcinoma organoids According to the method described in section (2) of Example 1, primary liver cancer cells were obtained from intraoperative tissue samples (numbers GL-003 and GL-004), and organoids were cultured using the culture medium formulation shown in Table 2 below.

[0052] [Table 4]

[0053] When using the culture medium for formulation 1, 200 μL of the prepared B27 was added to the 96-well plate containing the organoids in the medium for formulation 1, so that the final concentrations of B27 were 1:25, 1:50, and 1:100, respectively. Blank control (BC) wells were also set up using the medium for formulation 1. The final concentrations of other factors added to this series of culture media were the same as those in the HC-3 culture medium. The following experiments for formulations 1 to 11 were performed in the same manner, so details are not provided.

[0054] When using the culture medium of Formula 2, 200 μL of the prepared HGF was added to each well of the 96-well plate containing the organoids, so that the final concentrations of HGF were 1 ng / mL, 5 ng / mL, and 25 ng / mL, respectively. Blank control (BC) wells were also prepared using the medium of Formula 2.

[0055] When using the culture medium of Formula 3, 200 μL of the prepared ITS cell culture additive was added to the 96-well plate seeded with organoids in addition to the medium of Formula 3 so that the final concentrations of the ITS cell culture additive were 1:300, 1:100, and 1:30, respectively. Blank control (BC) wells were also set up using the medium of Formula 3.

[0056] When using the culture medium of Formula 4, 200 μL of the prepared Y27632 was added to each well of the 96-well plate containing the organoids, so that the final concentrations of Y27632 were 3 μM, 10 μM, and 30 μM, respectively. Blank control (BC) wells were also set up using the medium of Formula 4.

[0057] When using the culture medium of Formula 5, 200 μL of the prepared dexamethasone was added to each well of the 96-well plate containing the organoids, so that the final dexamethasone concentrations were 0.01 μM, 0.1 μM, and 1 μM, respectively. Blank control (BC) wells were also prepared using the medium of Formula 5.

[0058] When using the culture medium of Formula 6, 200 μL of the prepared NRG1 was added to each well of the 96-well plate containing the organoids, so that the final concentrations of NRG1 were 1 ng / mL, 5 ng / mL, and 25 ng / mL, respectively. Blank control (BC) wells were also set up using the medium of Formula 6.

[0059] When using the culture medium of Formula 7, 200 μL of prepared insulin was added per well to the 96-well plate seeded with organoids, so that the final insulin concentrations were 1 μg / mL, 3 μg / mL, and 10 μg / mL, respectively. Blank control (BC) wells were also set up using the medium of Formula 7.

[0060] When using the culture medium of Formula 8, 200 μL of the prepared EGF was added to each well of the 96-well plate containing the organoids, so that the final concentrations of EGF were 2 ng / mL, 6 ng / mL, and 18 ng / mL, respectively. Blank control (BC) wells were also set up using the medium of Formula 8.

[0061] When using Formula 9 culture medium, 200 μL of the prepared GlutaMAX was added to each well of the 96-well plate containing the organoids, resulting in final GlutaMAX concentrations of 1:300, 1:100, and 1:30, respectively. Blank control (BC) wells were also set up using Formula 9 medium.

[0062] When using the culture medium of Formula 10, 200 μL of the prepared Compound 1 was added to the 96-well plate inoculated with organoids in addition to the medium of Formula 10 so that the final concentrations of Compound 1 were 2.5 μM, 5 μM, and 10 μM, respectively. Blank control (BC) wells were set up using the medium of Formula 10.

[0063] When using the culture medium of Formula 11, 200 μL of the prepared non-essential amino acids were added to each well of the 96-well plate containing the organoids, so that the final concentrations of the non-essential amino acids were 50 μM, 100 μM, and 200 μM, respectively. Blank control (BC) wells were also prepared using the medium of Formula 11.

[0064] After 10 days, the cultured organoids were photographed, and their diameters were measured and tabulated to compare the promoting effects of various concentrations of factors on the growth of HCC organoids. Data collected from the two samples are summarized and shown in Figures 1A-1K. In Figures 1A-1K, the ratios represent the ratio of the diameter of organoids cultured for 10 days in each culture medium to the diameter of organoids cultured for 10 days in the corresponding culture medium in the BC wells. A ratio greater than 1 indicates that the culture medium containing a different concentration of factor or small molecule compound has a better growth-promoting effect than the culture medium in the blank control wells. A ratio less than 1 indicates that the culture medium containing a different concentration of factor or small molecule compound has a lesser growth-promoting effect than the culture medium in the blank control wells.

