A culture medium for liver cancer organoid culture, and its culture method and application

Abstract

The present invention relates to a culture medium for culturing liver cancer organoids, 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 also relates to methods for culturing liver cancer organoids and their uses. By using the liver cancer organoid culture medium of the present invention, efficient and rapid expansion of liver cancer organoids can be achieved. The expanded organoids retain the patient's pathological characteristics, improving the culture success rate and expansion rate of liver cancer organoids, and providing a research basis for personalized treatment of patients.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a culture medium for culturing liver cancer organoids, a method for culturing liver cancer organoids using the culture medium, and an application of the culture medium in evaluating and screening drug efficacy. Background Art

[0002] In recent years, adjuvant chemotherapy after liver cancer surgery has gradually gained the attention and recognition of clinicians as a new adjuvant treatment method, including postoperative TACE treatment, oral drug treatment, etc. However, due to the lack of standardized chemotherapy regimens, conventional chemotherapy is based on experience, ignoring individual differences, and has a certain degree of blindness. Therefore, the effect has been poor, and the efficacy of single drugs and combination drugs 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 emerging targeted drugs have reduced toxic side effects to a certain extent, the number is too small, the treatment cost is expensive, and the efficacy 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, precise chemotherapy cannot be achieved. Therefore, matching the in vitro drug sensitivity results of liver cancer with the clinical in vivo response has become the key to treatment.

[0003] Traditional clinical drug sensitivity testing mostly uses two-dimensional cell culture. However, these cells only simulate tissue physiological conditions to a limited extent and lack authentic in vivo tissue structure, which can easily lead to low differentiation levels and loss of cellular physiological function. This, in turn, makes the experimental results difficult to predict actual clinical outcomes. Organoids, a three-dimensional (3D) cell culture, are primarily derived from human embryonic stem cells, induced pluripotent stem cells, and adult stem cells with the ability to differentiate. Endogenous tissue stem cells exist in various tissues and organs and play a vital role in maintaining the functional morphology of each organ. Under certain inductive conditions in vitro, these stem cells can self-organize into miniature structures measuring only a few millimeters in diameter. Tumor organoids are miniature 3D tumor cell models cultured in the laboratory using primary tumors taken from patients. Tumor organoids closely mimic the characteristics of the original tumor tissue, preserving the heterogeneity between individual tumors. They can be used for functional testing, such as high-throughput drug screening and personalized precision medicine.

[0004] Currently, liver cancer organoid culture methods often rely on basal culture media (DMEM or DMEM / F12), R-spondin-1, Noggin, and other expensive protein factors, resulting in high costs. Furthermore, the technology is complex and technically challenging, limiting its large-scale commercial application. Therefore, there is a need to develop a low-cost, simple, and highly successful organoid culture method and culture medium. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a culture medium and a culture method for rapidly expanding liver cancer organoids in vitro.

[0006] One aspect of the present invention is to provide a culture medium for liver cancer 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 non-essential amino acids. The MST1 / 2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.

[0007]

[0008] in,

[0009] 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 by 1-2 independently R6, aryl C1-C6 alkyl (e.g., benzyl, etc.) and heteroaryl (e.g., thienyl, etc.);

[0010] R2 and R3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

[0011] 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 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, and C3-C6 heterocyclylC1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyranyl, etc.);

[0012] 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).

[0013] In a preferred embodiment, the MST1 / 2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,

[0014]

[0015] in,

[0016] R1 is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R6, thienyl optionally substituted by 1-2 independently R6, and benzyl optionally substituted by 1-2 independently R6, more preferably phenyl optionally substituted by 1-2 independently R6;

[0017] R5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R5 is more preferably hydrogen;

[0018] R6 are each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R6 is more preferably fluorine, methyl or trifluoromethyl.

[0019] Preferably, the MST1 / 2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.

[0020]

[0021]

[0022]

[0023]

[0024]

[0025] Most preferably, the MST1 / 2 kinase inhibitor of the present invention is Compound 1.

