WO2023060643A1 - 肠癌类器官的培养基及培养方法 - Google Patents

肠癌类器官的培养基及培养方法 Download PDF

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WO2023060643A1
WO2023060643A1 PCT/CN2021/126235 CN2021126235W WO2023060643A1 WO 2023060643 A1 WO2023060643 A1 WO 2023060643A1 CN 2021126235 W CN2021126235 W CN 2021126235W WO 2023060643 A1 WO2023060643 A1 WO 2023060643A1
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concentration
intestinal cancer
alkyl
culture medium
growth factor
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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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  • 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 for intestinal cancer organoids and a method for cultivating intestinal cancer organoids using the culture medium.
  • Colon cancer is one of the top ten malignant tumors with high incidence in my country, with an incidence rate of about 24/100,000, and it is also one of the tumors with the fastest-rising incidence rate in the past 20 to 30 years.
  • Bowel cancer includes colon cancer and rectal cancer. The incidence of bowel cancer from high to low is rectum, sigmoid colon, cecum, ascending colon, descending colon, and transverse colon. In recent years, there is a trend of developing to the proximal end (right colon). In the early stages of colon cancer, there are no symptoms, or the symptoms are not obvious, only discomfort, indigestion, and occult blood in the stool.
  • Organoids are organ-specific collections of cells derived from stem or precursor cells. Organoids cultured in vitro are highly similar to corresponding organs in terms of cellular components and tissue architecture, and have corresponding functional characteristics.
  • organoid culture is to cultivate a variety of cell groups contained in specific tissues and organs in a three-dimensional environment, and its culture system is more similar to the microenvironment in vivo. Therefore, it has shown great application prospects in basic research on the physiology and pathology of various organs, precision medicine, drug screening and development, gene therapy, and regenerative medicine.
  • the present invention provides a culture medium and in vitro culture method of intestinal cancer organoids.
  • One aspect of the present invention is to provide a culture medium for intestinal cancer organoids, said culture medium comprising MST1/2 kinase inhibitor; B27 supplement; N2 supplement; insulin; A8301; human fibroblast growth factor-10; Protein-1; Diregulin; Rho protein kinase inhibitor selected from at least one of Y27632, Fasudil, and H-1152; Fetal bovine serum; Basic fibroblast growth factor; Insulin-like growth factor-1 ; Forsklin; bovine pituitary gland extract; and fibroblast growth factor 7.
  • the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,
  • R 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by 1-2 independent R (such as phenyl and naphthyl, etc.), aryl C1-C6 alkyl (such as benzyl, etc.) and heteroaryl (such as thienyl, etc.);
  • R 2 and R 3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
  • R 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyran base, etc.);
  • R is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoro methyl).
  • halogen preferably fluorine and chlorine, more preferably fluorine
  • C1-C6 alkyl preferably methyl
  • C1-C6 alkoxy preferably methoxy
  • C1-C6 haloalkyl preferably trifluoro methyl
  • the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,
  • R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R6 , thienyl optionally substituted by 1-2 independently R6 , and optionally substituted by 1 -2 independently R6 substituted benzyl, R1 is more preferably optionally 1-2 independently R6 substituted phenyl;
  • R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R 5 is more preferably hydrogen;
  • R 6 is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R 6 is more preferably fluorine, methyl or trifluoromethyl.
  • the MST1/2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.
  • the MST1/2 kinase inhibitor of the present invention is Compound 1.
  • the content of each component in the culture medium of the present invention satisfies any one or more or all of the following:
  • the concentration range of the MST1/2 kinase inhibitor is usually 1.25-10 ⁇ M, preferably 2.5-10 ⁇ M;
  • the volume ratio range of the B27 additive to the medium is usually 1:12.5 to 1:200, more preferably 1:50 to 1:100;
  • the volume ratio range of N2 additive to the medium is usually 1:25 to 1:400, more preferably 1:100 to 1:400;
  • the concentration range of insulin is usually 0.25-1 ⁇ g/mL, more preferably 0.25-0.5 ⁇ g/mL;
  • the concentration range of A8301 is usually 1-9nM, more preferably 1-3nM;
  • the concentration range of human fibroblast growth factor-10 is usually 2.5-40 ng/mL, more preferably 5-20 ng/mL;
  • the concentration range of neuregulin-1 is usually 2.5-40 ng/mL, more preferably 2.5-10 ng/mL;
  • the concentration range of amphiregulin is usually 1-27 ng/mL, more preferably 1-3 ng/mL;
  • the concentration range of the Rho protein kinase inhibitor is usually 1.25-20 ⁇ M, more preferably 2.5-20 ⁇ M, and the Rho protein kinase inhibitor is preferably Y27632;
  • the volume concentration range of fetal bovine serum is usually 1.25% (v/v) to 20% (v/v), more preferably 1.25% (v/v) to 2.5% (v/v);
  • the concentration range of basic fibroblast growth factor is usually 2.5-40 ng/mL, more preferably 10-20 ng/mL;
  • the concentration range of insulin-like growth factor-1 is usually 12.5-100 ng/mL, more preferably 12.5-25 ng/mL;
  • the concentration range of Forsklin is usually 0.625-2.5 ⁇ M, more preferably 0.625-1.25 ⁇ M;
  • the volume ratio of bovine pituitary extract to medium is usually 1:2000 ⁇ 1:500;
  • the concentration range of fibroblast growth factor 7 is usually 0.625-2.5 ng/mL.
  • 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 intestinal cancer organoids in vitro.
  • primary intestinal cancer cells are cultured in vitro using the colon cancer organoid culture medium of the present invention.
  • the in vitro culture method of intestinal cancer organoids 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 Digestion is carried out in a shaking table, the digestion temperature range is 4-37°C, and the digestion speed range 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 cultured intestinal cancer organoids are not interfered by mesenchymal cells such as fibroblasts and adipocytes;
  • the amplification efficiency is high, and intestinal cancer organoids can be successfully cultured in about a week, and the amplified intestinal cancer organoids can also be continuously passaged;
  • the culture cost is controllable, and there is no need to add expensive Wnt agonists, R-spondin family proteins and other factors to the culture medium;
  • the colorectal cancer organoids obtained by the technique are large in quantity and high in homogeneity, and are suitable for high-throughput screening of new candidate compounds and providing patients with high-throughput in vitro sensitive function tests of drugs.
  • Figure 1 is a graph showing the effects of different combinations of factors added in intestinal cancer organoid culture medium on the growth of intestinal cancer organoids.
