WO2023035299A1 - 一种用于肝癌类器官培养的培养基、及其培养方法和应用 - Google Patents
一种用于肝癌类器官培养的培养基、及其培养方法和应用 Download PDFInfo
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- WO2023035299A1 WO2023035299A1 PCT/CN2021/118657 CN2021118657W WO2023035299A1 WO 2023035299 A1 WO2023035299 A1 WO 2023035299A1 CN 2021118657 W CN2021118657 W CN 2021118657W WO 2023035299 A1 WO2023035299 A1 WO 2023035299A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
Definitions
- the invention belongs to the field of biotechnology, and in particular relates to a medium for culturing liver cancer organoids, a method for cultivating liver cancer organoids using the medium, and its application in evaluating and screening the curative effect of drugs.
- Organoids which belong to three-dimensional (3D) cell cultures, are mainly derived from human embryonic stem cells, induced pluripotent stem cells and adult stem cells with differentiation ability. Endogenous tissue stem cells exist in different tissues and organs, and play an important role in maintaining the functional morphology of various organs. Under certain induction conditions in vitro, these stem cells can self-organize to form a miniature structure with a diameter of only a few millimeters.
- Tumor organoids are obtained from primary tumors in patients, and some miniature 3D tumor cell models are cultivated in the laboratory. Tumor organoids highly simulate the characteristics of the source tumor tissue, retain the tumor heterogeneity among individuals, and can be used for functional testing, such as high-throughput drug screening and individualized precision therapy.
- liver cancer organoid culture methods mostly use basal medium (DMEM or DMEM/F12), R-spondin-1, Noggin and some expensive protein factors, resulting in high cost of organoid culture;
- basal medium DMEM or DMEM/F12
- R-spondin-1 R-spondin-1
- Noggin R-spondin-1
- Noggin some expensive protein factors
- the present invention provides a culture medium and a culture method for rapid expansion of liver cancer organoids in vitro.
- One aspect of the present invention is to provide a culture medium for liver cancer organoids, said culture medium comprising 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 nonessential amino acids.
- the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,
- R 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by 1-2 independent R (such as phenyl and naphthyl, etc.), aryl C1-C6 alkyl (such as benzyl, etc.) and heteroaryl (such as thienyl, etc.);
- R 2 and R 3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
- R 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyran base, etc.);
- R 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 trifluoro methyl).
- halogen preferably fluorine and chlorine, more preferably fluorine
- C1-C6 alkyl preferably methyl
- C1-C6 alkoxy preferably methoxy
- C1-C6 haloalkyl preferably trifluoro methyl
- the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,
- R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R6 , thienyl optionally substituted by 1-2 independently R6 , and optionally substituted by 1 -2 independently R6 substituted benzyl, R1 is more preferably optionally 1-2 independently R6 substituted phenyl;
- R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R 5 is more preferably hydrogen;
- R 6 is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R 6 is more preferably fluorine, methyl or trifluoromethyl.
- the MST1/2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.
- the MST1/2 kinase inhibitor of the present invention is Compound 1.
- the content of each component in the culture medium of the present invention satisfies any one or more or all of the following:
- the concentration of the MST1/2 kinase inhibitor is preferably 2.5-10 ⁇ M
- the concentration of hepatocyte growth factor is preferably 1-25 ng/mL
- the volume ratio of the ITS cell culture additive to the medium is preferably 1:30 to 1:300;
- the concentration of Y27632 is preferably 3-30 ⁇ M
- the concentration of dexamethasone is preferably 0.1-1 ⁇ M
- the concentration of neuregulin 1 is preferably 1-25 ng/mL
- the concentration of insulin is preferably 1-10 ⁇ g/mL
- the concentration of epidermal growth factor is preferably 2 ⁇ 18ng/mL
- the volume ratio of GlutaMAX to the medium is preferably 1:30 to 1:300;
- Non-essential amino acids are one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the concentration of non-essential amino acids is preferably 50- 200 ⁇ M.
- the medium also contains an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one selected from streptomycin/penicillin, amphotericin B and Primocin one or more antibiotics.
- the streptomycin concentration ranges from 25 to 400 ⁇ g/mL, and the penicillin concentration ranges from 25 to 400 U/mL; when the antibiotic is selected from amphotericin B, The concentration range is 0.25-4 ⁇ g/mL, and when the antibiotic is selected from Primocin, the concentration range is 25-400 ⁇ g/mL.