[0026] In an embodiment of the present invention, the content of each component in the culture medium of the present invention satisfies any one, multiple or all of the following:

[0027] (1) The concentration of MST1 / 2 kinase inhibitor is preferably 2.5-10 μM;

[0028] (2) The volume ratio of B27 or N2 cell culture supplement to culture medium is 1:25 to 1:100;

[0029] (3) The concentration of hepatocyte growth factor is preferably 1 to 25 ng / mL;

[0030] (4) The volume ratio of ITS cell culture additive to culture medium is preferably 1:30 to 1:300;

[0031] (5) The concentration of Y27632 is preferably 3 to 30 μM;

[0032] (6) The concentration of dexamethasone is preferably 0.1 to 1 μM;

[0033] (7) The concentration of neuregulin 1 is preferably 1 to 25 ng / mL;

[0034] (8) The concentration of insulin is preferably 1 to 10 μg / mL;

[0035] (9) The concentration of epidermal growth factor is preferably 2 to 18 ng / mL;

[0036] (10) The volume ratio of GlutaMAX to culture medium is preferably 1:30 to 1:300;

[0037] (11) The non-essential amino acid is one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine. The concentration of the non-essential amino acid is preferably 50 to 200 μM.

[0038] In an embodiment of the present invention, the culture medium further contains 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.

[0039] In a preferred embodiment, when the antibiotic is selected from streptomycin / penicillin, the concentration of streptomycin is in the range of 25 to 400 μg / mL, and the concentration of penicillin is in the range of 25 to 400 U / mL. When the antibiotic is selected from amphotericin B, the concentration range is 0.25 to 4 μg / mL. When the antibiotic is selected from Primocin, the concentration range is 25 to 400 μg / mL.

[0040] The present invention also provides a method for culturing liver cancer organoids. In the method for culturing liver cancer organoids of the present invention, liver cancer organoids are cultured using the liver cancer organoid culture medium of the present invention.

[0041] The liver cancer organoid culture method of the present invention comprises the following steps.

[0042] 1. Isolate samples from solid liver cancer tissue to obtain primary liver cancer cells. This process includes the following steps:

[0043] (1) Isolate liver cancer tissue samples, add basal culture medium and tissue digestion solution at a ratio of 1:3 (approximately 10 mL of tissue digestion solution is added per 1 g of tumor tissue), and place in a constant temperature shaker for digestion at a temperature of 4-37°C, a shaker speed of 200-350 rpm, and a digestion time of 3-6 hours;

[0044] (2) After digestion, centrifuge and discard the supernatant. The centrifugation speed is 1200-1600 rpm and the centrifugation time is 2-6 minutes.

[0045] The basal culture medium 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 includes 1640 culture medium, collagenase II (1-2 mg / mL), collagenase IV (1-2 mg / mL), DNase (50-100 U / mL), hyaluronidase (0.5-1 mg / mL), calcium chloride (1-5 mM), and bovine serum albumin (BSA) (5-10 mg / mL).

[0046] 2. Prepare the liver cancer organoid culture medium of the present invention and culture the primary liver cancer cells obtained in the above steps.

[0047] The primary liver cancer cells obtained in step 1 were resuspended in the liver cancer organoid culture medium of the present invention and counted, and the cell density was diluted to 5-10×10 6 / mL, take out the diluted cell suspension and add it to an equal volume of Matrigel matrix gel to mix well, then inoculate the mixture into a multi-well plate, put the inoculated multi-well plate into the incubator for 30-60 minutes, wait for Matrigel to completely solidify, and then add liver cancer organoid culture medium for expansion culture.

[0048] The present invention also provides a method for evaluating or screening drugs for treating liver cancer, comprising the following steps:

[0049] (1) Cultivating liver cancer organoids using the liver cancer organoid cultivation method of the present invention;

[0050] (2) Select the drug to be tested and dilute it according to the required concentration gradient;

[0051] (3) adding the diluted drug to the organoids cultured in (1);

[0052] (4) Conduct organoid size or organoid viability tests.

