WO2023060642A1 - 一种肠癌原代细胞的培养基及体外培养方法和用途 - Google Patents

一种肠癌原代细胞的培养基及体外培养方法和用途 Download PDF

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WO2023060642A1
WO2023060642A1 PCT/CN2021/126234 CN2021126234W WO2023060642A1 WO 2023060642 A1 WO2023060642 A1 WO 2023060642A1 CN 2021126234 W CN2021126234 W CN 2021126234W WO 2023060642 A1 WO2023060642 A1 WO 2023060642A1
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culture medium
intestinal cancer
cells
alkyl
primary
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French (fr)
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刘青松
赫玉影
黄涛
陈程
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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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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • the invention belongs to the technical field of biomedicine, specifically relates to a culture medium and its application, and more specifically relates to a culture medium, a culture method and an application of intestinal cancer primary cells.
  • Colon cancer is a disease caused by genetic and environmental factors, and is one of the most common digestive tract tumors worldwide.
  • Survey data show (Zheng Shu et al., Prevention of Colorectal Cancer, Chinese Journal of Oncology, 2004, Vol.13, No.1, pp1-2), colon cancer is one of the most common tumors, and the overall morbidity and mortality On the rise, it is a malignant tumor that seriously threatens life and health.
  • Surgery combined with postoperative chemotherapy is currently the main treatment for colorectal cancer. Although surgical techniques have improved in recent years and the survival rate of colorectal cancer patients has improved, tumor metastasis and recurrence still bring poor prognosis to patients.
  • Xuefeng Liu et al. used irradiated mouse fibroblasts and a Rho-associated kinase inhibitor (Y-27632) to expand epithelial-derived cells.
  • This system has the ability to achieve unlimited growth of epithelial-derived cells without genetic manipulation.
  • Ability Xuefeng Liu et al., Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens.Nat.Protoc.2017,12,439).
  • the method established by Xuefeng Liu et al. has a long culture cycle and cannot achieve rapid expansion of cells.
  • the co-cultivation of primary cells and stromal cells is inevitable, and it cannot solve a series of problems in the culture schemes such as co-cultivation with stromal cells. problems, which limit the application of this technology.
  • the present invention provides a culture medium, in vitro culture method and application of intestinal cancer primary cells.
  • One aspect of the present invention is to provide a culture medium for primary intestinal cancer cells, the culture medium comprising MST1/2 kinase inhibitors; insulin; at least one selected from Y27632, Fasudil, and H-1152 Rho protein kinase inhibitor; insulin-transferrin-selenium supplement; glutamine supplement; neuregulin 1; bovine pituitary extract; basic fibroblast growth factor; R-spondin1; prostaglandin E2; and B27 .
  • the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,
  • R 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by 1-2 independent R (such as phenyl and naphthyl, etc.), aryl C1-C6 alkyl (such as benzyl, etc.) and heteroaryl (such as thienyl, etc.);
  • R 2 and R 3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
  • R 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyran base, etc.);
  • R is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoro methyl).
  • halogen preferably fluorine and chlorine, more preferably fluorine
  • C1-C6 alkyl preferably methyl
  • C1-C6 alkoxy preferably methoxy
  • C1-C6 haloalkyl preferably trifluoro methyl
  • the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,
  • R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R6 , thienyl optionally substituted by 1-2 independently R6 , and optionally substituted by 1 -2 independently R6 substituted benzyl, R1 is more preferably optionally 1-2 independently R6 substituted phenyl;
  • R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R 5 is more preferably hydrogen;
  • R 6 is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R 6 is more preferably fluorine, methyl or trifluoromethyl.
  • the MST1/2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.
  • the MST1/2 kinase inhibitor of the present invention is Compound 1.
  • the content of each component in the culture medium of the present invention satisfies any one or more or all of the following:
  • the content of MST1/2 kinase inhibitor in the medium is 1.25-5 ⁇ M;
  • the content of insulin in the medium is 1-9 ⁇ g/mL
  • Rho protein kinase inhibitor is preferably Y27632, and its content in the medium is 1.25-20 ⁇ M;
  • the volume ratio of insulin-transferrin-selenium supplement to the culture medium is 1:400 ⁇ 1:100;
  • the content of glutamine additive in the culture medium is 0.5-2mM
  • the content of neuregulin-1 in the medium is 2.5-40 ng/ml;
  • the volume ratio of bovine pituitary extract to medium is 1:2000 ⁇ 1:125;
  • the content of basic fibroblast growth factor in the culture medium is 2.5 ⁇ 20ng/ml
  • the content of prostaglandin E2 in the culture medium is 5-80nM
  • the medium also contains an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one selected from streptomycin/penicillin, amphotericin B and Primocin one or more antibiotics.
  • the streptomycin concentration ranges from 25 to 400 ⁇ g/mL, and the penicillin concentration ranges from 25 to 400 U/mL; when the antibiotic is selected from amphotericin B, The concentration range is 0.25-4 ⁇ g/mL, and when the antibiotic is selected from Primocin, the concentration range is 25-400 ⁇ g/mL.
  • the present invention also provides a method for culturing primary intestinal cancer cells in vitro.
  • primary intestinal cancer cells are cultured in vitro using the culture medium for primary intestinal cancer cells of the present invention.
  • the in vitro culture method of intestinal cancer primary cells of the present invention comprises the following steps:
  • tissue samples Separate colon cancer tissue samples, add basal medium and tissue digestion solution at a volume ratio of 1:3 (Note: the amount of tissue digestion solution is about 5-10mL tissue digestion solution for 1g of tumor tissue), and place at constant temperature Carry out digestion in a shaker, the digestion temperature is 4-37°C, and the digestion speed is 200rpm-350rpm;
  • the basal medium formula includes an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin/penicillin, amphotericin B and Primocin.
  • the formula of tissue digestion solution includes 1640 medium, collagenase II (1-2mg/mL), collagenase IV (1-2mg/mL), DNase (50-100U/mL), hyaluronidase (0.5-1mg/mL) mL), calcium chloride (1 ⁇ 5mM), bovine serum albumin BSA (5 ⁇ 10mg/mL).
  • the present invention also provides a method for evaluating or screening a drug for the treatment of intestinal cancer, comprising the following steps:
  • the primary intestinal cancer cells cultured in vitro can maintain the pathological characteristics of the patient
  • the cultured primary intestinal cancer cells are not disturbed by interstitial cells such as fibroblasts and adipocytes;
  • the amplification efficiency is high, and primary intestinal cancer cells can be successfully cultured in about a week, and the amplified primary intestinal cancer cells can also be continuously passaged;
  • the medium does not contain serum, and can avoid the co-cultivation of primary cells and stromal cells, and also solve a series of problems in the traditional culture schemes containing serum and co-cultivating with stromal cells;
  • the number of intestinal cancer primary cells obtained by the above-mentioned technology is large and the degree of homogeneity is high, which is suitable for high-throughput screening of new candidate compounds and providing patients with high-throughput in vitro sensitive function tests of drugs.
  • Fig. 1 is a graph showing the effects of different combinations of factors added in the culture medium of primary intestinal cancer cells on the growth of primary intestinal cancer cells.
  • 2A-2K are graphs showing the effects of different concentrations of factors added to the culture medium of primary intestinal cancer cells on the growth of primary intestinal cancer cells.
  • 3A-3F are microscopic observations of primary intestinal cancer cells cultured with the primary intestinal cancer cell culture medium of the present invention.
  • Figures 4A-4D are the immunohistochemical results of primitive intestinal cancer tissue cells.
  • 5A-5D are the immunohistochemical results of primary intestinal cancer cells obtained by culturing primary intestinal cancer tissue cells to the fifth passage using the primary intestinal cancer cell culture medium of the present invention.
  • Fig. 6 shows the cell growth curve of primary intestinal cancer cells cultured in vitro using the culture medium for primary intestinal cancer cells of the present invention.
  • Figure 7 shows the comparative results of the proliferation efficiency of intestinal cancer primary cells cultured by respectively using the primary intestinal cancer cell culture medium of the present invention and the literature medium of the prior art
  • 8A-8F show the results of drug screening of primary intestinal cancer cells cultured to different passages using the primary intestinal cancer cell culture medium of the present invention.
  • an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signal transduction.
  • MST1/2 kinase inhibitors for example, bind to MST1/2 kinase and reduce its activity. Due to the similarity in the structures of MST1 and MST2, MST1/2 kinase inhibitors can also be, for example, compounds that bind to MST1 or MST2 and reduce their activity.