- the invention also provides a method for culturing liver cancer organoids.
- liver cancer organoids are cultured using the liver cancer organoid culture medium of the present invention.
- the method for culturing liver cancer organoids of the present invention includes the following steps.
- the process includes the following steps:
- the basal medium formula includes an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin/penicillin, amphotericin B and Primocin.
- the formula of tissue digestion solution includes 1640 medium, collagenase II (1-2mg/mL), collagenase IV (1-2mg/mL), DNase (50-100U/mL), hyaluronidase (0.5-1mg/mL) mL), calcium chloride (1 ⁇ 5mM), bovine serum albumin BSA (5 ⁇ 10mg/mL).
- the liver cancer organoid medium of the present invention Resuspend and count the primary liver cancer cells obtained in the above step 1 with the liver cancer organoid medium of the present invention, dilute the cell density to 5-10 ⁇ 106 cells/mL, take out the diluted cell suspension and add an equal volume Mix well in the Matrigel matrigel, then inoculate the mixture on a multi-well plate, put the inoculated multi-well plate in the incubator for 30-60 minutes, wait until the Matrigel is completely solidified, and then add the liver cancer organoid medium to expand the culture.
- the present invention also provides a method for evaluating or screening a drug for treating liver cancer, which comprises the following steps:
- liver cancer organoids can be rapidly cultured, and the amplified liver cancer organoids can also be continuously passaged;
- the culture cost is controllable, and the 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;
- the number of hepatocellular carcinoma organoids obtained by the technique is large, which is suitable for high-throughput screening of candidate compounds and providing patients with high-throughput drug sensitivity functional tests in vitro.
- 1A-1K are graphs showing the effects of different concentrations of factors added to the liver cancer organoid medium of the present invention on the proliferation of liver cancer organoids.
- Figures 2A-2D are photographs of liver cancer organoids cultured using the liver cancer organoid medium of the present invention using a microscope, wherein Figure 2A shows photos of organoids cultured from sample GL-003 after 10 days; Figure 2B shows photos of organoids cultured after 10 days Photos of organoids cultured from sample GL-006; Figure 2C shows photos of organoids cultured from sample GL-008 after 12 days; Figure 2D shows photos of organoids cultured from sample GL-013 after 15 days.
- Figure 3A is the result of pathological and immunohistochemical identification of the liver cancer organoids cultured in the liver cancer organoid medium of the present invention for sample GL-006;
- Figure 3B is the result of pathological and immunohistochemical identification of the sample GL-006 tissue result.
- Fig. 4 is the comparison result of using the liver cancer organoid culture medium of the present invention and the existing culture medium to cultivate liver cancer organoids, wherein Fig. 4A shows the photos after culturing for 25 days with the HC-3 medium of the present invention; Photos after 25 days of culture in Laura medium; Figure 4C shows a bar graph comparing the relative size of organoids cultured in HC-3 medium and Laura medium.
- Figure 5 shows the results of different drug sensitivity tests of liver cancer organoids obtained by using the liver cancer organoid culture medium of the present invention, wherein Figure 5A shows the photos of organoid growth without drug treatment and the photos of organoid growth after drug treatment for 5 days Photo; FIG. 5B shows a histogram of the inhibition rate of different concentrations of test drugs to inhibit the growth of liver cancer organoids.
- an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signal transduction.
- MST1/2 kinase inhibitors for example, bind to MST1/2 kinase and reduce its activity. Due to the similarity in the structures of MST1 and MST2, MST1/2 kinase inhibitors can also be, for example, compounds that bind to MST1 or MST2 and reduce their activity.
- 2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): In a round bottom flask was added 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) Methanol (30 mL) was then added, followed by the dropwise addition of thionyl chloride (1.2 mL) under ice-cooling. The reaction system was reacted overnight at 85°C. After the reaction, the system was evaporated to dryness under reduced pressure to obtain a white solid, which was directly used in the next step.
- MST1/2 inhibitor compounds of the present invention were synthesized according to a method similar to compound 1, and their structures and mass spectrometry data are shown in the table below.
- the initial medium can be selected from DMEM/F12, DMEM, F12 or RPMI-1640 commonly used in the art.
- the formulation of the basal medium is: DMEM/F12 medium (purchased from Corning Company)+100 ⁇ g/mL Primocin (purchased from InvivoGen Company, 0.2% (v/v), commercially available product concentration 50mg/ml ).
