WO2021208129A1 - 一种用于乳腺上皮干细胞的培养基和培养方法 - Google Patents

一种用于乳腺上皮干细胞的培养基和培养方法 Download PDF

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WO2021208129A1
WO2021208129A1 PCT/CN2020/086366 CN2020086366W WO2021208129A1 WO 2021208129 A1 WO2021208129 A1 WO 2021208129A1 CN 2020086366 W CN2020086366 W CN 2020086366W WO 2021208129 A1 WO2021208129 A1 WO 2021208129A1
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cells
epithelial stem
culture medium
stem cell
cell culture
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刘青松
刘飞扬
王俊杰
梅沪生
王文超
任涛
王黎
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合肥中科普瑞昇生物医药科技有限公司
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    • G01N33/5073—Stem cells

Definitions

  • the present invention relates to a medium for culturing epithelial stem cells, especially mammary gland epithelial stem cells in vitro, and a medium and culture method for culturing organoids containing the stem cells.
  • the invention also relates to the use of the cell progeny and organoids cultured with the culture medium and the culture method of the invention in the efficacy evaluation and screening of drugs, toxicity determination and regenerative medicine.
  • Breast disease is one of the most important diseases affecting women's health.
  • the current standard treatment drugs for breast diseases, especially breast cancer are still quite scarce, and there is a lack of personalized and precise medication guidance.
  • the key to this problem is that there is currently a lack of cell models that can be continuously expanded in vitro and that can represent the biological characteristics of patients with breast diseases for drug efficacy evaluation and screening, toxicity determination, etc.
  • epithelial stem cells and precursor cells located in the basal layer of the tissue have unlimited self-renewal ability and carry the biological characteristics of the individual (Blanpain C. et al., Science, 344(6189): 1222281 ,2014; Donati G. et al., Cell Stem Cell, 16(5), 465-476, 2015).
  • CDKN2A-dependent cell cycle arrest mechanism it is difficult to achieve sustainable culture of epithelial stem cells in vitro.
  • Cell reprogramming technology is a technology that co-cultures the patient's autologous primary epithelial cells and murine-derived feeder cells.
  • the presence of these murine feeder cells can interfere with the detection, analysis and downstream applications of patients’ autologous cells (Lipsitz Y. et al., Nat.
  • Organoid technology is a technology that embeds the patient’s autologous primary epithelial cells in an extracellular matrix for three-dimensional culture in vitro. The technical principle is to add specific epithelial stem cell markers Lgr5 and (or) Lgr6 to the culture medium.
  • Ligands such as Wnt agonists and R-spondin (R-spondin) family proteins, stimulate the Wnt signaling pathway in cells, thereby promoting the in vitro self-renewal of epithelial stem cells (Sato et al., Gastroenterology, 141:1762-1772, 2011).
  • This technology does not require feeder cells, so there is no interference problem of mouse-derived feeder cells, but the medium of organoid technology needs to add a variety of specific factors, especially the essential components of the medium Wnt protein and R-spondin family protein, As a result, the cost of organoid culture and testing is expensive, and it is not suitable for popularization in clinics for large-scale applications.
  • this technology needs to embed the organoids in Matrigel during the whole process of culture and detection, and the size of the formed organoids is not easy to control, which leads to the poor operability and reproducibility of this technology, thus limiting Large-scale application of this technology in clinical in vitro drug efficacy and evaluation, high-throughput drug screening and toxicity testing (Nick Barker et al., Nat Cell Biol, 18(3):246-54, 2016; Huch M. et al., Development, 144,938-941, 2017).
  • the inventors unexpectedly discovered that the application of MST1/2 kinase inhibitors to the culture of breast epithelial stem cells can achieve the effect of significantly promoting the sustainable proliferation of breast epithelial stem cells, and the technology is applied to construct patient-derived stem cells.
  • the cultured breast epithelial cells can represent the biological characteristics of patients with breast diseases.
  • the present invention aims to provide an improved medium for culturing mammary epithelial stem cells and a culture method using the medium.
  • the objectives of sustainable in vitro culture, controllable cost, convenient operation and no interference from exogenous cells can be achieved.
  • this technology is applied to construct a patient-derived breast cell model, cells with the biological characteristics of patients with breast disease can be obtained, and it can be used in the fields of drug efficacy evaluation and screening, toxicity testing, and regenerative medicine.
  • One aspect of the present invention is to provide a medium for culturing patient-derived mammary epithelial stem cells and/or organoids containing mammary epithelial stem cells, which contains a TGF- ⁇ inhibitor, B27 and/or N2, insulin, receptor Tyrosine kinase ligand, Rock kinase inhibitor, P38 signal transduction inhibitor, bone morphogenetic protein (BMP) inhibitor, and MST1/2 kinase inhibitor.
  • the MST1/2 kinase inhibitor includes 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 aryl optionally substituted with 1-2 independently R 6 Group (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 heterocyclic group C1-C6 alkyl (the heterocyclic group is selected from, for example, piperidinyl, tetrahydropyran Base, etc.);
  • R 6 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
  • the MST1/2 kinase inhibitor includes a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,
  • R 1 is selected C1-C6 alkyl, optionally substituted with 1-2 R 6 independently substituted phenyl, optionally substituted with 1-2 R 6 independently substituted thienyl, and optionally substituted with 1 -2 R 6 independently substituted benzyl, R 1 is more preferably optionally substituted with 1-2 R 6 independently substituted with phenyl;
  • R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, and 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 is preferably compound 1 and compound 25 described above.
  • the concentration of the MST1/2 kinase inhibitor in the medium is 100 nM or more and 10 ⁇ M or less, more preferably 300 nM or more and 3 ⁇ M or less.
  • the culture medium of the present invention further contains one or more of sugar, Nicotinamide and N-Acetylcysteine.
  • the TGF- ⁇ inhibitor may be at least one selected from A8301, SB431542, Repsox, SB505124, SB525334, SD208, LY36494, and SJN2511.
  • the TGF- ⁇ inhibitor is preferably A8301.
  • the concentration of the TGF- ⁇ inhibitor is preferably 50 nM or more and 100 ⁇ M or less, more preferably 100 nM or more and 50 ⁇ M or less, and still more preferably 100 nM or more and 10 ⁇ M or less.
  • the B27 and/or N2 is diluted into the culture medium at a final concentration of 1:25 to 1:100; preferably, B27 is diluted 1:50 into the culture medium, and N2 is diluted 1:100 into the culture medium. ⁇ .
  • Insulin is added to the culture medium at a final concentration of 2-20 ⁇ g/ml, preferably 5 ⁇ g/ml-10 ⁇ g/ml; for example, a commercially available product (10 mg/ml) is added to the culture medium at a dilution of 1:500 to 1:5000 Preferably, it is added to the culture medium at a dilution of 1:1000 to 1:2000.
  • the receptor tyrosine kinase ligand is selected from epidermal growth factor (EGF), biregulin (Amphiregulin), transforming growth factor- ⁇ (TGF- ⁇ ), basic fibroblast growth factor (bFGF), At least one of brain-derived nerve growth factor (BDNF), neurotonin 1 (Neuregulin1), hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7), and fibroblast growth factor 10 (FGF10) .
  • the receptor tyrosine kinase ligands preferably include EGF, Neuregulin1 and FGF7.
  • the final concentration of the receptor tyrosine kinase ligand is 1 ng/ml to 1000 ng/ml, preferably 5 ng/ml to 500 ng/ml, and more preferably 10 ng/ml to 100 ng/ml.
  • the Rock kinase inhibitor is selected from at least one of Y27632, Fasudil, and H-1152.
  • the preferred Rock inhibitor is Y27632.
  • the final concentration of the Rock inhibitor is 1 ⁇ M to 100 ⁇ M, preferably 2 ⁇ M to 50 ⁇ M, more preferably 5 ⁇ M to 10 ⁇ M.
  • the P38 signal transduction inhibitor is selected from at least one of SB202190, SB203580, VX-702, VX-745, PD169316, RO4402247, and BIRB796.
  • P38 signal transduction inhibitor is preferably SB202190.
  • the concentration of the P38 signal transduction inhibitor is preferably 50 nM or more and 100 ⁇ M or less, more preferably 100 nM or more and 50 ⁇ M or less, and still more preferably 100 nM or more and 10 ⁇ M or less.
  • the BMP inhibitor is selected from tenascin-like proteins including Noggin, Grayling, tenascin, and tenascin domains, and follistatin-related proteins including follistatin and follistatin domains, including DAN, DAN-like protein of DAN cysteine domain, at least one of sclerostin/SOST, decorin, ⁇ 2-macroglobulin, and DMH1.
  • the BMP inhibitor is preferably noggin.
  • the final concentration of the BMP inhibitor is 1 ng/ml to 1000 ng/ml, preferably 10 ng/ml to 500 ng/ml, more preferably 20 ng/mL to 100 ng/mL.
  • sugars contained in the cell culture medium of the present embodiment include glucose, galactose, mannose, fructose, and the like. Among them, as the sugar, glucose is preferred, and D-glucose (dextrose) is particularly preferred.
  • the final concentration of sugar in the medium is 10 mM to 100 mM, preferably 15 mM to 40 mM.
  • the final concentration of nicotinamide in the culture medium is 1 mM to 10 mM, preferably 2 mM to 5 mM.
  • the final concentration of N-acetylcysteine in the medium is 0.1 mM to 5 mM, preferably 0.5 mM to 2 mM.
  • the culture medium of the present invention does not contain any protein in the Wnt protein and R-spondin family proteins that are necessary components in the organoid culture medium.
  • the medium of the present invention also does not contain uncertain components such as fetal bovine serum (FBS) or fetal calf serum, bovine pituitary extract (BPE).
  • FBS fetal bovine serum
  • BPE bovine pituitary extract
  • the second aspect of the present invention relates to a method for culturing epithelial stem cells, epithelial cells, epithelial tumor cells, or tissues or organoids derived from normal breast tissue or diseased breast tissue, or tissues or organoids containing at least any of these cells, wherein the The culture method includes: (1) the step of preparing an extracellular matrix; (2) making epithelial stem cells, epithelial cells, epithelial tumor cells, or tissues containing at least any of these cells adhere to the extracellular matrix or The step of embedding in an extracellular matrix; (3) adding the medium described in the first aspect above to treat the epithelial stem cells, epithelial cells, epithelial tumor cells, or tissues containing at least any of these cells The step of culturing to obtain the corresponding progeny of epithelial stem cells, epithelial cells, epithelial tumor cells or organoid progeny.
  • the extracellular matrix in the above-mentioned culture method uses a low growth factor type extracellular matrix glue.
  • a low growth factor type extracellular matrix glue for example, commercially available Matrigel TM (manufactured by BD Biosciences) or BME (manufactured by Trevigen) can be used. More specifically, the extracellular matrigel is diluted with a serum-free medium.
  • the medium may be the epithelial stem cell medium of the present invention, or DMEM/F12 (manufactured by Corning), DMEM medium (manufactured by Corning), and
  • the RPMI1640 medium (manufactured by Corning Corporation) may be a modified DMEM/F12 medium (manufactured by Thermo Corporation) or a modified RPMI1640 medium (manufactured by Thermo Corporation) or the like.
  • the dilution ratio of extracellular matrigel is 1:50-400, preferably 1:50-200.
  • the step of culturing epithelial stem cells or organoids is carried out under conditions of an oxygen concentration of 0.1%-25%, preferably 20% normal oxygen conditions, or 0.1%-15% hypoxic conditions.
  • the third aspect of the present invention covers the use of the culture medium and culture method of the present invention to obtain expanded epithelial stem cells, epithelial cells, epithelial tumor cell progeny or organoid progeny, and is applied to methods and uses of drug efficacy evaluation and screening, especially It is the method and application of in vitro curative effect evaluation and screening of anti-tumor drugs.
  • the present invention relates to a method for evaluating or screening the efficacy of breast cancer drugs, which is characterized in that it comprises the following steps:
  • the number of culture generations can reach more than 10 generations, preferably more than 12 generations.
  • the in vitro cell expansion time can last at least 3 months, and the differentiation ability can be maintained for a long time. , The frequency of gene mutations during in vitro culture is extremely low;
  • the cultured mammary epithelial stem cells are not interfered by fibroblasts, adipocytes and other mesenchymal cells, and purified mammary epithelial stem cells and their progeny can be obtained;
  • the medium composition does not contain uncertain components such as serum and bovine pituitary extract, so it is not affected by the quality and quantity of different batches of serum or bovine pituitary extract;
  • the amplified mammary epithelial cells can also be passaged continuously Amplification
  • the culture cost is controllable: the primary breast cancer culture medium does not need to add expensive Wnt agonist protein, R-spondin family protein and other ingredients. It is a simplification and improvement of the existing breast epithelial cell and organoid culture medium. Save the cost of breast epithelial stem cell culture medium;
  • the number of mammary epithelial cells obtained by culturing the technology is large, the degree of homogenization is high, and the cost is controllable. It is more suitable for high-throughput screening of new candidate compounds and providing patients with high-throughput screening of new candidate compounds than cell condition reprogramming technology and organoid technology. Flux drug in vitro sensitivity function test and other drug efficacy evaluation, screening and toxicity test field.
  • mammary epithelial stem cells, epithelial cells, epithelial tumor cells, or tissues containing at least any of these cells derived from humans or other mammals can be cultured to obtain expanded, Corresponding progeny of epithelial stem cells, epithelial cells, and epithelial tumor cells.
  • organoids can also be formed from at least one of the cells and the tissues.
  • the cells obtained by the culture method of this embodiment can be used in regenerative medicine, toxicity testing, basic medical research of breast epithelial cells, screening of drug responses, determination of drug in vitro metabolic stability and metabolic profile, and new drugs for breast diseases R&D, etc.
  • Figures 1A to 1C are drawings for explaining the effect of MST1/2 kinase inhibitors on prolonging the culture passage number of human breast cancer tumor cells.
  • Fig. 2 is a diagram for explaining the proliferation promoting effect of different concentrations of MST1/2 kinase inhibitors on human breast cancer tumor cells in vitro.
  • Figures 3A and 3B are diagrams for explaining the sustained promotion effect of MST1/2 kinase inhibitor on the proliferation of breast cancer tumor cells in vitro.
  • FIGS. 4A and 4B are drawings for explaining the sustained and reversible proliferation-promoting effect of MST1/2 kinase inhibitor on breast cancer tumor cells in the culture medium of the present invention.
  • Fig. 5A is a diagram for explaining the continuous promotion effect of the medium of the present invention on the proliferation of normal breast epithelial cells
  • Fig. 5B is a diagram for explaining the continuous promotion of the proliferation of breast cancer tumor cells by the medium of the present invention under hypoxic conditions Figure of the effect
  • Figure 5C is a drawing for explaining the effect of the culture medium of the present invention on the culture of breast tumor organoids.
  • 6A to 6C are drawings for explaining the effect of MST1/2 kinase inhibitor on the expression of stem markers in human breast epithelial cells and the signal pathway mediated by MST kinase.
  • Figures 7A and 7B are drawings for explaining the consistency analysis of gene copy number variation between breast cancer tumor cells cultured using the technology of the present invention and the original tissue from which the corresponding cells are derived.
  • Fig. 8 is a diagram for explaining the genetic mutation consistency analysis of breast cancer tumor cells cultured using the technology of the present invention and the original tissue from which the corresponding cells are derived.
  • Fig. 9 is a diagram for illustrating the comparison between the immunohistochemical results of breast cancer tumor cells cultured using the technique of the present invention and the immunohistochemical results of the original tissue section of the tissue sample itself.
  • Figures 10A and 10B are graphs for illustrating the dose-response curves of breast cancer tumor cells cultured using the technology of the present invention on different drugs.
  • epithelial cells include differentiated epithelial cells and epithelial stem cells obtained from epithelial tissues.
  • Epithelial stem cells refer to cells that have long-term self-renewal capabilities and differentiate into epithelial cells, and refer to stem cells derived from epithelial tissues.
  • epithelial tissues include cornea, oral mucosa, skin, conjunctiva, bladder, renal tubules, kidneys, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), large intestine (including colon)) , Liver, pancreas, breast, salivary gland, lacrimal gland, prostate, hair root, trachea, lung, etc.
  • the cell culture medium of the present embodiment is preferably used for the culture of mammary gland epithelial cells.
  • epithelial tumor cell refers to a cell obtained by tumorigenesis of a cell derived from the above-mentioned epithelial tissue.
  • organs refers to a three-dimensional, organ-like cell organization formed by spontaneously organizing and gathering cells at a high density in a controlled space.
  • the medium of the present embodiment also contains selected from TGF- ⁇ inhibitor, B27 and/or N2, insulin, receptor tyrosine kinase ligand, Rock kinase inhibitor, P38 signal One or more or all of transduction inhibitors, bone morphogenetic protein (BMP) inhibitors, glucose, nicotinamide, and N-acetylcysteine.
  • TGF- ⁇ inhibitor selected from TGF- ⁇ inhibitor, B27 and/or N2
  • insulin insulin
  • receptor tyrosine kinase ligand ligand
  • Rock kinase inhibitor Rock kinase inhibitor
  • P38 signal One or more or all of transduction inhibitors, bone morphogenetic protein (BMP) inhibitors, glucose, nicotinamide, and N-acetylcysteine.
  • BMP bone morphogenetic protein
  • glucose nicotinamide
  • the mammary epithelial stem cell culture medium of the present embodiment includes any serum-free cell culture medium.
  • the cell culture medium of this embodiment is preferably used for animal cells or human cells.