[0053] The beneficial effects of the present invention include:

[0054] (1) Improve the success rate of liver cancer organoid culture to over 90%;

[0055] (2) Ensure that the primary cultured liver cancer organoids in vitro can maintain the patient's pathological characteristics;

[0056] (3) High amplification efficiency, capable of rapidly culturing liver cancer organoids, and the amplified liver cancer organoids can be continuously passaged;

[0057] (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, fibroblast growth factor 10 (FGF10) and other factors;

[0058] (5) The technology described above can produce a large number of liver cancer organoids, which are suitable for high-throughput screening of candidate compounds and providing high-throughput in vitro drug sensitivity functional testing for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figures 1A-1K This is a graph showing the effects of different concentrations of factors added to the hepatic cancer organoid culture medium of the present invention on the proliferation of hepatic cancer organoids.

[0060] Figures 2A-2D This is a photograph of liver cancer organoids cultured using the liver cancer organoid culture medium of the present invention observed under a microscope. Figure 2A A photograph showing organoids obtained from sample GL-003 culture after 10 days; Figure 2B A photograph showing organoids obtained from sample GL-006 culture after 10 days; Figure 2C A photograph showing organoids obtained from sample GL-008 culture after 12 days; Figure 2D A photograph showing organoids obtained from sample GL-013 after 15 days of culture.

[0061] Figure 3A Results of pathological and immunohistochemical identification of liver cancer organoids obtained by culturing sample GL-006 using the liver cancer organoid culture medium of the present invention; Figure 3B These are the results of pathological and immunohistochemical identification of sample GL-006 tissue.

[0062] FIG4 is a comparison of the results of culturing liver cancer organoids using the liver cancer organoid culture medium of the present invention and the existing culture medium, wherein Figure 4A A photograph showing the cultured cells after 25 days using the HC-3 medium of the present invention; Figure 4B The photograph shows the cultured cells after 25 days using Laura's medium; Figure 4C A bar graph comparing the relative sizes of organoids cultured in HC-3 medium and Laura medium.

[0063] FIG5 shows the results of different drug sensitivity tests of liver cancer organoids obtained by culturing liver cancer organoid culture medium of the present invention, wherein Figure 5A Photos showing organoid growth without drug treatment and organoid growth 5 days after drug treatment; Figure 5B A bar graph showing the inhibition rate of liver cancer organoid growth at different concentrations of the test drugs. DETAILED DESCRIPTION

[0064] For a better understanding of the present invention, the present invention will be further described below in conjunction with the embodiments and drawings. The following embodiments are merely illustrative of the present invention and are not intended to limit the present invention.

[0065] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0066] 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, for example, binds to and reduces the activity of MST1 / 2 kinases. Due to the structural similarities between MST1 and MST2, an MST1 / 2 kinase inhibitor may also be a compound that binds to and reduces the activity of either MST1 or MST2.

[0067] 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)benzenesulfonamide 1

[0068]

[0069] Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): Add 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) to a round-bottom flask, followed by methanol (30 mL). Thionyl chloride (1.2 mL) was then added dropwise under an ice bath. The reaction was allowed to react at 85°C overnight. After completion of the reaction, the solvent was evaporated under reduced pressure to yield a white solid, which was used directly in the next step.

[0070] Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (A3): To a round-bottom flask, add methyl 2-amino-2-(2,6-difluorophenyl)acetate (2 g), followed by acetone (30 ml) and potassium carbonate (2.2 g). The mixture was then cooled to -10°C in an ice-salt bath. An acetone solution of 2,4-dichloro-5-nitropyrimidine (3.1 g) was then slowly added. The reaction mixture was stirred at room temperature overnight. After completion of the reaction, the 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 yield compound A3. LC / MS: M+H 359.0.

[0071] 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): To a round-bottom flask, add methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g), followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction system was stirred at 60°C for two hours. After completion of the reaction, the solvent was evaporated under reduced pressure, and the resulting product was neutralized with saturated sodium bicarbonate until alkaline. Extraction was performed with ethyl acetate, and 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 obtain the crude product. The crude product was washed with diethyl ether to obtain compound A4. LC / MS: M+H 297.0.

[0072] 2-Chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (A5): 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (2 g) and N,N-dimethylacetamide (10 mL) were added to a round-bottom flask. The mixture was cooled to -35°C, and iodomethane (0.9 mL) was added, followed by sodium hydride (615 mg). The reaction system was stirred for two hours. After completion of the reaction, the mixture was quenched with water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, then dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness 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.