  • 2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): In a round bottom flask was added 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) Methanol (30 mL) was then added, followed by the dropwise addition of thionyl chloride (1.2 mL) under ice-cooling. The reaction system was reacted overnight at 85°C. After the reaction, the system was evaporated to dryness under reduced pressure to obtain a white solid, which was directly used in the next step.
  • MST1/2 inhibitor compounds of the present invention were synthesized according to a method similar to compound 1, and their structures and mass spectrometry data are shown in the table below.
  • Example 1 Effects of various added factors in the culture medium of primary intestinal cancer cells on the growth of primary intestinal cancer cells
  • the initial medium can be selected from DMEM/F12, DMEM, F12 or RPMI-1640 commonly used in the art.
  • the formulation of the basal medium is: DMEM/F12 medium (purchased from Corning Company)+100 ⁇ g/mL Primocin (purchased from InvivoGen Company, 0.2% (v/v), commercially available product concentration 50mg/ml ).
  • DMEM/F12 medium purchased from Corning Company
  • Primocin purchased from InvivoGen Company, 0.2% (v/v), commercially available product concentration 50mg/ml .
  • Different types of additives were added to the basal medium to prepare the culture medium for primary intestinal cancer cells containing different additive components.
  • Colon cancer solid tumor tissue samples (intraoperative/endoscopic) were obtained from patients by professional medical staff from professional medical institutions, and all patients signed informed consent.
  • the intraoperative sample is 0.25cm 3
  • the endoscopic sample is 0.025cm 3 ; commercialized tissue preservation solution (manufacturer: Miltenyi Biotec) is used for storage and transportation.
  • Tissue digestion solution 1640 medium (Corning, 10-040-CVR), collagenase II (2mg/mL), collagenase IV (2mg/mL), DNase (50U/mL), hyaluronidase (0.75mg /mL), calcium chloride (3.3mM), BSA (10mg/mL).
  • Collagenase II, collagenase IV, DNase, and hyaluronidase mentioned above were all purchased from Sigma Company; calcium chloride was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; BSA was purchased from Biofroxx Company.
  • “+” means that compared with the basal medium, the medium added with this additive has the effect of promoting the proliferation of two cases of primary intestinal cancer cells isolated from intestinal cancer tissue; “-” means that the medium with this additive added
  • the culture medium showed an inhibitory effect on at least one of the primary intestinal cancer cells isolated from intestinal cancer tissue;
  • “ ⁇ ” indicates that the medium added with this additive has Proliferation was not significantly affected in at least two cases.
  • Example 2 Effect of the combination of different added factors in the culture medium of primary intestinal cancer cells on the proliferation of primary intestinal cancer cells
  • the colon cancer primary cell culture medium with different additive factor combinations was prepared, and the proliferation-promoting effect of different additive factor combinations on the intestinal cancer primary cells was investigated.
  • Colon cancer primary cells were obtained from colon cancer tissues (numbered OE(O)001, OE(O)002, OE(E)060, OE(E)071) according to the method of step (2)-3 of Example 1 , the obtained cell suspension was evenly divided into 16 parts, and centrifuged at 1500 rpm for 4 minutes. After centrifugation, use 200 ⁇ L of BM and No.1-15 medium to resuspend. Use basal medium at a ratio of 50:1 with Matrigel ( 356231) and mix evenly on ice, take 300 ⁇ L and plate a 48-well plate, incubate in a 37° C., 5% CO 2 incubator for 25 to 35 minutes, preferably 30 minutes, and discard the supernatant.
  • Inoculate in a 48-well plate (20,000 cells per well ) according to the living cell density of 2 ⁇ 104 cells/cm2, fill up the volume of each well in the 48-well plate to 1 mL with the corresponding medium, and mix thoroughly. After surface disinfection, they were cultured in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher).
  • the above-mentioned No.1-No.15 medium can all promote the proliferation of primary intestinal cancer cells to varying degrees.
  • factors A8301 (No.2), noggin (No.12) and Forsklin (No.15) were omitted, the growth-promoting effect of the medium formula was more obvious. Therefore, in the use of compounds containing insulin, Y-27632, insulin-transferrin-selenium supplement, glutamine supplement, neuregulin-1, bovine pituitary extract, basic fibroblast growth factor, R-spondin1, 1.
  • the proliferation effect is the best.
  • step (2) of embodiment 1 from endoscopic tissue sample (numbering is OE (E) 060, OE (E) 078, OE (O) 014, OE (O) 015, OE (E) 071 ) to obtain primary intestinal cancer cells.
  • the obtained primary intestinal cancer cells were inoculated in T12.5 well plates (300,000 cells per well) according to the viable cell density of 2.4 ⁇ 10 4 cells/cm 2 , and placed at 37°C and 5% CO 2 after surface disinfection. Incubator (purchased from Thermo Fisher) for cultivation.
  • the combination medium of the effective factors determined in Example 2 (containing basal medium BM, 3 ⁇ g/mL insulin, 10 ⁇ M Y-27632, 1:400 insulin-transferrin-selenium supplement, 2mM glutamine additive, 27ng /mL neuregulin-1, 1:500 bovine pituitary extract, 20ng/mL basic fibroblast growth factor, 5ng/mL R-spondin1, 5 ⁇ M compound 1, 20nM prostaglandin E2, 1:100B27) based on After culture and expansion, until the cells grow to more than 85%, add 500 ⁇ L of 0.05% trypsin (purchased from Gibco) to rinse for 1 minute, then add 500 ⁇ L of 0.05% trypsin to each well, and place at 37°C, 5% React in a CO 2 incubator for 2 to 10 minutes until the cells are completely digested, then the digestion is terminated.
  • basal medium BM 3 ⁇ g/mL insulin, 10 ⁇ M Y-27632, 1:
  • the medium of the following 11 formulations was prepared for experiments.
  • Insulin is not included in the above-mentioned components of the culture medium for primary intestinal cancer cells;
  • Formula 2 Y-27632 is not included in the above-mentioned components of the primary intestinal cancer cell culture medium;
  • Insulin-transferrin-selenium supplements are not included in the above-mentioned components of the culture medium for primary intestinal cancer cells;
  • Formula 4 the above-mentioned colon cancer primary cell culture medium components do not contain glutamine additives
  • Neuregulin-1 is not contained in the above-mentioned components of the primary intestinal cancer cell culture medium
  • Bovine pituitary gland extract is not included in the above-mentioned components of the culture medium for primary intestinal cancer cells;
  • R-spondin1 is not included in the above-mentioned components of the culture medium for primary intestinal cancer cells;
  • Formula 9 Compound 1 is not contained in the above-mentioned components of the primary intestinal cancer cell culture medium;
  • Prostaglandin E2 is not contained in the above-mentioned components of the culture medium for primary intestinal cancer cells;
  • B27 is not included in the above-mentioned components of the culture medium for primary intestinal cancer cells
  • Y-27632 When the medium of formula 2 was used, 1 mL of prepared Y-27632 was added to each well of the 48-well plate inoculated with primary cells, and the final concentrations of Y-27632 were 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M; and set up control wells (BC) using the medium of recipe 2.
  • the medium of formula 3 When using the medium of formula 3, add the prepared insulin-transferrin-selenium supplement 1mL per well to the 48-well plate inoculated with primary cells, and the final concentration of insulin-transferrin-selenium supplement 1:800(V/V), 1:400(V/V), 1:200(V/V), 1:100(V/V), 1:50(V/V); and use the formula 3 medium set control wells (BC).
  • the medium of formula 4 When using the medium of formula 4, add the prepared glutamine supplement 1mL per well to the 48-well plate inoculated with primary cells, and the final concentration of glutamine supplement is 0.5mM, 1mM, 2mM, 4mM respectively , 8 mM; and use the medium of formula 4 to set up control wells (BC).
  • bovine pituitary extract When the medium of formula 6 was used, 1 mL of prepared bovine pituitary extract was added to each well of the 48-well plate inoculated with primary cells, and the final concentration of bovine pituitary extract was 1:2000 (V/ V), 1:1000(V/V), 1:500(V/V), 1:250(V/V), 1:125(V/V); and use the medium of formula 6 to set up control wells ( BC).
  • the medium of formula 7 When using the medium of formula 7, add the prepared basic fibroblast growth factor 1mL per well to the 48-well plate inoculated with primary cells, and the final concentration of basic fibroblast growth factor is 1.25ng/mL , 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL; and use the medium of formula 7 to set up control wells (BC).