  • 2A-2O are graphs showing the effects of different concentrations of factors added to intestinal cancer organoid medium on the growth of intestinal cancer organoids.
  • 3A-3F are microscopic observations of intestinal cancer organoids cultured using the intestinal cancer organoid medium of the present invention.
  • 4A-4B are photographs of intestinal cancer organoids cultured to different passages using the intestinal cancer organoid medium of the present invention observed under a microscope.
  • Figures 5A-5D are the immunohistochemical results of primitive intestinal cancer tissue cells.
  • 6A-6D are the immunohistochemical results of intestinal cancer organoids obtained by culturing primitive intestinal cancer tissue cells to the sixth passage using the intestinal cancer organoid medium of the present invention.
  • Fig. 7 is micrographs of intestinal cancer organoids obtained by culturing intestinal cancer organoids using the intestinal cancer organoid medium of the present invention, literature medium, and commercial medium, respectively.
  • 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 may also be, for example, compounds that bind to MST1 or MST2 and reduce their activity.
  • 2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): In a round bottom flask was added 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) Methanol (30 mL) was then added, followed by the dropwise addition of thionyl chloride (1.2 mL) under ice-cooling. The reaction system was reacted overnight at 85°C. After the reaction, the system was evaporated to dryness under reduced pressure to obtain a white solid, which was directly used in the next step.
  • MST1/2 inhibitor compounds of the present invention were synthesized according to a method similar to compound 1, and their structures and mass spectrometry data are shown in the table below.
  • the initial medium can be selected from DMEM/F12, DMEM, F12 or RPMI-1640 commonly used in the art.
  • the formulation of the basal medium is: DMEM/F12 medium (purchased from Corning Company)+100 ⁇ g/mL Primocin (purchased from InvivoGen Company, 0.2% (v/v), commercially available product concentration 50mg/ml ).
  • 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 intestinal cancer organoid culture medium containing different additive components.
  • Colon cancer solid tumor tissue samples (intraoperative/endoscopic) were obtained from patients by professional medical staff of professional medical institutions, and all patients signed informed consent.
  • the intraoperative sample was 0.25cm 3
  • the endoscopic sample was 0.025cm 3 ; commercialized tissue preservation solution (manufacturer: Miltenyi Biotec) was 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.
  • the primary intestinal cancer cells isolated from 3 cases of intestinal cancer tissues (numbered OE(E)067, OE(E)071, OE(E)084) in basal medium according to the above step (2) , according to the volume ratio of 1:1 with Matrigel ( 356231) and mix evenly on ice, the final cell density is 5 ⁇ 10 5 cells/mL, take 7 ⁇ L Matrigel and cell suspension to form solidified droplets in the center of each well of a 96-well culture plate, and place the culture plate at 37°C Let stand for 30 minutes until the matrigel is completely set. The media with different components in Table 1 were added to the 96-well plate at a volume of 100 ⁇ L/well.
  • “+” means that compared with the basal medium, the medium added with this supplement has the effect of promoting the proliferation of at least two cases of primary intestinal cancer cells isolated from intestinal cancer tissue; “-” means that the addition of this The culture medium with added components showed an effect of inhibiting the proliferation of at least one of the primary intestinal cancer cells isolated from intestinal cancer tissues; The proliferation of primary cells was not significantly affected.
  • vitamin E vitamin E, SB202190, compound 1, B27, N2, insulin, insulin-transferrin-selenium supplement, A8301, human fibroblast growth factor-10, neuregulin-1, amphiregulin , Y-27632, fetal bovine serum, basic fibroblast growth factor, insulin-like growth factor-1, Forsklin, bovine pituitary gland extract, fibroblast growth factor 7 and other ingredients for further culture experiments.
  • Example 2 Effect of the combination of different added factors in intestinal cancer organoid culture medium on the growth of intestinal cancer organoids
  • intestinal cancer organoid culture media with different additive factor combinations were prepared, and the growth-promoting effects of different additive factor combinations on intestinal cancer organoids were investigated.
  • Colon cancer primary cells were obtained from colon cancer tissues (numbered OE(E)067, OE(E)071, OE(E)084, OE(E)099) according to the method of step (2)-3 of Example 1 , resuspended with basal medium and counted, according to the volume ratio of 1:1 with Matrigel ( 356231) and mix well on ice, the final cell density is 5 ⁇ 10 5 cells/mL, take 20 ⁇ L Matrigel and cell suspension to form solidified droplets in the center of each well of the 48-well culture plate, and place the culture plate at 37°C Let stand for 30 minutes until the Matrigel is completely set. Finally, 1 mL of BM medium and No.1-No.19 corresponding medium were added to the 48-well plate.
  • the relative cell viability (%) the chemiluminescence value of the experimental well/the chemiluminescence value of the control well ⁇ 100% is calculated to obtain the promoting effect of different components of the culture medium on the growth of intestinal cancer organoids.
  • the experimental results are shown in FIG. 1 .
  • the above-mentioned No.1 to No.19 mediums can all promote the proliferation of primary intestinal cancer cells to varying degrees.
  • the addition of vitamin E (No.2), SB202190 (No.3), and insulin-transferrin-selenium supplement (No.8) the growth-promoting effect of the medium formula was more obvious.
  • intestinal cancer primary cells from intestinal cancer tissue (numbering is OE (E) 071, OE (O) 015, OE (E) 113), regenerate with basal medium Suspended and counted, according to the volume ratio of 1:1 with Matrigel ( 356231) and mix well on ice, the final cell density is 5 ⁇ 10 5 cells/mL, take 7 ⁇ L Matrigel and cell suspension to form solidified droplets in the center of each well of the 96-well culture plate, and place the culture plate at 37°C Leave it for 30 minutes until the matrigel is completely solidified for the following culture experiments.
  • basal medium BM 10 ⁇ M compound 1, 1:50 (v/v) B27, 1:100 (v/v) N2, 1 ⁇ g/mL insulin, 9 nM A8301, 5 ng/mL human fibroblast growth factor -10, 5ng/mL neuregulin-1, 9ng/mL amphiregulin, 2.5 ⁇ M Y-27632, 1.25% (v/v) fetal bovine serum, 10ng/mL basic fibroblast growth factor, 50 ng/mL Intestinal cancer organoid medium with IGF-1, 2.5 ⁇ M Forsklin, 1:500 (v/v) bovine pituitary gland extract, and 1.25ng/mL fibroblast growth factor 7 and the following 15 formulations conduct experiment.