[0073] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzenesulfonamide (1): 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (100 mg), sulfonamide (53 mg), p-toluenesulfonic acid (53 mg), and sec-butanol (5 mL) were added to a round-bottom flask. The reaction system was stirred at 120°C overnight. After completion of the reaction, the mixture was filtered and washed with methanol and diethyl ether to obtain compound 1. LC / MS: M+H 461.1.

[0074] 2. Preparation of other MST1 / 2 inhibitor compounds of the present invention

[0075] Other MST1 / 2 inhibitor compounds of the present invention were synthesized in a similar manner to compound 1, and their structures and mass spectrometry data are shown in the following table.

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] Example 1 Effects of various added factors in liver cancer organoid culture medium on liver cancer organoid proliferation

[0082] (1) Preparation of liver cancer organoid culture medium

[0083] First, a basal medium containing an initial culture medium is prepared. The initial culture medium can be selected from DMEM / F12, DMEM, F12, or RPMI-1640, which are commonly used in the art. In this embodiment, the basal medium is formulated as follows: DMEM / F12 medium (purchased from Corning) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available at a concentration of 50 mg / ml).

[0084] Different types of additives (see Table 1) were added to the basal culture medium to prepare liver cancer organoid culture medium containing different additives.

[0085] (2) Isolation and processing of primary liver cancer cells

[0086] 1. Sample selection

[0087] Liver cancer solid tumor tissue samples (intraoperative) were obtained from patients by professional medical staff of professional medical institutions, and all patients signed informed consent. 3 Commercial tissue preservation solution (manufacturer: Miltenyi Biotec) was used for storage and transportation.

[0088] 2. Material Preparation

[0089] After disinfecting the surfaces of sterile 15mL centrifuge tubes, pipettes, 10mL pipettes, and sterile pipette tips, place them in a clean bench and irradiate with UV light for 30 minutes. Remove the basal culture medium from the 4°C refrigerator 30 minutes in advance, and remove the tissue digestion solution from the -20°C refrigerator 30 minutes in advance.

[0090] Tissue digestion solution formula: 1640 culture 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), and bovine serum albumin (BSA) (10 mg / mL).

[0091] The above-mentioned collagenase II, collagenase IV, DNA enzyme, and hyaluronidase were all purchased from Sigma; calcium chloride was purchased from Sangon Biotech (Shanghai) Co., Ltd.; and BSA was purchased from Biofroxx.

[0092] 3. Sample separation

[0093] 3.1 Take the tissue sample in the clean bench and place it in a culture dish. Remove the blood-stained tissue and rinse it twice with basal culture medium. Transfer the tissue to another culture dish and perform mechanical separation with a sterile scalpel to cut the tissue into 1*1*1mm blocks. 3 size;

[0094] 3.2 Aspirate the cut tissue into a 15 mL centrifuge tube, add 5 mL of basal culture medium, mix well, and centrifuge at 1500 rpm for 4 minutes;

[0095] 3.3 Discard the supernatant and add basal culture medium and tissue digestion solution in a 1:3 ratio (Note: the amount of tissue digestion solution added is approximately 10 mL for 1 g of tumor tissue). Label the sample name and number, seal with sealing film, and digest at 37°C in a shaker (Zhichu Instrument ZQLY-180N) at 300 rpm. Observe the digestion completion every 30 minutes, judging by the absence of visible particulate matter.

[0096] 3.4 After digestion is complete, filter the undigested tissue clumps through a 100 μm filter. Rinse the tissue clumps on the filter with basal culture medium into a centrifuge tube to reduce cell loss and centrifuge at 1500 rpm for 4 minutes at 25°C.

[0097] 3.5 Discard the supernatant and observe whether there are blood cells. If there are blood cells, add 8 mL of blood cell lysis buffer (purchased from Sigma), mix well, and lyse at 4°C for 20 minutes, inverting once during the process. Centrifuge at 1500 rpm at 25°C for 4 minutes.

[0098] 3.6 Discard the supernatant and add 2 mL of basal culture medium to resuspend the cells for later use.

[0099] 4. Cell Counting and Processing

[0100] 4.1 Microscopic observation: Pipette a small amount of resuspended cells and spread them flatly in a culture dish. Observe the density and morphology of cancer cells under a microscope (CNOPTEC, BDS400).