  • R-spondin1 When the medium of formula 8 is used, 1 mL of prepared R-spondin1 is added to each well of the 48-well plate inoculated with primary cells, and the final concentrations of R-spondin1 factors are 1.25 ng/mL and 2.5 ng/mL respectively. , 5ng/mL, 10ng/mL, 20ng/mL; and set up control wells (BC) using the medium of formula 8.
  • the ratio is the ratio of the number of cells obtained by using each medium for one generation of culture to the number of cells obtained by the corresponding control well for one generation of culture.
  • a ratio greater than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds has a better effect on promoting proliferation than the culture medium of the control well; a ratio less than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds promotes proliferation The effect was weaker than that of the culture medium in the control well.
  • insulin, Y-27632, insulin-transferrin-selenium supplement, glutamine supplement, neuregulin-1, bovine pituitary extract, basic fibroblast growth factor, R- Spondin1, compound 1, prostaglandin E2, and B27 have obvious proliferative effects on primary intestinal cancer cells.
  • the content of insulin is preferably 1 ⁇ g/mL-9 ⁇ g/mL, and the cell proliferation effect is the most obvious when the concentration is 9 ⁇ g/mL;
  • the content of Y-27632 is preferably 1.25 ⁇ M-20 ⁇ M, more preferably 5 ⁇ M-20 ⁇ M , the cell proliferation effect is the most obvious when the concentration is 10 ⁇ M;
  • the content of insulin-transferrin-selenium supplement is preferably 1:400(V/V) ⁇ 1:100(V/V), and the concentration is 1:200(V/V) V) the cell proliferation effect is the most obvious;
  • the content of glutamine additive is preferably 0.5mM ⁇ 2mM, and the cell proliferation effect is the most obvious when the concentration is 2mM;
  • the content of neuregulin-1 is preferably 2.5ng/ml ⁇ 40ng/ml,
  • the cell proliferation effect is the most obvious when the concentration is 10ng/mL;
  • the content of bovine pituitary gland extract is preferably 1:2
  • the most preferred concentration of each added factor in the above-mentioned medium is used as the intestinal cancer primary cell culture medium of the present invention used in the following examples, which contains: basal medium BM, 9 ⁇ g/mL insulin, 10 ⁇ M Y-27632, 1 : 200 Insulin-Transferrin-Selenium Supplement, 2mM Glutamine Supplement, 10ng/mL Neuregulin-1, 1:500 Bovine Pituitary Extract, 5ng/mL Basic Fibroblast Growth Factor, 2.5ng/mL R-spondin1, 2.5 ⁇ M compound 1, 40 nM prostaglandin E2, 1:50B27 (hereinafter referred to as "CA-2" medium).
  • basal medium BM 9 ⁇ g/mL insulin
  • 10 ⁇ M Y-27632 1 : 200 Insulin-Transferrin-Selenium Supplement
  • 2mM Glutamine Supplement 10ng/mL Neuregulin-1
  • Bovine Pituitary Extract 5ng/mL Basic Fibroblast
  • step (2) of embodiment 1 from endoscopic tissue sample (numbering is OE (E) 088, OE (E) 089, OE (E) 099, OE (E) 100, OE (E) 101 , OE (E) 104) to obtain primary colon cancer cells and use CA-2 medium for culture.
  • the obtained primary intestinal cancer cells were seeded in a 6-well plate (250,000 cells per well) according to the living cell density of 2 ⁇ 10 4 cells/cm 2 , and 4 ml of CA-2 medium was added. After surface disinfection, they were cultured in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher).
  • FIGS. 3A-3F are photos taken under a 10x objective lens. The cells were closely arranged under the microscope, with slightly irregular shapes.
  • a cancer tissue with a size of about 0.25 cm3 was taken from the intraoperative tissue of a patient with colorectal cancer (sample number OE(O)015), soaked in 1 mL of 4% paraformaldehyde and fixed.
  • the sample OE(O)015 was continuously cultured to the fifth generation using the intestinal cancer primary cell culture medium CA-2 of the present invention.
  • the primary intestinal cancer cells fixed with 4% paraformaldehyde were embedded in paraffin and cut into 4 ⁇ m thick tissue sections with a microtome. Then routine immunohistochemical detection was performed (see Li et al., Nature Communication, (2016) 9:2983 for specific steps).
  • the primary antibodies used were ki-67, CK20, CDX-2, and villin (all purchased from CST).
  • Figures 4A-4D and 5A-5D are comparison diagrams of immunohistochemical results of primitive tissue cells and primary intestinal cancer cells cultured with the primary intestinal cancer medium CA-2 of the present invention, respectively.
  • Figure 4A and Figure 5A are pictures of the ki-67 antibody labeled with colon cancer tissue and cultured primary colon cancer cells, respectively, and
  • Figure 4B and Figure 5B are the marker CK20 of colon cancer tissue and cultured colon cancer primary cells, respectively.
  • Figure 4C and Figure 5C are the pictures of the labeled CDX-2 antibody of the colon cancer tissue and the cultured primary colon cancer cells respectively, and
  • Figure 4D and Figure 5D are the colon cancer tissue and the cultured colon cancer primary cells respectively Picture of cells labeled with villin antibody.
  • the expression of the biomarkers related to intestinal cancer on the primary intestinal cancer cells is the same as that of the original tissue from which the primary intestinal cancer cells originate.
  • the expression of markers in slices was basically the same. It shows that the intestinal cancer primary cells cultivated by the technology of the present invention maintain the original pathological characteristics of the cancer tissues of intestinal cancer patients.
  • Example 6 Colon cancer primary cell culture cycle and cell number statistics and Population Doubling (PD) value calculation
  • Intestinal cancer was obtained from 4 cases of intestinal cancer endoscopic samples (numbered OE(E)067, OE(E)071, OE(E)090, OE(E)099) according to the method of step (2)-3 of Example 1 Primary cells.
  • the obtained primary intestinal cancer cells were inoculated in T12.5 culture flasks (300,000 cells per flask) according to the viable cell density of 2.4 ⁇ 10 4 cells/cm 2 .
  • Fig. 6 shows the growth curves of 4 examples of primary cells that were drawn using the intestinal cancer primary cell culture medium CA-2 of the present invention, drawn by Graphpad Prism software, the abscissa represents the days of cell culture, and the ordinate is the cumulative cell proliferation Multiple, which indicates the multiple of cell expansion during the culture period. The larger the value, the more times the cells are expanded within a certain period, that is, the more cells are expanded. The slope represents the rate of cell expansion. . It can be confirmed from FIG. 6 that when the intestinal cancer primary cells cultured in the medium of the present invention are continuously cultured and expanded for at least 80 days, the cell expansion rate remains basically unchanged and still has the ability to continue to expand.
  • Embodiment 7 and the comparison of literature culture medium culture effect
  • Literature medium (Seungil Kim et al., Original Research, 2020, Vol.25(7)744–754), its formula is DMEM/F12+10% (v/v) fetal bovine serum+1% penicillin/streptomycin+ 1% (v/v) glutamine additive (purchased from thermo) + 1% (v/v) HEPES (purchased from Gibco) + N2 1:100 (v/v) (purchased from Gibco) + B27 1:50 (v/v) (purchased from Gibco)+1mM N-acetylcysteine (purchased from Taosu Biochemical)+50ng/mL epidermal growth factor (purchased from R&D)+100ng/mL Noggin (purchased from R&D)+ 10 mM nicotinamide (purchased from MCE) + 500 nM A8301 (purchased from MCE) + 10 ⁇ M SB202190 (purchased from MCE) + 0.01 ⁇ M prostaglandin E2 (purchase
  • Colon cancer primary cells were obtained from the endoscopic tissue sample (numbered OE(E)124) according to the method of step (2)-3 of Example 1.
  • the obtained primary intestinal cancer cells were inoculated in T12.5 culture flasks (300,000 cells per flask) according to the viable cell density of 2.4 ⁇ 10 4 cells/cm 2 .
  • Example 8 The intestinal cancer primary cells expanded using the culture medium of the present invention are used for drug screening and curative effect evaluation
  • Colon cancer primary cells (numbered OE(E)113) were isolated according to the method of step (2) of Example 1, as a first generation, and cultured using the intestinal cancer primary cell culture medium CA-2 of the present invention , when the cells expanded to 85%, subcultured.
  • step (2)-4 of Example 1 the cell subculture was counted, and the cells were placed in the sample tank (purchased from Corning Corporation) according to the viable cell density of 1 ⁇ 105 cells/mL and mixed thoroughly, and then placed in the 384-well The cells were cultured in an opaque white cell culture plate (purchased from Corning Corporation), with a volume of 50 ⁇ L per well, and the number of cells was 5000 per well.