  • B27 is not included in the above intestinal cancer organoid medium components
  • N2 is not included in the above intestinal cancer organoid medium components
  • Insulin is not included in the above intestinal cancer organoid culture medium components
  • Formula 4 A8301 is not included in the above intestinal cancer organoid medium components
  • Neuregulin-1 is not included in the above intestinal cancer organoid medium components
  • Formula 7 Amphiregulin is not included in the above intestinal cancer organoid medium components
  • Formula 8 Y-27632 is not included in the above intestinal cancer organoid medium components
  • Formula 9 fetal bovine serum is not included in the above intestinal cancer organoid medium components
  • Insulin-like growth factor-1 is not included in the above intestinal cancer organoid culture medium components;
  • Formula 12 Forsklin is not included in the above intestinal cancer organoid medium components
  • Bovine pituitary extract is not included in the above intestinal cancer organoid culture medium components
  • Formula 14 the above intestinal cancer organoid medium components do not contain fibroblast growth factor 7;
  • Formula 15 Compound 1 was not included in the above intestinal cancer organoid culture medium components.
  • the medium of formula 6 When the medium of formula 6 is used, 100 ⁇ L of prepared neuregulin-1 is added to each well of the 96-well plate inoculated with primary cells, and the final concentration of neuregulin-1 is 2.5 ng/mL and 5 ng respectively. /mL, 10ng/mL, 20ng/mL, 40ng/mL; and use the medium of formula 6 to set up control wells (BC).
  • the medium of Formula 10 When using the medium of Formula 10, add the prepared basic fibroblast growth factor 100 ⁇ L per well to the 96-well plate inoculated with primary cells, and the final concentration of basic fibroblast growth factor is 2.5 ng/mL , 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL; and use the culture medium of formula 10 to set up control wells (BC).
  • the medium of formula 14 When using the medium of formula 14, add the prepared fibroblast growth factor 7 to each well of 96-well plates inoculated with primary cells at 100 ⁇ L, and the final concentration of fibroblast growth factor 7 is 0.625 ng/mL , 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL; and use the medium of formula 14 to set up control wells (BC).
  • the relative viability is the ratio of the viability of intestinal cancer organoids cultured in each medium for 7-10 days to the viability of intestinal cancer organoids in the corresponding control wells after 7-10 days 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.
  • the volume ratio range of B27 additive relative to the medium is preferably 1:12.5 ⁇ 1:200, more preferably 1:50 ⁇ 1:100, most preferably 1:50; N2 additive relative to The volume ratio range of the medium is preferably 1:25-1:400, more preferably 1:100-1:400, most preferably 1:200;
  • the concentration range of insulin is preferably 0.25-1 ⁇ g/mL, more preferably 0.25 ⁇ 0.5 ⁇ g/mL, most preferably 0.5 ⁇ g/mL;
  • the concentration range of A8301 is preferably 1 ⁇ 9 nM, more preferably 1 ⁇ 3 nM, most preferably 3 nM;
  • the concentration range of human fibroblast growth factor-10 is preferably 2.5 ⁇ 40ng/mL, more preferably 5 ⁇ 20ng/mL, most preferably 10ng/mL;
  • the concentration range of neuregulin-1 is preferably 2.5 ⁇ 40ng/mL, more preferably 2.5 ⁇ 10ng/mL, most preferably 5ng /mL
  • the most preferred concentration of each added factor in the above medium is used as the intestinal cancer organoid medium of the present invention used in the following examples, which contains: basal medium BM, 5 ⁇ M compound 1, 1:50 (v/v) B27, 1:200(v/v) N2, 0.5 ⁇ g/mL insulin, 3nM A8301, 10ng/mL human fibroblast growth factor-10, 5ng/mL neuregulin-1, 3ng/mL amphiregulin, 10 ⁇ M Y-27632, 2.5% (v/v) fetal bovine serum, 10ng/mL basic fibroblast growth factor, 12.5ng/mL insulin-like growth factor-1, 1.25 ⁇ M Forsklin, 1:1000(v/v) bovine brain pituitary extract, and 0.625ng/mL fibroblast growth factor 7.
  • basal medium BM 5 ⁇ M compound 1, 1:50 (v/v) B27, 1:200(v/v) N2, 0.5 ⁇ g/mL insulin, 3nM A
  • the method of 3 of the step (2) of embodiment 1 from endoscopic tissue sample (numbering is OE (E) 050, OE (E) 071, OE (E) 078, OE (E) 120, OE (O) 012 , OE (O) 015) to obtain primary intestinal cancer cells, and resuspended with basal medium and counted, according to the volume ratio of 1:1 with matrigel ( 356231) and mix well on ice, the final cell density is 5 ⁇ 10 5 cells/mL, take 50 ⁇ L Matrigel and cell suspension to form solidified droplets in the center of each well of the 24-well culture plate, and place the culture plate at 37°C Leave it for 30 minutes until the Matrigel is completely solidified, and gently add 1 mL of the intestinal cancer organoid culture medium of the present invention at room temperature to each well along the side wall of the well using a pipette gun.
  • FIGS. 3A-3F are photographs of the intestinal cancer organoids recorded under a 10x objective lens. Intestinal cancer organoids are spherical in shape under the microscope, with a smooth surface and intestinal structure.
  • step (2) of Example 1 Obtain intestinal cancer primary cells from endoscopic tissue samples OE(E)113 and OE(E)120 according to the method of step (2) of Example 1, resuspend and count with basal medium, according to the ratio of 1:1 Volume ratio to Matrigel ( 356231) and mix evenly on ice, the final cell density is 5 ⁇ 105 cells/mL, take 50 ⁇ L Matrigel and cell suspension to form solidified droplets in the center of each well of the 24-well culture plate, and place the culture plate at 37°C Let stand for 30 minutes until the matrigel is completely solidified. Add 1 mL of the intestinal cancer organoid culture medium of the present invention, culture for 8 days, and subculture.
  • 4A-4B are photographs of intestinal cancer organoids of different generations (P1 to P5) recorded under a 10x objective lens. The results showed that OE(E)113 and OE(E)120 intestinal cancer organoids were continuously passaged to the fifth generation, and their growth was stable, and they still proliferated stably.
  • a cancer tissue with a size of about 0.25 cm3 was removed from the intraoperative tissue of a patient with intestinal 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 sixth passage using the intestinal cancer organoid medium of the present invention.
  • the primary antibodies used were ki-67, CK20, CDX-2, and villin (all purchased from CST).