[0101] 4.2 Live cell counting: Take 12 μL of the resuspended cell suspension and 12 μL of trypan blue dye (manufacturer: Sangon Biotech (Shanghai) Co., Ltd.) and mix thoroughly. Then, take 20 μL and add it to a cell counting plate (manufacturer: Countstar, specification: 50 plates / box). Using a cell counter (Countstar, IC1000), calculate the percentage of live large cells (cell size >10 μm) = number of live cells / total number of cells*100%.

[0102] (3) Culture of liver cancer organoids

[0103] The primary liver cancer cells obtained in the above steps were resuspended in pre-cooled DMEM / F12 and counted, and the cell density was diluted to 5-10×10 6 / mL, take out 400μL of the diluted cell suspension and add it to an equal volume of Matrigel matrix gel (Corning) and mix gently, then inoculate the mixture into a 96-well plate at 5μL / well. Place the inoculated culture plate in the incubator for 30 minutes, wait for the Matrigel to completely solidify, and then add the culture medium shown in Table 1 that has been restored to room temperature in advance, and expand the culture by replacing the culture medium every three days. After 7 days, the cultured organoids were photographed, and the diameter of the organoids was measured and statistically analyzed to compare the promoting effect of each factor on the proliferation of liver cancer organoids. Among them, as an experimental control, a basal culture medium without any additives was used, and the experimental results are shown in Table 1.

[0104] Table 1 Additives in culture medium and their effects on promoting organoid proliferation

[0105]

[0106]

[0107] Among them, "+" indicates that compared with the basic culture medium, the culture medium with the addition of this additive has a proliferation-promoting effect on at least two of the liver cancer organoids isolated from liver cancer tissue; "-" indicates that the culture medium with the addition of this additive has an inhibitory effect on the proliferation of at least one of the liver cancer organoids isolated from liver cancer tissue; "○" indicates that the culture medium with the addition of this additive has no obvious effect on the proliferation of at least two of the liver cancer organoids isolated from liver cancer tissue.

[0108] Based on the above results, factors such as B27, hepatocyte growth factor (HGF), ITS cell culture supplement, Y27632, dexamethasone, neuregulin 1 (NRG1), insulin, epidermal growth factor (EGF), GlutaMAX, compound 1, and non-essential amino acids are planned to be selected for further culture experiments.

[0109] Example 2 Effects of different concentrations of culture medium additives on the proliferation of liver cancer organoids

[0110] Primary liver cancer cells were obtained from intraoperative tissue samples (numbered GL-003 and GL-004) according to the method of Example 1 (2), and organoid culture was performed using the culture medium formula in Table 2 below.

[0111] Table 2 Culture medium formula (concentration is final concentration)

[0112]

[0113]

[0114] When using medium from Recipe 1, add 200 μL of B27 prepared in Recipe 1 to each well of a 96-well plate seeded with organoids, achieving final B27 concentrations of 1:25, 1:50, and 1:100, respectively. Control wells (BC) are also set up using medium from Recipe 1. The final concentrations of other added factors in this series of media are the same as those in HC-3 medium. Experiments with Recipes 1-11 were performed in the same manner and are not further detailed here.

[0115] When using the culture medium of Formula 2, add 200 μL of prepared HGF to each well of the 96-well plate seeded with organoids based on Formula 2, with final HGF concentrations of 1 ng / mL, 5 ng / mL, and 25 ng / mL, respectively; and set up control wells (BC) using the culture medium of Formula 2.

[0116] When using the medium of Formula 3, add 200 μL of the prepared ITS cell culture supplement based on Formula 3 to each well of the 96-well plate seeded with organoids. The final concentrations of ITS cell culture supplement are 1:300, 1:100, and 1:30, respectively. Set up control wells (BC) using the medium of Formula 3.

[0117] When using the culture medium of Formula 4, 200 μL of the prepared Y27632 was added to each well of the 96-well plate seeded with organoids based on Formula 4. The final concentrations of Y27632 were 3 μM, 10 μM, and 30 μM, respectively. Control wells (BC) were set up using the culture medium of Formula 4.