  • a predetermined synthetic medium buffered to pH 7.2 or higher and pH 7.6 or lower with a carbonic acid buffer can be used. More specifically, the serum-free minimal medium is selected from, for example, DMEM/F-12 (manufactured by Corning Corporation) medium.
  • RPMI1640 medium manufactured by Corning Corporation
  • DMEM medium manufactured by Corning Corporation
  • modified DMEM/F-12 manufactured by Thermo Corporation
  • modified RPMI1640 medium manufactured by Thermo Corporation
  • Mammalian Sterile 20-like Kinase 1 and 2 are the upstream regulators of the mitogen-activated protein kinase (MAPK) signal transduction pathway with a molecular weight of 56-60 KD. It can regulate a variety of cell processes, including proliferation, apoptosis, migration and cytoskeleton rearrangement.
  • MST1 mitogen-activated protein kinase
  • the MST1 gene was cloned from the lymphatic cDNA library by PCR when studying the homologue of Saccharomyces cerevisiae Ste20 and human in 1995. Its coded product is similar in structure and function to yeast Ste20, so it was named MST1.
  • MST1 also has three paralogues of MST2, MST3 and MST4 (Dan et al., Trends Cell Biol. 11, 220-230, 2001). The homology of MST2 and MST1 protein is 76%. At present, its physiological function is not clear. Some studies suggest that MST2 may have a compensatory effect on the function mediated by MST1 (Wu S. et al., Cell, 114, 445-456, 2003 ).
  • MST1/2 is an orthologous protein of Drosophila Hippo (Hpo), which is a core regulatory protein in the Hippo signal transduction pathway.
  • Hpo Drosophila Hippo
  • This evolutionary conservative program can control tissue growth and organ size by regulating cell proliferation, apoptosis and stem cell self-renewal.
  • the mammalian Hippo signal transduction pathway involves a kinase cascade in which MST1/2 kinase and SAV1 scaffold protein form a phosphorylated and activate LATS1/2 complex.
  • LATS1/2 kinase phosphorylates YAP and TAZ, thereby promoting the retention and functional inhibition of these transcriptional co-activators in the cytoplasm (Zhao.B et al., Nat Cell Biol, 13, 877-883, 2011).
  • an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signaling.
  • MST1/2 kinase inhibitors for example, bind to MST1/2 kinase and reduce its activity. Since the structures of MST1 and MST2 are similar, the MST1/2 kinase inhibitor may also be, for example, a compound that binds to MST1 or MST1/2 and reduces its activity.
  • the MST1/2 kinase inhibitor has preferably 50% or more, more preferably 70% or more, further more preferably 80% or more, particularly preferably More than 90% inhibitory activity.
  • the inhibitory effect of MST1/2 inhibitors can be evaluated by methods known to those skilled in the art.
  • As the evaluation system there can be exemplified the specific antibody detection method of Thr183 phosphorylation site of MST1, the in vitro experiment of recombinant protein kinase, the DiscoverRx high-throughput screening platform for MST1/2 kinase inhibitor, and the activity of MST1/2 kinase. Detection kit (manufactured by Promega), etc.
  • the compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof may be included.
  • R 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl optionally substituted with 1-2 independently R 6 Group (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 heterocyclic group C1-C6 alkyl (the heterocyclic group is selected from, for example, piperidinyl, tetrahydropyran Base, etc.);
  • R 6 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
  • the MST1/2 kinase inhibitor includes a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,
  • R 1 is selected C1-C6 alkyl, optionally substituted with 1-2 R 6 independently substituted phenyl, optionally substituted with 1-2 R 6 independently substituted thienyl, and optionally substituted with 1 -2 R 6 independently substituted benzyl, R 1 is more preferably optionally substituted with 1-2 R 6 independently substituted with phenyl;
  • R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, and 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 is preferably compound 1 and compound 25 described above.
  • the concentration of the MST1/2 kinase inhibitor contained in the medium is preferably 100 nM or more and 10 ⁇ M or less, more preferably 300 nM or more and 3 ⁇ M or less.
  • an MST1/2 kinase inhibitor to the medium every 2 days, or to change the medium to a fresh medium every 4 days.
  • Transforming growth factor- ⁇ (transforming growth factor- ⁇ , TGF- ⁇ ) is a kind of growth factor, which is produced in almost all cells such as the kidney, bone marrow, and platelets. There are five subtypes of TGF- ⁇ ( ⁇ 1 ⁇ 5). In addition, it is known that TGF- ⁇ promotes the proliferation of osteoblasts and the synthesis and proliferation of connective tissues such as collagen, and has an inhibitory effect on the proliferation of epithelial cells and osteoclasts.
  • a TGF- ⁇ inhibitor is, for example, a compound that prevents or inhibits the binding of TGF- ⁇ to the TGF- ⁇ receptor, and is a compound that binds to TGF- ⁇ in order to form a complex that neutralizes the activity of TGF- ⁇ .
  • a TGF- ⁇ inhibitor is, for example, a compound that binds to the TGF- ⁇ receptor, prevents or inhibits the binding of TGF- ⁇ to the receptor, and acts as an antagonist or inverse agonist.
  • the TGF- ⁇ inhibitor has preferably 50% or more, more preferably 70% or more, further more preferably 80% or more, particularly preferably 90%. % Or more inhibitory activity.
  • the TGF- ⁇ inhibitory activity can be evaluated by a method known to those skilled in the art.
  • As the evaluation system a human PAI-1 promoter using a luciferase reporter gene or a cell experiment using a reporter construct containing a Smad binding site to stably transfect cells (De Gouville et al., Br J Pharmacol, 145 ( 2):166-177, 2005).
  • the TGF- ⁇ inhibitor contained in the medium for culturing mammary epithelial stem cells of the present embodiment includes at least one of A8301, SB431542, Repsox, SB505124, SB525334, SD208, LY36494, and SJN2511.
  • the TGF- ⁇ inhibitor contained in the cell culture medium for culturing mammary epithelial stem cells of the present embodiment is preferably A8301.
  • the concentration of the TGF- ⁇ inhibitor contained in the medium of the present embodiment is preferably 50 nM or more and 100 ⁇ M or less, more preferably 100 nM or more and 50 ⁇ M or less, and still more preferably 100 nM or more and 10 ⁇ M or less.
  • TGF- ⁇ inhibitor In the culture of epithelial stem cells, it is preferable to add a TGF- ⁇ inhibitor to the medium every 2 days, or to change the medium to a fresh medium every 4 days.
  • the receptor tyrosine kinase ligand contained in the culture medium of the present embodiment for example, selected from epidermal growth factor (EGF), biregulin (Amphiregulin), transforming growth factor- ⁇ (TGF- ⁇ ), Basic fibroblast growth factor (bFGF), brain-derived nerve growth factor (BDNF), neurotonin 1 (Neuregulin1), hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7) and fibroblasts At least one of growth factor 10 (FGF10).
  • the receptor tyrosine kinase ligands preferably include EGF, Neuregulin1 and FGF7.
  • the final concentration of the receptor tyrosine kinase ligand is 1 ng/ml to 1000 ng/ml, preferably 5 ng/ml to 500 ng/ml, and more preferably 10 ng/ml to 100 ng/ml.
  • EGF is a powerful division and proliferation factor for culturing various ectodermal cells and mesodermal cells, and has a significant effect on the specific cell differentiation of some fibroblasts.
  • the EGF precursor is cleaved by proteolysis and exists as a membrane-bound molecule that stimulates cell production of 53-amino acid peptide hormone.
  • the receptor tyrosine kinase ligand contained in the medium used in the culture method of the present embodiment is preferably EGF.
  • the concentration of EGF contained in the cell culture medium of the present embodiment is preferably 1 ng/mL or more and 1000 ng/mL or less, more preferably 5 ng/mL or more and 500 ng/mL or less, still more preferably 5 ng/mL or more and 100 ng/mL or less .
  • the cell culture medium of the present embodiment preferably contains EGF and Amphiregulin or Neuregulin 1 or hepatocyte growth factor (HGF) or fibroblast growth factor 7 (FGF7) or fibroblasts.
  • EGF hepatocyte growth factor
  • FGF7 fibroblast growth factor 7
  • FGF10 cell growth factor 10
  • receptor tyrosine kinase ligands are used, preferably 1 ng/mL or more and 1000 ng/mL or less, more preferably 5 ng/mL or more and 500 ng/mL or less, still more preferably 10 ng/mL or more and Below 100ng/mL.
  • receptor tyrosine kinase ligand to the medium every 2 days, or to replace the medium with a fresh medium every 4 days.
  • p38 signaling inhibitor or “p38 inhibitor” refers to any inhibitor that directly or indirectly negatively regulates p38 signaling.
  • inhibitors of p38 signaling for example, bind to p38 and reduce its activity.
  • p38 protein kinase is part of the mitogen-activated protein kinase (MAPK) family.
  • MAPK is a serine/threonine-specific protein kinase that responds to various cell activities such as environmental stress and inflammatory cytokines, conditional gene expression, mitosis, differentiation, proliferation, and cell survival/programmed death.
  • p38 MAPK exists as ⁇ , ⁇ , ⁇ 2, ⁇ , and ⁇ homologous isomers.
  • p38 signaling inhibitors are also agents that, for example, bind to at least one p38 homoisomer and reduce its activity.
  • the p38 signaling inhibitor has preferably 50% or more, more preferably 70% or more, further more preferably 80% or more, particularly preferably 90% or more The inhibitory activity.
  • the inhibitory effect of the p38 signaling inhibitor can be evaluated by a method known to those skilled in the art.
  • examples include the detection method of phosphorylation site-specific antibody of Thr180/Tyr182 phosphorylation, biochemical recombinant kinase test, tumor necrosis factor alpha (TNF- ⁇ ) secretion test, and DiscoverRx high for p38 inhibitors. Throughput screening platform, p38 activity test kit (manufactured by Sigma-aldrich), etc.
  • p38 signaling inhibitor contained in the medium of the present embodiment for example, at least one of SB202190, SB203580, VX-702, VX-745, PD169316, RO4402247, and BIRB796 can be mentioned.
  • P38 signal transduction inhibitor is preferably SB202190.
  • the concentration of the p38 signaling inhibitor contained in the medium is preferably 50 nM or more and 100 ⁇ M or less, more preferably 100 nM or more and 50 ⁇ M or less, and still more preferably 100 nM or more and 10 ⁇ M or less.
  • a p38 signaling inhibitor to the medium every 2 days, or to change the medium to a fresh medium every 4 days.
  • Bone Morphogeneitc Protein acts as a dimer ligand and binds to a receptor complex formed by two different receptor serine/threonine kinases, type I and type II receptors.
  • the type II receptor phosphorylates the type I receptor, so the receptor kinase is activated.
  • the type I receptor then phosphorylates the specific receptor substrate (SMAD), thereby generating transcriptional activity through the signaling pathway.
  • a BMP inhibitor is, for example, an agent that prevents or inhibits the binding of BMP molecules to BMP receptors, and is an agent that binds to BMP molecules in order to form a complex that neutralizes the activity of BMP.
  • a BMP inhibitor is, for example, a drug that binds to a BMP receptor, prevents or inhibits the binding of BMP molecules to the receptor, and acts as an antagonist or an inverse agonist.
  • the BMP inhibitor Compared with the BMP activity level in the absence of the inhibitor, the BMP inhibitor has an inhibition of preferably 50% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% or more active.
  • BMP inhibitory activity can be evaluated by measuring the transcriptional activity of BMP using a method known to those skilled in the art (Zilberberg et al., BMC Cell Biol, 8:41, 2007).
  • the BMP inhibitor contained in the medium of the present embodiment is preferably a natural BMP binding protein, and examples thereof include Noggin, Gremlin, Chordin, and Tenascin domains.
  • Tenascin-like proteins including follistatin, follistatin-related proteins, including DAN, DAN-like proteins containing DAN cysteine domain, sclerostatin/SOST, Decorin, and ⁇ 2-macroglobulin, etc. It can also be a small molecule inhibitor, such as DMH1.
  • tenascin-like protein or DAN-like protein is preferred, and tenascin-like protein is more preferred.
  • tenascin-like protein noggin is preferred.
  • Tenascin-like protein and DAN-like protein are diffusive proteins that bind to BMP molecules with various affinities and can inhibit the proximity of BMP molecules to signal transduction receptors.
  • the concentration of the BMP inhibitor contained in the medium of the present embodiment is preferably 1 ng/ml to 1000 ng/ml, preferably 10 ng/ml to 500 ng/ml, and preferably 20 ng/mL or more and 100 ng/mL or less.
  • a BMP inhibitor to the medium every 2 days, or to replace the medium with a fresh medium every 4 days.
  • the cell culture medium of this embodiment may further include a Rock kinase (Rho-kinase) inhibitor.
  • a Rock kinase (Rho-kinase) inhibitor for example, at least one of Y27632, Fasudil, and H-1152 can be selected, and Y27632 is preferably used.
  • the final concentration of the Rock inhibitor is 1 ⁇ M to 100 ⁇ M, preferably 2 ⁇ M to 50 ⁇ M, more preferably 5 ⁇ M to 10 ⁇ M.
  • Y27632 When Y27632 is used, it is preferably added during the first two days of culturing of single-cell-dispersed stem cells.
  • the final concentration of Y27632 contained in the medium of this embodiment is preferably 1 ⁇ M to 100 ⁇ M, preferably 2 ⁇ M to 50 ⁇ M, and more preferably 5 ⁇ M to 10 ⁇ M.
  • the cell culture medium of the present embodiment may further include purified, natural, semi-synthetic, and/or synthetic supplements that stimulate cell proliferation, and do not include components of unknown components, such as serum.
  • the supplement contained in the cell culture medium for culturing mammary epithelial stem cells of the present embodiment can use commercially available supplements, such as B27 (manufactured by Gibco) and/or N2 (manufactured by Gibco), preferably at a ratio of 1:25 to 1: It is added at a final concentration of 100, more preferably at a final concentration of 1:50 to 1:100.
  • the cell culture medium of this embodiment also contains insulin.
  • Insulin is recognized as an essential component for maintaining cell growth in vitro.
  • the insulin contained in the culture medium of this embodiment can use commercially available insulin, such as purified human insulin solution (manufactured by Sigma), or a supplement containing insulin, such as insulin-transferrin-sodium selenite (ITS- X, made by Thermo Fisher Scientific).
  • the final concentration of insulin in the culture medium is 2 ⁇ g/ml-20 ⁇ g/ml; the preferred concentration is 5 ⁇ g/ml-10 ⁇ g/ml.
  • the cell culture medium of this embodiment may further include at least one sugar that can be a source of carbon energy.
  • sugars contained in the cell culture medium of the present embodiment include glucose, galactose, mannose, fructose, and the like. Among them, as the sugar, glucose is preferred, and D-glucose (dextrose) is particularly preferred.
  • the final concentration of sugar contained in the cell culture medium for culturing mammary epithelial stem cells of the present embodiment in the culture medium is 10 mM to 100 mM, preferably 15 mM to 40 mM.
  • the cell culture medium of this embodiment may further contain nicotinamide and/or N-acetylcysteine.
  • Niacinamide functions as an antioxidant in this embodiment, which is beneficial to the formation of organoids.
  • the final concentration of nicotinamide contained in the improved organoid culture medium of this embodiment in the medium is 1 mM to 10 mM, preferably 2 mM to 5 mM.
  • N-acetylcysteine is a stimulant that promotes the proliferation of organoids and also has the effect of promoting the growth of organoids.
  • the final concentration of N-acetylcysteine contained in the improved organoid culture medium of this embodiment in the medium is 0.1 mM to 5 mM, preferably 0.5 mM to 2 mM.
  • the culture method according to Embodiment 1 of the present invention is a method for culturing epithelial stem cells, epithelial cells, epithelial tumor cells, or tissues containing at least any of these cells derived from normal breast tissue or diseased breast tissue .
  • the method includes the following steps:
  • epithelial stem cells, epithelial cells, epithelial tumor cells, or tissues containing at least any of these cells derived from mammals including humans can be cultured for a long time to obtain expanded and corresponding Progeny of epithelial stem cells, epithelial cells, epithelial tumor cells or organoids.
  • ECM Extracellular Matrix
  • ECM contains a variety of polysaccharides, water, elastin, and glycoproteins.
  • glycoproteins include collagen, nestin, fibronectin, laminin, and the like.
  • polysaccharides include proteoglycan and glycosaminoglycan.
  • elastin, elastic fiber etc. are mentioned.
  • ECM As the ECM of this embodiment, a commercially available ECM can be used.
  • extracellular matrix protein manufactured by Invitrogen
  • basement membrane preparation Matrigel TM (manufactured by BD Biotech)
  • EHS Engelbreth-Holm-Swarm
  • ProNectin SigmaZ378666
  • a mixture of natural ECM and synthetic ECM can be used.
  • a basement membrane preparation (Matrigel TM (manufactured by BD Biotech)) derived from EHS mouse sarcoma cells is preferred.
  • ECM ECM to culture epithelial stem cells can enhance the long-term survival of stem cells and the persistence of undifferentiated stem cells.
  • stem cell cultures cannot be cultured for a long time, and the continued existence of undifferentiated stem cells cannot be observed.
  • epithelial stem cells that cannot be cultured without ECM can be cultured.
  • ECM can be mixed with epithelial stem cells and the cells are embedded in the ECM.
  • the ECM sinks at the bottom of a culture dish with cells suspended.
  • the above-mentioned epithelial stem cell culture medium can be added to allow it to diffuse into the ECM for use.
  • Cells in the culture medium can be immobilized on the ECM by interacting with the surface structure of the ECM, for example by interacting with integrins.