[0065] According to the results of Figures 1A to 1K, the volume concentration of B27 is preferably 1:25 to 1:100, the amount of hepatocyte growth factor is preferably 1 ng / mL to 25 ng / mL, the volume concentration of ITS cell culture additive is preferably 1:30 to 1:300, the amount of Y27632 is preferably 3 μM to 30 μM, the amount of dexamethasone is preferably 0.1 μM to 1 μM, the amount of NRG1 is preferably 1 ng / mL to 25 ng / mL, the amount of insulin is preferably 1 μg / mL to 10 μg / mL, the amount of epidermal growth factor is preferably 2 ng / mL to 18 ng / mL, the volume concentration of GlutaMAX is preferably 1:30 to 1:300, the amount of MST1 / 2 kinase inhibitor compound 1 is preferably 2.5 μM to 10 μM, and the amount of non-essential amino acid is preferably 50 μM to 200 μM.

[0066] Example 3. Cultivation and identification of hepatocellular carcinoma organoids Primary hepatoma cells (GL-003, GL-006, GL-008, GL-013) obtained by the method described in Section (2) of Example 1 were resuspended in the HC-3 culture medium for hepatocellular carcinoma organoids of the present invention and counted. 6 cells / mL~10×10 6 The cells were diluted to 1000 cells / mL, and an equal volume of Matrigel matrix gel (Corning) was added to 400 μL of the diluted cell suspension and mixed gently. The mixture was then seeded into a 24-well plate at 50 μL per well. The plate was placed in an incubator for 30 minutes until the Matrigel solidified completely. Next, 500 μL of HC-3 culture medium for hepatocellular carcinoma organoids, which had been warmed to room temperature, was added per well. The culture medium was refreshed every 3 days for expansion and culture.

[0067] On days 10 to 15, the cultured hepatocellular carcinoma organoids were observed using a microscope (Invitrogen, EVOS M500). Figures 2A to 2D show photographs of hepatocellular carcinoma organoids cultured from samples GL-003 (day 10), GL-006 (day 10), GL-008 (day 12), and GL-013 (day 15) taken under a 4x objective lens. Hepatocellular carcinoma organoids appear as regular spheres with relatively smooth surfaces under the microscope.

[0068] Cultured hepatocellular carcinoma organoids were subjected to pathological and immunohistochemical characterization, and corresponding tissue samples were also sent for pathological and immunohistochemical characterization to compare the consistency of results between organoids and tissues.

[0069] Figure 3A shows the pathological and immunohistochemical results of hepatocellular carcinoma organoids cultured in vitro from sample GL-006, each photographed under a 20x objective lens. As shown in Figure 3A, the HE results indicate that the structural morphology of the organoids is that of cancer tissue. CK19, Heppar-1, and Ki67 are expressed, indicating that the sample is hepatocellular carcinoma. Figure 3B shows the pathological and immunohistochemical results of the corresponding tissue from GL-006 before culture, demonstrating that the diagnostic results of hepatocellular carcinoma organoids cultured using the HC-3 culture medium of the present invention are consistent with those of the hepatocellular carcinoma tissue before culture.

[0070] Example 4. Comparison of culture effects with existing culture media (1) Preparation of control culture medium The culture medium used in the literature (Laura et al., Nat Med. 2017, 23(12): 1424-1435) was prepared. This was prepared using Advanced DMEM / F12 medium (purchased from Corning) + 1:100 penicillin / streptomycin (purchased from Corning) + 1:100 GlutaMAX (purchased from Corning) + 10 mM HEPES (purchased from Thermo Fisher) + 1:50 B27 (purchased from Gibco) + 1:100 N2 (purchased from Gibco) + 1.25 mmol / L N-acetylcysteine ​​(purchased from MCE) + 10 mmol / L nicotinamide (purchased from MCE) + 10 nM gastrin (purchased from MCE) + 50 ng / ml epidermal growth factor (purchased from R&D) + 100 ng / ml fibroblast growth factor 10 (purchased from sino biological) + 25 ng / ml hepatocyte growth factor (purchased from R&D) + 10 μmol / L forskolin (purchased from MCE) + 5 μmol / L The formulation is A8301 (purchased from MCE) + 10 μmol / L Y27632 (purchased from MCE) + 3 nmol / L dexamethasone (purchased from MCE). Hereinafter, this will be referred to as Laura culture medium.