[0118] When using the culture medium of Formula 5, 200 μL of prepared dexamethasone was added to each well of the 96-well plate seeded with organoids based on Formula 5, with final concentrations of dexamethasone of 0.01 μM, 0.1 μM, and 1 μM, respectively; and control wells (BC) were set up using the culture medium of Formula 5.

[0119] When using the culture medium of Formula 6, 200 μL of the prepared NRG1 was added to each well of the 96-well plate seeded with organoids based on Formula 6, with final NRG1 concentrations of 1 ng / mL, 5 ng / mL, and 25 ng / mL, respectively; and control wells (BC) were set up using the culture medium of Formula 6.

[0120] When using the culture medium of Formula 7, 200 μL of prepared insulin was added to each well of the 96-well plate seeded with organoids based on Formula 7, with final insulin concentrations of 1 μg / mL, 3 μg / mL, and 10 μg / mL, respectively; and control wells (BC) were set up using the culture medium of Formula 7.

[0121] When using the culture medium of Formula 8, 200 μL of prepared EGF was added to each well of the 96-well plate seeded with organoids based on Formula 8. The final concentrations of EGF were 2 ng / mL, 6 ng / mL, and 18 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 8.

[0122] When using the medium of Formula 9, add 200 μL of the prepared GlutaMAX based on Formula 9 to each well of the 96-well plate seeded with organoids. The final concentrations of GlutaMAX are 1:300, 1:100, and 1:30, respectively. Set up control wells (BC) using the medium of Formula 9.

[0123] When using the culture medium of Formula 10, 200 μL of compound 1 prepared on the basis of Formula 10 was added to each well of a 96-well plate seeded with organoids, with the final concentrations of compound 1 being 2.5 μM, 5 μM, and 10 μM, respectively; and control wells (BC) were set using the culture medium of Formula 10.

[0124] When using the culture medium of Formula 11, add 200 μL of the prepared non-essential amino acids based on Formula 12 to each well of a 96-well plate seeded with organoids. The final concentrations of the non-essential amino acids are 50 μM, 100 μM, and 200 μM, respectively. Set up control wells (BC) using the culture medium of Formula 12.

[0125] After 10 days, the cultured organoids were photographed, and the diameter of the organoids was measured and statistically analyzed to compare the promoting effect of each factor concentration on the proliferation of liver cancer organoids. The data collected from the two samples are summarized and shown in Figures 1A to 1K . Figures 1A to 1K The ratio is the ratio of the organoid diameter obtained after 10 days of culture using each medium to the diameter of the organoid obtained after 10 days of culture using the corresponding BC control well. 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.

[0126] according to Figures 1A to 1KAccording to the results, the volume concentration of B27 is preferably 1:25 to 1:100; the content of hepatocyte growth factor is preferably 1 to 25 ng / mL; the volume concentration of ITS cell culture additive is preferably 1:30 to 1:300; the content of Y27632 is preferably 3 to 30 μM; the content of dexamethasone is preferably 0.1 to 1 μM; the content of neuregulin 1 is preferably 1 to 25 ng / mL; the content of insulin is preferably 1 to 10 μg / mL; the content of epidermal growth factor is preferably 2 to 18 ng / mL; the volume concentration of GlutaMAX is preferably 1:30 to 1:300; the content of MST1 / 2 kinase inhibitor compound 1 is preferably 2.5 to 10 μM; and the content of non-essential amino acids is preferably 50 to 200 μM.

[0127] Example 3 Liver cancer organoid culture and identification

[0128] The primary liver cancer cells (GL-003, GL-006, GL-008, GL-013) obtained according to the method described in Example 1 (2) were resuspended in the liver cancer organoid culture medium HC-3 of the present invention and counted. The cell density was diluted to 5-10×10 6 Cells were diluted to a volume of 400 μL / mL. 400 μL of the diluted cell suspension was added to an equal volume of Matrigel (Corning) and gently mixed. The mixture was then seeded into a 24-well plate at a rate of 50 μL / well. The seeded plate was placed in an incubator for 30 minutes to allow the Matrigel to completely solidify. HC-3, a liver cancer organoid culture medium previously brought to room temperature, was then added at a rate of 500 μL per well. The culture medium was replaced every three days for expansion.