  • ECM can be coated on the surface of the culture vessel. More specifically, the extracellular matrigel is diluted with a serum-free medium.
  • the medium may be the cell culture medium of the present invention or the above-mentioned cell culture medium.
  • the dilution ratio of the extracellular matrigel is, for example, 1:20-400, and a more preferable range is 1:50-200.
  • the coating method is to add the diluted extracellular matrigel to the culture vessel to completely cover the bottom of the culture vessel. It is allowed to stand at 37°C, and the coating time is at least 30 minutes, and a more preferable range is 30 to 60 minutes. After the coating is finished, aspirate and discard the excess extracellular matrigel diluent, and incubate the vessel for use.
  • breast epithelial stem cells, epithelial cells, epithelial tumor cells, or tissues containing at least any of these cells are prepared.
  • breast epithelial cells can be derived from breast cancer tissue samples and para-cancerous tissue samples.
  • Breast cancer tissue samples are, for example, derived from surgically removed cancer tissue samples from breast tumor patients who have explained and obtained consent, and the adjacent tissue samples are collected from breast tissue that is at least 5 cm away from the breast cancer tissue. Collect the above-mentioned tissue samples within half an hour after the patient's surgical resection or biopsy.
  • DMEM/F12 medium contains 50-200U/mL (such as 100U/mL) penicillin and 50-200 ⁇ g/mL (such as 100 ⁇ g/mL) streptomycin (below Referred to as transport fluid).
  • tissue sample In the biological safety cabinet, transfer the tissue sample to a cell culture dish, rinse the tissue sample with transport fluid, wash off the blood cells on the surface of the tissue sample, and remove unnecessary tissues such as skin and fascia on the surface of the tissue sample.
  • tissue sample fragments to the centrifuge tube, centrifuge at least 1000 rpm with a benchtop centrifuge for 3-10 minutes; then use a pipette to carefully remove the supernatant in the centrifuge tube, and then use 5-25mL collagenase II (0.5 ⁇ 5mg/mL, such as 1mg/mL) and collagenase IV (0.5 ⁇ 5mg/mL, such as 1mg/mL) serum-free DMEM/F12 medium resuspended, placed on a 37 °C constant temperature shaker for shaking digestion, the time is at least 1 hour (digestion time depends on the sample size; if the sample is larger than 1g, the digestion time is increased to 1.5-2 hours); after that, centrifuge at least 300g/min in a benchtop centrifuge for 3-10 minutes, discard the supernatant, and after digestion
  • the tissue cells are resuspended in 5-25mL DMEM/F12 medium containing, for example, 10% calf serum
  • the cell suspension is centrifuged in a centrifuge at at least 300 g/min for 3-10 minutes, the supernatant is discarded, and then resuspended in the primary cell culture medium of the present invention.
  • the epithelial stem cells, epithelial cells, epithelial tumor cells, or tissue containing at least any of these cells isolated by the above-mentioned method are seeded on the extracellular matrix obtained in the preparation step and left to stand.
  • the seeded cells can adhere to the ECM by interacting with the surface structure of the ECM, for example by interacting with integrins.
  • the culture medium of the present invention is added and cultured before the cells are not dried.
  • the culture temperature is preferably from 30°C to 40°C, more preferably about 37°C.
  • the culture time can be adjusted appropriately according to the cells used. Approximately 1 to 2 weeks after the start of the culture, the corresponding progeny of epithelial stem cells, epithelial cells, epithelial tumor cells or organoids can be obtained.
  • the culture method of this embodiment can achieve a long culture time of 3 months or more, and the cells can also be maintained cultured.
  • the self-renewal and differentiation ability of the cells can be maintained for a long time, and the frequency of gene mutations in in vitro culture is extremely low.
  • the cell culture step of this embodiment can be cultured not only under normoxia conditions but also under hypoxia conditions.
  • it can be expanded from mammalian epithelial stem cells, epithelial cells, or epithelial tumor cells including humans, or tissues containing at least any of these cells, or tissues that could not be cultured in the past, Corresponding epithelial stem cells, epithelial cells, epithelial tumor cell progeny or organoid progeny.
  • the oxygen concentration is preferably 0.1% or more and 15% or less, more preferably 0.3% or more and 10% or less, and still more preferably 0.5% or more and 5% or less.
  • Organoids can be obtained by the culture method according to Embodiment 1 of the present invention.
  • the organoid of the present embodiment can be applied to regenerative medicine, basic medical research of epithelial cells, screening of drug response, development of new drugs using disease-derived epithelial organoids, and the like.
  • the present invention provides the use of the above-mentioned epithelial stem cells for drug response screening, toxicity testing, or regenerative medicine.
  • the above-mentioned epithelial stem cells are cultured in a multi-well plate such as a 96-well plate or a 384-well plate.
  • a multi-well plate such as a 96-well plate or a 384-well plate.
  • molecular libraries include antibody fragment libraries, peptide phage display libraries, peptide libraries (for example, LOPAP (trademark), manufactured by Sigma-Aldrich), lipid libraries (manufactured by BioMol), and synthetic compound libraries (manufactured by MCE) Or natural compound library (Specs, manufactured by TimTec), etc.
  • gene libraries can be used.
  • gene libraries include cDNA libraries, antisense libraries, siRNA, or other non-coding RNA libraries.
  • a method of exposing cultured cells to multiple concentrations of a test agent for a certain period of time, and evaluating the culture at the end of the exposure time can be exemplified.
  • the epithelial stem cells obtained in this embodiment can also be used to identify drugs that specifically target epithelial tumor cells, but not normal cells.
  • epithelial stem cells obtained in this embodiment can be used instead of cell lines such as Caco-2 cells in toxicity experiments of new drug candidates or known or new nutritional supplement foods.
  • epithelial stem cells obtained in this embodiment can be used for culturing pathogens such as norovirus for which no suitable tissue culture or animal model is currently available.
  • epithelial stem cells obtained in this embodiment can be used in regenerative medicine, for example, in the repair of breast tissue after radiation exposure or after surgery.
  • Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): Add 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0g) in a round bottom flask Then methanol (30 mL) was added, and then thionyl chloride (1.2 mL) was added dropwise under an ice bath. The reaction system was reacted overnight at 85°C. After the reaction, the solvent was evaporated to dryness under reduced pressure, and the obtained white solid was directly used in the next step.
  • MST1/2 inhibitor compounds of the present invention were synthesized according to a method similar to that of compound 1, and their structures and mass spectral data are shown in the following table.
  • insulin manufactured by Sigma-Aldrich, a commercial product with a concentration of 10 mg/mL, and a volume ratio of 1:1000
  • DMEM/F-12 medium manufactured by Corning
  • B27 was added at a final concentration of 1:50
  • Noggin Noggin, manufactured by R&D
  • TGF- ⁇ inhibitor A8301 manufactured by MCE
  • the P38 signal transduction inhibitor SB202190 was added at a concentration of 500 nM
  • the ROCK kinase inhibitor Y27632 was added at a final concentration of 10 ⁇ M.
  • EGF epidermal growth factor
  • FGF7 fibroblast growth factor 7 FGF7
  • neurotonin 1 at a final concentration of 10ng/mL Neuregulin1 ( Peprotech Co.) and glucose with a final concentration of 15.8 mM (Peprotech Co.) were prepared as a basic medium for culturing mammary epithelial stem cells. (Hereinafter also referred to as "basic medium” or “basic culture”).
  • compound 1 or compound 25 was added at a final concentration of 1 ⁇ M.
  • a medium with the following combination of constituent components was prepared.
  • HMFL-XN26 diseased tissue
  • the dilution ratio of Matrigel is 1:50-400, preferably 1:100-200.
  • Figure 1A shows the comparison of cytoscopy photographs of samples HMFL-XN26 at the 4th, 5th, 6th and 7th passages after the initial culture (passage 0) under each culture condition (100-fold inverted phase difference). Under the microscope). The sample HMFL-XN26 was cultured in a basic medium to the 6th generation, but growth arrested. The medium with basal medium + compound 1 can still continue to culture mammary epithelial cells after the cells are cultured to the sixth generation.
  • Fig. 1B is a graph obtained by quantifying the number of culture passages of breast tumor cells in each medium.
  • Figure 1C uses the number of days of culture as the abscissa and the population doubling number as the ordinate. The growth curves of breast tumor cells under each culture condition are drawn using Graphpad Prism7.0 software. The formula for calculating the population doubling number is:
  • N is the number of cells at passage
  • X 0 is the number of cells at the initial seeding
  • HMFL-XN40 epithelial tumor cell sample
  • the epithelial tumor cells were seeded on a 6-well plate coated with Matrigel TM (manufactured by BD Biosciences).
  • a basal medium was added to the wells inoculated with the above-mentioned epithelial tumor cells, and culture was performed at 37°C with an oxygen concentration of 20%. Digestion, passage, culture and count were carried out according to the step 4 of Example 1, and the culture was continued for 3 generations.
  • the epithelial tumor cells were uniformly inoculated into each well of a 24-well plate coated with Matrigel TM (manufactured by BD Biosciences) at a cell density of 3 ⁇ 10 4 cells/well. Inoculate, add 1 mL each of the medium containing basal medium + DMSO and the medium containing basal medium + compound 1 to each well. The concentration of compound 1 in the medium from low to high is 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, 3 ⁇ M and 10 ⁇ M.
  • Figure 2 shows the cell count results of HMFL-XN40 continuously cultured from the 4th generation to the 6th generation, and each generation of breast tumor cells under various culture conditions.
  • DMSO basal medium + solvent control
  • Example 1 According to the same manner as in Example 1, a basic medium and a medium having the following combination of constituent components were prepared.
  • HMFL-XN34 and HMFL-XN35 cancer tissue samples from breast tumor patients who were explained and obtained consent were obtained.
  • Embodiment 1 follow the steps in Embodiment 1 to obtain epithelial tumor cells.
  • the epithelial tumor cells obtained from HMFL-XN34 were seeded on a 24-well plate coated with MatrigelTM (manufactured by BD Biosciences).
  • HMFL-XN35 was inoculated in a 24-well plate in the same way, and basal medium, basal medium + compound 1 (final concentration of 1 ⁇ M) medium were added respectively, and cultured at 37°C with an oxygen concentration of 20%. The medium was replaced every 2 days after the start of the culture.
  • Figure 3A is a comparison of cytoscopy photographs of mammary epithelial cells derived from HMFL-XN34 at the fourth generation (34 days of co-cultivation) after the start of primary culture (passage 0) under culture conditions with or without compound 25 ( Under a 100-fold inverted phase contrast microscope), and breast epithelial cells derived from HMFL-XN35 at the sixth passage (42 days of co-cultivation) after the start of the primary culture (passage 0) under the culture conditions with or without compound 1
  • Fig. 3B is a statistical result after normalization of the cell count results of different breast tumor cells cultured under various culture conditions.
  • Figures 1 and 2 of Figure 4A respectively show that breast epithelial cells derived from HMFL-XN35 were cultured under the culture conditions of compound 1 from the beginning of the primary culture (passage 0) and continued to be cultured until the 18th passage. conventional digestion, and press again the density of 3 ⁇ 10 4 passaging inoculum, time of inoculation the cells were cultured in the original medium (basal medium + 1 ⁇ M compound 1, FIG. No. 1 of FIG. 4A) and the compound was removed and culture medium (basal medium 1 Base, Fig. 4A, Fig. 2), the cytomicroscopic photograph after continuing the culture for 8 days.
  • Figure 4A Figure 3 is a cytomicroscopic photograph (under a 100-fold inverted phase contrast microscope) of the cells in Figure 2 after adding compound 1 to the culture medium and culturing for another 8 days.
  • Figure 4B is the normalized statistical results of the cell counts of different breast tumor cells cultured under each culture condition, wherein the cell counts of 8 days of culture with basal medium + 1 ⁇ M compound 1 are taken as the reference 100%, After 8 days of culture, the culture medium without compound 1 produced a cell count lower than 50% of the baseline, and after adding compound 1 to the culture medium for another 8 days, the cell count increased significantly.
  • MST1/2 kinase inhibitor compound 1 is an essential component for realizing the medium of the present invention to maintain the continuous proliferation of breast epithelial cells in vitro, and compound 1 realizes the effect of continuously promoting the proliferation of breast cancer tumor cells in vitro It is reversible.
  • Insulin manufactured by Sigma-Aldrich
  • B27 was added at a final concentration of 1:50
  • a final concentration of 100 ng/mL Noggin manufactured by Peprotech
  • compound 1 is added at a final concentration of 1 ⁇ M
  • A8301 manufactured by MCE
  • SB202190 manufactured by MCE
  • compound 1 is added at a final concentration of 10 ⁇ M Y27632 (manufactured by MCE Corporation).
  • EGF epidermal growth factor
  • HMFL-XN28 breast tissue samples from the paracancerous tissues of breast tumor patients who have been explained and approved. Collect the part at least 5 cm away from the breast tumor as normal breast tissue. Then, according to the same procedure as in embodiment 1, freshly isolated normal breast epithelial cells were obtained, and then cultured in vitro according to the procedure of embodiment 2-2.
  • Figure 5A shows that normal breast epithelial cells derived from HMFL-XN28 paracancerous tissues were cultured to the 2nd day and continued to the 18th day after the start of the primary culture (passage 0) under the culture condition of basal medium + compound 1 Photo of cytoscopy (under a 100x inverted phase contrast microscope).
  • Example 2-2 breast tumor cells derived from HMFL-XN34 were isolated and seeded on a 24-well plate coated with Matrigel TM (manufactured by BD Biosciences). Next, 1 mL of a medium of basal medium + compound 1 was added to the wells inoculated with the above-mentioned epithelial tumor cells, and culture was performed at 37°C under the condition of a low oxygen concentration of 2%.
  • Fig. 5B is a cytomicroscopic photograph showing HMFL-XN34-derived breast tumor cells cultured to the 4th day and cultured to the 32nd day after the primary culture (passage 0) under the culture condition of basal medium + compound 1 ( 100 times inverted phase contrast microscope).
  • the medium of the present invention can continuously promote the proliferation of normal breast epithelial cells in vitro and can continue to culture breast tumor cells under hypoxic conditions.
  • Example 2-2 the breast tumor cells derived from HMFL-XN35 were isolated.
  • 40 ⁇ L of Matrigel TM (manufactured by BD Biosciences) was seeded onto a 24-well plate with epithelial tumor cells.
  • the modified organoid culture medium was added to the wells inoculated with the above-mentioned epithelial tumor cells, and cultured at 37°C under the condition of an oxygen concentration of 20%.
  • the culture medium was replaced every 2 days after the start of the culture.
  • Fig. 5C shows images (under a 100-fold inverted phase contrast microscope) on the 4th day and the 38th day after the start of the culture of the primary culture (passage 0).
  • the improved organoid culture medium of the present invention can realize the high-efficiency culture of breast tumor organoids in vitro.
  • Example 1 According to the same method as in Example 1, a basic medium and a medium composed of the following composition were prepared.
  • HMFL-XN40 epithelial tumor cell sample
  • the epithelial tumor cells were seeded on a 6-well plate coated with Matrigel TM (manufactured by BD Biosciences).
  • Matrigel TM manufactured by BD Biosciences.
  • 3 mL each of basal medium, basal medium + compound 25 medium was added, and the concentration of compound 25 was 1 ⁇ M and 3 ⁇ M from low to high.
  • Cultivation was performed at 37°C with an oxygen concentration of 20%.
  • the culture medium was replaced every 2 days after the start of the culture.
  • Lgr5 is a Wnt-dependent stem cell marker (Barker et al., Mol Cell Biol, 22:1184-93, 2002), and Lgr5 plays an important role in the self-renewal and continuous proliferation of breast cells (Plaks et al., Cell Reports, 3, 70-78, 2013), this result suggests that compound 25 can maintain the characteristics of breast cancer cell stem proliferation by inhibiting the MST1/2-mediated signaling pathway of breast tumor cells, and it can continue to promote breast cancer in vitro The role of cell proliferation.
  • Example 3 Following the steps in Example 1 to obtain a freshly isolated epithelial tumor cell sample (HMFL-XN41).
  • the isolated epithelial tumor cells were divided into three equal parts. The first aliquot was collected directly; the second and third aliquots of epithelial tumor cells were respectively seeded on a 6-well plate coated with Matrigel TM (manufactured by BD Biosciences).
  • Matrigel TM manufactured by BD Biosciences.
  • 3 mL of basal medium and 3 mL of compound 1 basal medium were added respectively, and the concentration of compound 1 was 1 ⁇ M. Cultivation was performed at 37°C with an oxygen concentration of 20%. The culture medium was replaced every 2 days after the start of the culture.
  • the test results showed that after this freshly isolated breast tumor cell (HMFL-XN41) was cultured in the basal medium for 96 hours, the signal pathway mediated by MST1/2 kinase was activated, while the ERK kinase related to cell growth and proliferation was affected. Inhibition, the dryness marker Lgr5 and its related proteins were also inhibited, indicating that the basal medium caused the activation of the MST1/2 kinase-mediated apoptosis pathway.
  • compound 1 can significantly inhibit the activation of the MST1/2 kinase-mediated signaling pathway, stimulate the activation of the pro-proliferation signaling molecule ERK, and significantly upregulate the stemness marker family members Lgr4, Lgr5 and Lgr6 of breast tumor cells .
  • compound 1 and compound 25 can inhibit the MST1/2-mediated signaling pathway of breast tumor cells and promote the continuous proliferation of tumor cells.
  • HMFL-XN42 epithelial tumor cell sample
  • HMFL-XN32 and HMFL-XN33 were continuously cultured in vitro.