  • Figures 5A-5D and Figures 6A-6D are comparisons of the immunohistochemical results of primitive tissue cells and intestinal cancer organoids obtained by culturing the cells with the intestinal cancer organoid medium of the present invention to passage 6, respectively.
  • Figure 5A and Figure 6A are pictures of the original intestinal cancer tissue and cultured intestinal cancer organoids labeled with ki-67 antibody
  • Figure 5B and Figure 6B are pictures of the original intestinal cancer tissue and cultured intestinal cancer organoids labeled with CK20
  • the pictures of the antibody, Figure 5C and Figure 6C are the pictures of the labeled CDX-2 antibody of the original intestinal cancer tissue and the cultured intestinal cancer organoid, respectively
  • Figure 5D and Figure 6D are the original intestinal cancer tissue and the cultured intestinal cancer organoid, respectively Pictures of organs labeled with villin antibodies.
  • the intestinal cancer organoids (sample number OE(O)015) cultivated by the technology of the present invention are cultured to the sixth passage, the expression of intestinal cancer-related biomarkers on the organoids is consistent with that of the organoids.
  • the expression of markers in the original tissue sections was basically the same. This shows that the organoids cultivated by the technology of the present invention maintain the original pathological characteristics of the cancer tissues of patients with intestinal cancer.
  • Embodiment 6 and the comparison of literature culture medium, commercialization culture medium culture effect
  • Literature medium (Seungil Kim et al., Original Research, 2020, Vol.25(7) 744–754): 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) + 1:100 (v/v) N2 (purchased from Gibco) + 1:50 (v/v) ) B-27 (purchased from Gibco)+1mM N-acetylcysteine (purchased from Tao Su Biochemical)+50ng/mL epidermal growth factor (purchased from R&D)+100ng/mL Noggin (purchased from R&D)+10mM Nicotinamide (purchased from MCE) + 500nM A8301 (purchased from MCE) + 10 ⁇ M SB202190 (purchased from MCE) + 0.01 ⁇ M prostaglandin E2 (purchased from To
  • step (2) of Example 1 primary intestinal cancer cells were obtained from endoscopic tissue samples (numbered OE(E)122), resuspended with basal medium and counted, according to the volume ratio of 1:1 with Matrigel ( 356231) and mix evenly on ice, the final cell density is 5 ⁇ 10 5 cells/mL, take 50 ⁇ L Matrigel and cell suspension to form solidified droplets in the center of each well of the 24-well culture plate, and place the culture plate at 37°C Leave it for 30 minutes until Matrigel is completely solidified, then gently add 1 mL of the intestinal cancer organoid culture medium of the present invention, literature culture medium and commercial culture medium to each well at room temperature using a pipette gun along the side wall of the well. base.
  • the surface of the 24-well culture plate was sterilized and cultured in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher). After 5 days of culture, the cultured primary intestinal cancer cells were observed with a microscope (EVOS M500 from Invitrogen Company).
  • Figure 7 is the intestinal cancer organoids recorded under a 10x objective lens.
  • the intestinal cancer organoid medium of the present invention has a short culture period, a large number of organoids and a complete organoid structure when culturing intestinal cancer organoids in vitro. Its effect is obviously better than literature culture medium and commercial culture medium.
  • the invention provides a medium and a culture method for culturing intestinal cancer organoids, and the cultured organoids can be applied to the efficacy evaluation and screening of drugs.