- Intraoperative samples were obtained from patients by professional medical staff from professional medical institutions, and all patients signed informed consent. Intraoperative samples of 0.25 cm 3 were stored and transported in commercial tissue preservation solution (manufacturer: Miltenyi Biotec).
- Tissue digestion solution formula 1640 medium (Corning, 10-040-CVR), collagenase II (2mg/mL), collagenase IV (2mg/mL), DNase (50U/mL), hyaluronidase (0.75 mg/mL), calcium chloride (3.3mM), bovine serum albumin BSA (10mg/mL).
- Collagenase II, collagenase IV, DNase, and hyaluronidase mentioned above were all purchased from Sigma; calcium chloride was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; BSA was purchased from Biofroxx.
- “+” means that compared with the basal medium, the medium added with this additive has the effect of promoting the proliferation of at least two cases of liver cancer organoids isolated from liver cancer tissue; “-” means that the medium added with this additive It has the effect of inhibiting the proliferation of at least one case of liver cancer organoids isolated from liver cancer tissues; “ ⁇ ” indicates that the medium supplemented with this additive has no obvious proliferation of at least two cases of liver cancer organoids isolated from liver cancer tissues Impact.
- B27 hepatocyte growth factor (HGF), ITS cell culture supplement, Y27632, dexamethasone, neuregulin 1 (NRG1), insulin, epidermal growth factor (EGF), GlutaMAX, compound 1, Factors such as non-essential amino acids were used for further cultivation experiments.
- HGF hepatocyte growth factor
- ITS cell culture supplement Y27632
- dexamethasone neuregulin 1 (NRG1)
- insulin insulin
- EGF epidermal growth factor
- GlutaMAX GlutaMAX
- compound 1 Factors such as non-essential amino acids
- Figures 1A-1K the ratios are the diameters of organoids cultured for 10 days using each medium to the diameters of organoids cultured for 10 days in the corresponding BC control wells.
- a ratio greater than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds has a better effect on promoting proliferation than the culture medium of the control well; a ratio less than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds promotes proliferation The effect was weaker than that of the culture medium in the control well.
- 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 ⁇ 30 ⁇ M; the content of dexamethasone is preferably 0.1 ⁇ 1 ⁇ M; the content of neuregulin 1 is preferably 1 ⁇ 25 ng/mL; the content of insulin is preferably 1 ⁇ 10 ⁇ g/mL; The content of growth factors is preferably 2-18ng/mL; the volume concentration of GlutaMAX is preferably 1:30-1:300; the content of MST1/2 kinase inhibitor compound 1 is preferably 2.5-10 ⁇ M; the content of non-essential amino acids is preferably 50 ⁇ 200 ⁇ M.
- liver cancer primary cells (GL-003, GL-006, GL-008, GL-013) obtained according to the method described in Example 1 (2) were resuspended with the liver cancer organoid medium HC-3 of the present invention and Count, dilute the cell density to 5-10 ⁇ 106 cells/mL, take out 400 ⁇ L of the diluted cell suspension and add it to an equal volume of Matrigel matrigel (Corning) to mix gently, and then inoculate the mixture at 50 ⁇ L/well in 24-well plate.
- Matrigel matrigel Cornning
- the inoculated culture plate into the incubator for 30 minutes, wait until the Matrigel is completely solidified, then add the liver cancer organoid medium HC-3 that has been returned to room temperature in advance, 500 ⁇ L per well, and expand the culture by replacing the medium every three days.
- liver cancer organoids were observed using a microscope (EVOS M500 from Invitrogen).
- Figures 2A-2D are taken under a 4x objective lens after culturing samples GL-003 (day 10), GL-006 (day 10), GL-008 (day 12), and GL-013 (day 15) Photographs of HCC organoids. Under the microscope, liver cancer organoids were spherical in shape with a smooth surface.
- Pathological and immunohistochemical identifications were performed on the cultured liver cancer organoids, and corresponding tissue samples were sent for pathological and immunohistochemical identifications to compare the consistency of organoids and tissue results.
- Figure 3A is the results of pathological and immunohistochemical identification of liver cancer organoids obtained from sample GL-006 cultured in vitro, which are pictures taken under a 20x objective lens. As shown in Figure 3A, HE results showed that the structural morphology of organoids was cancerous; the expression of CK19, Heppar-1, and Ki67 suggested that the sample was liver cancer.