  • the intestinal cancer primary cell culture medium of the present invention from the edge of the hole plate to seal the plate, and label the sample name, drug addition time and CellTiter-Glo (purchased from Promega Company) detection time on the plate.
  • the surface was sterilized with 75% alcohol (purchased from Lierkang), placed in a 37°C, 5% CO 2 incubator for cultivation, and the drug was added after 24 hours.
  • the cultured first generation, second generation, third generation, fourth generation and fifth generation cells were respectively obtained for drug screening, and the drug sensitivity of the continuous passage of intestinal cancer primary cells cultured with the medium of the present invention was tested.
  • the CellTiter-Glo luminescent reagent (purchased from Promega) was taken out from the refrigerator at 4°C, and 10 ml of the reagent was placed in the sampling tank. Take out the 384-well plate to be tested in the incubator, add 10 ⁇ L CellTiter-Glo luminescent reagent to each well, let it stand for 10 minutes, mix well, and use a multifunctional microplate reader (Envision of Perkin Elmer Company) to detect.
  • cell inhibition rate (%) 100%-the chemiluminescence value of the drug-dosing well/the chemiluminescence value of the control well ⁇ 100%, calculate the cell inhibition rate after different drugs act on the cells, and use the graphpad prism software to calculate the effect of the drug on the cells Half inhibition rate (IC 50 ).
  • IC 50 Half inhibition rate
  • the intestinal cancer primary cells cultivated by the primary intestinal cancer cell culture medium CA-2 of the present invention are used for drug screening, and the inhibitory effects of the same drug on the cultured cells of different generations are basically consistent (inhibition The curves are basically the same).
  • Cells from the same patient differ in their sensitivity to different drugs at their maximum blood concentration in the human body. According to the results, it can be judged that the efficacy of the drug is used clinically for intestinal cancer patients, and at the same time, it can be shown that the sensitivity of the tumor cells of different generations obtained according to the culture method of the present invention is stable to the drug.
  • the invention provides a primary cell culture medium and a culture method for culturing intestinal cancer primary cells in vitro, and the cultured cells can be applied to the curative effect evaluation and screening of drugs.
  • the present invention is suitable for industrial applications.

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Abstract

一种肠癌原代细胞的培养基及体外培养方法及其应用。该培养基包含MST1/2激酶抑制剂；胰岛素，选自Y27632、法舒地尔、和H-1152中的至少一种的Rho蛋白激酶抑制剂；胰岛素-转铁蛋白-硒补充剂；谷氨酰胺添加剂；神经调节蛋白1；牛脑垂体提取物；碱性成纤维生长因子；R-spondin1；前列腺素E2；和B27。与现有培养方式相比，使用所述培养基进行的体外培养具有更高的扩增效率；用该培养基进行肠癌原代的培养，能够维持原发组织的形态结构和病理特征，提高肠癌原代细胞培养的成功率和存活率。

Description

一种肠癌原代细胞的培养基及体外培养方法和用途 技术领域
本发明属于生物医药技术领域，具体涉及培养基及其应用，更具体涉及一种肠癌原代细胞的培养基及培养方法和用途。
背景技术
肠癌是由遗传和环境因素作用引起的疾病，是世界范围内最常见的消化道肿瘤之一。调查数据表明(郑树等，结直肠癌的预防，中华肿瘤杂志，2004，Vol.13，No.1，pp1～2)，肠癌为最常见的肿瘤之一，且发病率和死亡率整体呈上升趋势，是当前严重威胁生命健康的恶性肿瘤。手术结合术后化疗是肠癌目前的主要治疗方式，虽然近些年来外科技术的不断进步，肠癌患者的生存率得到了提高，但是肿瘤的转移和复发仍给病人带来不良预后。在结直肠癌的精准治疗方面，靶向药物的出现为晚期结直肠癌患者带来了希望，未来的发展方向是如何合理的选择靶向药物和制定个体化治疗方案。而药敏试验技术的不断革新为靶向药物、化疗药物以及靶向药物联合的疗效预测提供了有力的技术支持，为实现肠癌患者的个体化治疗奠定坚实的基础。
现有的体外培养的肠癌细胞系主要通过将正常细胞长期培养而自发永生化或者转染促使正常细胞永生化的癌基因获得。传统方法建立的细胞系依然是细胞、分子和癌生物学研究的主要支柱。但是，这些方法改变了细胞的遗传背景，长期培养的细胞系也容易造成基因组不稳定，可能导致肿瘤细胞系的表型和体内肿瘤细胞发生人为的改变。这些细胞系中通常缺乏原发肿瘤的复杂异质性，从而限制了这些细胞系对于预测肿瘤细胞反应的应用，影响肠癌的科学研究和药物研发的准确性。另外，从肠癌组织获得的细胞培养成癌细胞的过程中，常规培养方法较难得到癌细胞，培养过程中存在被成纤维细胞干扰，形成的克隆无法传代等问题，限制了人肠癌原代细胞的应用。
2017年，Xuefeng Liu等人使用辐照的小鼠成纤维细胞和Rho相关激酶抑制剂(Y-27632)来扩增上皮来源的细胞，该体系具有无需基因操作就能实现上皮来源细胞无限增长的能力(Xuefeng Liu等， Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens.Nat.Protoc.2017,12,439)。但是，Xuefeng Liu等人建立的方法的培养周期较长，不能实现细胞的快速扩增，原代细胞与基质细胞的共培养不可避免，无法解决与基质细胞共培养等培养方案所存在的一系列问题，限制了该技术的应用。
发明内容
为了解决上述技术问题，本发明提供一种肠癌原代细胞的培养基及体外培养方法和应用。
本发明的一个方面在于提供一种肠癌原代细胞的培养基，所述培养基包含MST1/2激酶抑制剂；胰岛素；选自Y27632、法舒地尔、和H-1152中的至少一种的Rho蛋白激酶抑制剂；胰岛素-转铁蛋白-硒补充剂；谷氨酰胺添加剂；神经调节蛋白1；牛脑垂体提取物；碱性成纤维生长因子；R-spondin1；前列腺素E2；和B27。