  • Breast cancer tumor cells (HMFL-XN32, P10) cultured and passaged 10 times in vitro and tumor tissues directly derived from breast cancer patients were used to extract the genomic DNA of the cells and corresponding tissues using DNeasyblood&tissuekit (manufactured by QIAGEN). Collect 2 mL of peripheral blood from patients with cell sources, and use the same method to extract genomic DNA as a background control.
  • Figure 8 shows the whole exome data of breast tumor cells (HMFL-XN34, HMFL-XN35, HMFL-XN38 and HMFL-XN39) obtained by culturing four other breast cancer tissue samples according to the above steps and corresponding tumor tissues. The results of the comparison of the number of high-frequency gene mutations. The analysis results are produced using https://bioinfogp.cnb.csic.es/tools/venny/index.html software. It can be confirmed from Figure 8 that the breast cancer tumor cells derived from the cultured cancer tissue and the high-frequency mutation genes in the corresponding tumor tissue are basically the same, indicating that the breast tumor cells cultured by the culture medium and the culture method of the present invention can maintain the patient The original gene mutation characteristics in cancer tissues.
  • HMFL-XN35 Obtain a breast tumor tissue sample of HMFL-XN35 according to the same procedure of Example 1 of 4.
  • the cancer tissues about the size of soybean grains were taken from samples of breast cancer patients, soaked in 10 mL 4% paraformaldehyde and fixed for the following hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) detection.
  • the remaining tissues used the steps of Example 3 to continuously culture HMFL-XN35 to the 10th generation.
  • the medium was basal medium + 1 ⁇ M compound 1, and the following (HE) staining method and immunohistochemistry (IHC) method were also performed for detection.
  • HE hematoxylin-eosin
  • IHC immunohistochemistry
  • HE staining was performed using an HE staining kit (manufactured by Solarbio). Refer to the instruction manual of the kit for specific experimental procedures.
  • IHC immunohistochemical detection
  • the primary antibodies used in IHC are ER (manufactured by Cell Signaling Technology), PR (manufactured by Cell Signaling Technology), and HER2 (manufactured by Cell Signaling Technology).
  • the secondary antibodies used are SignalStain Boost IHC Detection Reagent (HRP, Rabbit) (manufactured by Cell Signaling Technology), SignalStain Boost IHC Detection Reagent (HRP, Mouse) (manufactured by Cell Signaling Technology).
  • ER and PR are important indicators for predicting whether a patient can accept endocrine therapy
  • HER2 is an important indicator for predicting whether a patient can accept anti-HER2 targeted therapy.
  • these indicators are also clinically used for molecular pathological diagnosis of breast cancer. Important reference.
  • HE staining is an important basis for clinical pathological diagnosis of breast cancer.
  • the HE staining and the expression of ER, PR, and HER2 of the original tissue and the cells cultured to the 10th passage were photographed (under a 200-fold biological microscope). The result is shown in Figure 9.
  • the results of HE staining showed that the morphology of the cells in the tissue and the cells cultured in vitro were more consistent, and they were all breast cancer cells.
  • the results of immunohistochemistry showed that the molecular typing of the original patient was ER(-), PR(-), HER2(+), and the molecular typing of breast cancer cells cultured to the 10th generation was also ER(-), PR( -), HER2(+).
  • breast cancer tumor cells cultured from a breast cancer tumor sample derived from a patient can be used to detect the sensitivity of the patient's tumor cells to different drugs.
  • the plating of breast cancer tumor cells the single cell suspension of breast cancer tumor cells (the 10th generation of HMFL-XN38 and the 11th generation of HMFL-XN39) obtained by the steps of Example 3 is 3,000-5,000/ The well density was seeded in a 384-well plate to allow the cells to adhere overnight.
  • the drug storage plate using the method of concentration gradient dilution: take the drug mother solution to be tested (the concentration of the drug mother solution is prepared at 20 ⁇ M), and dilute it according to 1:3 to obtain 7 concentrations of drugs in sequence. Add different concentrations of drugs into a 384-well drug storage plate in a volume of 10 ⁇ L. In the solvent control group, an equal volume of DMSO was added to each well.
  • the drugs to be tested are afatinib (manufactured by MCE), lapatinib (manufactured by MCE), docetaxel (manufactured by MCE), and tamoxifen (manufactured by MCE).
  • Cell viability detection 72 hours after administration, use Cell Titer-Glo detection reagent (manufactured by Promega) to detect the chemiluminescence value of the cells after the drug is added.
  • the value of the chemiluminescence value reflects the cell viability and the effect of the drug on the cell viability For influence, add the prepared Cell Titer-Glo detection solution to each well, and use the microplate reader to detect the chemiluminescence value after mixing.
  • Figures 10A and B respectively show the effects of breast cancer tumor cell samples (HMFL-XN38 and HMFL-XN39) obtained from surgically removed cancer tissue samples from two different breast cancer patients against the chemotherapy drug docetaxel and endocrine therapy drugs. Sensitivity of moxifen, targeted drugs lapatinib and afatinib. The results show that the cells of the same patient have different sensitivities to different drugs, and the cells of different patients have different sensitivities to the same drug.
  • breast cancer tumor cells derived from hormone receptor-negative and HER2 receptor-positive breast cancer patients are sensitive to HER2-targeted drugs lapatinib and afatinib; and to the endocrine drug tamoxicil The sensitivity of fen is low.
  • the breast cancer cell (HMFL-XN39) of another triple-negative breast cancer patient was sensitive to the chemotherapy drug docetaxel, but not to the three targeted drugs tested.
  • the present invention provides a medium for culturing epithelial stem cells, especially mammary epithelial stem cells in vitro, and a medium and culture method for culturing organoids containing the stem cells.
  • Cell progeny and organoids can be used for drug efficacy evaluation and screening, toxicity determination and regenerative medicine. Therefore, the present invention is suitable for industrial applications.

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Abstract

本发明提供一种用于培养乳腺上皮干细胞的培养基，其含有TGF-β抑制剂、B27和/或N2、胰岛素、受体酪氨酸激酶配体、Rock激酶抑制剂、P38信号转导抑制剂、骨形态发生蛋白抑制剂、以及MST1/2激酶抑制剂。本发明还提供采用上述乳腺上皮干细胞培养基培养细胞的方法，以及采用所述培养方法得到的扩增细胞群或类器官在药物筛选、毒性测试和再生医疗中的应用和方法。

Description

一种用于乳腺上皮干细胞的培养基和培养方法 技术领域
本发明涉及用于体外培养上皮干细胞、尤其是乳腺上皮干细胞的培养基以及用于培养包含所述干细胞的类器官的培养基和培养方法。本发明还涉及用本发明的培养基和培养方法所培养的细胞后代和类器官在药物的疗效评估和筛选、毒性测定和再生医学中的用途。
背景技术
乳腺疾病，特别是乳腺癌，是影响女性健康的最主要的疾病之一。近年来，尽管人们对乳腺疾病的分类和发病机制的研究取得了很多进展，目前针对乳腺疾病，特别是乳腺癌的标准治疗药物仍相当匮乏，更缺乏个性化的精准用药指导。导致这一问题的关键在于目前尚缺乏体外可持续扩增且能够代表乳腺疾病患者自身生物学特性的细胞模型来进行药物的疗效评估和筛选、毒性测定等。
近年来，研究发现在人体的许多组织内，位于组织基底层的上皮干细胞及前体细胞具有无限自我更新的能力并携带个体的生物学特性(Blanpain C.等，Science，344(6189)：1242281,2014；Donati G.等，Cell Stem Cell，16(5),465-476，2015)。然而，由于CDKN2A依赖性的细胞周期阻滞机制，体外可持续培养上皮干细胞较难实现。
目前有两种体外培养上皮干细胞的技术在药物的疗效评估和筛选、毒性测定、再生医学领域发展得相对成熟。一种是使用经辐射的饲养细胞和ROCK激酶抑制剂Y27632来促进上皮细胞的生长以考察个体患者的药物敏感性的技术，即细胞条件重编程技术(Liu等，Am J Pathol，180:599-607，2012)。另一种技术是体外3D培养成体干细胞从而获得类似于组织器官的类器官技术(Hans Clevers等，Cell，172:1-14,2018)。
然而，这两种技术都存在一定的局限性。细胞重编程技术是一种将患者自体原代上皮细胞与鼠源性饲养细胞共培养的技术。然而，在对患者细胞进行药物疗效评估或信号通路分析时，这些鼠源性饲养细胞的存在会干扰患者自体细胞的检测、分析及下游应用(Lipsitz Y.等，Nat.Biotechnol.，34，393-400,2016)；但如果撤除鼠源性饲养细胞， 病人自体原代细胞就脱离了重编程环境，细胞的增殖速率和细胞内信号通路会发生明显的改变(Liu等，Am J Pathol，183(6):1862-1870，2013；Liu等，Cell Death Dis.，9(7):750，2018)。类器官技术是将患者自体原代上皮细胞包埋在细胞外基质内进行体外三维立体培养的技术，其技术原理主要是通过在培养基中添加特定的上皮干细胞标志物Lgr5和(或)Lgr6的配体，例如Wnt激动剂和R-海绵硬蛋白(R-spondin)家族蛋白，从而激动细胞内的Wnt信号通路，进而促进上皮干细胞的体外自我更新(Sato等，Gastroenterology，141:1762-1772，2011)。该技术无需饲养细胞，因此不存在鼠源性饲养细胞的干扰问题，但是类器官技术的培养基内需添加多种特定的因子，特别是该培养基的必需成分Wnt蛋白和R-spondin家族蛋白，导致类器官培养及检测成本昂贵，不适于普及到临床进行大规模应用。此外，该技术在培养和检测全过程都需要将类器官包埋在基质胶内，且所形成的类器官大小尺寸不易控制，导致该技术的可操作性和可重复性不强，从而限制了该技术在临床体外药物疗效和评估、高通量药物筛选和毒性测试上的大规模应用(Nick Barker等，Nat Cell Biol，18(3):246-54，2016；Huch M.等，Development，144,938-941，2017)。