  • the present invention is suitable for industrial applications.

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Abstract

一种肠癌类器官的培养基及培养方法。该培养基包含MST1/2激酶抑制剂；B27添加剂；N2添加剂；胰岛素；A8301；人成纤维细胞生长因子-10；神经调节蛋白-1；双调节蛋白；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；胎牛血清；碱性成纤维生长因子；胰岛素样生长因子-1；Forsklin；牛脑垂体提取物；和成纤维细胞生长因子7。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类器官的培养，能够维持原发组织的形态结构和病理特征，提高肠癌类器官培养的成功率和存活率。

Description

肠癌类器官的培养基及培养方法 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类器官的培养基及使用该培养基培养肠癌类器官的方法。
背景技术
肠癌是我国高发的十大恶性肿瘤之一，发病率约十万分之二十四，也是近二三十年来发病率上升最快的肿瘤之一。肠癌包括结肠癌和直肠癌，肠癌的发病率从高到低依次为直肠、乙状结肠、盲肠、升结肠、降结肠及横结肠，近年有向近端(右半结肠)发展的趋势。肠癌早期无症状，或症状不明显，仅感不适、消化不良、大便潜血等。随着癌肿发展，症状逐渐出现，表现为大便习惯改变、腹痛、便血、腹部包块、肠梗阻等，伴或不伴贫血、发热和消瘦等全身症状。近年来，欧美肠癌发病率和死亡率出现下降，而我国却出现增长趋势。目前，肠癌的治疗仍旧以手术为主，同时辅以放疗及化疗等以巩固治疗效果。虽然近年来业界在肠癌靶向治疗、免疫治疗等综合治疗方面取得了长足的进展，但总体预后并不乐观。
肠癌的病因尚未完全清楚，目前认为主要是环境因素与遗传因素综合作用的结果。肠癌的发生发展机制错综复杂，建立一套科学严谨的研究模型不但有利于肠癌的基础研究，而且有助于肠癌的诊断与治疗，有助于提高肠癌的生存率。类器官是衍生于干细胞或前体细胞的器官特异性细胞集合。体外培养的类器官在细胞成分和组织架构上与对应器官高度相似，并具备相应的功能学特征。与常规细跑培养在二维环境中培养单一细胞类群不同，类器官培养是在三维环境中培养出特定组织器官包含的多种细胞类群，其培养体系与体内微环境更为相似。因此，在各种器官生理病理的基础研究、精准医疗、药物筛选和开发、基因治疗、再生医学等方面，显示出巨大的应用前景。
虽然多种肿瘤组织使用不同的方法、不同的培养条件下可在体外成功培养为类器官，但是目前关于肠癌类器官的培养方法的研究及报 道较少，尤其是具体的试验流程、操作步骤、培养条件、即培养基配方尚无太多报道。
因此，本领域需要一种肠癌类器官体外培养成功率高、并能够维持原有组织病理特性的类器官培养基配方与培养方法。
发明内容
为了解决上述技术问题，本发明提供一种肠癌类器官的培养基及体外培养方法。
本发明的一个方面在于提供一种肠癌类器官的培养基，所述培养基包含MST1/2激酶抑制剂；B27添加剂；N2添加剂；胰岛素；A8301；人成纤维细胞生长因子-10；神经调节蛋白-1；双调节蛋白；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；胎牛血清；碱性成纤维生长因子；胰岛素样生长因子-1；Forsklin；牛脑垂体提取物；和成纤维细胞生长因子7。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 PCTCN2021126235-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 PCTCN2021126235-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 PCTCN2021126235-appb-000003
Figure PCTCN2021126235-appb-000004
Figure PCTCN2021126235-appb-000005
Figure PCTCN2021126235-appb-000006
Figure PCTCN2021126235-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激酶抑制剂的浓度范围通常为1.25～10μM，优选为2.5～10μM；
(2)B27添加剂相对于培养基的体积比范围通常为1:12.5～1:200，更优选为1:50～1:100；
(3)N2添加剂相对于培养基的体积比范围通常为1:25～1:400，更优选为1:100～1:400；
(4)胰岛素的浓度范围通常为0.25～1μg/mL，更优选0.25～0.5μg/mL；
(5)A8301的浓度范围通常为1～9nM，更优选为1～3nM；
(6)人成纤维细胞生长因子-10的浓度范围通常为2.5～40ng/mL，更优选为5～20ng/mL；
(7)神经调节蛋白-1的浓度范围通常为2.5～40ng/mL，更优选为2.5～10ng/mL；
(8)双调节蛋白的浓度范围通常为1～27ng/mL，更优选为1～3ng/mL；
(9)Rho蛋白激酶抑制剂的浓度范围通常为1.25～20μM，更优选为2.5～20μM，Rho蛋白激酶抑制剂优选为Y27632；
(10)胎牛血清的体积浓度范围通常为1.25％(v/v)～20％(v/v)，更优选为1.25％(v/v)～2.5％(v/v)；
(11)碱性成纤维生长因子的浓度范围通常为2.5～40ng/mL，更优选为10～20ng/mL；
(12)胰岛素样生长因子-1的浓度范围通常为12.5～100ng/mL，更优选为12.5～25ng/mL；
(13)Forsklin的浓度范围通常为0.625～2.5μM，更优选为0.625～1.25μM；
(14)牛脑垂体提取物相对于培养基的体积比通常为1:2000～1:500；
(15)成纤维细胞生长因子7的浓度范围通常为0.625～2.5ng/mL。
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类器官的体外培养方法。本发明的肠癌类器官的体外培养方法中，使用本发明的肠癌类器官培养基对肠癌原代细胞进行体外培养。
本发明的肠癌类器官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，消化温度范围为4～37℃，消化转速范围为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，离心转速范围为1200～1600rpm，离心时间为2～6分钟，加入本发明的肠癌类器官培养基重悬备用。
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.使用本发明的肠癌类器官培养基进行培养
将上述步骤1中获得的肠癌原代细胞用基础培养基重悬并计数，按照1:1的体积比与基质胶冰上混合均匀，细胞终密度为1～8×10 5个/mL，取基质胶与细胞悬液在培养板中形成凝固液滴，将培养板在4～37℃下静置10～60分钟以待基质胶完全凝固，加入本发明的肠癌类器官培养基，置培养箱中培养。
本发明的技术方案能够取得以下技术效果：
(1)提高肠癌类器官培养的成功率，能够培养来源于结肠、直肠等多来源样本的肿瘤组织，培养成功率达到80％以上；
(2)体外培养的肠癌类器官能够保持病人的病理特性；
(3)所培养的肠癌类器官不受成纤维细胞、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类器官，扩增出的肠癌类器官还可以连续传代；
(5)培养成本可控，培养基无需加入价格昂贵的Wnt激动剂、R-spondin家族蛋白等因子；
(6)所述技术培养获得的肠癌类器官数量大，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为表示肠癌类器官培养基中不同添加因子组合对肠癌类器官生长的影响的图。
图2A-2O为显示肠癌类器官培养基的添加因子的不同浓度对肠癌类器官生长影响的图。
图3A-3F为利用显微镜观察使用本发明的肠癌类器官培养基培养得到的肠癌类器官的照片。
