Method for constructing liver cancer brain metastatic tumor organoid

Abstract

The invention discloses a method for constructing a liver cancer brain metastatic tumor organ. The invention provides a liver cancer brain metastatic tumor organoid culture medium, a culture method of a liver cancer brain metastatic tumor organoid, application of the corresponding organoid in screening of drugs for treating liver cancer brain metastatic tumor or construction of a liver cancer brain metastatic tumor drug screening model, and application in research of pathogenesis or pathogenic factors of liver cancer brain metastatic tumor. The organoid cultured by the culture medium and the method provided by the invention is higher in success rate, can stably grow, provides a new thought for high-throughput drug screening and auxiliary clinical treatment decision-making of the liver cancer brain metastatic tumor, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for constructing liver cancer brain metastasis tumor organoids; specifically, the present invention relates to a culture medium for liver cancer brain metastasis tumor organoids and application thereof. Background Art

[0002] Brain metastasis from liver cancer is one of the serious complications of liver cancer in the late stage. It refers to the metastasis of liver cancer cells to the brain through the bloodstream to form new tumor lesions. The appearance of brain metastasis from liver cancer not only increases the complexity of treatment, but also significantly affects the quality of life and prognosis of patients. Common symptoms of brain metastasis from liver cancer include headache, nausea, vomiting, decreased vision or hearing, limb dysfunction, language disorders, etc. Headache usually manifests as paroxysmal aggravation, more common in the morning and evening, mostly in the forehead and temporal region. Vomiting often occurs with severe headache, and in severe cases, it is even impossible to eat. Optic nerve head edema is an important sign of increased intracranial pressure, which can lead to decreased vision and narrowed visual field. In addition, brain metastasis from liver cancer may also cause epileptic seizures, mild aphasia, slow pulse, and increased blood pressure. It can be seen that the clinical manifestations of brain metastasis from liver cancer are diverse and the treatment methods are complex.

[0003] With the development of technology, organoids have gradually become known as an emerging in vitro disease research model. Organoids are a 3D cell culture model that is closer to the physiological or pathological state in vivo. Organoids can not only serve as a preclinical model for a wider range of cancer research, but also provide personalized treatment recommendations for patients with advanced diseases. However, because liver cancer brain metastases are less malignant and have poorer proliferation ability than primary glioblastoma, there is currently no method to successfully culture liver cancer brain metastases organoids. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a liver cancer brain metastasis culture medium and a method for constructing liver cancer brain metastasis organoids.

[0005] To achieve the above object, the present invention adopts the following technical solution: A first aspect of the present invention provides a liver cancer brain metastasis organoid culture medium.

[0006] Furthermore, the culture medium includes a basal culture medium and additives.

[0007] Furthermore, the basic culture medium includes DMEM / F-12 and Neurobasal.

[0008] Furthermore, the additives include nutritional additives, non-essential amino acids, antibiotics, insulin, β-mercaptoethanol, mitogen-activated growth factors, fibroblast growth factor, HGF, Forskolin, vitamins, Rock inhibitors, and BMP inhibitors.

[0009] Furthermore, the nutritional additives include GlutaMax additives, B-27 additives and N2 additives.

[0010] Furthermore, the non-essential amino acids are selected from MEM non-essential amino acid solution.

[0011] Furthermore, the antibiotics include Primocin, Penicillin-Streptomycin, and Metronidazole.

[0012] Furthermore, the antibiotic is selected from penicillin-streptomycin.

[0013] Furthermore, the mitogenic proliferation factors include EGF, BDNF, and KG.

[0014] Furthermore, the mitogenic proliferation factor is selected from EGF.

[0015] Furthermore, the fibroblast growth factor is selected from FGF-10.

[0016] Furthermore, the vitamin is selected from Nicotinamide.

[0017] Furthermore, the Rock inhibitors include Y-27632, HA1077, and H-1152.

[0018] Furthermore, the Rock inhibitor is selected from Y-27632.