鉴于以上技术的局限性，临床上亟待开发一种体外可持续培养乳腺上皮干细胞的培养技术，其培养可持续，成本可控，操作便捷，不受外源性细胞干扰。
本发明人曾在专利(PCT/CN2019/119116)中记载了一种无需饲养细胞、成本可控、操作便捷的体外培养乳腺上皮干细胞的方法。这是首个无需饲养细胞，培养成分中不含Wnt蛋白、R-spondin家族蛋白等Wnt激动剂的二维体外培养乳腺上皮干细胞的培养系统。这个培养系统可维持乳腺上皮细胞至少1个月的体外持续增殖。本发明人在原有发明的基础上，意外地发现将MST1/2激酶抑制剂应用于培养乳腺上皮干细胞可实现明显地促进乳腺上皮干细胞可持续增殖的效果，且将该技术应用于构建病人源性乳腺细胞模型时，所培养的乳腺上皮细胞能代表乳腺疾病患者自身的生物学特性。
发明内容
本发明旨在提供一种改进的用于培养乳腺上皮干细胞的培养基及使用该培养基的培养方法。采用本发明的培养基和培养方法，能够达到体外培养可持续、成本可控、操作便捷并不受外源性细胞干扰的目的。在该技术应用于构建病人源性乳腺细胞模型时，能够获得具有乳腺疾病患者自身生物学特性的细胞，并能够应用于药物疗效评估和筛选、毒性测试和再生医学等领域。
本发明的一个方面在于提供一种用于培养病人源性乳腺上皮干细胞和/或包含乳腺上皮干细胞的类器官的培养基，其含有TGF-β抑制剂、B27和/或N2、胰岛素、受体酪氨酸激酶配体、Rock激酶抑制剂、P38信号转导抑制剂、骨形态发生蛋白(BMP)抑制剂、以及MST1/2激酶抑制剂。其中，该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。
Figure PCTCN2020086366-appb-000001
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(例如苯基和萘基等)、芳基C1-C6烷基(例如苯甲基等)和杂芳基(例如噻吩基等)；
R 2和R 3各自独立地选自C1-C6烷基，优选C1-C3烷基，更优选甲基；
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(所述杂环基选自例如哌啶基、四氢吡喃基等)；
R 6选自卤素(优选氟和氯，更优选氟)、C1-C6烷基(优选甲基)、C1-C6烷氧基(优选甲氧基)、和C1-C6卤代烷基(优选三氟甲基)。
优选的实施方式中，MST1/2激酶抑制剂包括式(Ia)的化合物或其 药学可接受的盐、或溶剂化物，
Figure PCTCN2020086366-appb-000002
其中，
R 1选自C1-C6烷基、任选地被1-2个独立地R 6取代的苯基、任选地被1-2个独立地R 6取代的噻吩基、和任选地被1-2个独立地R 6取代的苯甲基，R 1更优选为任选地被1-2个独立地R 6取代的苯基；
R 5选自氢、C1-C6烷基、和C3-C6环烷基，R 5更优选为氢；
R 6各自独立地选自卤素、C1-C6烷基、和C1-C6卤代烷基，R 6更优选为氟、甲基或三氟甲基。
优选地，所述MST1/2抑制剂是选自以下化合物或其药学可接受的盐、或溶剂化物中的至少一种。
Figure PCTCN2020086366-appb-000003
Figure PCTCN2020086366-appb-000004
Figure PCTCN2020086366-appb-000005
Figure PCTCN2020086366-appb-000006
Figure PCTCN2020086366-appb-000007
MST1/2激酶抑制剂优选上述化合物1和化合物25。
MST1/2激酶抑制剂在培养基中的浓度是100nM以上且10μM以下，进一步优选300nM以上且3μM以下。
进一步优选地，本发明的培养基还含有：糖、烟酰胺(Nicotinamide)和N-乙酰半胱氨酸(N-Acetylcysteine)中的一种或多种。
其中，所述TGF-β抑制剂可以是选自A8301、SB431542、Repsox、SB505124、SB525334、SD208、LY36494、和SJN2511的至少一种。TGF-β抑制剂优选A8301。TGF-β抑制剂的浓度较好是50nM以上且 100μM以下，更好是100nM以上且50μM以下，进一步优选100nM以上且10μM以下。
其中，所述B27和/或N2以1:25～1:100的终浓度稀释至培养基中；优选地，B27以1:50倍稀释至培养基中，N2以1:100倍稀释至培养基中。
胰岛素以终浓度2～20μg/ml，优选5μg/ml～10μg/ml添加至培养基中；例如，将市售产品(10mg/ml)以1:500～1:5000倍稀释添加至培养基中，优选地，以1:1000～1:2000倍稀释添加至培养基中。
其中，所述受体酪氨酸激酶配体选自表皮生长因子(EGF)、双向调节素(Amphiregulin)、转化生长因子-α(TGF-α)、碱性成纤维细胞生长因子(bFGF)、脑源性神经生长因子(BDNF)、神经调节素1(Neuregulin1)、肝细胞生长因子(HGF)、成纤维细胞生长因子7(FGF7)以及成纤维细胞生长因子10(FGF10)中的至少一种。受体酪氨酸激酶配体优选包括EGF、Neuregulin1和FGF7。其中，受体酪氨酸激酶配体的终浓度为1ng/ml至1000ng/ml，优选5ng/ml至500ng/ml，进一步优选10ng/ml～100ng/ml。
其中，所述Rock激酶抑制剂选自Y27632、法舒地尔、H-1152中的至少一种。优选的Rock抑制剂为Y27632。其中所述Rock抑制剂的终浓度为1μM至100μM，优选2μM至50μM，更优选5μM至10μM。
其中，所述P38信号转导抑制剂选自SB202190、SB203580、VX-702、VX-745、PD169316、RO4402247、和BIRB796的至少一种。P38信号转导抑制剂优选SB202190。所述P38信号转导抑制剂的浓度较好是50nM以上且100μM以下，更好是100nM以上且50μM以下，进一步优选100nM以上且10μM以下。
其中，所述BMP抑制剂选自包括头蛋白(Noggin)、格雷林、腱蛋白、腱蛋白结构域的腱蛋白样蛋白，包括卵泡抑素、卵泡抑素结构域的卵泡抑素相关蛋白，包括DAN、DAN半胱氨酸结构域的DAN样蛋白，硬骨素/SOST、核心蛋白聚糖、α2-巨球蛋白、及DMH1中的至少一种。BMP抑制剂优选头蛋白。其中，BMP抑制剂的终浓度为1ng/ml至1000ng/ml，优选10ng/ml至500ng/ml，更优选20ng/mL至100ng/mL。
作为本实施方式的细胞培养基所含的糖，可例举例如葡萄糖、半乳糖、甘露糖、果糖等。其中，作为糖，较好是葡萄糖，特别好是D-葡萄糖(右旋糖)。糖在培养基内的终浓度为10mM～100mM，优选15mM～40mM。
烟酰胺在培养基内的终浓度为1mM～10mM，优选2mM～5mM。N-乙酰半胱氨酸在培养基内的终浓度为0.1mM～5mM，优选0.5mM～2mM。
实质上，本发明培养基不含类器官培养基内的必加成分Wnt蛋白、R-spondin家族蛋白中的任何蛋白。此外，本发明培养基也不含胎牛血清(fetal bovine serum(FBS)或fetal calf serum)、牛垂体提取物(BPE)等不确定的成分。
本发明的第二方面涉及培养正常乳腺组织来源或病变乳腺组织来源的上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含这些细胞中的至少任一种的组织或类器官的培养方法，其中，该培养方法包括：(1)制备细胞外基质的步骤；(2)使上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含这些细胞中的至少任一种的组织粘附于所述细胞外基质上或包埋于细胞外基质的步骤；(3)添加上述第一方面所述的培养基，对所述上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含所述这些细胞中的至少任一种的组织进行培养，得到扩增的、相应的上皮干细胞、上皮细胞、上皮肿瘤细胞后代或类器官后代的步骤。
其中，上述培养方法中的细胞外基质使用低生长因子型细胞外基质胶，例如，可采用市售的Matrigel TM(BD Biosciences公司制)或BME(Trevigen公司制)。更具体而言，用无血清的培养基稀释细胞外基质胶，培养基可以是本发明的上皮干细胞培养基，也可以是DMEM/F12(Corning公司制)、DMEM培养基(Corning公司制)和RPMI1640培养基(Corning公司制)，也可以是改良的DMEM/F12培养基(Thermo公司制)或改良的RPMI1640培养基(Thermo公司制)等。细胞外基质胶的稀释比例为1:50-400，优选为1:50-200。
在所述培养方法中，在培养上皮干细胞或类器官步骤中，在氧浓度0.1％～25％的条件下进行，优选20％的正常氧气条件，或0.1％～15％的低氧条件。
本发明的第三方面涵盖使用本发明的培养基和培养方法获得扩增的上皮干细胞、上皮细胞、上皮肿瘤细胞后代或类器官后代，并应用于药物的疗效评估和筛选的方法和用途，特别是抗肿瘤药物的体外疗效评估和筛选的方法和用途。
优选地，本发明涉及一种乳腺癌药物的疗效评估或筛选的方法，其特征在于，包括以下步骤：
(1)使用本发明的培养基和培养方法培养乳腺上皮干细胞、上皮细胞、或上皮肿瘤细胞；
(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)中培养得到的细胞添加稀释后的所述药物；和
(4)进行细胞活性测试。
本发明的有益效果包括：
(1)延长原代乳腺上皮细胞培养的体外可持续增殖的代数和时间，培养代数达到10代以上，优选12代以上，体外细胞扩增时间可持续至少3个月，并且可长期维持分化能力，体外培养过程中发生基因突变的频率极低；
(2)保持体外培养的乳腺上皮细胞能够维持细胞来源病人的病理表型和异质性，可应用于再生医学领域；
(3)所培养的乳腺上皮干细胞不受成纤维细胞、脂肪细胞等间质细胞的干扰，能得到纯化的乳腺上皮干细胞及其后代；
(4)培养基成分不含血清、牛垂体提取物等不确定成分，所以不受不同批次血清或牛垂体提取物的质量和数量的影响；
(5)扩增乳腺上皮细胞效率高，只要有10 4级别的细胞数量就可在两周左右时间内成功扩增出10 6数量级的乳腺上皮细胞，扩增出的乳腺上皮细胞还可以持续传代扩增；
(6)培养成本可控：原代乳腺癌培养基无需加入价格昂贵的Wnt激动剂蛋白、R-spondin家族蛋白等成分，是对已有乳腺上皮细胞及类器官培养基的简化和改进，大大节约了乳腺上皮干细胞培养基的成本；
(8)操作便捷，该技术相比条件重编程技术，无需培养饲养细胞并对饲养细胞进行辐射，避免了不同批次饲养细胞的质量和数量影响原代细胞培养效率的问题，药物筛选的铺板和检测的对象只有乳腺上 皮细胞，而不受细胞条件重编程技术所述的共培养体系中饲养细胞的干扰；相比类器官技术，本发明采用的细胞外基质胶的包被方法，培养器皿可预先准备，无需像类器官技术一样将细胞包埋于基质胶内，所述技术操作步骤简便易行；若使用本技术培养乳腺类器官，由于培养基成分已简化且成本大大降低，所以该技术比类器官技术更适合体外大规模应用和推广；
(9)所述技术培养获得的乳腺上皮细胞数量大，均一化程度高，成本可控，比细胞条件重编程技术和类器官技术更适合应用于高通量筛选新候选化合物和为病人提供高通量药物体外敏感性功能测试等药物疗效的评估、筛选以及毒性测试领域。
采用本实施方式的细胞培养基，可培养来源于包括人的或其他哺乳动物的乳腺上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含这些细胞中的至少任一种的组织，得到扩增的、相应的上皮干细胞、上皮细胞、上皮肿瘤细胞后代。此外，还可从所述细胞和所述组织中的至少一方形成类器官。
另外，通过本实施方式的培养方法获得的细胞可应用于再生医疗、毒性测试、乳腺上皮细胞的基础医学研究、药物应答的筛选、药物体外代谢稳定性和代谢谱的测定和针对乳腺疾病的新药研发等。
附图说明
图1A至1C是用于说明MST1/2激酶抑制剂延长人源性乳腺癌肿瘤细胞的培养代数的效果的附图。
图2是用于说明不同浓度的MST1/2激酶抑制剂在体外对人源性乳腺癌肿瘤细胞的增殖促进效果的附图。
图3A和3B是用于说明MST1/2激酶抑制剂在体外对乳腺癌肿瘤细胞的增殖持续促进效果的附图。
图4A和4B是用于说明本发明培养基中，MST1/2激酶抑制剂主导的对乳腺癌肿瘤细胞的持续的且可逆的促增殖作用的附图。
图5A是用于说明本发明的培养基对正常乳腺上皮细胞的增殖持续促进效果的附图；图5B是用于说明本发明的培养基在缺氧条件下对乳腺癌肿瘤细胞的增殖持续促进效果的附图；图5C是用于说明本发明 的培养基对乳腺肿瘤类器官的培养效果的附图。
图6A至6C是用于说明MST1/2激酶抑制剂对人源性乳腺上皮细胞内干性标志物表达以及MST激酶介导的信号通路的影响的附图。
图7A和7B是用于说明采用本发明技术培养获得的乳腺癌肿瘤细胞和对应细胞来源的原始组织的基因拷贝数变异一致性分析的附图。
图8是用于说明采用本发明技术培养获得的乳腺癌肿瘤细胞和对应细胞来源的原始组织的基因突变一致性分析的附图。
图9是用于说明采用本发明技术培养获得的乳腺癌肿瘤细胞的免疫组化结果与该组织样本自身原始的组织切片的免疫组化结果对比的附图。
图10A和10B是用于说明采用本发明技术培养的乳腺癌肿瘤细胞对不同药物的剂量-效应曲线图。
具体实施方式
本说明书中，上皮细胞包括从上皮组织获取的已分化的上皮细胞及上皮干细胞。“上皮干细胞”是指具有长期的自我更新能力和向上皮细胞分化的细胞，是指来源于上皮组织的干细胞。作为上皮组织，可例举例如角膜、口腔粘膜、皮肤、结膜、膀胱、肾小管、肾脏、消化器官(食道、胃、十二指肠、小肠(包括空肠及回肠)、大肠(包括结肠))、肝脏、胰脏、乳腺、唾液腺、泪腺、前列腺、毛根、气管、肺等。其中，本实施方式的细胞培养基较好是用于来源于乳腺上皮细胞的培养。
此外，本说明书中，“上皮肿瘤细胞”是指来源于上述的上皮组织的细胞肿瘤化而得的细胞。
本说明书中，“类器官”是指通过使细胞在受控的空间内高密度地自发组织和聚集而成的三维立体的、类似于器官的细胞组织体。
本实施方式的培养基除了包括MST1/2激酶抑制剂以外，还包含选自TGF-β抑制剂、B27和/或N2、胰岛素、受体酪氨酸激酶配体、Rock激酶抑制剂、P38信号转导抑制剂、骨形态发生蛋白(BMP)抑制剂、葡萄糖、烟酰胺和N-乙酰半胱氨酸中的一种或多种或全部。包含所述成分的哪一种或哪几种可根据培养的细胞或组织的种类等适当选择。 以下，对本实施方式的乳腺干细胞培养基的构成成分进行详细说明。
细胞培养基本培养基
本实施方式的乳腺上皮干细胞培养基中包含任意的无血清的细胞培养基本培养基。本实施方式的细胞培养基较好是用于动物细胞或人的细胞。作为所述的无血清的基本培养基，可使用例如用碳酸类缓冲液缓冲化至pH7.2以上且pH7.6以下的规定的合成培养基等。更具体来说，无血清的基本培养基选自，例如DMEM/F-12(Corning公司制)培养基。此外，作为替代还可使用RPMI1640培养基(Corning公司制)、DMEM培养基(Corning公司制)、以及改良型DMEM/F-12(Thermo公司制)和改良型RPMI1640培养基(Thermo公司制)等。
MST1/2激酶抑制剂
哺乳动物不育系20样激酶1和2(Mammalian Sterile 20-like Kinase，MST1/2)激酶是分子量为56-60 KD的丝裂原活化蛋白激酶(MAPK)信号转导通路的上游调控因子，可调节多种细胞进程，包括增殖、凋亡、迁移和细胞骨架重排。MST1基因是1995年在研究酿酒酵母Ste20与人的同系物时通过PCR从淋巴cDNA文库中克隆出来的，其编码产物与酵母Ste20的结构和功能类似，因此命名为MST1。MST1还有MST2、MST3和MST4三个旁系同源物(Dan等，Trends Cell Biol.11,220-230,2001)。MST2和MST1蛋白的同源性有76％，目前其生理功能并不明确，有研究认为MST2对MST1介导的功能可能有代偿作用(Wu S.等，Cell，114，445-456，2003)。
研究表明，MST1/2是果蝇Hippo(Hpo)的直系同源蛋白，后者是Hippo信号转导通路中的一种核心调节蛋白。这种在进化方面较保守的程序可通过调控细胞增殖、凋亡及干细胞自我更新来控制组织生长和器官大小。哺乳动物Hippo信号转导通路涉及一个激酶级联反应，其中，MST1/2激酶和SAV1支架蛋白会形成一个磷酸化并激活LATS1/2的复合体。LATS1/2激酶会磷酸化YAP和TAZ，进而促进对这些转录共激活因子在细胞质的滞留以及功能抑制(Zhao.B等，Nat Cell Biol，13，877-883，2011)。
本说明书中，MST1/2激酶抑制剂是指直接或间接地对MST1/2信号传导进行负调节的任意的抑制剂。一般来说，MST1/2激酶抑制剂例如 与MST1/2激酶结合并降低其活性。由于MST1和MST2的结构具有相似性，MST1/2激酶抑制剂也可以是例如与MST1或MST1/2结合并降低其活性的化合物。
MST1/2激酶抑制剂与不存在该抑制剂的条件下的MST1/2活性水平相比，具有较好是50％以上、更好是70％以上、进一步更好是80％以上、特别好是90％以上的抑制活性。MST1/2抑制剂产生的抑制效果可通过本领域技术人员公知的方法进行评价。作为所述的评价体系，可例举MST1的Thr183磷酸化位点的特异性抗体检测方法、重组蛋白激酶体外实验、MST1/2激酶抑制剂用的DiscoverRx高通量筛选平台、MST1/2激酶活性检测试剂盒(Promega公司制)等。
作为本实施方式的上皮干细胞或类器官培养用的培养基所含的MST1/2抑制剂，可包含式(I)的化合物或其药学可接受的盐或溶剂化物，
Figure PCTCN2020086366-appb-000008