[0129] On days 10-15, the cultured liver cancer organoids were observed using a microscope (Invitrogen EVOS M500). Figures 2A-2D The images above are taken using a 4x objective lens. The liver cancer organoids obtained after culture were cultured using samples GL-003 (Day 10), GL-006 (Day 10), GL-008 (Day 12), and GL-013 (Day 15). Under the microscope, the liver cancer organoids appear spherical with a smooth surface.

[0130] The cultured liver cancer organoids were subjected to pathological and immunohistochemical identification, and the corresponding tissue samples were sent for pathological and immunohistochemical identification to compare the consistency of the organoid and tissue results.

[0131] Figure 3A The results of pathological and immunohistochemical identification of liver cancer organoids obtained by in vitro culture of sample GL-006 are pictures taken under a 20x objective lens. Figure 3AAs shown, HE results showed that the structural morphology of the organoid was that of cancer tissue; the expression of CK19, Heppar-1, and Ki67 suggested that the sample was liver cancer. Figure 3B 1 are the pathological and immunohistochemical results of the corresponding tissue of GL-006 before culture. The results show that the liver cancer organoids cultured using the culture medium HC-3 of the present invention are consistent with the diagnostic results of the liver cancer tissue before culture.

[0132] Comparison of Example 4 with existing culture medium culture effects

[0133] (1) Preparation of control culture medium

[0134] The culture medium used in the preparation literature (Laura et al., Nat Med. 2017, 23(12): 1424-1435) was prepared, and its formula was Advanced DMEM / F12 culture medium (purchased from Corning) + 1:100 Penicillin / Streptomycin (purchased from Corning) + 1:100 GlutaMAX (purchased from Corning) + 10 mM HEPES (purchased from Thermo Fisher Scientific) + 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 Company) + 25ng / ml hepatocyte growth factor (purchased from R&D Company) + 10μmol / L forskolin (purchased from MCE Company) + 5μmol / L A8301 (purchased from MCE Company) + 10μmol / L Y27632 (purchased from MCE Company) + 3nmol / L dexamethasone (purchased from MCE Company). Hereinafter referred to as Laura medium.

[0135] (2) Liver cancer organoid culture

[0136] Primary liver cancer cells were obtained from the intraoperative tissue sample GL-018 according to the method of Example 1 (2), and organoid culture was performed using HC-3 medium and Laura medium according to the method of Example 3, respectively.

[0137] On the 25th day of culture, the cultured liver cancer organoids were observed using a microscope (Invitrogen EVOS M500). Figure 4A and 4B These are photos of organoids cultured in HC-3 medium and Laura medium, taken under a 4x objective lens. Figure 4C This is a bar graph comparing the relative sizes of organoids cultured in two culture media.

[0138] according to Figures 4A-4C The results showed that compared with Laura medium, HC-3 medium can significantly promote the expansion and culture of liver cancer organoids.

[0139] Example 5: Liver cancer organoids expanded using the culture medium of the present invention for drug screening

[0140] (1) Liver cancer organoid culture

[0141] Primary liver cancer cells were isolated from a liver cancer intraoperative sample (GL-006) according to the method of Example 1 (2), and organoids were cultured using HC-3 medium. Drug screening was performed when the diameter of the liver cancer organoids exceeded 50 μm.

[0142] (2) Screening drug preparation

[0143] Three drugs (bortezomib, aclarubicin, doxorubicin; all purchased from MCE) with two concentration gradients were prepared according to the table below and stored for later use.

[0144] Table 3 Preparation of aclarubicin, doxorubicin, and bortezomib drug additives

[0145] Aclarubicin Preparation concentration (mM) 1 0.1 Doxorubicin Preparation concentration (mM) 1 0.1 Bortezomib Preparation concentration (mM) 1 0.1

[0146] (3) Dosing

[0147] Remove the prepared drug, place it at room temperature, and dilute it 1000-fold with HC-3 medium for later use. Remove the organoids cultured according to step (1) from the incubator, remove the medium in the culture wells, and slowly pour the medium containing the drug into the culture wells along the well walls. After the addition of the drug, disinfect the surface of the 96-well plate and move it to the incubator for further culture. After 5 days, measure the viability of the organoids.