[0019] Furthermore, the BMP inhibitors include Noggin, Gremlin, Chordin, Follistatin, Sclerostin, and Decorin.

[0020] Furthermore, the BMP inhibitor is selected from Noggin.

[0021] Furthermore, the usage ratio of DMEM / F-12 and Neurobasal is 1:1.

[0022] Furthermore, the final concentration of the DMEM / F-12 is 2×; the final concentration of the Neurobasal is 2×.

[0023] Furthermore, the final concentration of the GlutaMax additive is 100×, the final concentration of the B-27 additive is 50×, and the final concentration of the N2 additive is 100×.

[0024] Furthermore, the final concentration of the MEM non-essential amino acid solution is 100×.

[0025] Furthermore, the concentration of the penicillin-streptomycin is 10,000 U / mL, and the final concentration of the penicillin-streptomycin is 100×.

[0026] Furthermore, the final concentration of the insulin is 2.5 μg / mL.

[0027] Furthermore, the final concentration of the β-mercaptoethanol is 1000×.

[0028] Furthermore, the final concentration of EGF is 50 ng / mL.

[0029] Furthermore, the final concentration of HGF is 20 ng / mL.

[0030] Furthermore, the final concentration of the FGF-10 is 100 ng / mL.

[0031] Furthermore, the final concentration of Forskolin is 10 μM.

[0032] Furthermore, the final concentration of Nicotinamide is 10 mM.

[0033] Furthermore, the final concentration of Y-27632 is 10 μM.

[0034] Furthermore, the final concentration of Noggin is 50 ng / mL.

[0035] In the present invention, organoids refer to cell clusters with 3D three-dimensional structures, and refer to miniaturized and simplified versions of organs prepared through artificial culture processes. The source of cells constituting organoids is not limited. Organoids can be derived from tissues and stem cells. Organoids may have an environment that allows interaction with the surrounding environment during cell growth. Therefore, the 3D organoids in the present invention almost completely simulate the organs that interact in vivo and can be an excellent model for observing the development of disease therapeutic agents. It can similarly replicate the physiological activity functions of the human body, and by constructing organ analogs from the patient's tissues, disease modeling can be performed based on the patient's genetic information, and drug screening can be performed through repeated testing.

[0036] In a specific embodiment of the present invention, the organoids are derived from liver cancer brain metastasis tissue.

[0037] In some embodiments, the basal culture medium includes but is not limited to DMEM / F-12, Neurobasal, Dulbecco's modified Eagle's medium (DMEM), basal medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow's basic medium (G-MEM), Eagle's basal medium (BME), Iscov's modified Dulbecco's medium, Ham's F-10, Ham's F -12, 199 medium, and RPMI1640 medium.

[0038] In a specific embodiment of the present invention, the basal culture medium is selected from a combination of DMEM / F-12 and Neurobasal, and the usage ratio of DMEM / F-12 to Neurobasal is 1:1.

[0039] In some embodiments, the nutritional additives include, but are not limited to, GlutaMax additives, B-27 additives, N2 additives, G-5 additives, Poloxamer 188 solution, yeast extract, and the like.

[0040] In a particular embodiment of the present invention, the nutritional additive is selected from the group consisting of GlutaMax additive, B-27 additive and N2 additive.

[0041] In some embodiments, the non-essential amino acids include, but are not limited to, L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine.

[0042] In a specific embodiment of the present invention, the non-essential amino acids are selected from MEM non-essential amino acid solution, which contains seven non-essential amino acids: L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine and glycine.

[0043] In some embodiments, the antibiotics include Primocin, penicillin-streptomycin, and metronidazole. Among them, penicillin-streptomycin is used as part of the culture medium for cell culture, which can effectively inhibit the growth of most Gram-positive bacteria and Gram-negative bacteria, thereby effectively controlling bacterial contamination of cells. Primocin has a killing effect on Gram-positive bacteria, Gram-negative bacteria, mycoplasma and fungi. Primocin TM It is not toxic to primary cells because its target is only present on microorganisms.