图4A-4B为利用显微镜观察使用本发明的肠癌类器官培养基培养至不同代数的肠癌类器官的照片。
图5A-5D为原始肠癌组织细胞的免疫组化结果。
图6A-6D为使用本发明的肠癌类器官培养基培养原始肠癌组织细胞至第六代得到的肠癌类器官的免疫组化结果。
图7为分别使用本发明的肠癌类器官培养基、文献培养基、和商品化培养基对肠癌类器官进行培养得到的肠癌类器官的显微镜照片。
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 PCTCN2021126235-appb-000008
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 PCTCN2021126235-appb-000009
Figure PCTCN2021126235-appb-000010
Figure PCTCN2021126235-appb-000011
Figure PCTCN2021126235-appb-000012
Figure PCTCN2021126235-appb-000013
实施例1 肠癌类器官培养基中各添加因子对肠癌类器官生长的影响
(1)肠癌类器官培养基的配制
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类器官培养基。
(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(配方如下)。
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，消化时间约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)肠癌类器官的培养
将按照上述步骤(2)从3例肠癌组织(编号为OE(E)067、OE(E)071、OE(E)084)分离得到的肠癌原代细胞用基础培养基重悬并计数，按照1:1的体积比与基质胶(
Figure PCTCN2021126235-appb-000014
356231)冰上混合均匀，细胞终密度为5×10 5个/mL，取7μL基质胶与细胞悬液在96孔培养板的每个孔的中心形成凝固液滴，将培养板在37℃下静置30分钟以待基质胶完全凝固。将表1中不同成分的培养基按100μL/孔体积加入96孔板内。其中，作为实验对照，使用未添加任何添加成分的基础培养基。培养7～10天后，每孔加入50μL CellTiter-Glo(购自Promega公司)发光试剂，静置10分钟后混匀，使用多功能酶标仪(PerkinElmer公司Envision)检测。按照公式计算细胞相对活率(％)＝实验孔化学发光数值/对照孔化学发光数值×100％，推测不同添加成分对肠癌类器官生长的促进作用。将实验结果示于表1。
表1 培养基中的添加成分及促细胞增殖效果
Figure PCTCN2021126235-appb-000015
Figure PCTCN2021126235-appb-000016
其中，“+”表示与基础培养基相比，加入该添加成分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有促进增殖的作用；“－”表示添加该添加成分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；“○”表示添加该添加成分的培养基对从肠癌组织分离出的肠癌原代细胞的增殖无明显的影响。
根据以上结果，拟选择维生素E、SB202190、化合物1、B27、N2、胰岛素、胰岛素-转铁蛋白-硒补充剂、A8301、人成纤维细胞生长因子-10、神经调节蛋白-1、双调蛋白、Y-27632、胎牛血清、碱性成纤维生 长因子、胰岛素样生长因子-1、Forsklin、牛脑垂体提取物、成纤维细胞生长因子7等添加成分进行进一步培养实验。
实施例2 肠癌类器官培养基中不同添加因子的组合对肠癌类器官生长的影响
根据表2中的成分配制不同添加因子组合的肠癌类器官培养基，考察不同添加因子组合对肠癌类器官的促生长作用。
表2 不同组分培养基的配制(浓度为终浓度)
Figure PCTCN2021126235-appb-000017
按照实施例1的步骤(2)之3的方法从肠癌组织(编号为OE(E)067、OE(E)071、OE(E)084、OE(E)099)获得肠癌原代细胞，用基础培养基重悬并计数，按照1:1的体积比与基质胶(
Figure PCTCN2021126235-appb-000018
356231)冰上混合均匀，细胞终密度为5×10 5个/mL，取20μL基质胶与细胞悬液在48孔培养板的每个孔的中心形成凝固液滴，将培养板37℃下静置30分钟以待基质胶完全凝固。最后分别加入BM培养基和No.1～No.19号对应的培养基1mL至48孔板。培养7～10天后，每孔加入20μL CellTiter-Glo(购自Promega公司)发光试剂，静置10分钟后混匀，使用多功能酶标仪(PerkinElmer公司Envision)检测。按照公式细胞计算相对活率(％)＝实验孔化学发光数值/对照孔化学发光数值×100％，得出不同组分培养基对肠癌类器官生长促进作用。将实验结果示于图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.19培养基时，均能够不同程度地促进肠癌原代细胞的增殖。当省略添加因子维生素E(No.2)、SB202190(No.3)、胰岛素-转铁蛋白-硒补充剂(No.8)时，培养基配方的促增殖作用更为明显。因此，在后续实施例中，使用含B27、N2、胰岛素、A8301、化合物1、人成纤维细胞生长因子-10、神经调节蛋白-1、双调蛋白、Y-27632、胎牛血清、碱性成纤维生长因子、胰岛素样生长因子-1、Forsklin、牛脑垂体提取物、和成纤维细胞生长因子7等因子作为培养肠癌类器官的培养基配方进行进一步研究。
实施例3 肠癌类器官培养基所添加因子的不同浓度对肠癌类器官的增殖作用
按照实施例1的步骤(2)之3的方法从肠癌组织(编号为OE(E)071、OE(O)015、OE(E)113)获得肠癌原代细胞，用基础培养基重悬并计数，按照1:1的体积比与基质胶(
Figure PCTCN2021126235-appb-000019
356231)冰上混合均匀，细胞终密度为5×10 5个/mL，取7μL基质胶与细胞悬液在96孔培养板的每个孔的中心形成凝固液滴，将培养板37℃下静置30分钟以待基质胶完全凝固，用于以下培养实验。
接着，配制含有基础培养基BM、10μM化合物1、1:50(v/v)B27、1:100(v/v)N2、1μg/mL胰岛素、9nM A8301、5ng/mL人成纤维细胞生长因子-10、5ng/mL神经调节蛋白-1、9ng/mL双调蛋白、2.5μM Y-27632、1.25％(v/v)胎牛血清、10ng/mL碱性成纤维生长因子、50 ng/mL胰岛素样生长因子-1、2.5μM Forsklin、1:500(v/v)牛脑垂体提取物、和1.25ng/mL成纤维细胞生长因子7的肠癌类器官培养基和以下15种配方培养基进行实验。
配方1：上述肠癌类器官培养基组分中不含B27；
配方2：上述肠癌类器官培养基组分中不含N2；
配方3：上述肠癌类器官培养基组分中不含胰岛素；
配方4：上述肠癌类器官培养基组分中不含A8301；
配方5：上述肠癌类器官培养基组分中不含人成纤维细胞生长因子-10；
配方6：上述肠癌类器官培养基组分中不含神经调节蛋白-1；
配方7：上述肠癌类器官培养基组分中不含双调蛋白；
配方8：上述肠癌类器官培养基组分中不含Y-27632；
配方9：上述肠癌类器官培养基组分中不含胎牛血清；
配方10：上述肠癌类器官培养基组分中不含碱性成纤维生长因子；
配方11：上述肠癌类器官培养基组分中不含胰岛素样生长因子-1；
配方12：上述肠癌类器官培养基组分中不含Forsklin；
配方13：上述肠癌类器官培养基组分中不含牛脑垂体提取物；
配方14：上述肠癌类器官培养基组分中不含成纤维细胞生长因子7；
配方15：上述肠癌类器官培养基组分中不含化合物1。