- Figure 3B is the pathological and immunohistochemical results of the corresponding tissue of GL-006 before culture, and the results show that the liver cancer organoid cultured with the medium HC-3 of the present invention is consistent with the diagnosis result of the liver cancer tissue before culture.
- the medium used in the preparation literature (Laura et al., Nat Med.2017,23(12):1424-1435), its formula is Advanced DMEM/F12 medium (purchased from Corning Company)+1:100 Penicillin/Streptomycin (purchased From Corning)+1:100 GlutaMAX (purchased from Corning)+10mM 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 company)+10mmol/L nicotinamide (purchased from MCE company)+10nM gastrin (purchased from MCE company)+50ng/ml epidermal growth factor (purchased from MCE company) R&D company)+100ng/ml fibroblast growth factor 10 (purchased from Sino biological company)+25ng/ml hepatocyte growth factor (purchased from R&D company)+10 ⁇ mol/L
- the primary liver cancer cells were obtained from the intraoperative tissue sample GL-018 according to the method of (2) of Example 1, and the organoids were cultured according to the method of Example 3 with HC-3 medium and Laura medium respectively.
- Figures 4A and 4B are photographs of organoids cultured in HC-3 medium and Laura medium respectively under a 4x objective lens
- Figure 4C is a bar graph comparing the relative sizes of organoids cultured in the two media.
- HC-3 medium can significantly promote the expansion and culture of liver cancer organoids.
- Example 5 Liver cancer organoids amplified using the medium of the present invention are used for drug screening
- Liver cancer primary cells were isolated from the intraoperative liver cancer sample (GL-006) according to the method of Example 1 (2), and organoids were cultured in HC-3 medium, and drug screening was performed when the diameter of the liver cancer organoids exceeded 50 ⁇ m .
- Arubicin Preparation concentration (mM) 1 0.1
- Doxorubicin Preparation concentration (mM) 1 0.1
- Bortezomib Preparation concentration (mM) 1 0.1
- the drug inhibition rate (%) 100%-(the chemiluminescence value drug treatment group of the culture well on the fifth day/the chemiluminescence value drug treatment group of the culture well on the zero day)/(the chemiluminescence value DMSO of the culture well on the fifth day/the The chemiluminescent value of the zero-day culture wells (DMSO )*100% was calculated to obtain the inhibition rates of different drugs, and the results are shown in FIGS. 5A and 5B .
- Figure 5A is a photo of organoid growth without drug treatment and a photo of organoid growth after 5 days of drug treatment taken under a microscope (Invitrogen EVOS M500) with a 4x objective lens
- Figure 5B is a test drug at different concentrations inhibiting liver cancer organoids Growth inhibition rate histogram.
- FIG. 5A It can be confirmed from FIG. 5A that the organoids cultured using the liver cancer organoid medium of the present invention grow well, and the growth of the organoids is significantly inhibited after being treated with bortezomib and arubicin.
- Fig. 5B is a histogram of the inhibition rate of three test drugs at different concentrations to inhibit the growth of liver cancer organoids. It can be seen from Figure 5B that the data error value of the drug treatment group is very small, indicating that using this system for drug screening, the data between the repeated wells of the same drug are basically consistent.
- bortezomib has a strong inhibitory effect on the growth of organoids at two concentrations, and the inhibitory effects of different concentrations of arubicin are significantly different. There was no inhibition of growth, suggesting that organoids from the same patient differ in their effectiveness and sensitivity to different drugs. According to the results, the effectiveness and effective dosage of the drug can be judged when the liver cancer patients are used clinically.
- the invention provides a culture medium and a culture method for liver cancer organoids, and the cultured organoids can be applied to the efficacy evaluation and screening of drugs.
- the present invention is suitable for industrial applications.