[0044] In a specific embodiment of the invention, the antibiotic is selected from penicillin-streptomycin.

[0045] In the present invention, insulin plays an important role in many cell activities and is a commonly used additive in mammalian cell culture medium. Insulin can accelerate the utilization of glucose and inhibit the production of glucose, inhibit the decomposition of fat, promote the synthesis and storage of fat, promote the synthesis of protein, prevent the decomposition of protein, promote potassium ions and magnesium ions to pass through the cell membrane and enter the cell; promote the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and adenosine triphosphate (ATP).

[0046] In the present invention, β-mercaptoethanol plays an important role in cell growth, can reduce sulfur-containing compounds in serum to glutathione, can induce cell proliferation and delay cell senescence, and at the same time avoid peroxide damage to cells.

[0047] In some embodiments, the mitogenic proliferation factors include, but are not limited to, a family of growth factors such as EGF (epidermal growth factor), BDNF (brain derived neurotrophic factor), and KGF (keratinocyte growth factor). These mitogenic proliferation factors can be used in various combinations.

[0048] In a specific embodiment of the present invention, the mitogenic proliferation factor is selected from EGF.

[0049] In some embodiments, fibroblast growth factor (FGF) is a family of growth factors involved in angiogenesis, wound healing and embryonic development. FGF is a heparin binding protein, and the interaction of the heparan sulfate proteoglycan associated with the cell surface is essential for FGF signal transduction. Those of ordinary skill in the art will readily understand suitable FGF pathway activators. FGF pathway activators include but are not limited to FGF1, FGF2, FGF3, FGF4, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23.

[0050] In a specific embodiment of the present invention, the fibroblast growth factor is selected from FGF-10.

[0051] In the present invention, the Forskolin is an effective adenylate cyclase activator, which can increase the intracellular cAMP level, thereby promoting cell proliferation or specific cell responses, such as the opening of CFTR channels.

[0052] In some embodiments, the vitamins include but are not limited to thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), D-calcium pantothenate (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-α-tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

[0053] In a specific embodiment of the present invention, the vitamin is selected from vitamin B3 (Nicotinamide).

[0054] In some embodiments, the Rock (Rho-kinase) inhibitors include but are not limited to Y-27632 ((R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridinyl)cyclohexanecarboxamide dihydrochloride monohydrate), HA1077 (Fasudil) (5-(1,4-diazepan-1-ylsulfonyl)isoquinoline), H-1152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride).

[0055] In a specific embodiment of the present invention, the Rock inhibitor is selected from Y-27632.

[0056] In some embodiments, the BMP (bone morphogenetic protein) inhibitor includes but is not limited to Noggin, Gremlin, Chordin, Tenasin-like proteins such as Tenasin domain, Follistatin, Follistatin-related proteins such as Follistatin domain, DAN, DAN-like proteins such as DAN cysteine ​​knot domain, Sclerostin / SOST, Decorin, and α2-macroglobulin.

[0057] In a specific embodiment of the present invention, the BMP inhibitor is selected from Noggin.

[0058] In the present invention, the organoid culture medium may further comprise inorganic salts, including but not limited to salts of calcium, copper, iron, magnesium, potassium, sodium, and zinc. Salts are generally used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. More specifically, salts include but are not limited to CaCl2, CuSO4·5H2O, Fe(NO3)·9H2O, FeSO4·7H2O, MgCl, MgSO4, KCl, NaHCO3, NaCl, Na2HPO4, Na2HPO4·H2O, and ZnSO4·7H2O.

[0059] In the present invention, the organoid culture medium may further contain sugars that can serve as a carbon energy source. Sugars include, but are not limited to, glucose, galactose, mannose, and fructose. Among them, as the sugar, glucose is preferred, and D-glucose (dextrose) is more preferred.

[0060] In the present invention, the organoid culture medium may further contain trace elements. The trace elements include barium, bromine, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum or their ions.