在使用配方1的培养基时，在接种有原代细胞的96孔板中分别添加配制好的B27每孔100μL，B27相对于培养基的体积比分别为1:200、1:100、1:50、1:25、1:12.5；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96孔板中分别添加配制好的N2每孔100μL，N2相对于培养基的体积比分别为1:400、1:200、1:100、1:50、1:25；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96孔板中分别添加配制好的胰岛素每孔100μL，胰岛素的终浓度分别为0.25μg/mL、0.5μg/mL、1μg/mL、2μg/mL、4μg/mL；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96孔板中分别添加配制好的A8301每孔100μL，A8301的终浓度分别为1nM、3nM、9nM、27nM、81nM；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96孔板中分别添加配制好的人成纤维细胞生长因子-10每孔100μL，人成纤维细胞生长因子-10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96孔板中分别添加配制好的神经调节蛋白-1每孔100μL，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96孔板中分别添加配制好的双调蛋白每孔100μL，双调蛋白的终浓度分别为1ng/mL、3ng/mL、9ng/mL、27ng/mL、81ng/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96孔板中分别添加配制好的Y-27632每孔100μL，Y-27632的终浓度分别为1.25μM、2.5μM、5μM、10μM、20μM；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96孔板中分别添加配制好的胎牛血清每孔100μL，胎牛血清相对于培养基的体积比分别为1.25％、2.5％、5％、10％、20％；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96孔板中分别添加配制好的碱性成纤维生长因子每孔100μL，碱性成纤维生长因子的终浓度分别为2.5ng/mL、5ng/mL、10ng/mL、20ng/mL、40ng/mL；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96孔板中分别添加配制好的胰岛素样生长因子-1每孔100μL，胰岛素样生长因子-1的终浓度分别为12.5ng/mL、25ng/mL、50ng/mL、100ng/mL、200ng/mL；并使用配方11的培养基设置对照孔(BC)。
在使用配方12的培养基时，在接种有原代细胞的96孔板中分别 添加配制好的Forsklin每孔100μL，Forsklin的终浓度分别为0.625μM、1.25μM、2.5μM、5μM、10μM；并使用配方12的培养基设置对照孔(BC)。
在使用配方13的培养基时，在接种有原代细胞的96孔板中分别添加配制好的牛脑垂体提取物每孔100μL，牛脑垂体提取物相对于培养基的体积比分别为1:2000、1:1000、1:500、1:250、1:125；并使用配方13的培养基设置对照孔(BC)。
在使用配方14的培养基时，在接种有原代细胞的96孔板中分别添加配制好的成纤维细胞生长因子7每孔100μL，成纤维细胞生长因子7的终浓度分别为0.625ng/mL、1.25ng/mL、2.5ng/mL、5ng/mL、10ng/mL；并使用配方14的培养基设置对照孔(BC)。
在使用配方15的培养基时，在接种有原代细胞的96孔板中分别添加配制好的化合物1每孔100μL，化合物1的终浓度分别为1.25μM、2.5μM、5μM、10μM、20μM；并使用配方15的培养基设置对照孔(BC)。
待类器官培养7-10天后，分别参比对照孔(BC)细胞数计算相对活率，将结果分别示于图2A～2O。图2A～2O中，相对活率为使用各培养基培养类器官培养7～10天后的肠癌类器官活率，与对应的对照孔培养7～10天后的肠癌类器官活率的比值。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～2O的结果，B27添加剂相对于培养基的体积比范围优选为1:12.5～1:200，更优选为1:50～1:100，最优选为1:50；N2添加剂相对于培养基的体积比范围优选为1:25～1:400，更优选为1:100～1:400，最优选为1:200；胰岛素的浓度范围优选为0.25～1μg/mL，更优选为0.25～0.5μg/mL，最优选为0.5μg/mL；A8301的浓度范围优选为1～9nM，更优选为1～3nM，最优选为3nM；人成纤维细胞生长因子-10的浓度范围优选为2.5～40ng/mL，更优选为5～20ng/mL，最优选为10ng/mL；神经调节蛋白-1的浓度范围优选为2.5～40ng/mL，更优选为2.5～10ng/mL，最优选为5ng/mL；双调节蛋白的浓度范围 优选为1～27ng/mL，更优选为1～3ng/mL，最优选为3ng/mL；Y27632的浓度范围优选为1.25～20μM，更优选为2.5～20μM，最优选为10μM；胎牛血清相对于培养基的体积比范围优选为1.25％(v/v)～20％(v/v)，更优选为1.25％(v/v)～2.5％(v/v)，最优选为2.5％(v/v)；碱性成纤维生长因子的浓度范围优选为2.5～40ng/mL，更优选10～20ng/mL，最优选为10ng/mL；胰岛素样生长因子-1的浓度范围优选为12.5～100ng/mL，更优选12.5～25ng/mL，最优选为12.5ng/mL；Forsklin的浓度范围优选为0.625～2.5μM，更优选为0.625～1.25μM，最优选为1.25μM；牛脑垂体提取物相对于培养基的体积比优选为1:2000～1:500，最优选为1:1000；成纤维细胞生长因子7的浓度范围优选为0.625～2.5ng/mL，最优选为0.625ng/mL；化合物1的浓度范围优选为1.25～10μM，更优选为2.5～10μM，最优选为5μM。
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类器官培养基，其含有：基础培养基BM、5μM化合物1、1:50(v/v)B27、1:200(v/v)N2、0.5μg/mL胰岛素、3nM A8301、10ng/mL人成纤维细胞生长因子-10、5ng/mL神经调节蛋白-1、3ng/mL双调蛋白、10μM Y-27632、2.5％(v/v)胎牛血清、10ng/mL碱性成纤维生长因子、12.5ng/mL胰岛素样生长因子-1、1.25μM Forsklin、1:1000(v/v)牛脑垂体提取物、和0.625ng/mL成纤维细胞生长因子7。
实施例4 肠癌类器官培养及传代
(1)肠癌类器官培养
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)050、OE(E)071、OE(E)078、OE(E)120、OE(O)012、OE(O)015)获得肠癌原代细胞，并用基础培养基重悬并计数，按照1:1的体积比与基质胶(
Figure PCTCN2021126235-appb-000020
356231)冰上混合均匀，细胞终密度为5×10 5个/mL，取50μL基质胶与细胞悬液在24孔培养板的每个孔的中心形成凝固液滴，将培养板37℃下静置30分钟以待基质胶完全凝固，使用移液枪沿着孔侧壁向每个孔中轻轻地加入1mL于室温的本发明的肠癌类器官培养基。24孔培养板表面消毒后置于37℃、5％CO 2培养箱(购自赛 默飞)培养。培养3～8天后使用显微镜(Invitrogen公司，EVOS M500)观察培养得到的肠癌类器官，图3A-3F是10倍物镜下拍摄记录的肠癌类器官的照片。肠癌类器官在镜下呈规则球状，表面较光滑，呈肠道结构。
(2)肠癌类器官传代
按照实施例1的步骤(2)之3的方法从内镜组织样本OE(E)113、OE(E)120获取肠癌原代细胞，并用基础培养基重悬并计数，按照1:1的体积比与基质胶(
Figure PCTCN2021126235-appb-000021
356231)冰上混合均匀，细胞终密度为5×10 5个/mL，取50μL基质胶与细胞悬液在24孔培养板的每个孔的中心形成凝固液滴，将培养板在37℃下静置30分钟以待基质胶完全凝固。加入本发明肠癌类器官培养基1mL，培养8天后后传代。
从上述步骤中培养获得的第一代类器官中获取肠癌原代细胞，用基础培养基重悬并计数，按照1:1的体积比与基质胶(
Figure PCTCN2021126235-appb-000022