[0071] (2) Cultivation of hepatocellular carcinoma organoids According to the method described in Section (2) of Example 1, primary liver cancer cells were obtained from intraoperative tissue sample GL-018, and cultured in HC-3 culture medium and Laura culture medium, respectively, according to the method described in Example 3 to obtain organoids.

[0072] On day 25 of culture, cultured hepatocellular carcinoma organoids were observed under a microscope (Invitrogen, EVOS M500). Figures 4A and 4B are photographs taken under a 4x objective of organoids cultured in HC-3 culture medium and Laura culture medium, respectively. Figure 4C is a bar graph comparing the relative sizes of organoids cultured using the two culture media.

[0073] The results in Figures 4A to 4C show that, compared with Laura culture medium, HC-3 culture medium can significantly promote the expansion and culture of hepatocellular carcinoma organoids.

[0074] Example 5. Drug screening using hepatocellular carcinoma organoids obtained by expansion and growth using the culture medium of the present invention (1) Cultivation of hepatocellular carcinoma organoids According to the method described in section (2) of Example 1, primary hepatoma cells were isolated from a hepatoma surgical sample (GL-006) and cultured in HC-3 culture medium until the diameter of the hepatocellular carcinoma organoids exceeded 50 μm, and the organoids were subsequently used for drug screening.

[0075] (2) Preparation of drugs for screening Two concentration gradients of three drugs (bortezomib, aclarubicin and doxorubicin; all purchased from MCE) were prepared according to the table below and stored for use.

[0076] [Table 5]

[0077] (3) Drug load The prepared drugs were removed and placed at room temperature. The drugs were diluted 1000-fold with HC-3 culture medium before use. The organoids cultured according to step (1) were removed from the incubator, the culture medium was removed from the wells, and the drug-containing culture medium was slowly added to the wells along the wall. After adding the drugs, the surface of the 96-well plate was sterilized and then transferred to the incubator for further culture. After 5 days, the viability of the organoids was measured.

[0078] (4) Detection of organoid viability CellTiter-Glo luminescent reagent (purchased from Promega) was removed from a 4°C refrigerator, 10 mL of the reagent was added to the loading slot, and the 96-well test plate was removed from the incubator. 20 μL of CellTiter-Glo luminescent reagent was added to each well. After allowing to stand for 10 minutes and mixing thoroughly, the assay was performed using a multifunction microplate reader (Envision, Perkin Elmer).

[0079] (5) Data processing The inhibition rates of different drugs were calculated according to the following formula: drug inhibition rate (%) = 100% - (chemiluminescence value of drug-treated wells on day 5 / chemiluminescence value of drug-treated wells on day 0) / (chemiluminescence value of DMSO wells on day 5 / chemiluminescence value of DMSO wells on day 0) × 100%. The results are shown in Figure 5A and Figure 5B. Figure 5A shows photographs of organoid growth without drug treatment and after 5 days of drug treatment, taken under a 4x objective microscope (Invitrogen, EVOS M500). Figure 5B shows a bar graph of the inhibition rate of hepatocellular carcinoma organoid growth by different concentrations of the test drugs.

[0080] Figure 5A shows that organoids cultured using the culture medium for hepatocellular carcinoma organoids of the present invention grew well, and that the growth of organoids was significantly inhibited after treatment with bortezomib and aclarubicin. Figure 5B shows a bar graph of the inhibition rate of hepatocellular carcinoma organoid growth by three test drugs at different concentrations. Figure 5B shows that the data error between drug-treated groups is extremely small, indicating that when this system is used for drug screening, the data between replicate wells of the same drug are substantially consistent. Of the three antitumor drugs, bortezomib had a strong inhibitory effect on organoid growth at two concentrations, while the inhibitory effect of aclarubicin varied greatly depending on the concentration, and doxorubicin had no inhibitory effect on the growth of this hepatocellular carcinoma organoid line. This indicates that the efficacy and sensitivity of organoids derived from the same patient vary depending on the drug. Based on these results, the efficacy and effective dose of drugs used clinically in liver cancer patients can be determined. [Industrial Applicability]

[0081] The present invention provides a culture medium and a culture method for hepatocellular carcinoma organoid culture, and the cultured organoids can be used for drug efficacy evaluation and screening. Therefore, the present invention is suitable for industrial application.