[0061] A second aspect of the present invention provides a method for culturing liver cancer brain metastasis organoids.

[0062] Furthermore, the method comprises culturing liver cancer brain metastasis tissue using the culture medium described in the first aspect of the present invention.

[0063] Furthermore, the method also includes cutting the liver cancer brain metastasis tissue into pieces before culturing.

[0064] Furthermore, the liver cancer brain metastasis tissue is cut into pieces with a diameter of about 0.5-1.0 mm.

[0065] Furthermore, the culture is carried out in a low-adsorption culture dish.

[0066] Furthermore, the specific conditions of the culture are: shaking culture at 37°C, 5% CO2, and 120rpm / min.

[0067] A third aspect of the present invention provides a liver cancer brain metastasis organoid.

[0068] Furthermore, the organoid is cultured using the culture medium described in the first aspect of the present invention or obtained by the method described in the second aspect of the present invention.

[0069] A fourth aspect of the present invention provides any of the following applications: (1) Use of the culture medium described in the first aspect of the present invention in culturing organoids of liver cancer brain metastases; (2) Use of the organoids described in the third aspect of the present invention in screening drugs for preventing and / or treating brain metastases of liver cancer; (3) Use of the organoids described in the third aspect of the present invention in establishing a drug sensitivity model of liver cancer brain metastases; (4) Use of the organoids described in the third aspect of the present invention in the study of the pathogenesis or pathogenic factors of brain metastases of liver cancer.

[0070] In the present invention, treatment and its grammatical variations refer to clinical interventions that attempt to change the natural course of the individual being treated, and can be performed for prevention or during clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

[0071] A fifth aspect of the present invention provides an in vitro drug screening model for liver cancer brain metastases.

[0072] Furthermore, the model comprises the organoid described in the third aspect of the present invention.

[0073] A sixth aspect of the present invention provides a method for predicting drug sensitivity of brain metastases from liver cancer.

[0074] Further, the method comprises contacting the organoid described in the third aspect of the present invention or the model described in the fifth aspect of the present invention with a test drug.

[0075] Furthermore, the drugs include targeted drugs and chemotherapy drugs.

[0076] Furthermore, the method further comprises exposing the organoid to radiation.

[0077] The method further includes measuring the organoids contacted with the test drug, and determining the sensitivity of the test drug based on the morphological changes of the organoids. The morphological changes of the organoids include a decrease in the number of organoids or a change in the size of the organoids. If the number of the organoids decreases or the growth of the organoids is inhibited, it indicates that the organoids are sensitive at the test drug concentration.

[0078] In the present invention, contacting the organoid with the test drug means placing the organoid and the test drug in contact. The contact between the organoid and the test drug may be, for example, adding the test drug to a solution containing the organoid. The test drug of the present invention may be a low molecular weight compound, a protein (such as an antibody), DNA, RNA, a low molecular weight interfering RNA, or an antisense oligonucleotide. The test drug may be, for example, a drug for treating a disease or cancer other than liver cancer brain metastases. The test drug may be, for example, a mixture of one or more. The test drug is preferably a substance.

[0079] A seventh aspect of the present invention provides a method for screening drugs for preventing and / or treating brain metastases from liver cancer.

[0080] Furthermore, the method comprises contacting the candidate drug with the organoid described in the third aspect of the present invention or the model described in the fifth aspect of the present invention.

[0081] The method further includes measuring the organoid or model after contact with the candidate substance; and comparing the measured value of the organoid or model after contact with the candidate drug with the control value. In one embodiment, the method includes determining whether the candidate substance is a drug capable of preventing and / or treating brain metastases of liver cancer based on the comparison result.

[0082] In the present invention, contacting the candidate drug with the organoid or model means placing the organoid or model and the candidate drug in contact with each other. The contacting of the organoid or model with the candidate drug may be, for example, adding the candidate drug to a solution containing the organoid or model.