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Abstract
Description
序号 | 培养基添加剂种类 | 供应商 | 终浓度 | 促增殖程度分级 |
1 | N2 | Gibco | 1:50 | + |
2 | 表皮细胞生长因子EGF | R&D | 5ng/mL | + |
3 | R-spondin1 | R&D | 20ng/mL | + |
4 | 前列腺素E2 | Tocris | 0.5μM | ○ |
5 | 胰岛素 | Peprotech | 1.5μg/mL | + |
6 | B27 | Gibco | 1:50 | + |
7 | A8301 | MCE | 100nM | - |
8 | SB202190 | MCE | 200nM | - |
9 | 碱性成纤维细胞生长因子bFGF | R&D | 10ng/mL | - |
10 | 氢化可的松 | Sigma | 10ng/mL | ○ |
11 | Noggin | R&D | 30ng/mL | ○ |
12 | 胎牛血清FBS | Excell | 5% | + |
13 | 胰岛素样生长因子-1IGF-1 | R&D | 45ng/mL | ○ |
14 | 角化细胞生长因子KGF | R&D | 5ng/mL | ○ |
15 | GlutaMAX | Gibco | 1:100 | + |
16 | 非必需氨基酸 | Corning | 100μM | + |
17 | 地塞米松 | MCE | 0.1μM | + |
18 | 神经调节蛋白1NRG1 | sino biological | 5ng/mL | + |
19 | Y27632 | MCE | 10μM | + |
20 | ITS细胞培养添加剂 | Gibco | 1:100 | + |
21 | 化合物1 | 制备例 | 5μM | + |
22 | CHIR99021 | MCE | 2.5μM | - |
23 | 肝细胞生长因子HGF | R&D | 5ng/mL | + |
阿柔比星 | 配制浓度(mM) | 1 | 0.1 |
多柔比星 | 配制浓度(mM) | 1 | 0.1 |
硼替佐米 | 配制浓度(mM) | 1 | 0.1 |
Claims (10)
- 一种用于肝癌类器官的培养基,其特征在于包括MST1/2激酶抑制剂、选自N2和B27的至少一种细胞培养添加剂、肝细胞生长因子、ITS细胞培养添加剂、Y27632、地塞米松、神经调节蛋白1、胰岛素、表皮细胞生长因子、GlutaMAX、和非必需氨基酸,其中,所述MST1/2激酶抑制剂包括式(I)的化合物或其药学可接受的盐、或溶剂化物,其中,R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的芳基、芳基C1-C6烷基和杂芳基;R 2和R 3各自独立地选自C1-C6烷基;R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、和C3-C6杂环基C1-C6烷基;R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。
- 如权利要求1所述的培养基,其中R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的苯基、萘基、苯甲基和噻吩基;R 2和R 3各自独立地选自C1-C3烷基;R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8 环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、哌啶基C1-C6烷基、和四氢吡喃基C1-C6烷基;R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。
- 如权利要求3所述的培养基,其中R 1为任选地被1-2个独立地R 6取代的苯基;R 5为氢;R 6优选为氟、甲基或三氟甲基。
- 如权利要求1~5中任一项所述的培养基,其特征在于所述培养基中各成分的含量满足以下任意一项或多项或全部满足:所述MST1/2激酶抑制剂的浓度为2.5~10μM;所述B27或N2细胞培养添加剂相对于培养基的体积比为1:25~1:100;所述肝细胞生长因子的浓度为1~25ng/mL;所述ITS细胞培养添加剂相对于培养基的体积比为1:30~1:300;所述Y27632的浓度为3~30μM;所述地塞米松的浓度为0.1~1μM;所述神经调节蛋白1的浓度为1~25ng/mL;所述胰岛素的浓度为1~10μg/mL;所述表皮细胞生长因子的浓度为2~18ng/mL;所述GlutaMAX相对于培养基的体积比为1:30~1:300;所述非必需氨基酸为选自甘氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、脯氨酸和丝氨酸中的一种或多种,其浓度为50~200μM。
- 如权利要求1~5中任一项所述的培养基,其特征在于还包括:选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基;和选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。
- 如权利要求1~5中任一项所述的培养基,其特征在于所述培养基不含Wnt激动剂、R-spondin家族蛋白、Noggin蛋白、BMP抑制剂、或成纤维细胞生长因子10。
- 一种肝癌类器官的培养方法,其特征在于包括以下步骤:(1)从肝癌实体瘤组织分离样本,获得肝癌原代细胞;(2)配制根据权利要求1-8中任一项所述的肝癌类器官的培养基,并对步骤(1)获得的肝癌原代细胞进行类器官培养。
- 一种用于评估或筛选治疗肝癌的药物的方法,其特征在于,包括以下步骤:(1)使用根据权利要求9所述的肝癌类器官的培养方法培养肝癌类器官;(2)选定需要检测的药物并按照所需浓度梯度进行稀释;(3)对(1)中培养得到的类器官添加稀释后的所述药物;(4)进行类器官大小或类器官活力检测。
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