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(例如苯基和萘基等)、芳基C1-C6烷基(例如苯甲基等)和杂芳基(例如噻吩基等)；
R 2和R 3各自独立地选自C1-C6烷基，优选C1-C3烷基，更优选甲基；
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(所述杂环基选自例如哌啶基、四氢吡喃基等)；
R 6选自卤素(优选氟和氯，更优选氟)、C1-C6烷基(优选甲基)、C1-C6烷氧基(优选甲氧基)、和C1-C6卤代烷基(优选三氟甲基)。
优选的实施方式中，MST1/2激酶抑制剂包括式(Ia)的化合物或其 药学可接受的盐、或溶剂化物，
Figure PCTCN2020086366-appb-000009
其中，
R 1选自C1-C6烷基、任选地被1-2个独立地R 6取代的苯基、任选地被1-2个独立地R 6取代的噻吩基、和任选地被1-2个独立地R 6取代的苯甲基，R 1更优选为任选地被1-2个独立地R 6取代的苯基；
R 5选自氢、C1-C6烷基、和C3-C6环烷基，R 5更优选为氢；
R 6各自独立地选自卤素、C1-C6烷基、和C1-C6卤代烷基，R 6更优选为氟、甲基或三氟甲基。
优选地，所述MST1/2抑制剂是选自以下化合物或其药学可接受的盐、或溶剂化物中的至少一种。
Figure PCTCN2020086366-appb-000010
Figure PCTCN2020086366-appb-000011
Figure PCTCN2020086366-appb-000012
Figure PCTCN2020086366-appb-000013
Figure PCTCN2020086366-appb-000014
MST1/2激酶抑制剂优选上述化合物1和化合物25。
所述培养基所含的MST1/2激酶抑制剂的浓度较好是100nM以上且10μM以下，进一步优选300nM以上且3μM以下。
上皮干细胞和类器官的培养中，较好是每2天向培养基中添加MST1/2激酶抑制剂一次，或每4天一次将培养基更换为新鲜的培养基。
TGF-β抑制剂
转化生长因子-β(transforming growth factor-β，TGF-β)是生长因子的一种，在肾脏、骨髓、血小板等几乎所有的细胞中产生。TGF-β 存在五种亚型(β1～β5)。此外，已知TGF-β促进成骨细胞的增殖以及如胶原等结缔组织的合成及增殖，对上皮细胞的增殖和破骨细胞起到抑制性作用。一般来说，TGF-β抑制剂例如为阻止或抑制TGF-β对TGF-β受体的结合，是为了形成中和TGF-β活性的复合物而结合于TGF-β的化合物。此外，TGF-β抑制剂例如为与TGF-β受体结合，阻止或抑制TGF-β对受体的结合，是作为拮抗剂或反向激动剂发挥作用的化合物。
TGF-β抑制剂与不存在该抑制剂的条件下的TGF-β活性水平相比，具有较好是50％以上、更好是70％以上、进一步更好是80％以上、特别好是90％以上的抑制活性。TGF-β抑制活性可通过本领域技术人员公知的方法进行评价。作为所述评价体系，可例举利用荧光素酶报告基因的人PAI-1启动子或使用含Smad结合部位的报告构建体稳定转染细胞的细胞实验(De Gouville等,Br J Pharmacol,145(2):166-177,2005)。
作为本实施方式的乳腺上皮干细胞培养用培养基所含的TGF-β抑制剂，可例举A8301、SB431542、Repsox、SB505124、SB525334、SD208、LY36494、及SJN2511中的至少一种。作为本实施方式的乳腺上皮干细胞培养用细胞培养基所含的TGF-β抑制剂，其中较好是A8301。
本实施方式的培养基所含的TGF-β抑制剂的浓度较好是50nM以上且100μM以下，更好是100nM以上且50μM以下，进一步优选100nM以上且10μM以下。
上皮干细胞的培养中，较好是每2天向培养基中添加TGF-β抑制剂一次，或者每4天一次将培养基更换为新鲜的培养基。
受体酪氨酸激酶配体
作为本实施方式的培养基所含的受体酪氨酸激酶配体，可例举例如选自表皮生长因子(EGF)、双向调节素(Amphiregulin)、转化生长因子-α(TGF-α)、碱性成纤维细胞生长因子(bFGF)、脑源性神经生长因子(BDNF)、神经调节素1(Neuregulin1)、肝细胞生长因子(HGF)、成纤维细胞生长因子7(FGF7)以及成纤维细胞生长因子10(FGF10)中的至少一种。受体酪氨酸激酶配体优选包括EGF、Neuregulin1和FGF7。其中，受体酪氨酸激酶配体的终浓度为1ng/ml至1000ng/ml，优选5ng/ml至500ng/ml，进一步优选10ng/ml～100ng/ml。
EGF是针对各种培养外胚层性细胞及中胚层性细胞的强力的分裂 增殖因子，对部分的成纤维细胞的特异性细胞分化具有显著的影响。EGF前体通过蛋白质分解被切割，作为使刺激细胞的53-氨基酸肽激素生成的膜结合分子存在。
作为本实施方式的培养方法中使用的培养基所含的受体酪氨酸激酶配体，其中较好是EGF。本实施方式的细胞培养基所含的EGF的浓度较好是1ng/mL以上且1000ng/mL以下，更好是5ng/mL以上且500ng/mL以下，进一步优选5ng/mL以上且100ng/mL以下。
此外，本实施方式的细胞培养基中较好是EGF与双向调节素(Amphiregulin)或者神经调节素1(Neuregulin1)或者肝细胞生长因子(HGF)或者成纤维细胞生长因子7(FGF7)或者成纤维细胞生长因子10(FGF10)的组合，或者更优选包括EGF、Neuregulin1和FGF7的生长因子组合。
使用多种受体酪氨酸激酶配体，较好是较好是1ng/mL以上且1000ng/mL以下，更好是5ng/mL以上且500ng/mL以下，进一步更好是10ng/mL以上且100ng/mL以下。
在本实施方式的乳腺上皮干细胞的培养中，较好是每2天向培养基中添加受体酪氨酸激酶配体一次，或者每4天一次将培养基更换为新鲜的培养基。
P38信号转导抑制剂
本说明书中，“p38信号转导抑制剂”或“p38抑制剂”是指直接或间接地对p38信号传导进行负调节的任意的抑制剂。一般来说，p38信号传导抑制剂例如与p38结合并降低其活性。p38蛋白激酶是丝裂原活化蛋白激酶(MAPK)家族的一部分。MAPK是对环境压力及炎症细胞因子等细胞外刺激进行应答、条件基因表达、有丝分裂、分化、增殖、及细胞生存/程序性死亡等各种细胞活性的丝氨酸/苏氨酸特异性蛋白激酶。p38 MAPK作为α、β、β2、γ、及δ同源异构体存在。此外，p38信号传导抑制剂也是例如与至少一种p38同源异构体结合并降低其活性的药剂。
p38信号传导抑制剂与不存在该抑制剂的条件下的p38活性水平相比，具有较好是50％以上、更好是70％以上、进一步更好是80％以上、特别好是90％以上的抑制活性。p38信号传导抑制剂产生的抑制效果可 通过本领域技术人员公知的方法进行评价。作为所述的评价体系，可例举Thr180/Tyr182磷酸化的磷酸化部位特异性抗体检测方法、生化学重组激酶试验、肿瘤坏死因子α(TNF-α)分泌试验、p38抑制剂用的DiscoverRx高通量筛选平台、p38活性试验试剂盒(Sigma-aldrich公司制)等。
作为本实施方式的培养基所含的p38信号传导抑制剂，可例举例如SB202190、SB203580、VX-702、VX-745、PD169316、RO4402247、以及BIRB796中的至少一种。P38信号转导抑制剂优选SB202190。
所述培养基所含的p38信号传导抑制剂的浓度较好是50nM以上且100μM以下，更好是100nM以上且50μM以下，进一步更好是100nM以上且10μM以下。
上皮干细胞的培养中，较好是每2天向培养基中添加p38信号传导抑制剂一次，或者每4天一次将培养基更换为新鲜的培养基。
BMP抑制剂
骨形态发生蛋白(Bone Morphogeneitc Protein，BMP)作为二聚体配体与由2种不同的受体丝氨酸/苏氨酸激酶、I型及II型受体形成的受体复合物结合。II型受体将I型受体磷酸化，因而该受体激酶被激活。该I型受体接着将特异性受体底物(SMAD)磷酸化，因而通过信号传导通路产生转录活性。一般来说，BMP抑制剂例如为阻止或抑制BMP分子对BMP受体的结合，是为了形成中和BMP活性的复合物而结合于BMP分子的药剂。此外，BMP抑制剂例如为与BMP受体结合，阻止或抑制BMP分子对受体的结合，是作为拮抗剂或反向激动剂发挥作用的药剂。
BMP抑制剂与不存在该抑制剂的条件下的BMP活性水平相比，具有较好是50％以上、更好是70％以上、进一步更好是80％以上、特别好是90％以上的抑制活性。BMP抑制活性可通过使用本领域技术人员公知的方法(Zilberberg等,BMC Cell Biol,8:41,2007)测定BMP的转录活性来进行评价。
作为本实施方式的培养基所含的BMP抑制剂，较好是天然的BMP结合蛋白质，可例举例如包括头蛋白(Noggin)、格雷林(Gremlin)、腱蛋白(Chordin)、腱蛋白结构域等的腱蛋白样蛋白，包括卵泡抑素 (Follistatin)、卵泡抑素结构域等的卵泡抑素相关蛋白，包括DAN、含DAN半胱氨酸结构域等的DAN样蛋白，硬骨素/SOST、核心蛋白聚糖(Decorin)、和α2-巨球蛋白等。也可为小分子抑制剂，例如DMH1。
作为本实施方式的培养基所含的BMP抑制剂，其中较好是腱蛋白样蛋白或DAN样蛋白，更好是腱蛋白样蛋白。作为腱蛋白样蛋白，较好是头蛋白。腱蛋白样蛋白和DAN样蛋白是扩散性蛋白质，以各种亲和度与BMP分子结合，可抑制BMP分子向信号传导受体的接近。通过将这些BMP抑制剂添加至上皮干细胞培养用细胞培养基中，可妨碍干细胞的丢失。
本实施方式的培养基所含的BMP抑制剂的浓度较好是1ng/ml至1000ng/ml，优选10ng/ml至500ng/ml，优选20ng/mL以上且100ng/mL以下。
干细胞的培养中，较好是每2天向培养基中添加BMP抑制剂一次，或者每4天一次将培养基更换为新鲜的培养基。
其他成分
本实施方式的细胞培养基可还包含Rock激酶(Rho-激酶)抑制剂。作为Rock激酶抑制剂，可以选择例如Y27632、法舒地尔、H-1152中的至少一种，较好的是使用Y27632。其中所述Rock抑制剂的终浓度为1μM至100μM，优选2μM至50μM，更优选5μM至10μM。在使用Y27632的情况下，较好是在呈单细胞分散的干细胞的培养的最初2天添加。本实施方式的培养基所含的Y27632的终浓度优选为1μM至100μM，较好的是2μM至50μM，更好是5μM至10μM。
本实施方式的细胞培养基可还包含纯化的、天然的、半合成的和/或合成的刺激细胞增殖的补充物，且不包含组分不明确成分，例如血清。本实施方式的乳腺上皮干细胞培养用细胞培养基所含的补充物可使用市售的补充物，例如B27(Gibco公司制)和/或N2(Gibco公司制)，优选以1:25～1:100的终浓度添加，更优选以1:50～1:100的终浓度添加。
本实施方式的细胞培养基还包含胰岛素。胰岛素是已公认的维持细胞体外生长的必须成分。本实施方式的培养基所含的胰岛素可使用市售的胰岛素，例如纯化的人胰岛素溶液(Sigma公司制)，或包含胰岛素的补充剂，例如胰岛素-转铁蛋白-亚硒酸钠(ITS-X，Thermo Fisher  Scientific公司制)。其中胰岛素在培养基内的终浓度为2μg/ml～20μg/ml；优选浓度为5μg/ml～10μg/ml。
本实施方式的细胞培养基可还包含至少一种可成为碳能量源的糖。作为本实施方式的细胞培养基所含的糖，可例举例如葡萄糖、半乳糖、甘露糖、果糖等。其中，作为糖，较好是葡萄糖，特别好是D-葡萄糖(右旋糖)。本实施方式的乳腺上皮干细胞培养用细胞培养基所含的糖在培养基内的终浓度为10mM～100mM，优选15mM～40mM。
本实施方式的细胞培养基可还包含烟酰胺和/或N-乙酰半胱氨酸。烟酰胺在本实施方式中的作用为抗氧化剂，有利于类器官的形成。本实施方式的改良类器官培养基所含的烟酰胺在培养基内的终浓度为1mM～10mM，优选2mM～5mM。N-乙酰半胱氨酸为促类器官增殖刺激物，也具有促进类器官生长的作用。本实施方式的改良类器官培养基所含的N-乙酰半胱氨酸在培养基内的终浓度为0.1mM～5mM，优选0.5mM～2mM。
[实施方式1]
本发明的实施方式1所涉及的培养方法是用于培养正常乳腺组织来源或病变乳腺组织来源的上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含这些细胞中的至少任一种的组织的培养方法。
其中，所述方法包括以下步骤：
(1)制备细胞外基质；
(2)将上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含这些细胞中的至少任一种的组织与细胞外基质粘附，添加于所述细胞外基质上或包埋于细胞外基质中；
(3)采用本发明的培养基对所述上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含这些细胞中的至少任一种的组织进行培养，得到扩增的、相应的上皮干细胞、上皮细胞、上皮肿瘤细胞后代或类器官后代。
如果采用本实施方式的培养方法，可长期培养来源于包括人的哺乳动物的上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含这些细胞中的至少任一种的组织，得到扩增的、相应的上皮干细胞、上皮细胞、上皮肿瘤细胞后代或类器官后代。
以下，对本实施方式的培养方法中的各步骤进行详细说明。
细胞外基质的制备
一般来说，“细胞外基质(Extracellular Matrix，ECM)”是指生物中存在于细胞外的超分子结构。该ECM成为用于上皮干细胞、上皮肿瘤细胞、或包含这些细胞的组织增殖的基础。
ECM包含各种各样的多糖、水、弹性蛋白、及糖蛋白。作为糖蛋白，可例举例如胶原、巢蛋白、纤连蛋白、层粘连蛋白等。作为多糖，可例举例如蛋白多糖、糖胺多糖等。作为弹性蛋白可例举弹力纤维等。
本实施方式的ECM可使用市售的ECM。例如细胞外基质蛋白质(Invitrogen公司制)、来源于Engelbreth-Holm-Swarm(EHS)小鼠肉瘤细胞的基底膜制备物(Matrigel TM(BD生物科技公司制))等。可使用ProNectin(SigmaZ378666)等合成ECM。此外，可使用天然ECM和合成ECM的混合物。本实施方式优选来源于EHS小鼠肉瘤细胞的基底膜制备物(Matrigel TM(BD生物科技公司制))。
使用ECM来培养上皮干细胞的情况下，可强化干细胞的长期生存及未分化干细胞的持续存在。在不存在ECM的条件下，无法长期培养干细胞培养物，观察不到未分化干细胞的持续存在。另外，如果存在ECM，则可培养在不存在ECM的条件下无法培养的上皮干细胞。
ECM可与上皮干细胞混匀并将细胞包埋于ECM内部。通常ECM沉于悬浮有细胞的培养皿的底部。例如，ECM在37℃凝固时，可加入上述的上皮干细胞培养基，使其扩散于ECM中使用。培养基中的细胞可通过与ECM的表面结构相互作用，例如通过与整联蛋白相互作用而固定于ECM。
ECM可包被于培养容器的表面使用。更具体而言，用无血清的培养基稀释细胞外基质胶，培养基可以是本发明的细胞培养基，也可以是上述的细胞培养基本培养基。细胞外基质胶的稀释比例为例如1:20～400，进一步更好的范围为1:50～200。包被方法为将稀释后的细胞外基质胶加入培养器皿内，使其完全覆盖培养器皿底部。在37℃条件下静置，包被时间为至少30分钟，进一步更好的范围为30～60分钟。包被结束后吸弃多余的细胞外基质胶稀释液，培养器皿备用。
细胞与ECM的粘附
接着，准备乳腺上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含这些细胞中的至少任一种的组织。
作为从乳腺上皮组织分离上皮细胞的方法，可例举本技术领域中公知的方法。例如，乳腺上皮细胞可以来源于乳腺癌组织样本和癌旁组织样本。乳腺癌组织样本例如来源于进行过说明并获得同意的乳腺肿瘤患者手术切除癌组织样本，癌旁组织样本采集自离乳腺癌组织距离至少5cm以上的乳腺组织。在患者手术切除或活检后的半小时内进行上述组织样本的收集。更具体而言，在无菌环境下，切取非坏死部位的组织样本，其体积在0.5cm 3以上，将其置于预冷的10～50mLDMEM/F12培养基中，培养基盛在塑料无菌带盖离心管内，冰上运输至实验室；其中，DMEM/F12培养基中含有50～200U/mL(例如100U/mL)青霉素和50～200μg/mL(例如100μg/mL)链霉素(以下简称运输液)。
在生物安全柜内，将组织样本转移至细胞培养皿内，用运输液润洗组织样本，将组织样本表面的血细胞清洗掉，并剔除组织样本表面的皮肤、筋膜等不需要的组织。
将润洗后的组织样本转移至另一个新的培养皿内，加入5～25mL运输液，用无菌手术刀片和手术镊将组织样本分割为直径小于1mm 3的组织碎块。
将组织样本碎块转移至离心管内，用台式离心机以至少1000转/分钟离心3～10分钟；然后用移液器小心移除离心管内上清，再用5～25mL含胶原酶II(0.5～5mg/mL，例如1mg/mL)和胶原酶IV(0.5～5mg/mL，例如1mg/mL)的无血清DMEM/F12培养基重悬，置37℃恒温摇床上进行振荡消化，时间为至少1小时(消化时间取决于样本大小；如果样本大于1g，则消化时间增至1.5～2小时)；之后用台式离心机以至少300g/分钟离心3～10分钟，弃去上清液，消化后的组织细胞用5～25mL含例如10％小牛血清的DMEM/F12培养基重悬，研磨过筛，细胞筛孔径为40～100μm(例如100μm)，将过筛的细胞悬液收集于离心管中；用血细胞计数板计数。
然后将细胞悬液在离心机中以至少300g/分钟离心3～10分钟，弃去上清，再用本发明的原代细胞培养基重悬。
将通过上述的方法分离的上皮干细胞、上皮细胞、上皮肿瘤细胞、 或包含这些细胞中的至少任一种的组织接种至所述制备步骤中得到的细胞外基质上并静置。接种的细胞可通过与ECM的表面结构相互作用，例如通过与整联蛋白相互作用而粘附于ECM。
细胞培养
接着，细胞接种后，在细胞未干燥之前，添加本发明的培养基进行培养。培养温度较好是30℃以上且40℃以下，更好是37℃左右。培养时间可根据使用的细胞适当调整。培养开始起1～2周左右后，可得到扩增的、相应的上皮干细胞、上皮细胞、上皮肿瘤细胞后代或类器官后代。此外，对于以往仅可维持培养1个月的细胞，通过本实施方式的培养方法，即可达到3个月以上的较长培养时间，也可进行细胞的维持培养。使用本实施方式的培养方法培养上皮干细胞的情况下，可长期维持细胞的自我更新和分化能力，体外培养发生基因突变的频率极低。