[0083] In the present invention, the organoid or model after contact with the above-mentioned candidate substance is measured, including the reduction of organoid viability, the reduction of organoid proliferation, the increase of organoid death, the change of organoid size, the number of organoids, and the expression of marker proteins of cells constituting the organoid. Among them, the change of organoid size can be, for example, the measurement of the size of the organoid, and the size of the organoid can be measured, for example, using a known device (such as a microscope or FACS). The size of the organoid can be, for example, the circumference, diameter (such as the major diameter and the minor diameter) or area of ​​the organoid. The expression of marker proteins of cells constituting the organoid can be measured using a known device (such as a microscope or FACS) and a known reagent (such as an antibody labeled with a fluorescent substance). The expression of marker proteins of cells constituting the organoid can be, for example, the fluorescence intensity corresponding to the amount of marker proteins expressed per unit area of ​​the organoid. The marker protein measured can be one or a combination of two or more. The marker protein measured is preferably a combination of two or more.

[0084] In the present invention, the measured value of an organoid or model may be, for example, a measured value measured once, an average value of multiple measurements, or an average value of measured values ​​of multiple organoids. The measured value of an organoid may be, for example, a change value (e.g., a difference or multiple) before and after contact with a test substance. The control value may be, for example, when the value after contact with a candidate drug is used as the measured value of the organoid, the measured value of a control organoid before contact with the candidate drug or not contacted with the candidate drug may be used as the control value.

[0085] Advantages and beneficial effects of the present invention: The organoid culture medium and construction method provided by the present invention have a high success rate, and the organoids are in good growth condition. They have built a technical platform for the realization of precision medicine for liver cancer brain metastases and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 This is a microscope image of an organoid after 10 days of culture using the culture medium components of the embodiment group provided by the present invention; Figure 2 This is a statistical diagram of cell activity after 10 days of culture using the culture medium components of the embodiment group provided by the present invention; Figure 3 This is a microscopic image of an organoid after 10 days of culture using the comparative culture medium components; Figure 3 A. Figure 3 B. Figure 3 C are microscope images of organoids after culturing for 10 days using the culture medium components of Comparative Example A, Comparative Example B, and Comparative Example C; Figure 4 This is a statistical chart of cell activity after 10 days of culture using the medium components of Comparative Example A. DETAILED DESCRIPTION

[0087] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0088] In addition, in the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0089] Example 1 Method for constructing liver cancer brain metastasis organoids 1. Composition of Hepatocellular Carcinoma Brain Metastasis Organoid Culture Medium Three groups of comparative culture medium components were set up in this experiment. The culture medium components of comparative example A are DMEM / F-12 culture medium 2×, Neurobasal culture medium 2×, B-27 additive (minus vitamin A) 50×, N-2 additive 100×, MEM non-essential amino acid solution 100×, GlutaMAX additive 100×, penicillin-streptomycin solution 100×, insulin 2.5μg / mL, β-mercaptoethanol 1000×; the usage ratio of DMEM / F-12 culture medium and Neurobasal culture medium is 1:1; The culture medium of comparative example B comprises 2× DMEM / F-12 culture medium, 2× Neurobasal culture medium, 50× B-27 additive (containing vitamin A), 100× N-2 additive, 100× MEM non-essential amino acid solution, 100× GlutaMAX additive, 100× penicillin-streptomycin solution, 2.5 μg / mL insulin, and 1000× β-mercaptoethanol; the dosage ratio of DMEM / F-12 culture medium to Neurobasal culture medium is 1:1; The culture medium of comparative example C comprises 2× DMEM / F-12 culture medium, 2× Neurobasal culture medium, 50× B-27 additive (containing vitamin A), 100× N-2 additive, 100× MEM non-essential amino acid solution, 100× GlutaMAX additive, 100× penicillin-streptomycin solution, 2.5 μg / mL insulin, 1000× β-mercaptoethanol, and 100 μm vitamin C; the dosage ratio of DMEM / F-12 culture medium to Neurobasal culture medium is 1:1; The culture medium components of the example group are DMEM / F-12 culture medium 2×, Neurobasa culture medium 2×, B-27 supplement (minus vitamin A) 50×, N-2 supplement 100×, MEM non-essential amino acid solution 100×, GlutaMAX supplement 100×, penicillin-streptomycin (10,000 U / mL) 100×, insulin 2.5ug / mL, B-mercaptoethanol 1000×, EGF 50ng / mL, FGF-10 100ng / mL, HGF 20ng / mL, Forskolin 10uM, Nicotinamide 10mM, Y-27632 10uM, and Noggin 50ng / mL.