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 PCTCN2021126234-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 PCTCN2021126234-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 PCTCN2021126234-appb-000003
Figure PCTCN2021126234-appb-000004
Figure PCTCN2021126234-appb-000005
Figure PCTCN2021126234-appb-000006
Figure PCTCN2021126234-appb-000007
最优选地，本发明的MST1/2激酶抑制剂为化合物1。
在本发明的实施方式中，本发明的培养基中各成分的含量满足以下任意一项或多项或全部满足：
(1)MST1/2激酶抑制剂在培养基中的含量为1.25～5μM；
(2)胰岛素在培养基中的含量为1～9μg/mL；
(3)Rho蛋白激酶抑制剂优选为Y27632，其在培养基中的含量为1.25～20μM；
(4)胰岛素-转铁蛋白-硒补充剂相对于培养基的体积比为1:400～1:100；
(5)谷氨酰胺添加剂在培养基中的含量为0.5～2mM；
(6)神经调节蛋白-1在培养基中的含量为2.5～40ng/ml；
(7)牛脑垂体提取物相对于培养基的体积比为1:2000～1:125；
(8)碱性成纤维生长因子在培养基中的含量为2.5～20ng/ml；
(9)R-spondin1在培养基中的含量为1.25～10ng/mL；
(10)前列腺素E2在培养基中的含量为5～80nM；
(11)B27相对于培养基的体积比为1:200～1:12.5。
在本发明的实施方式中，所述培养基还含有选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基；和选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。
在优选的实施方式中，当抗生素选自链霉素/青霉素时，链霉素浓度范围为25～400μg/mL，青霉素浓度范围为25～400U/mL，当抗生素选自两性霉素B时，浓度范围为0.25～4μg/mL，当抗生素选自Primocin时，浓度范围为25～400μg/mL。
根据第二个方面，本发明还提供一种肠癌原代细胞的体外培养方法。在本发明的肠癌原代细胞的体外培养方法中，使用本发明的肠癌原代细胞培养基对肠癌原代细胞进行体外培养。
本发明的肠癌原代细胞的体外培养方法包括以下步骤：
1.肠癌原代细胞的分离
(1)分离肠癌组织样本，按1:3的体积比加入基础培养基和组织消化液(注：组织消化液的加入量是1g肿瘤组织使用约5-10mL组织消化液)，置于恒温摇床中进行消化，消化温度为4～37℃，消化转速为200rpm～350rpm；
(2)消化充分直至未见明显组织块即可终止消化，消化时间为3～6小时；
(3)离心后弃去上清液，离心转速为1200～1600rpm，离心时间为2～6分钟，加入本发明的肠癌原代细胞培养基重悬备用。
其中，基础培养基配方包括选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基；和选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。组织消化液配方包括1640培养基、胶原酶Ⅱ(1～2mg/mL)、胶原酶Ⅳ(1～2mg/mL)、DNA酶(50～100U/mL)、透明质酸酶(0.5～1mg/mL)、氯化钙(1～5mM)、牛血清白蛋白BSA(5～10mg/mL)。
2.使用本发明的肠癌原代细胞培养基进行培养
使用基础培养基按照50:1的体积比与基质胶(
Figure PCTCN2021126234-appb-000008
356231)冰上混合均匀，铺板后在37℃、5％CO 2培养箱孵育25～35分钟，弃上清。将上述步骤1中获得的肠癌原代细胞用本发明的肠癌原代细胞培养基重悬并计数，调整细胞密度为2～4×10 4个细胞/mL，加入本发明的肠癌原代细胞培养基，置培养箱中培养。
在又一方面，本发明还提供一种评估或筛选用于治疗肠癌疾病的药物的方法，其包括以下步骤：
(1)使用本发明的肠癌原代细胞的培养方法培养肠癌原代细胞；
(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)中培养得到的肠癌原代细胞添加各浓度梯度的所述药物；
(4)进行细胞活性测试。
本发明的技术方案能够取得以下技术效果：
(1)提高肠癌原代细胞培养的成功率，能够培养来源于结肠、直肠、十二指肠癌等多来样本源的肿瘤组织，成功率达到80％以上；
(2)体外培养的肠癌原代细胞能够保持病人的病理特性；
(3)所培养的肠癌原代细胞不受成纤维细胞、脂肪细胞等间质细胞的干扰；
(4)扩增效率高，可在一周左右时间内成功培养出肠癌原代细胞，扩增出的肠癌原代细胞还可以连续传代；
(5)培养成本可控，培养基无需加入价格昂贵的Wnt激动剂等因子；
(6)培养基不含血清，并且可避免原代细胞与基质细胞的共培养，也解决了传统的含血清、与基质细胞共培养等培养方案所存在的一系列问题；
(7)所述技术培养获得的肠癌原代细胞数量大，均一化程度高，适合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功能测试。
附图说明
图1为表示肠癌原代细胞培养基中不同添加因子组合对肠癌原代细胞生长的影响的图。
图2A-2K为显示肠癌原代细胞培养基的添加因子的不同浓度对肠癌原代细胞生长影响的图。
图3A-3F为利用显微镜观察使用本发明的肠癌原代细胞培养基培养得到的肠癌原代细胞的照片。
图4A-4D为原始肠癌组织细胞的免疫组化结果。
图5A-5D为使用本发明的肠癌原代细胞培养基培养原始肠癌组织细胞至第五代得到的肠癌原代细胞的免疫组化结果。
图6显示使用本发明的肠癌原代细胞培养基对肠癌原代细胞进行体外培养的细胞生长曲线。
图7显示分别使用本发明的肠癌原代细胞培养基和现有技术的文献培养基对肠癌原代细胞进行培养的增殖效率比较结果
图8A-8F显示使用本发明的肠癌原代细胞培养基培养至不同代数的肠癌原代细胞用于药物筛选的结果。
具体实施方式
为更好地理解本发明，下面结合实施例及附图对本发明作进一步描述，以下实施例仅是对本发明进行说明而非对其加以限定。
[MST1/2激酶抑制剂的制备实施例]
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MST2结合并降低其活性的化合物。
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 PCTCN2021126234-appb-000009
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.本发明的其他MST1/2抑制剂化合物的制备
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 PCTCN2021126234-appb-000010
Figure PCTCN2021126234-appb-000011
Figure PCTCN2021126234-appb-000012
Figure PCTCN2021126234-appb-000013
Figure PCTCN2021126234-appb-000014
实施例1 肠癌原代细胞培养基中各添加因子对肠癌原代细胞生长的影响
(1)肠癌原代细胞培养基的配制
首先配制含有初始培养基的基础培养基。初始培养基可选自本领域常用的DMEM/F12、DMEM、F12或RPMI-1640。在本实施例中，基础培养基的配方为：DMEM/F12培养基(购自Corning公司)+100μg/mL Primocin(购自InvivoGen公司，0.2％(v/v)，市售产品浓度50mg/ml)。在基础培养基内分别加入不同种类的添加剂(参见表1)配制成含有不同添加成分的肠癌原代细胞培养基。
(2)肠癌原代细胞的分离和处理
1样品选择
肠癌实体瘤组织样品(术中/内镜)由专业医疗机构的专业医务人员从患者获取，患者均签署了知情同意书。术中样本0.25cm 3，内镜样本0.025cm 3；采用商品化组织保存液(生产厂家：Miltenyi Biotec)存储运输。
2材料准备
15mL无菌离心管、移液枪、10mL移液管、无菌枪头等表面消毒后放入超净工作台中紫外照射30分钟。提前30分钟从4℃冰箱取出基础培养基，提前30分钟从-20℃冰箱取出组织消化液。
组织消化液：1640培养基(Corning，10-040-CVR)、胶原酶Ⅱ(2mg/mL)、胶原酶Ⅳ(2mg/mL)、DNA酶(50U/mL)、透明质酸酶(0.75mg/mL)、氯化钙(3.3mM)、BSA(10mg/mL)。
以上提及的胶原酶Ⅱ、胶原酶Ⅳ、DNA酶、和透明质酸酶均购自Sigma公司；氯化钙购自生工生物工程(上海)股份有限公司；BSA购自Biofroxx公司。
3.肠癌原代细胞的分离
3.1超净台中取组织样品于培养皿中，去除带血液的组织，用基础培养基冲洗2次，将组织转移至另一培养皿中用无菌手术刀进行机械分离，将组织块分割为1×1×1mm 3大小；