此外，本实施方式的细胞培养步骤，不但可在常氧条件下，还可在低氧条件下进行培养。通过在低氧条件下进行，可由包括人的哺乳动物的上皮干细胞、上皮细胞、或上皮肿瘤细胞、或者包含这些细胞中的至少任一种的组织、或以往无法培养的组织得到扩增的、相应的上皮干细胞、上皮细胞、上皮肿瘤细胞后代或类器官后代。
本实施方式中的低氧条件下是指氧浓度较好是0.1％以上且15％以下，更好是0.3％以上且10％以下，进一步更好是0.5％以上且5％以下。
类器官
通过本发明的实施方式1所涉及的培养方法可获得类器官。
本实施方式的类器官可应用于再生医疗、上皮细胞的基础医学研究、药物应答的筛选、使用来源于疾病的上皮类器官的新药研发等。
用途
本实施方式中，本发明提供用于药物应答的筛选、毒性试验、或者再生医疗的上述的上皮干细胞的用途。
药物应答的筛选中，使用上述培养方法培养的上皮干细胞的情况下，将上述上皮干细胞在例如96孔板或384孔板等多孔板中进行培养。使用分子库，鉴定对该上皮干细胞产生影响的分子。作为分子库，可例举例如抗体片段库、肽噬菌体展示库、肽库(例如LOPAP(商标)， Sigma-Aldrich公司制)、脂质库(BioMol公司制)、合成化合物库(MCE公司制)或者天然化合物库(Specs，TimTec公司制)等。另外，可使用基因文库。作为基因文库，可例举例如cDNA文库、反义文库、siRNA、或者其他非编码RNA文库等。作为具体方法，可例举将培养获得的细胞在试验药剂的多个浓度暴露一定的时间，暴露时间结束时，对培养物进行评价的方法。此外，本实施方式获得的上皮干细胞也可用于鉴定特异性靶向上皮肿瘤细胞，但不靶向正常细胞的药物。
另外，本实施方式获得的上皮干细胞可在新候选药物或者已知或新的营养补充食品的毒性实验中代替Caco-2细胞等细胞株使用。
另外，本实施方式获得的上皮干细胞可用于培养目前没有适当的组织培养或动物模型的例如诺如病毒等病原体。
此外，本实施方式获得的上皮干细胞可用于再生医疗中，例如应用于放射线照射后或术后的乳腺组织修复中。
以下，通过实施例对本发明进行说明，但本发明并不受到以下的实施例的限制。
[实施例1]
1. MST1/2激酶抑制剂化合物1的制备
4-((7-(2,6-二氟苯基)-5,8-二甲基-6-氧代-5,6,7,8-四氢蝶啶-2-基)氨基)苯 磺酰胺1
Figure PCTCN2020086366-appb-000015
2-氨基-2-(2,6-二氟苯基)乙酸甲酯(A2)：在圆底烧瓶中加入2-氨基-2-(2,6-二氟苯基)乙酸(2.0克)后加入甲醇(30毫升)，随后冰浴下滴加二氯亚砜(1.2毫升)。反应体系在85℃反应过夜。反应结束后，体系在减压下蒸干溶剂，所得白色固体，直接用于下一步。
2-((2-氯-5-硝基嘧啶-4-基)氨基)-2-(2,6-二氟苯基)乙酸甲酯(A3)： 在圆底烧瓶中加入2-氨基-2-(2,6-二氟苯基)乙酸甲酯(2克)后加入丙酮(30毫升)和碳酸钾(2.2克)，然后用冰盐浴使体系冷却到-10℃，接着缓慢加入2,4-二氯-5-硝基嘧啶(3.1克)的丙酮溶液。反应体系在室温搅拌过夜。反应结束后，过滤，滤液在减压下除去溶剂，残留物经加压硅胶柱层析提纯后得化合物A3。LC/MS：M+H 359.0。
2-氯-7-(2,6-二氟苯基)-7,8-二氢蝶啶-6(5H)-酮(A4)：在圆底烧瓶中加入2-((2-氯-5-硝基嘧啶-4-基)氨基)-2-(2,6-二氟苯基)乙酸甲酯(2.5克)后加入醋酸(50毫升)和铁粉(3.9克)。反应体系在60℃搅拌两小时。反应结束后，体系在减压下蒸干溶剂，所得物用饱和碳酸氢钠中和至碱性。乙酸乙酯萃取，有机相分别用水、饱和食盐水洗涤后用无水硫酸钠干燥。有机相经过滤，减压蒸干后得粗品。粗品经乙醚洗涤后得化合物A4。LC/MS：M+H 297.0。
2-氯-7-(2,6-二氟苯基)-5,8-二甲基-7,8-二氢蝶啶-6(5H)-酮(A5)：在圆底烧瓶中加入2-氯-7-(2,6-二氟苯基)-7,8-二氢蝶啶-6(5H)-酮(2克)和N,N-二甲基乙酰胺(10毫升)，冷却至-35℃，加入碘甲烷(0.9毫升)，随后加入氢化钠(615毫克)，反应体系继续搅拌两小时。反应结束后，加水淬灭，乙酸乙酯萃取，有机相分别用水、饱和食盐水洗涤后用无水硫酸钠干燥。有机相经过滤，减压蒸干后得粗品。粗品经乙醚洗涤后得化合物A5。LC/MS：M+H 325.0。
4-((7-(2,6-二氟苯基)-5,8-二甲基-6-氧代-5,6,7,8-四氢蝶啶-2-基)氨基)苯磺酰胺(1)：在圆底烧瓶中加入2-氯-7-(2,6-二氟苯基)-5,8-二甲基-7,8-二氢蝶啶-6(5H)-酮(100毫克)、磺胺(53毫克)、对甲苯磺酸(53毫克)和仲丁醇(5毫升)。反应体系在120℃搅拌过夜。反应结束后，过滤，甲醇和乙醚洗涤得化合物1。LC/MS：M+H 461.1。
2.化合物25及本发明的其他MST1/2抑制剂化合物的制备
4-((5,8-二甲基-6-氧代-7-(邻甲苯基)-5,6,7,8-四氢蝶啶-2-基)氨基)苯磺酰 胺25
Figure PCTCN2020086366-appb-000016
化合物25的合成通过使用类似于化合物1中所述的步骤完成。MS(ESI)m/z(M+1)+：439.15。
本发明的其他MST1/2抑制剂化合物按照与化合物1类似的方法合成，其结构及质谱数据如下表所示。
Figure PCTCN2020086366-appb-000017
Figure PCTCN2020086366-appb-000018
Figure PCTCN2020086366-appb-000019
Figure PCTCN2020086366-appb-000020
Figure PCTCN2020086366-appb-000021
3.乳腺上皮干细胞培养用细胞培养基的制备
首先，向市售的DMEM/F-12培养基(Corning公司制)以最终浓度10μg/ml加入胰岛素(Sigma-Aldrich公司制，市售产品浓度10mg/mL，以1:1000的体积比加入培养基)，以1:50的终浓度加入B27，以100ng/mL的条件添加头蛋白(Noggin，R&D公司制)，以最终浓度500nM条件添加TGF-β抑制剂A8301(MCE公司制)，以最终浓度500nM条件添加P38信号转导抑制剂SB202190(MCE公司制)，以最终浓度10μM条件添加ROCK激酶抑制剂Y27632(MCE公司制)。
然后，添加最终浓度20ng/mL的表皮生长因子EGF(R&D公司制)、最终浓度5ng/mL的成纤维细胞生长因子7 FGF7(R&D公司制)、最终浓度10ng/mL的神经调节素1 Neuregulin1(Peprotech公司制)、最终浓度15.8mM的葡萄糖(Peprotech公司制)，制备成培养乳腺上皮干细胞的基础培养基。(以下也称“基础培养基”或“基培”)。
接着，以最终浓度1μM条件添加化合物1或化合物25。制备构成成分为以下组合的培养基。
·基础培养基+化合物1
4.来源于乳腺肿瘤的上皮肿瘤细胞的培养
基于中国科学院合肥物质科学研究院医学伦理委员会认可的伦理研究计划，从进行说明并获得同意的乳腺肿瘤患者的乳腺癌组织中获取病变组织样本(HMFL-XN26)。接着，按照实施方式1中的步骤获得新鲜分离的上皮肿瘤细胞。然后，将上皮肿瘤细胞接种至包被有Matrigel TM(BD Biosciences公司制)的24孔板。包被方式为用无血清的培养基稀释Matrigel，培养基可以是本发明的原代细胞培养基，也可以是DMEM/F12(Corning：R10-092-CV)。Matrigel的稀释比例为1:50-400，优选为1:100-200。将稀释后的Matrigel加入培养器皿内，使其完全覆盖培养器皿底部，静置包被30分钟以上，优选在37℃条件下静置包被，优选包被30～60分钟。包被结束后吸弃多余的Matrigel稀释液，培养器皿备用。
向接种了上述上皮肿瘤细胞的孔中分别添加基础培养基、基础培养基+化合物1的培养基各1mL，在37℃以氧浓度20％的条件进行培养。培养开始后每2天进行一次培养基的更换。待培养6天后，弃去原24孔板内的培养基上清，加入0.5mL 0.05％胰酶(Thermo Fisher公司制)对细胞进行消化，37℃下孵育15分钟后，用含有10％(v/v)小牛血清、100U/mL青霉素和100μg/mL链霉素的DMEM/F12培养液5mL重悬消化处理后的细胞并收集至离心管内，以300g/分钟转速离心5分钟。使用本发明的培养基重悬离心后的细胞沉淀，细胞计数板对细胞悬液进行计数。按4×10 4个/孔密度将细胞分别接种至另一包被有Matrigel TM(BD Biosciences公司制)的24孔培养板中继续培养。
当传代后的细胞在培养板内继续生长至铺满培养板底部约80％后，再次按上述操作方法消化收集所培养获得的细胞并计数。同样按4×10 4个/孔密度接种并持续培养。
图1A是表示样本HMFL-XN26分别在各培养条件下自初代培养(传代0)开始后培养第4代、第5代、第6代和第7代的细胞镜下照片对比(100倍倒置相差显微镜下)。样本HMFL-XN26在基础培养基 的条件下培养至第6代则出现生长停滞现象。而采用基础培养基+化合物1的培养基在细胞培养至第6代后仍然能够对乳腺上皮细胞持续进行培养。图1B是对各培养基中的乳腺肿瘤细胞的培养代数定量化而得的图表。图1C是以培养天数为横坐标，群体倍增数为纵坐标，采用Graphpad Prism7.0软件绘制的各培养条件下乳腺肿瘤细胞的生长曲线。群体倍增数的计算公式为：
群体倍增数＝[log(N/X 0)]/log2
其中，N为传代时的细胞数目，X 0为初始接种时的细胞数目(Greenwood等，Environ Mol Mutagen 2004，43(1):36-44)。根据图1A～C确认，与基础培养基相比，将化合物1添加至培养基中，乳腺上皮细胞在体外可持续培养的代数至少提高一倍。
[实施例2]
1.根据实施例1相同的方式制备基础培养基。
2.按照实施方式1中的步骤获得新鲜分离的上皮肿瘤细胞样品(HMFL-XN40)。然后，将上皮肿瘤细胞接种至包被有Matrigel TM(BD Biosciences公司制)的6孔板。向接种了上述上皮肿瘤细胞的孔中添加基础培养基，在37℃以氧浓度20％的条件进行培养。按实施例1的4的步骤进行消化、传代、培养和计数并持续培养3代。在进行第4代肿瘤细胞接种时，将上皮肿瘤细胞按3×10 4个/孔的细胞密度均匀地接种至包被有Matrigel TM(BD Biosciences公司制)的24孔板的各孔中。接种，分别向各孔加入含基础培养基+DMSO和含基础培养基+化合物1的培养基各1mL，化合物1的在培养基内的浓度由低到高分别是0.1μM、0.3μM、1μM、3μM和10μM。
图2是HMFL-XN40自第4代持续培养至第6代，每一代的乳腺肿瘤细胞在各培养条件下的细胞计数结果。根据图2确认，与基础培养基+溶剂对照(DMSO)组相比，将0.3μM～3μM的化合物1添加至培养基中，乳腺上皮细胞在体外持续扩增的效果较为明显；10μM的化合物1则不能实现促进乳腺肿瘤细胞体外持续扩增的效果，可能是化合物在培养基内的浓度过高影响了细胞内其他蛋白靶点所致。
[实施例3]
1.根据与实施例1相同的方式制备基础培养基和构成成分为以下组合的培养基。
·基础培养基+1μM化合物1
·基础培养基+1μM化合物25
2.根据实施例1之4进行来源于乳腺肿瘤细胞的培养
基于中国科学院合肥物质科学研究院医学伦理委员会认可的伦理研究计划，分别获取两例进行说明并获得同意的乳腺肿瘤患者的癌组织样本(HMFL-XN34和HMFL-XN35)。接着，按照实施方式1中的步骤获得上皮肿瘤细胞。然后，将由HMFL-XN34获得的上皮肿瘤细胞接种至包被有Matrigel TM(BD Biosciences公司制)的24孔板。向接种了上述上皮肿瘤细胞的孔中分别添加基础培养基、基础培养基+化合物25(终浓度为1μM)的培养基各1mL，在37℃以氧浓度20％的条件进行培养。HMFL-XN35按同样的方式接种于24孔板中，分别添加基础培养基、基础培养基+化合物1(终浓度为1μM)的培养基，在37℃以氧浓度20％的条件进行培养。分别在培养开始后每2天进行一次培养基的更换。
按实施例1之4的步骤进行消化、传代、培养和计数。
图3A是表示源自HMFL-XN34的乳腺上皮细胞分别在添加或不添加化合物25的培养条件下自初代培养(传代0)开始后第4代(共培养34天)的细胞镜下照片对比(100倍倒置相差显微镜下)，以及源自HMFL-XN35的乳腺上皮细胞分别在添加或不添加化合物1的培养条件下自初代培养(传代0)开始后第6代(共培养42天)的细胞镜下照片对比(100倍倒置相差显微镜下)。图3B是对各培养条件下培养的不同的乳腺肿瘤细胞的细胞计数结果进行归一化后的统计结果。
根据图3A和B确认，与基础培养基相比，将化合物1或化合物25添加至培养基中，能实现体外对乳腺癌肿瘤细胞增殖的持续促进的效果。
[实施例4]
1.根据与实施例1相同的方法制备基础培养基和构成成分为以下 组合的培养基：
·基础培养基+1μM化合物1
2.根据实施例2之2进行来源于HMFL-XN35的乳腺肿瘤细胞的培养、消化、传代和计数。
图4A的1和2号图分别表示的是源自HMFL-XN35的乳腺上皮细胞在添加化合物1的培养条件下自初代培养(传代0)开始后不断传代持续培养至第18代后，将细胞进行常规消化，并按3×10 4密度再次传代接种，接种时将细胞分别培养在原培养基(基础培养基+1μM化合物1，图4A之1号图)和撤去化合物1的培养基(基础培养基，图4A之2号图)中，继续培养8天后的细胞镜下照片。图4A的3号图是表示在2号图中的细胞中，又重新添加化合物1入培养基中，再培养8天后的细胞镜下照片(100倍倒置相差显微镜下)。图4B是对各培养条件下培养的不同的乳腺肿瘤细胞的细胞计数结果进行归一化后的统计结果，其中以基础培养基+1μM化合物1进行培养8天的细胞计数结果为基准100％，撤去化合物1的培养基在培养8天后产生的细胞计数低于基准的50％，而重新将化合物1加入培养基再培养8天后，细胞计数得以显著增加。
根据图4A和B确认，MST1/2激酶抑制剂化合物1是实现本发明的培养基在体外保持乳腺上皮细胞持续增殖的必须成分，且化合物1实现体外对乳腺癌肿瘤细胞增殖的持续促进的效果是可逆的。
[实施例5]
1.根据与实施例1相同的方法制备基础培养基和构成成分为以下组合的培养基：
·基础培养基+1μM化合物1
2.制备改良类器官培养基
向市售的DMEM/F-12培养基(Corning公司制)以最终浓度10μg/ml加入胰岛素(Sigma-Aldrich公司制)，以1:50的终浓度加入B27，以最终浓度100ng/mL的条件添加头蛋白(Peprotech公司制)，以最终浓度1μM条件添加化合物1，以最终浓度500nM条件添加A8301(MCE公司制)，以最终浓度500nM条件添加SB202190(MCE公司 制)，以最终浓度10μM条件添加Y27632(MCE公司制)。然后，添加最终浓度5ng/mL的表皮生长因子(EGF)(R&D公司制)、最终浓度5ng/mL的FGF7(R&D公司制)、最终浓度5ng/mL的FGF10(R&D公司制)、最终浓度10ng/mL的Neuregulin1(Peprotech公司制)、最终浓度1.25mM的N-乙酰半胱氨酸(Sigma-Aldrich公司制)、最终浓度5mM的烟酰胺(Sigma-Aldrich公司制)，制备成培养乳腺类器官的培养基。由于该培养基内没有加入公认的类器官培养基中的必加成分R-海绵硬蛋白这个昂贵的成分，从而降低了类器官成本。以下也称“改良类器官培养基”。
3.基于中国科学院合肥物质科学研究院医学伦理委员会认可的伦理研究计划，从进行说明并获得同意的乳腺肿瘤患者的癌旁组织中获取乳腺组织样本(HMFL-XN28)。作为正常的乳腺组织采集距离乳腺肿瘤至少5cm以上的部分。接着，按照实施方式1中的相同步骤获得新鲜分离的正常乳腺上皮细胞，然后按照实施例2之2的步骤进行体外培养。
图5A是表示源自HMFL-XN28的癌旁组织的正常乳腺上皮细胞在基础培养基+化合物1的培养条件下自初代培养(传代0)开始后培养至第2天和持续培养至第18天的细胞镜下照片(100倍倒置相差显微镜下)。
4.根据实施例2之2分离得到来源于HMFL-XN34的乳腺肿瘤细胞并接种于包被有Matrigel TM(BD Biosciences公司制)的24孔板。接着，在接种了上述上皮肿瘤细胞的孔中添加基础培养基+化合物1的培养基1mL，在37℃以低氧浓度2％的条件进行培养。图5B是表示源自HMFL-XN34的乳腺肿瘤细胞在基础培养基+化合物1的培养条件下自初代培养(传代0)开始后培养至第4天和培养至第32天的细胞镜下照片(100倍倒置相差显微镜下)。
根据图5A和B可以确认，本发明的培养基能实现体外对正常乳腺上皮细胞增殖的持续促进效果并且在缺氧条件下也能够持续培养乳腺肿瘤细胞。
5.根据实施例2之2分离得到来源于HMFL-XN35的乳腺肿瘤细胞。接着，将上皮肿瘤细胞与40μL的Matrigel TM(BD Biosciences公 司制)一起接种至24孔板。向接种了上述上皮肿瘤细胞的孔中添加改良类器官培养基，在37℃以氧浓度20％的条件进行培养。培养开始后每2天进行一次培养基的更换。图5C是表示自初代培养(传代0)的培养开始后第4天的以及第38天的图像(100倍倒置相差显微镜下)。
根据图5C可以确认，本发明的改良类器官培养基能实现体外对乳腺肿瘤类器官的高效培养。
[实施例6]
1.根据与实施例1相同的方法制备基础培养基和构成成分为以下组合的培养基。
·基础培养基+化合物1
·基础培养基+化合物25
2.按照实施方式1中的步骤获得新鲜分离的上皮肿瘤细胞样品(HMFL-XN40)。然后，将上皮肿瘤细胞接种至包被有Matrigel TM(BD Biosciences公司制)的6孔板。向接种了上述上皮肿瘤细胞的孔中分别添加基础培养基、基础培养基+化合物25的培养基各3mL，化合物25的浓度由低到高分别是1μM和3μM。在37℃以氧浓度20％的条件进行培养。培养开始后每2天进行一次培养基的更换。待培养72小时后，收集各组细胞，采用公认的免疫印迹法检测化合物25对MST1/2的直接底物MOB1的磷酸化，以及对干细胞标志物Lgr5的影响。结果如图6A所示。检测结果显示，MST1/2激酶抑制剂化合物25能够剂量依赖性地抑制MST1/2激酶直接底物的磷酸化，并且对乳腺肿瘤细胞的干性标志物Lgr5有明显上调。
由于Lgr5是Wnt-依赖性干细胞标志物(Barker等，Mol Cell Biol，22:1184-93，2002)，且Lgr5对乳腺细胞的自我更新和持续增殖具有重要作用(Plaks等，Cell Reports，3，70-78，2013)，这一结果提示化合物25能够通过抑制乳腺肿瘤细胞的MST1/2介导的信号通路，从而维持了乳腺癌细胞干性增殖的特性，并起到在体外持续促进乳腺癌细胞增殖的作用。