[0090] 2. Organoid culture method for brain metastases from liver cancer I. Place the obtained fresh ex vivo liver cancer brain metastasis specimens into pre-cooled liver cancer brain metastasis preservation solution, and transport them to the laboratory in an ice bath and incubator box; II. Pour the liver cancer brain metastasis specimen into a large dish in a clean bench under aseptic operation; III. Use surgical blades, autoclaved microscissors and microtweezers to remove the red vascular tissue and black necrotic tissue produced by surgical electrocoagulation. Carefully cut the specimen into pieces with a diameter of about 0.5-1.0 mm. Transfer the pieces to a 15 mL centrifuge tube, add 10 mL of DPBS and mix thoroughly. Let it stand for 1 minute to remove the less active tissue and impurities in the supernatant. Repeat 3 times. IV. After removing the supernatant, add 5 mL of 1× RBC lysis buffer to remove the remaining red blood cells. During the removal process, place the centrifuge tube on a shaker in a 37°C, 5% CO2 incubator with the shaker speed set to 120 rpm / min for 10 minutes; V. After the removal is completed, the centrifuge tube is allowed to stand for about 1 minute, and the shredded tissue fragments are deposited at the bottom. The supernatant is removed by suction, and 10 mL of DPBS is added to mix well. The supernatant is removed by standing. Repeat 2 times, and then 10 mL of the comparative example or example culture medium provided above is added, and the supernatant is removed by standing for 1 minute. VI. Transfer the organoids obtained above to low-adhesion six-well plates, with about 50-60 organoids per six-well plate, and add 2 mL of culture medium to each six-well plate; VII. Place the above six-well plate on a shaker in a 37°C, 5% CO2 incubator. Set the shaker speed to 120rpm / min. Change the culture medium in the dish every 2-3 days according to the color change of the culture medium. Replace 2 / 3 of the culture medium each time. Observe the growth status of the organoids every day, take photos to record the growth rate and morphological changes.

[0091] 3. Cell activity assay method: Cover the matrix gel with an appropriate amount of cell recovery solution matrix gel digestion solution, place the culture plate at 2-8°C or on ice, incubate for 30-90 minutes, and shake gently, add PBS to wash 2-3 times, place in a 15ml tube and let stand for 1-2 minutes, add an appropriate amount of trypsin to the 15ml tube after the organoids settle, digest at 37°C for 10 minutes, and after the organoids are digested into single cells, use a 70um sieve to remove incompletely digested organoids, leaving the cell suspension, 1000r, centrifuge for 5min, remove the supernatant; add 2ml PBS to resuspend, take 20ul of the cell suspension, add an equal amount of AOPI dye solution and mix well, count and analyze the cell activity with a cell counter.

[0092] 4. Experimental results The microscopic image of the organoid after 10 days of culture using the culture medium components of the Example group is as follows Figure 1 As shown, the organoids are in good growth condition, with high cell survival rate and high aggregation rate ( Figure 2 ); while the growth state of the control organoids was poor, the cell survival rate was low, and the aggregation rate was 0% ( Figure 3 , 4 ).

[0093] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A liver cancer brain metastasis organoid culture medium, characterized in that: The culture medium comprises a basic culture medium and additives; The basal culture medium includes DMEM / F-12 and Neurobasal; The additives include nutritional additives, non-essential amino acids, antibiotics, insulin, beta-mercaptoethanol, mitogen-activated growth factors, fibroblast growth factor, HGF, Forskolin, vitamins, Rock inhibitors, and BMP inhibitors.