3.2将切割后的术中或内镜组织吸至15mL离心管中，加入5mL基础培养基，混匀，于1500rpm离心4分钟；
3.3弃上清，按1:3比例加入基础培养基和组织消化液(注：组织消化液的加入量是1g肿瘤组织使用约10mL组织消化液)，标记样品名称及编号，用封口膜密封，在37℃下于300rpm摇床(知楚仪器ZQLY-180N)中进行消化，期间每30分钟观察消化是否完成，判断依据为无肉眼可见的颗粒物，消化时间4小时；
3.4消化完成后，经100μm滤网过滤掉未消化的组织团块，滤网上的组织团块用基础培养基冲洗入离心管中以减少细胞损失，于25℃下1500rpm离心4分钟；
3.5弃上清，观察是否有血细胞，若有血细胞，加8mL血细胞裂解液(购自Sigma公司)，混匀，4℃裂解20分钟，期间颠倒混匀一次，25℃下1500rpm离心4分钟；
3.6弃上清，加入2mL基础培养基重悬细胞，备用。
4细胞计数及处理
4.1镜下观察：移取少量重悬细胞平铺于培养皿中，显微镜(CNOPTEC，BDS400)下观察癌细胞密度和形态；
4.2活细胞计数：取重悬的细胞悬液12μL，12μL台盼蓝染液(生工生物工程(上海)股份有限公司)充分混合后，取20μL加入细胞计数板(Countstar，规格：50片/盒)，细胞计数仪(Countstar，IC1000)下计算出活的大细胞(细胞粒径>10μm)百分率＝活细胞数/总细胞数×100％。
(3)肠癌原代细胞的培养
将按照上述步骤(2)从3例肠癌组织(编号为OE(O)001、OE(O)002、OE(E)003)分离得到的肠癌原代细胞用基础培养基重悬并计数。使用基础培养基按照50:1的比例与基质胶(
Figure PCTCN2021126234-appb-000015
356231)冰上混合均匀，取300μL铺板，37℃、5％CO 2培养箱孵育25～35分钟，优选30分钟，弃上清。将含有肠癌原代细胞的表1中不同成分的培养基按每孔4×10 4个细胞、500μL/孔体积加入48孔板内。其中，作为实验对照，使用未添加任何添加剂的基础培养基。培养7～10天后，细胞长至85％，弃培养基，使用每孔100μL 0.05％胰蛋白酶(购自Gibco公司)润洗1遍，吸去后再每孔加入200μL 0.05％胰蛋白酶。置于37℃、5％CO 2培养箱中反应10分钟，显微镜(CNOPTEC，BDS400)下观察细胞已完全消化，加入300μL含10％血清(Excell Bio，FND500)的DMEM/F12培养基终止消化，取20μL加入细胞计数板(Countstar，规格：50片/盒)，细胞计数仪(Countstar，IC1000)计出细胞总数。将实验结果示于表1。
表1 各添加因子及其促细胞增殖程度分级
Figure PCTCN2021126234-appb-000016
Figure PCTCN2021126234-appb-000017
其中，“+”表示与基础培养基相比，加入该添加剂的培养基对从肠癌组织分离出的肠癌原代细胞中的两例有促进增殖的作用；“－”表示添加该添加剂的培养基对从肠癌组织分离出的肠癌原代细胞中的至少一例显示有抑制增殖的作用；“○”表示添加该添加剂的培养基对从肠癌组织分离出的肠癌原代细胞中的至少两例的增殖没有明显的影响。
根据以上结果，拟选A8301、胰岛素、Y-27632、胰岛素-转铁蛋白-硒补充剂、谷氨酰胺添加剂、神经调节蛋白-1、牛脑垂体提取物、碱性成纤维生长因子、noggin、R-spondin1、化合物1、前列腺素E2、B27、Forsklin等因子进行进一步培养实验。
实施例2 肠癌原代细胞培养基中不同添加因子的组合对肠癌原代细胞增殖的影响
根据表2中的成分配制不同添加因子组合的肠癌原代细胞培养基，考察不同添加因子组合对肠癌原代细胞的促增殖作用。
表2 不同组分培养基的配制(浓度为终浓度)
Figure PCTCN2021126234-appb-000018
按照实施例1的步骤(2)之3的方法从肠癌组织(编号为OE(O)001、OE(O)002、OE(E)060、OE(E)071)获得肠癌原代细胞，将所获得的细胞悬液平均分成16份，1500rpm离心4分钟。离心后分别使用200μL BM和No.1～15号培养基重悬。使用基础培养基按照50:1的比例与基质胶(
Figure PCTCN2021126234-appb-000019
356231)冰上混合均匀，取300μL铺板48孔板，37℃、5％CO 2培养箱孵育25～35分钟，优选30分钟，弃上清。分别按照活细胞密度2×10 4个/cm 2接种于48孔板中(每孔2万细胞数)，分别使用对应的培养基补齐48孔板中各孔体积至1mL，充分混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。
48孔板中细胞长至85％以上，弃培养基，使用100μL 0.05％胰蛋白酶(购自Gibco公司)润洗1遍，吸去后再每孔加入200μL 0.05％胰蛋白酶。置于37℃、5％CO 2培养箱中反应10分钟，显微镜(CNOPTEC，BDS400)下观察细胞已完全消化，加入300μL含10％ 胎牛血清DMEM培养基终止消化，取20μL加入细胞计数板(Countstar，规格：50片/盒)，细胞计数仪(Countstar，IC1000)计出细胞总数。将由分离自术中、内镜组织的样本OE(O)001、OE(O)002、OE(E)060、OE(E)071的肠癌原代细胞得到的结果示于图1。
根据图1的结果可知，与基础培养基相比，在使用上述No.1～No.15培养基时，均能够不同程度地促进肠癌原代细胞的增殖。当省略添加因子A8301(No.2)、noggin(No.12)、Forsklin(No.15)时，培养基配方的促增殖作用更为明显。因此，在使用含有胰岛素、Y-27632、胰岛素-转铁蛋白-硒补充剂、谷氨酰胺添加剂、神经调节蛋白-1、牛脑垂体提取物、碱性成纤维生长因子、R-spondin1、化合物1、前列腺素E2、B27等添加成分的培养基培养肠癌原代细胞时，增殖效果最好。
实施例3 肠癌原代细胞培养基所含添加因子的不同浓度对肠癌原代细胞的增殖作用
按照实施例1的步骤(2)之3的方法从内镜组织样本(编号为OE(E)060、OE(E)078、OE(O)014、OE(O)015、OE(E)071)获得肠癌原代细胞。使用实施例2中的有效因子的组合培养基进行培养。使用基础培养基按照50:1的比例与基质胶(
Figure PCTCN2021126234-appb-000020
356231)冰上混合均匀，取4mL铺板T12.5培养瓶，37℃、5％CO 2培养箱孵育25～35分钟，优选为30分钟，弃上清。
所获得的肠癌原代细胞，按照活细胞密度2.4×10 4个/cm 2接种于T12.5孔板中(每孔30万细胞数)，表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。在实施例2中确定的有效因子的组合培养基(含有基础培养基BM、3μg/mL胰岛素、10μM Y-27632、1:400胰岛素-转铁蛋白-硒补充剂、2mM谷氨酰胺添加剂、27ng/mL神经调节蛋白-1、1:500牛脑垂体提取物、20ng/mL碱性成纤维生长因子、5ng/mL R-spondin1、5μM化合物1、20nM前列腺素E2、1:100B27)的基础之上培养扩增，至细胞长至85％以上，加入500μL 0.05％胰蛋白酶(购自Gibco公司)润洗1分钟，吸去后再每孔加入500μL 0.05％胰蛋白酶，置于37℃、5％CO 2培养箱中反应2～10分钟，直至细胞已经消化完全即终止消化。1500rpm离心4分钟后，弃上清。DMEM/F12 重悬细胞沉淀。取20μL加入细胞计数板(生产厂家：Countstar，规格：50片/盒)，细胞计数仪(Countstar，IC1000)计出细胞总数。所得细胞用于以下培养实验。
配制以下11种配方的培养基进行实验。
配方1：上述肠癌原代细胞培养基组分中不含胰岛素；
配方2：上述肠癌原代细胞培养基组分中不含Y-27632；
配方3：上述肠癌原代细胞培养基组分中不含胰岛素-转铁蛋白-硒补充剂；
配方4：上述肠癌原代细胞培养基组分中不含谷氨酰胺添加剂；
配方5：上述肠癌原代细胞培养基组分中不含神经调节蛋白-1；
配方6：上述肠癌原代细胞培养基组分中不含牛脑垂体提取物；
配方7：上述肠癌原代细胞培养基组分中不含碱性成纤维生长因子；
配方8：上述肠癌原代细胞培养基组分中不含R-spondin1；
配方9：上述肠癌原代细胞培养基组分中不含化合物1；
配方10：上述肠癌原代细胞培养基组分中不含前列腺素E2；
配方11：上述肠癌原代细胞培养基组分中不含B27；
每孔加入20μl含1x10 4个细胞的细胞重悬液，分别使用1mL上述配方1～11的培养基来稀释细胞悬液。
在使用配方1的培养基时，在接种有原代细胞的48孔板中分别添加配制好的胰岛素每孔1mL，胰岛素的终浓度分别为1μg/mL、3μg/mL、9μg/mL、27μg/mL、81μg/mL；并使用配方1的培养基设置对照孔(BC)。
在使用配方2的培养基时，在接种有原代细胞的48孔板中分别添加配制好的Y-27632每孔1mL，Y-27632的终浓度分别为1.25μM、2.5μM、5μM、10μM、20μM；并使用配方2的培养基设置对照孔(BC)。
在使用配方3的培养基时，在接种有原代细胞的48孔板中分别添加配制好的胰岛素-转铁蛋白-硒补充剂每孔1mL，胰岛素-转铁蛋白-硒补充剂的终浓度分别为1:800(V/V)、1:400(V/V)、1:200(V/V)、1:100(V/V)、1:50(V/V)；并使用配方3的培养基设置对照孔(BC)。