3.按照实施例1中的步骤获得新鲜分离的上皮肿瘤细胞样本(HMFL-XN41)。将分离得到的上皮肿瘤细胞分成三等分。第一等分 直接收集；第二和第三等分的上皮肿瘤细胞分别接种至包被有Matrigel TM(BD Biosciences公司制)的6孔板。向接种了上述上皮肿瘤细胞的孔中分别添加基础培养基、添加化合物1基础培养基各3mL，化合物1的浓度为1μM。在37℃以氧浓度20％的条件进行培养。培养开始后每2天进行一次培养基的更换。待培养96小时后，收集各组细胞，采用公认的免疫印迹法检测化合物1对乳腺肿瘤细胞内的MST1/2、MST1/2的直接底物MOB1、以及下游和细胞生长增殖相关的激酶ERK1/2的磷酸化的影响，同时检测对乳腺癌干细胞标志物Lgr5和其同家族与Lgr5功能类似的干性标志物Lgr4和Lgr6的影响。结果如图6B所示。检测结果显示，这例新鲜分离的乳腺肿瘤细胞(HMFL-XN41)在基础培养基中培养96小时后，MST1/2激酶介导的信号通路发生活化，而与细胞生长和增殖相关的ERK激酶受到抑制，干性标记物Lgr5及其相关蛋白也受到抑制，表明基础培养基导致了MST1/2激酶介导的细胞凋亡通路的激活。添加化合物1后能够明显抑制MST1/2激酶介导的信号通路的活化，刺激促增殖信号分子ERK的活化，并且对乳腺肿瘤细胞的干性标志物家族成员Lgr4、Lgr5和Lgr6均有明显地上调。这表明化合物1和化合物25一样，能够抑制乳腺肿瘤细胞的MST1/2介导的信号通路并能够促进肿瘤细胞的持续增殖。
4.按照上述步骤获得新鲜分离的上皮肿瘤细胞样本(HMFL-XN42)。将分离得到的上皮肿瘤细胞分成五等分。第一等分直接收集；第二和第三等分的上皮肿瘤细胞分别接种至包被有Matrigel TM(BD Biosciences公司制)的6孔板。向接种了上述上皮肿瘤细胞的孔中分别加入的基础培养基各3mL，分别培养48小时和96小时后收集；第四和第五等分的上皮肿瘤细胞分别接种至包被有Matrigel TM(BD Biosciences公司制)的6孔板，并向孔中分别加入添加了1μM化合物1的基础培养基各3mL，分别培养48小时和96小时后收集。在37℃以氧浓度20％的条件进行培养。培养开始后每2天进行一次培养基的更换。待培养48和96小时后，分别收集各组细胞，采用公认的免疫印迹法检测化合物1在不同时间点对乳腺肿瘤细胞内的Wnt信号通路下游的重要信号分子TCF4/TCF7L2的影响，同时检测对乳腺癌干细胞标志物Lgr5和其同家族与Lgr5功能类似的干性标志 物Lgr4和Lgr6的影响。结果如图6C所示。检测结果显示，这例新鲜分离的乳腺肿瘤细胞(HMFL-XN42)在基础培养基中分别培养48小时和96小时后，干性标记物Lgr5及其相关蛋白Lgr4和Lgr6可时间依赖性地表达上调，同时Wnt下游的重要信号分子TCF4/TCF7L2的表达也上调，提示基础培养基本身对这一例病人源性乳腺肿瘤细胞的干性增殖有一定的促进作用。在添加化合物1后，乳腺肿瘤细胞的干性标志物家族成员Lgr4、Lgr5和Lgr6以及TCF4/TCF7L2呈现时间依赖性地上调，且对乳腺组织的自我更新和持续增殖具有重要作用的Lgr5上调得比不加化合物1时更加明显。这表明化合物1能够时间依赖性的促进肿瘤细胞在体外的持续增殖。
[实施例7]
1.根据与实施例1相同的方法制备基础培养基和构成成分为以下组合的培养基：
·基础培养基+1μM化合物1
2.采用与实施例3的相同步骤分别将乳腺肿瘤细胞样本(HMFL-XN32和HMFL-XN33)进行体外持续培养。将体外培养并传代10次的乳腺癌肿瘤细胞(HMFL-XN32，P10)和乳腺癌病人直接来源的肿瘤组织，分别使用DNeasy blood&tissue kit(QIAGEN公司制)提取细胞和相应组织的基因组DNA。收集细胞来源患者的外周血2mL，采用同样的方法提取基因组DNA作为背景对照。随后，对细胞和血液样本的基因组DNA进行全外显子组测序(具体操作步骤参见HansClevers等，Cell，11；172(1-2):373-386，2018)，并对测序结果进行基因拷贝数变异分析。HMFL-XN33(P12)及相应组织的数据采用和上述同样的方法获得。图7A和B表示体外培养的乳腺肿瘤细胞和对应组织的全外显子组的基因拷贝数变异的散点图。
经图7A和B可以确认，体外持续培养的乳腺肿瘤细胞的基因拷贝数的获得和缺失的位置和数目与病人原始肿瘤组织基本保持一致。
图8表示按照上述步骤对另外4例乳腺癌组织样本培养获得的乳腺肿瘤细胞(HMFL-XN34、HMFL-XN35、HMFL-XN38和HMFL-XN39)与相应肿瘤组织的全外显子组数据进行肿瘤高频基因突变数目分析比 对的结果。分析结果使用https://bioinfogp.cnb.csic.es/tools/venny/index.html软件制作。经图8可以确认培养的癌组织来源的乳腺癌肿瘤细胞和相对应的肿瘤组织内的的高频突变基因基本一致，说明本发明的培养基和培养方法所培养出的乳腺肿瘤细胞能够保持病人癌组织内原始的基因突变特性。
[实施例8]
1.根据实施例1之4的同样步骤获得HMFL-XN35的乳腺肿瘤组织样本。从乳腺癌患者的样本中取出约黄豆粒大小的癌组织，浸泡在10mL 4％多聚甲醛中固定，用于下面的苏木素-伊红(HE)染色法和免疫组织化学(IHC)法检测。其余组织使用实施例3的步骤将HMFL-XN35持续培养至第10代，培养基为基础培养基+1μM化合物1，同样进行下面的(HE)染色法和免疫组织化学(IHC)法检测。
2.采用苏木素-伊红(HE)染色法和免疫组织化学(IHC)法检测HMFL-XN35细胞及细胞来源的原始组织内乳腺癌细胞形态及与乳腺癌相关的重要生物标记物的表达情况。
4％多聚甲醛固定后的组织，经石蜡包埋，用切片机切成4μm厚的组织切片。随后进行常规的HE染色和IHC检测。HE染色使用HE染色试剂盒(Solarbio公司制)操作。具体实验步骤参照试剂盒的使用说明书。
免疫组织化学检测(IHC)具体步骤参见Yu等，Science，345(6193):216-220，2014。IHC所用的一抗为ER(Cell Signaling Technology公司制)、PR(Cell Signaling Technology公司制)、HER2(Cell Signaling Technology公司制)。所用的二抗为SignalStain Boost IHC Detection Reagent(HRP,Rabbit)(Cell Signaling Technology公司制)、SignalStain Boost IHC Detection Reagent(HRP,Mouse)(Cell Signaling Technology公司制)。其中，ER、PR是预测患者是否可接受内分泌治疗的重要指标；HER2是预测患者是否可接受抗HER2靶向治疗的重要指标，目前这些指标也是临床上用于乳腺癌的分子分型病理诊断的重要依据。
HE染色是临床上用于乳腺癌病理诊断的重要依据。分别拍摄原始组织和培养至第10代的细胞的HE染色情况和ER、PR、HER2的表达 情况(200倍生物显微镜下)。结果如图9所示。其中，HE染色结果显示组织内的细胞和体外培养的细胞形态较为一致，均为乳腺癌细胞。免疫组化结果显示原始患者的分子分型为ER(-)、PR(-)、HER2(+)，培养至第10代时的乳腺癌细胞的分子分型也为ER(-)、PR(-)、HER2(+)。
由图9可以确认，采用本发明技术培养至第10代时细胞与原始组织内细胞病理形态保持一致，细胞上与乳腺癌相关的生物标记物的表达情况与原始组织的标记物表达情况一致。说明采用本发明技术所培养的乳腺肿瘤细胞保持了乳腺癌病人原始的病理特性。
[实施例9]
下面以乳腺癌患者手术切除样本为例，说明由病人来源的乳腺癌肿瘤样本培养得到的乳腺癌肿瘤细胞可以用于检测病人肿瘤细胞对不同药物的敏感性。
1.根据与实施例1相同的方法制备构成成分为以下组合的培养基：
·基础培养基+1μM化合物1
2.乳腺癌肿瘤细胞的铺板：将通过实施例3的步骤培养得到的乳腺癌肿瘤细胞(HMFL-XN38第10代和HMFL-XN39第11代)的单细胞悬液，按3000～5000个/孔密度接种于384孔板中，使细胞贴壁过夜。
3.药物梯度实验：
(1)采用浓度梯度稀释的方法配制药物贮存板：取待测药物母液(药物母液浓度按20μM配制)，按照1:3稀释，依次得到7种浓度的药物。将不同浓度的药物按10μL体积加入384孔药物储存板中。溶剂对照组各孔加入等体积的DMSO。本实施例中，待测药物为阿法替尼(MCE公司制)、拉帕替尼(MCE公司制)、多西他赛(MCE公司制)和他莫昔芬(MCE公司制)。
(2)使用高通量自动化工作站(购自Perkin Elmer公司)将384孔药物贮存板内的不同浓度药物和溶剂对照加入到铺有乳腺癌肿瘤细胞的384孔细胞培养板中，药物组和溶剂对照组都各设3个复孔。每孔加入药物体积为100 nL。
(3)细胞活性检测：给药72小时后，用Cell Titer-Glo检测试剂 (Promega公司制)检测加药培养后细胞的化学发光数值，化学发光数值的大小反映细胞活力以及药物对细胞活力的影响，每孔加入配制好的Cell Titer-Glo检测液，混匀后使用酶标仪检测化学发光数值。
使用Graphpad Prism 7.0软件作图并计算半数抑制率IC 50。
图10A和B分别表示从两个不同的乳腺癌患者的手术切除癌组织样本所培养获得的乳腺癌肿瘤细胞样本(HMFL-XN38和HMFL-XN39)对化疗药物多西他赛、内分泌治疗药物他莫昔芬、和靶向药物拉帕替尼以及阿法替尼的敏感性。结果显示，同一病人的细胞对不同药物具有不同的敏感性，不同病人的细胞对同一药物的敏感性也不同。
其中，源自激素受体阴性、HER2受体阳性的乳腺癌患者的乳腺癌肿瘤细胞(HMFL-XN38)对HER2靶向药物拉帕替尼和阿法替尼敏感；而对内分泌药物他莫昔芬的敏感性较低。而另一例三阴性乳腺癌患者的乳腺癌肿瘤细胞(HMFL-XN39)则对化疗药物多西他赛敏感，而对三种所测试的靶向药物均不敏感。
根据图10确认，本发明技术所培养的乳腺癌病人癌组织来源的乳腺癌肿瘤细胞对化疗药物和靶向药物的敏感性测试结果与病人临床的分子分型相符，提示本发明技术所培养得到的乳腺癌肿瘤细胞在预测乳腺癌患者临床药物疗效方面具有应用潜力。
工业应用性
本发明提供一种用于体外培养上皮干细胞、尤其是乳腺上皮干细胞的培养基以及用于培养包含所述干细胞的类器官的培养基和培养方法，使用本发明的培养基和培养方法所培养的细胞后代和类器官可用于药物的疗效评估和筛选、毒性测定和再生医学。因而，本发明适于工业应用。
尽管本文对本发明作了详细说明，但本发明不限于此，本技术领域的技术人员可以根据本发明的原理进行修改，因此，凡按照本发明的原理进行的各种修改都应当理解为落入本发明的保护范围。

Claims (19)

  1. 一种乳腺上皮干细胞培养基，其特征在于：
    含有TGF-β抑制剂、B27和/或N2、胰岛素、受体酪氨酸激酶配体、Rock激酶抑制剂、P38信号转导抑制剂、骨形态发生蛋白抑制剂、以及MST1/2激酶抑制剂。
  2. 如权利要求1所述的乳腺上皮干细胞培养基，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，
    Figure PCTCN2020086366-appb-100001
    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。
  3. 如权利要求2所述的乳腺上皮干细胞培养基，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。
  4. 如权利要求2所述的乳腺上皮干细胞培养基，其中所述MST1/2激酶抑制剂包括式(Ia)的化合物或其药学可接受的盐、或溶剂化物，
    Figure PCTCN2020086366-appb-100002
    其中，
    R 1选自C1-C6烷基、任选地被1-2个独立地R 6取代的苯基、任选地被1-2个独立地R 6取代的噻吩基、和任选地被1-2个独立地R 6取代的苯甲基；
    R 5选自氢、C1-C6烷基、和C3-C6环烷基；
    R 6各自独立地选自卤素、C1-C6烷基、和C1-C6卤代烷基。
  5. 如权利要求4所述的乳腺上皮干细胞培养基，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。
  6. 如权利要求1所述的乳腺上皮干细胞培养基，其中所述MST1/2激酶抑制剂选自以下化合物或其药学可接受的盐中的至少一种：
    Figure PCTCN2020086366-appb-100003
    Figure PCTCN2020086366-appb-100004
    Figure PCTCN2020086366-appb-100005
    Figure PCTCN2020086366-appb-100006
    Figure PCTCN2020086366-appb-100007
  7. 根据权利要求1～6中任一项所述的乳腺上皮干细胞培养基，其中所述MST1/2激酶抑制剂在所述乳腺上皮干细胞培养基中的浓度是100nM以上且10μM以下，优选300nM以上且3μM以下。
  8. 如权利要求1～6中任一项所述的乳腺上皮干细胞培养基，其中所述TGF-β抑制剂选自A8301、SB431542、Repsox、SB505124、SB525334、SD208、LY36494、和SJN2511中的至少一种。
  9. 如权利要求1～6中任一项所述的乳腺上皮干细胞培养基，其中所述受体酪氨酸激酶配体选自表皮生长因子、双向调节素、转化生长因子-α、碱性成纤维细胞生长因子、脑源性神经生长因子、神经调节素1、肝细胞生长因子、成纤维细胞生长因子7、和成纤维细胞生长因子10中的至少一种；所述受体酪氨酸激酶配体优选包括表皮生长因子、神经调节素1、和成纤维细胞生长因子7。
  10. 如权利要求1～6中任一项所述的乳腺上皮干细胞培养基，其中所述Rock激酶抑制剂选自Y27632、法舒地尔、H-1152中的至少一种。
  11. 如权利要求1～6中任一项所述的乳腺上皮干细胞培养基，其中所述P38信号转导抑制剂选自SB202190、SB203580、VX-702、VX-745、PD169316、RO4402247、和BIRB796中的至少一种。
  12. 如权利要求1～6中任一项所述的乳腺上皮干细胞培养基，其中所述骨形态发生蛋白抑制剂选自包括头蛋白、格雷林、腱蛋白、腱蛋白结构域的腱蛋白样蛋白，包括卵泡抑素、卵泡抑素结构域的卵泡 抑素相关蛋白，包括DAN、DAN半胱氨酸结构域的DAN样蛋白，硬骨素/SOST、核心蛋白聚糖、α2-巨球蛋白、和DMH1中的至少一种。
  13. 如权利要求1～6中任一项所述的乳腺上皮干细胞培养基，其中
    所述TGF-β抑制剂在所述乳腺上皮干细胞培养基中的浓度是50nM以上且100μM以下，更好是100nM以上且50μM以下，进一步优选100nM以上且10μM以下；
    所述B27和/或所述N2在所述乳腺上皮干细胞培养基中的浓度是1:25～1:100，优选1:50～1:100；
    所述胰岛素在所述乳腺上皮干细胞培养基中的浓度是2μg/ml～20μg/ml，优选5μg/ml～10μg/ml；
    所述受体酪氨酸激酶配体在所述乳腺上皮干细胞培养基中的浓度是1ng/ml至1000ng/ml，优选5ng/ml至500ng/ml，进一步优选10ng/ml～100ng/ml；
    所述Rock激酶抑制剂在所述乳腺上皮干细胞培养基中的浓度是1μM至100μM，优选2μM至50μM，更优选5μM至10μM；
    所述P38信号转导抑制剂在所述乳腺上皮干细胞培养基中的浓度是50nM以上且100μM以下，更好是100nM以上且50μM以下，进一步优选100nM以上且10μM以下；
    所述骨形态发生蛋白抑制剂在所述乳腺上皮干细胞培养基中的浓度是1ng/ml至1000ng/ml，优选10ng/ml至500ng/ml，更优选20ng/mL至100ng/mL。
  14. 如权利要求1～6中任一项所述的乳腺上皮干细胞培养基，其还包括选自糖、烟酰胺和N-乙酰半胱氨酸中的一种或多种，其中所述糖选自葡萄糖、半乳糖、甘露糖、和果糖。
  15. 如权利要求14所述的乳腺上皮干细胞培养基，其中所述糖在所述乳腺上皮干细胞培养基中的浓度为10mM～100mM，优选15mM～40mM；
    所述烟酰胺在所述乳腺上皮干细胞培养基中的浓度为1mM～10mM，优选2mM～5mM；
    所述N-乙酰半胱氨酸在所述乳腺上皮干细胞培养基中的浓度为0.1mM～5mM，优选0.5mM～2mM。
  16. 如权利要求1～6中任一项所述的乳腺上皮干细胞培养基，其特征在于：用于培养正常乳腺组织来源或病变乳腺组织来源的上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含这些细胞中的至少任一种的组织或类器官。
  17. 一种培养正常乳腺组织来源或病变乳腺组织来源的上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含这些细胞中的至少任一种的组织或类器官的培养方法，其特征在于，包括以下步骤：
    (1)制备细胞外基质；
    (2)将上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含这些细胞中的至少任一种的组织粘附于所述细胞外基质上或包埋于细胞外基质中；
    (3)添加如权利要求1～16中的任一项所述的乳腺上皮干细胞培养基，对所述上皮干细胞、上皮细胞、上皮肿瘤细胞、或包含这些细胞中的至少任一种的组织进行培养，得到扩增的、相应的上皮干细胞、上皮细胞、上皮肿瘤细胞后代或类器官后代。
  18. 如权利要求17所述的培养方法，其中，在培养上皮干细胞或类器官步骤中，在氧浓度0.1％～25％的条件下进行培养，优选的正常氧气条件，或0.1％～15％的低氧条件。
  19. 一种乳腺癌药物的疗效评估或筛选的方法，其特征在于，包括以下步骤：
    (1)使用权利要求17或18所述的培养方法培养乳腺上皮干细胞、上皮细胞、或上皮肿瘤细胞；
    (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)中培养得到的细胞添加稀释后的所述药物；和
    (4)进行细胞活性测试。
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