2. The culture medium according to claim 1, characterized in that The nutritional additives include GlutaMax additives, B-27 additives, and N2 additives; Preferably, the non-essential amino acids are selected from MEM non-essential amino acid solution; Preferably, the antibiotics include Primocin, penicillin-streptomycin, and metronidazole; Preferably, the antibiotic is selected from penicillin-streptomycin; Preferably, the mitogenic proliferation factors include EGF, BDNF, and KG; Preferably, the mitogenic proliferation factor is selected from EGF; Preferably, the fibroblast growth factor is selected from FGF-10; Preferably, the vitamin is selected from Nicotinamide; Preferably, the Rock inhibitor includes Y-27632, HA1077, H-1152; Preferably, the Rock inhibitor is selected from Y-27632; Preferably, the BMP inhibitors include Noggin, Gremlin, Chordin, Follistatin, Sclerostin, and Decorin; Preferably, the BMP inhibitor is selected from Noggin.

3. The culture medium according to claim 1 or claim 2, characterized in that The usage ratio of DMEM / F-12 and Neurobasal is 1:1; Preferably, the final concentration of the DMEM / F-12 is 2×; the final concentration of the Neurobasal is 2×; Preferably, the final concentration of the GlutaMax additive is 100×, the final concentration of the B-27 additive is 50×, and the final concentration of the N2 additive is 100×; Preferably, the final concentration of the MEM non-essential amino acid solution is 100×; Preferably, the concentration of penicillin-streptomycin is 10,000 U / mL, and the final concentration of penicillin-streptomycin is 100×; Preferably, the final concentration of the insulin is 2.5 μg / mL; Preferably, the final concentration of β-mercaptoethanol is 1000×; Preferably, the final concentration of EGF is 50 ng / mL; Preferably, the final concentration of FGF-10 is 100 ng / mL; Preferably, the final concentration of HGF is 20 ng / mL; Preferably, the final concentration of Forskolin is 10 μM; Preferably, the final concentration of Nicotinamide is 10 mM; Preferably, the final concentration of Y-27632 is 10 μM; Preferably, the final concentration of Noggin is 50 ng / mL.

4. A method for culturing organoids of liver cancer brain metastases, characterized in that: The method comprises culturing liver cancer brain metastasis tissue using the culture medium described in any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The method further comprises cutting and processing the liver cancer brain metastasis tissue into pieces before culturing; Preferably, the liver cancer brain metastasis tissue is cut into pieces with a diameter of about 0.5-1.0 mm; Preferably, the culturing is carried out in a low-adsorption culture dish; Preferably, the specific conditions of the culture are: shaking culture at 37°C, 5% CO2, and 120rpm / min.

6. A liver cancer brain metastasis organoid, characterized in that: The organoid is cultured using the culture medium described in any one of claims 1 to 3 or is obtained by the method described in claim 4 or 5.

7. Applications as described in any of the following: (1) Use of the culture medium according to any one of claims 1 to 3 in culturing organoids of liver cancer brain metastases; (2) Use of the organoid according to claim 6 in screening drugs for preventing and / or treating brain metastases of liver cancer; (3) Use of the organoid described in claim 6 in establishing a drug sensitivity model of liver cancer brain metastases; (4) Use of the organoid described in claim 6 in the study of the pathogenesis or pathogenic factors of brain metastases of liver cancer.

8. An in vitro drug screening model for liver cancer brain metastases, characterized in that: The model comprises the organoid of claim 6.

9. A method for predicting drug sensitivity of liver cancer brain metastases, characterized in that: The method comprises contacting the organoid of claim 6 or the model of claim 8 with a test drug; Preferably, the drugs include targeted drugs and chemotherapy drugs.

10. A method for screening drugs for preventing and / or treating brain metastases from liver cancer, characterized in that: The method comprises contacting a candidate drug with the organoid of claim 6 or the model of claim 8.

Images