在使用配方4的培养基时，在接种有原代细胞的48孔板中分别添 加配制好的谷氨酰胺添加剂每孔1mL，谷氨酰胺添加剂的终浓度分别为0.5mM、1mM、2mM、4mM、8mM；并使用配方4的培养基设置对照孔(BC)。
在使用配方5的培养基时，在接种有原代细胞的48孔板中分别添加配制好的神经调节蛋白-1每孔1mL，神经调节蛋白-1的终浓度分别为2.5ng/mL、5ng/mL、10ng/mL、20ng/mL、40ng/mL；并使用配方5的培养基设置对照孔(BC)。
在使用配方6的培养基时，在接种有原代细胞的48孔板中分别添加配制好的牛脑垂体提取物每孔1mL，牛脑垂体提取物的终浓度分别为1:2000(V/V)、1:1000(V/V)、1:500(V/V)、1:250(V/V)、1:125(V/V)；并使用配方6的培养基设置对照孔(BC)。
在使用配方7的培养基时，在接种有原代细胞的48孔板中分别添加配制好的碱性成纤维生长因子每孔1mL，碱性成纤维生长因子的终浓度分别为1.25ng/mL、2.5ng/mL、5ng/mL、10ng/mL、20ng/mL；并使用配方7的培养基设置对照孔(BC)。
在使用配方8的培养基时，在接种有原代细胞的48孔板中分别添加配制好的R-spondin1每孔1mL，R-spondin1因子的终浓度分别为1.25ng/mL、2.5ng/mL、5ng/mL、10ng/mL、20ng/mL；并使用配方8的培养基设置对照孔(BC)。
在使用配方9的培养基时，在接种有原代细胞的48孔板中分别添加配制好的化合物1每孔1mL，化合物1的终浓度分别为1.25μM、2.5μM、5μM、10μM、20μM；并使用配方9的培养基设置对照孔(BC)。
在使用配方10的培养基时，在接种有原代细胞的48孔板中分别添加配制好的前列腺素E2每孔1mL，前列腺素E2的终浓度分别为5nM、10nM、20nM、40nM、80nM；并使用配方10的培养基设置对照孔(BC)。
在使用配方11的培养基时，在接种有原代细胞的48孔板中分别添加配制好的B27每孔1mL，B27的终浓度分别为1:200(V/V)、1:100(V/V)、1:50(V/V)、1:25(V/V)、1:12.5(V/V)；并使用配方11的培养基设置对照孔(BC)。
待细胞扩增至48孔的85％左右消化计数，分别参比对照孔(BC) 细胞数计算增殖倍数，将结果分别示于图2A～2K。图2A～2K中，比值为使用各培养基培养一代得到的细胞数与对应的对照孔培养一代得到的细胞数的比。比值大于1说明配制的含不同浓度的因子或小分子化合物的培养基促增殖效果优于对照孔培养基；比值小于1，则说明配制的含不同浓度的因子或小分子化合物的培养基促增殖效果较对照孔培养基促增殖效果弱。
根据图2A～2K的结果，胰岛素、Y-27632、胰岛素-转铁蛋白-硒补充剂、谷氨酰胺添加剂、神经调节蛋白-1、牛脑垂体提取物、碱性成纤维生长因子、R-spondin1、化合物1、前列腺素E2、B27对肠癌原代细胞有明显的促增殖作用。根据本实施例的结果，胰岛素的含量优选为1μg/mL～9μg/mL，浓度为9μg/mL时细胞增殖效果最明显；Y-27632的含量优选为1.25μM～20μM，更优选为5μM～20μM，浓度为10μM时细胞增殖效果最明显；胰岛素-转铁蛋白-硒补充剂的含量优选为1:400(V/V)～1:100(V/V)，浓度为1:200(V/V)时细胞增殖效果最明显；谷氨酰胺添加剂的含量优选为0.5mM～2mM，浓度为2mM时细胞增殖效果最明显；神经调节蛋白-1的含量优选为2.5ng/ml～40ng/ml，浓度为10ng/mL时细胞增殖效果最明显；牛脑垂体提取物含量优选为1:2000(V/V)～1:125(V/V)，更优选为1:1000(V/V)～1:250(V/V)，浓度为1:500(V/V)时细胞增殖效果最明显；碱性成纤维生长因子的含量优选为2.5ng/ml～20ng/ml，浓度为5ng/mL时细胞增殖效果最明显；R-spondin1的含量优选为1.25ng/ml～10ng/ml，更优选为1.25ng/ml～5ng/ml，浓度为2.5ng/mL时细胞增殖效果最明显；化合物1的含量优选为1.25μM～5μM，浓度为2.5μM时细胞增殖效果最明显；前列腺素E2的含量优选为5nM～80nM，更优选为20nM～80nM，浓度为40nM时细胞增殖效果最明显；B27含量优选为1:200(V/V)～1:12.5(V/V)，更优选为1:50(V/V)～1:25(V/V)，浓度为1:50(V/V)时细胞增殖效果最明显。
采用上述培养基中各添加因子的最优选浓度，作为下面实施例中使用的本发明的肠癌原代细胞培养基，其含有：基础培养基BM、9μg/mL胰岛素、10μM Y-27632、1:200胰岛素-转铁蛋白-硒补充剂、2mM谷氨酰胺添加剂、10ng/mL神经调节蛋白-1、1:500牛脑垂体提取物、 5ng/mL碱性成纤维生长因子、2.5ng/mL R-spondin1、2.5μM化合物1、40nM前列腺素E2、1:50B27(以下称为“CA-2”培养基)。
实施例4 肠癌原代细胞的培养
按照实施例1的步骤(2)之3的方法从内镜组织样本(编号为OE(E)088、OE(E)089、OE(E)099、OE(E)100、OE(E)101、OE(E)104)获得肠癌原代细胞，并使用CA-2培养基进行培养。使用基础培养基按照50:1的比例与基质胶(
Figure PCTCN2021126234-appb-000021
356231)冰上混合均匀，取2mL铺板6孔培养板，37℃、5％CO 2培养箱孵育25～35分钟，优选为30分钟，弃上清。所获得的肠癌原代细胞，按照活细胞密度2×10 4个/cm 2接种于6孔板中(每孔25万细胞数)，加入4ml CA-2培养基。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。
使用显微镜(Invitrogen公司EVOS M500)观察培养得到的肠癌原代细胞，图3A～3F是在10倍物镜下拍摄得到的照片，细胞在镜下呈紧密排列，形态略不规则。
实施例5 肠癌原代细胞培养的组化鉴定
从一例结直肠癌患者的术中组织(样本编号OE(O)015)取出约0.25cm 3大小的癌组织，浸泡在1mL 4％多聚甲醛中固定。使用实施例4的方法采用本发明的肠癌原代细胞培养基CA-2将样本OE(O)015持续培养至第5代。4％多聚甲醛固定后的肠癌原代细胞，经石蜡包埋，用切片机切成4μm厚的组织切片。随后进行常规的免疫组织化学检测(具体步骤参见Li等，Nature Communication，(2018)9:2983)。所使用的一抗为ki-67、CK20、CDX-2、villin(均购自CST)。
图4A-4D和5A-5D分别是原始组织细胞和采用该细胞以本发明的肠癌原代培养基CA-2培养而获得的肠癌原代细胞的免疫组化结果对比图。图4A和图5A分别是肠癌组织和培养后的肠癌原代细胞的标记ki-67抗体的图片，图4B和图5B分别是肠癌组织和培养后的肠癌原代细胞的标记CK20抗体的图片，图4C和图5C分别是肠癌组织和培养后的肠癌原代细胞的标记CDX-2抗体的图片，图4D和图5D分别是肠癌组织和培养后的肠癌原代细胞的标记villin抗体的图片。由此可以 确认，采用本发明技术培养的肠癌原代细胞培养至第5代时，肠癌原代细胞上与肠癌相关的生物标记物的表达情况与肠癌原代细胞来源的原始组织切片的标记物表达情况基本一致。说明采用本发明技术所培养的肠癌原代细胞保持了肠癌病人癌组织的原始病理特性。
实施例6 肠癌原代细胞培养周期和细胞数统计及Population Doubling(PD)值计算
按照实施例1步骤(2)之3的方法从4例肠癌内镜样本(编号为OE(E)067、OE(E)071、OE(E)090、OE(E)099)获得肠癌原代细胞。所获得的肠癌原代细胞，按照活细胞密度2.4×10 4个/cm 2接种于T12.5培养瓶(每瓶30万细胞数)。使用移液枪分别向每个T12.5培养瓶中轻轻地加入4mL于室温的本发明肠癌原代细胞培养基CA-2。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。待细胞培养5～9天后传代计数，同时记录直至传代时培养的天数，将该直至传代时培养的天数作为一个培养周期。在该实验条件下持续培养，将扩增所得的细胞进行不同代数扩增，每一代进行传代后计数并记录相应培养的周期，根据公式Population Doubling(PD)＝3.32×log10(消化后细胞总数/初始种入细胞数)计算PD，公式参见Chapman等，Stem Cell Research & Therapy 2014,5:60。
图6显示采用Graphpad Prism软件绘制的、使用本发明的肠癌原代细胞培养基CA-2培养的4例原代细胞的生长曲线，横坐标表示细胞培养的天数，纵坐标是累计的细胞增殖倍数，表示细胞在培养周期内扩增的倍数，数值越大表示细胞在一定周期内扩增的次数越多，即扩增得到的细胞数也就越多，斜率代表的是细胞扩增的速率。从图6中可以确认，本发明的培养基培养的肠癌原代细胞持续培养扩增至少80天时，细胞扩增速率基本保持不变，仍具有继续扩增的能力。
实施例7 与文献培养基培养效果的比较
(1)培养基的配制