[0082] Although the present invention has been described in detail in this specification, the present invention is not limited thereto, and those skilled in the art can make modifications according to the principles of the present invention. Therefore, it should be understood that any modifications made according to the principles of the present invention fall within the protection scope of the present invention.

Claims

1. A culture medium for hepatocellular carcinoma organoids, comprising: A culture medium comprising an MST1 / 2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, ITS cell culture additive, Y27632, dexamethasone, neuregulin-1, insulin, epidermal growth factor, GlutaMAX, and a non-essential amino acid, The MST1 / 2 kinase inhibitor has the formula (I): 【Chemistry 1】 (In the formula, R １ is C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and one to two independent R ６ aryl optionally substituted with one to two independent R ６ aryl C1-C6 alkyl optionally substituted with, and one to two independent R ６ heteroaryl optionally substituted with R ２ and R ３ are each independently selected from C1-C6 alkyl; R ４ and R ５ are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, hydroxylC1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, and C3-C6 heterocyclylC1-C6 alkyl; R ６ is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl, or a pharmaceutically acceptable salt or solvate thereof; the concentration of the MST1 / 2 kinase inhibitor is 2.5 μM to 10 μM; the volume ratio of the B27 or N2 cell culture additive in the culture medium is 1:25 to 1:100; the concentration of the hepatocyte growth factor is 1 ng / mL to 25 ng / mL; the volume ratio of the ITS cell culture additive in the culture medium is 1:30 to 1:300; the concentration of Y27632 is 3 μM to 30 μM; the concentration of dexamethasone is 0.1 μM to 1 μM; the concentration of neuregulin-1 is 1 ng / mL to 25 ng / mL; the concentration of the insulin is 1 μg / mL to 10 μg / mL; the concentration of the epidermal growth factor is 2 ng / mL to 18 ng / mL; the volume ratio of GlutaMAX in the culture medium is 1:30 to 1:300; A culture medium in which the non-essential amino acids are one or more selected from the group consisting of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine, and have a concentration of 50 μM to 200 μM.

2. R １ is C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and one to two independent R ６ phenyl optionally substituted with one to two independent R ６ naphthyl optionally substituted with one to two independent R ６ phenylmethyl optionally substituted with, and one to two independent R ６ thienyl optionally substituted with R ２ and R ３ are each independently selected from C1-C3 alkyl; R ４ and R ５ are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, hydroxylC1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, piperidylC1-C6 alkyl, and tetrahydropyranylC1-C6 alkyl; R ６ is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl; The culture medium of claim 1 .

3. The MST1 / 2 kinase inhibitor is represented by formula (Ia): 【Chemistry 2】 (In the formula, R １ is C1-C6 alkyl, 1 to 2 independent R ６ phenyl optionally substituted with one to two independent R ６ thienyl optionally substituted with, and one to two independent R ６ phenylmethyl optionally substituted with R ５ is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; R ６ is independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl), or a pharmaceutically acceptable salt or solvate thereof.

4. R １ There are one to two independent R ６ is phenyl optionally substituted with R ５ is hydrogen, R ６ The culture medium of claim 3 , wherein is fluoro, methyl, or trifluoromethyl.

5. The culture medium according to claim 1, wherein the MST1 / 2 kinase inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts thereof: Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5

6. an initial medium selected from the group consisting of DMEM / F12, DMEM, F12, or RPMI-1640; one or more antibiotics selected from the group consisting of streptomycin / penicillin, amphotericin B, and primocin; The culture medium according to any one of claims 1 to 5, further comprising:

7. The culture medium according to any one of claims 1 to 5, characterized in that it does not contain a Wnt agonist, an R-spondin family protein, a Noggin protein, a BMP inhibitor or fibroblast growth factor 10.

8. A method for culturing hepatocellular carcinoma organoids, comprising the steps of: (1) isolating a sample from solid liver cancer tissue to obtain primary liver cancer cells; (2) A culture medium for hepatocellular carcinoma organoids according to any one of claims 1 to 7 is prepared, and the primary hepatocellular carcinoma cells obtained in step (1) are used to culture the organoids on the culture medium for hepatocellular carcinoma organoids according to any one of claims 1 to 7.

9. A method for evaluating or screening a therapeutic agent for liver cancer, comprising the steps of: (1) A step of culturing hepatocellular carcinoma organoids using the method for culturing hepatocellular carcinoma organoids according to claim 8; (2) selecting a test drug and diluting it to a required concentration gradient; (3) adding the diluted drug to the organoid obtained in step (1); (4) A step of detecting the size or viability of the organoid.

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