[0148] (4) Organoid viability test

[0149] Take out the CellTiter-Glo luminescent reagent (purchased from Promega) from the 4°C refrigerator, take 10 ml of the reagent into the sample tank, take out the 96-well plate to be tested from the incubator, add 20 μL of CellTiter-Glo luminescent reagent to each well, let it stand for 10 minutes, mix it, and use a multi-function microplate reader (Perkin Elmer Envision) for detection.

[0150] (5) Data processing

[0151] According to the formula, drug inhibition rate (%) = 100% - (chemiluminescence value of culture well on the fifth day 药物处理组 / Chemiluminescence value of culture wells on day 0 药物处理组 ) / (chemiluminescence value of culture wells on the fifth day DMSO / Chemiluminescence value of culture wells on day 0 DMSO )*100%, calculate the inhibition rate of different drugs, and show the results in Figure 5A and 5B . Figure 5A The photos of organoid growth taken under a 4x objective microscope (Invitrogen EVOS M500) were taken before drug treatment and after drug treatment for 5 days. Figure 5B The figure shows the inhibition rate of liver cancer organoid growth at different concentrations of test drugs.

[0152] Depend on Figure 5A It was confirmed that organoids cultured using the liver cancer organoid culture medium of the present invention had good growth status, and that organoid growth was significantly inhibited after treatment with bortezomib and aclarubicin. Figure 5B The figure is a bar graph showing the inhibition rate of liver cancer organoid growth by three test drugs at different concentrations. Figure 5B As can be seen, the data error for the drug-treated group is very small, indicating that using this system for drug screening, data between replicate wells for the same drug are largely consistent. Of the three anti-cancer drugs, bortezomib exhibited a strong inhibitory effect on organoid growth at both concentrations. Aclarubicin exhibited significant differences in inhibitory effect at different concentrations, while doxorubicin had no inhibitory effect on the growth of this liver cancer organoid. This suggests that organoids from the same patient may have varying efficacy and sensitivity to different drugs. These results can be used to assess the effectiveness and effective dosage of these drugs in the clinical use of liver cancer patients.

[0153] Industrial Applicability

[0154] The present invention provides a culture medium and method for culturing liver cancer organoids. The cultured organoids can be used for evaluating and screening drug efficacy. Therefore, the present invention is suitable for industrial applications.

[0155] Although the present invention is described in detail herein, the present invention is not limited thereto. Those skilled in the art may make modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the scope of protection of the present invention.

Claims

1. A culture medium for liver cancer organoids, characterized in that: The invention is prepared from the following ingredients: an MST1 / 2 kinase inhibitor; a B27 supplement; a hepatocyte growth factor; an ITS cell culture supplement; Y27632; dexamethasone; a neuregulin 1; an insulin; an epidermal growth factor; GlutaMAX; non-essential amino acids; an initial culture medium selected from DMEM / F12, DMEM, F12, or RPMI-1640; and an antibiotic selected from one or more of streptomycin / penicillin, amphotericin B, and primocin. Wherein, the MST1 / 2 kinase inhibitor is selected from Compound 1 or a pharmaceutically acceptable salt or solvate thereof, in, The concentration of the MST1 / 2 kinase inhibitor is 2.5 to 10 μM; The volume ratio of the B27 additive to the culture medium of the liver cancer organoid is 1:25 to 1:100; The concentration of the hepatocyte growth factor is 1 to 25 ng / mL; The volume ratio of the ITS cell culture additive to the culture medium of the liver cancer organoid is 1:30 to 1:300; The concentration of Y27632 is 3 to 30 μM; The concentration of dexamethasone is 0.1-1 μM; The concentration of neuregulin 1 is 1 to 25 ng / mL; The concentration of the insulin is 1 to 10 μg / mL; The concentration of the epidermal growth factor is 2 to 18 ng / mL; The volume ratio of the GlutaMAX to the culture medium of the liver cancer organoid is 1:30 to 1:300; 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 50-200 μM.

2. A method for culturing liver cancer 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 organoids according to claim 1, and performing organoid culture on the primary liver cancer cells obtained in step (1).

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

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