文献培养基(Seungil Kim等，Original Research，2020,Vol.25(7)744–754)，其配方为DMEM/F12+10％(v/v)胎牛血清+1％青霉 素/链霉素+1％(v/v)谷氨酰胺添加剂(购自thermo)+1％(v/v)HEPES(购自Gibco)+N2 1:100(v/v)(购自Gibco)+B27 1:50(v/v)(购自Gibco)+1mM N-乙酰半胱氨酸(购自陶素生化)+50ng/mL表皮细胞长因子(购自R&D)+100ng/mL Noggin(购自R&D)+10mM烟酰胺(购自MCE)+500nM A8301(购自MCE)+10μM SB202190(购自MCE)+0.01μM前列腺素E2(购自Tocris)(以下简称为“文献”培养基)。
(2)肠癌原代细胞获取与培养
按照实施例1的步骤(2)之3的方法从内镜组织样本(编号为OE(E)124)获得肠癌原代细胞。使用基础培养基按照50:1的比例与基质胶(
Figure PCTCN2021126234-appb-000022
356231)冰上混合均匀，取4mL铺板T12.5培养瓶，37℃、5％CO 2培养箱孵育25～35分钟，优选为30分钟，弃上清。所获得的肠癌原代细胞，按照活细胞密度2.4×10 4个/cm 2接种于T12.5培养瓶(每瓶30万细胞数)。使用移液枪分别向每个T12.5培养瓶中轻轻地加入4mL于室温的本发明肠癌原代细胞培养基CA-2、或文献培养基。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。培养7天后，细胞计数，图7显示分别使用2种培养基培养的原代细胞的增殖效率。
根据图7的结果可知，与文献培养基相比，本发明的肠癌原代细胞培养基体外培养肠癌细胞的增殖效率明显更优。
实施例8 使用本发明的培养基扩增得到的肠癌原代细胞用于药物筛选和疗效评估
1、细胞培养和铺板
按照实施例1的步骤(2)之3的方法分离得到肠癌原代细胞(编号为OE(E)113)，作为一代，并使用本发明的肠癌原代细胞培养基CA-2进行培养，待细胞扩增至85％，进行传代。按照实施例1中步骤(2)之4将细胞传代计数，将细胞按照活细胞密度1×10 5个/mL细胞置于加样槽(购自康宁公司)中充分混匀后，在384孔不透明白色细胞培养板(购自康宁公司)中进行培养，每孔体积50μL，细胞数目为5000个/孔。从孔板边缘加入本发明的肠癌原代细胞培养基封板，板上标注 样品名称、加药时间及CellTiter-Glo(购自Promega公司)检测时间。表面用75％酒精(购自利尔康)消毒，置于37℃、5％CO 2培养箱培养，24小时后加药。分别获取培养第1代、第2代、第3代、第4代、第5代细胞进行药物筛选，测试使用本发明的培养基培养的肠癌原代细胞连续传代的药物敏感性。
2、筛选药物配制
按照下表配制6个浓度梯度的6种药物(阿糖胞苷、多柔比星、硼替佐米、帕比司他、阿扎胞苷、高三尖杉酯碱；均购自MCE公司)，在384孔药板(购自赛默飞公司)每孔中添加30μL，保存待用。
表3 药物作用浓度设置
Figure PCTCN2021126234-appb-000023
3、高通量加药
取出配制好的药板，置于室温，于离心机(贝克曼)中室温1000rpm离心1分钟后取出。采用高通量自动化加样系统(Perkin Elmer公司JANUS)进行高通量加药。对培养有人肠癌原代细胞的384孔板在每孔加入0.1μL对应浓度的筛选药物。加药结束后，384孔板表面消毒后移至培养箱中，72小时后测定细胞活性。
4、细胞活性测试
4℃冰箱取出CellTiter-Glo发光试剂(购自Promega公司)，取10毫升试剂置于加样槽中。培养箱中取出待检测384孔板，每孔加入10μL CellTiter-Glo发光试剂，静置10分钟后混匀，使用多功能酶标仪(Perkin Elmer公司Envision)检测。
5、数据处理
按照公式细胞抑制率(％)＝100％-加药孔化学发光数值/对照孔化学发光数值×100％，计算得到不同药物作用细胞后的细胞抑制率，使用graphpad prism软件计算药物对细胞作用的半数抑制率(IC 50)。 将结果示于图8A-8F。
由图8A-8F可以确认，使用本发明的肠癌原代细胞培养基CA-2培养得到的肠癌原代细胞进行药物筛选，相同药物对于培养的不同代数细胞的抑制效果基本保持一致(抑制曲线基本保持一致)。同一病人的细胞对不同药物在人体内最大血药浓度时的敏感性不同。根据结果可以判断肠癌患者在临床使用该种药物时的有效性，同时可以说明根据本发明培养方法得到不同代数的肿瘤细胞对药物的敏感性是稳定的。
工业应用性
本发明提供一种用于体外培养肠癌原代细胞的原代细胞培养基及培养方法，可将培养得到的细胞应用于药物的疗效评估和筛选。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 (9)

  1. 一种肠癌原代细胞的培养基，其特征在于，包含：
    培养基包含MST1/2激酶抑制剂；胰岛素；选自Y27632、法舒地尔、和H-1152中的至少一种的Rho蛋白激酶抑制剂；胰岛素-转铁蛋白-硒补充剂；谷氨酰胺添加剂；神经调节蛋白1；牛脑垂体提取物；碱性成纤维生长因子；R-spondin1；前列腺素E2；和B27，
    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 PCTCN2021126234-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。
  2. 如权利要求1所述的培养基，其中
    R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的苯基、萘基、苯甲基和噻吩基；
    R 2和R 3各自独立地选自C1-C3烷基；
    R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、哌啶基C1-C6烷基、和四氢吡喃基C1-C6烷基；
    R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。
  3. 如权利要求1所述的培养基，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 PCTCN2021126234-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。
  4. 如权利要求3所述的培养基，其中
    R 1为任选地被1-2个独立地R 6取代的苯基；
    R 5为氢；
    R 6优选为氟、甲基或三氟甲基。
  5. 如权利要求1所述的培养基，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 PCTCN2021126234-appb-100003
    Figure PCTCN2021126234-appb-100004
    Figure PCTCN2021126234-appb-100005
    Figure PCTCN2021126234-appb-100006
    Figure PCTCN2021126234-appb-100007
  6. 如权利要求1～5中任一项所述的培养基，其特征在于所述培养基中各成分的含量满足以下任意一项或多项或全部满足：
    (1)所述MST1/2激酶抑制剂在培养基中的含量为1.25～5μM；
    (2)所述胰岛素在培养基中的含量为1～9μg/mL；
    (3)所述Rho蛋白激酶抑制剂在培养基中的含量为1.25～20μM；
    (4)所述胰岛素-转铁蛋白-硒补充剂相对于培养基的体积比为1:400～1:100；
    (5)所述谷氨酰胺添加剂在培养基中的含量为0.5～2mM；
    (6)所述神经调节蛋白-1在培养基中的含量为2.5～40ng/ml；
    (7)所述牛脑垂体提取物相对于培养基的体积比为1:2000～1:125；
    (8)所述碱性成纤维生长因子在培养基中的含量为2.5～20ng/ml；
    (9)所述R-spondin1在培养基中的含量为1.25～10ng/mL；
    (10)所述前列腺素E2在培养基中的含量为5～80nM；
    (11)所述B27相对于培养基的体积比为1:200～1:12.5。
  7. 如权利要求1～6中任一项所述的培养基，其特征在于还包括：
    选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基；和选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。
  8. 一种肠癌原代细胞的培养方法，其特征在于，包括以下步骤：
    (1)配制如权利要求1～7中任一项所述的肠癌原代细胞的培养基；
    (2)从肠癌组织样本获取肠癌原代细胞；
    (3)在步骤(2)中获得的肠癌原代细胞中加入步骤(1)中获得的肠癌原代细胞的培养基进行培养。
  9. 一种评估或筛选用于治疗肠癌疾病的药物的方法，其包括以下步骤：
    (1)根据权利要求8所述的培养方法培养肠癌原代细胞；
    (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)中培养得到的肠癌原代细胞添加各浓度梯度的所述药物；
    (4)进行细胞活性测试。
PCT/CN2021/126234 2021-10-11 2021-10-26 一种肠癌原代细胞的培养基及体外培养方法和用途 WO2023060642A1 (zh)

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