356231)冰上混合均匀，细胞终密度为5×10 5个/mL，取50μL基质胶与细胞悬液在24孔培养板的每个孔的中心形成凝固液滴，将培养板在37℃下静置30分钟以待基质胶完全凝固。加入本发明肠癌类器官培养基1mL，继续培养8天后获得下一代类器官。以下第三至第五代类器官按照相同的方法获得。
图4A-4B是10倍物镜下拍摄记录的不同代数(P1至P5)的肠癌类器官的照片。结果显示，OE(E)113、OE(E)120两例肠癌类器官连续传代至第五代长势稳定，仍稳定增殖。
实施例5 培养的肠癌类器官组化鉴定
从一例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类器官培养基将样本OE(O)015持续培养至第6代。4％多聚甲醛固定后的肠癌类器官，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5A-5D和图6A-6D分别是原始组织细胞和采用该细胞以本发明 的肠癌类器官培养基培养至第6代而获得的肠癌类器官的免疫组化结果对比图。图5A和图6A分别是肠癌原始组织和培养后的肠癌类器官的标记ki-67抗体的图片，图5B和图6B分别是肠癌原始组织和培养后的肠癌类器官的标记CK20抗体的图片，图5C和图6C分别是肠癌原始组织和培养后的肠癌类器官的标记CDX-2抗体的图片，图5D和图6D分别是肠癌原始组织和培养后的肠癌类器官的标记villin抗体的图片。由此可以确认，采用本发明技术培养的肠癌类器官(样本编号OE(O)015)培养至第6代时，类器官上与肠癌相关的生物标记物的表达情况与类器官来源的原始组织切片的标记物表达情况基本一致。这说明采用本发明技术所培养的类器官保持了肠癌病人癌组织的原始病理特性。
实施例6 与文献培养基、商品化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)：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)+1:100(v/v)N2(购自Gibco)+1:50(v/v)B-27(购自Gibco)+1mM N-乙酰半胱氨酸(购自陶素生化)+50ng/mL表皮细胞生长因子(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)。
商品化培养基：IntestiCult TM Organoid Growth Medium(Human)(购自STEMCELL，06010)。
(2)肠癌原代细胞获取与肠癌类器官培养
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)122)获得肠癌原代细胞，并用基础培养基重悬并计数，按照1:1的体积比与基质胶(
Figure PCTCN2021126235-appb-000023
356231)冰上混合均匀，细胞终密度为5×10 5个/mL，取50μL基质胶与细胞悬液在24孔培养板的每个孔的中心形成凝固液滴，将培养板37℃下静置30分钟以待基质胶完全凝固，使用移液枪沿着孔侧壁分别向每个孔中轻轻地加入1mL于室温的 本发明的肠癌类器官培养基、文献培养基和商品化培养基。24孔培养板表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。培养5天后使用显微镜(Invitrogen公司EVOS M500)观察培养得到的肠癌原代细胞，图7是10倍物镜下拍摄记录的肠癌类器官。
根据图7的结果可知，与文献培养基、商品化培养基相比，本发明的肠癌类器官培养基体外培养肠癌类器官时，培养周期短，类器官数量多且类器官结构完整，其效果明显优于文献培养基和商品化培养基。
工业应用性
本发明提供一种用于肠癌类器官培养的培养基及培养方法，可将培养得到的类器官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. 一种肠癌类器官的培养基，其特征在于，包含：
    包含MST1/2激酶抑制剂；B27添加剂；N2添加剂；胰岛素；A8301；人成纤维细胞生长因子-10；神经调节蛋白-1；双调节蛋白；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；胎牛血清；碱性成纤维生长因子；胰岛素样生长因子-1；Forsklin；牛脑垂体提取物；和成纤维细胞生长因子7，
    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 PCTCN2021126235-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 PCTCN2021126235-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 PCTCN2021126235-appb-100003
    Figure PCTCN2021126235-appb-100004
    Figure PCTCN2021126235-appb-100005
    Figure PCTCN2021126235-appb-100006
    Figure PCTCN2021126235-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激酶抑制剂的浓度为1.25～10μM；
    (2)所述B27添加剂相对于培养基的体积比为1:12.5～1:200；
    (3)所述N2添加剂相对于培养基的体积比为1:25～1:400；
    (4)所述胰岛素的浓度为0.25～1μg/mL；
    (5)所述A8301的浓度为1～9nM；
    (6)所述人成纤维细胞生长因子-10的浓度为2.5～40ng/mL；
    (7)所述神经调节蛋白-1的浓度为2.5～40ng/mL；
    (8)所述双调节蛋白的浓度为1～27ng/mL；
    (9)所述Rho蛋白激酶抑制剂的浓度为1.25～20μM；
    (10)所述胎牛血清的体积浓度为1.25％(v/v)～20％(v/v)；
    (11)所述碱性成纤维生长因子的浓度为2.5～40ng/mL；
    (12)所述胰岛素样生长因子-1的浓度为12.5～100ng/mL；
    (13)所述Forsklin的浓度为0.625～2.5μM；
    (14)所述牛脑垂体提取物相对于培养基的体积比为1:2000～1:500；
    (15)所述成纤维细胞生长因子7的浓度为0.625～2.5ng/mL。
  7. 如权利要求1～6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激酶抑制剂的浓度为2.5～10μM；
    (2)所述B27添加剂相对于培养基的体积比为1:50～1:100；
    (3)所述N2添加剂相对于培养基的体积比为1:100～1:400；
    (4)所述胰岛素的浓度为0.25～0.5μg/mL；
    (5)所述A8301的浓度为1～3nM；
    (6)所述人成纤维细胞生长因子-10的浓度为5～20ng/mL；
    (7)所述神经调节蛋白-1的浓度为2.5～10ng/mL；
    (8)所述双调节蛋白的浓度为1～3ng/mL；
    (9)所述Rho蛋白激酶抑制剂为Y27632，其浓度为2.5～20μM；
    (10)所述胎牛血清的体积浓度为1.25％(v/v)～2.5％(v/v)；
    (11)所述碱性成纤维生长因子的浓度为10～20ng/mL；
    (12)所述胰岛素样生长因子-1的浓度为12.5～25ng/mL；
    (13)所述Forsklin的浓度为0.625～1.25μM；
    (14)所述牛脑垂体提取物相对于培养基的体积比为1:2000～1:500；
    (15)所述成纤维细胞生长因子7的浓度为0.625～2.5ng/mL。
  8. 如权利要求1～7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。
  9. 一种肠癌类器官的培养方法，其特征在于，包括以下步骤：
    (1)配制如权利要求1～8中任一项所述的肠癌类器官的培养基；
    (2)从肠癌组织样本获取肠癌原代细胞，将获取的肠癌原代细胞的悬液与基质胶混合；
    (3)在步骤(2)中获得的肠癌原代细胞与基质胶的混合物中加入步骤(1)中获得的肠癌类器官的培养基进行培养。
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