WO2023060764A1 - 胃癌原代细胞的培养基和培养方法 - Google Patents

胃癌原代细胞的培养基和培养方法 Download PDF

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WO2023060764A1
WO2023060764A1 PCT/CN2021/140029 CN2021140029W WO2023060764A1 WO 2023060764 A1 WO2023060764 A1 WO 2023060764A1 CN 2021140029 W CN2021140029 W CN 2021140029W WO 2023060764 A1 WO2023060764 A1 WO 2023060764A1
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gastric cancer
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刘青松
汪文亮
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 field of biotechnology, and in particular relates to a culture medium for gastric cancer primary cells and a method for cultivating gastric cancer primary cells using the medium.
  • Gastric cancer is a malignant tumor originating from the gastric mucosal epithelium, and its incidence rate ranks first among various malignant tumors in my country. There are obvious regional differences in the incidence of gastric cancer. The incidence of gastric cancer in the northwest and eastern coastal areas of my country is significantly higher than that in the south. The age of onset is more than 50 years old, and the ratio of male to female incidence is 2:1. Due to changes in diet structure, increased work pressure, and Helicobacter pylori infection, gastric cancer tends to be younger. Gastric cancer can occur in any part of the stomach, and more than half of them occur in the gastric antrum. The greater curvature, the lesser curvature, and the anterior and posterior walls can be involved.
  • gastric cancer The vast majority of gastric cancer is adenocarcinoma, with no obvious symptoms in the early stage, or non-specific symptoms such as epigastric discomfort and belching, which are often similar to the symptoms of chronic gastric diseases such as gastritis and gastric ulcer, and are easily overlooked. Therefore, the early diagnosis rate of gastric cancer in my country is still low. At present, the diagnosis rate of early gastric cancer in China is still lower than 20%, and the 5-year survival rate of gastric cancer patients is only 27.4%.
  • Cell reprogramming technology is a technique in which the patient's own primary epithelial cells are co-cultured with mouse-derived feeder cells. The presence of these murine cells interferes with the results of drug susceptibility testing of patient primary cells; however, if the murine feeder cells are withdrawn, the patient's own primary cells detach In addition to the reprogramming environment, the cell proliferation rate and intracellular signaling pathways will be significantly changed (Liu et al., Am J Pathol, 183(6):1862-1870, 2013; Liu et al., Cell Death Dis., 9(7) : 750, 2018), so that the response of the patient's own primary cells to the drug is greatly affected.
  • Organoid technology is a technology in which the patient's own primary epithelial cells are embedded in the extracellular matrix for three-dimensional culture in vitro. This technology does not require feeder cells, so there is no interference from mouse-derived feeder cells. However, a variety of specific growth factors (such as Wnt proteins and R-spondin family proteins) need to be added to the medium of organoid technology, which is expensive and not suitable for widespread clinical application.
  • specific growth factors such as Wnt proteins and R-spondin family proteins
  • organoids In addition, during the whole culture process of organoids, cells need to be embedded in extracellular matrix gel, and the plating steps of cell seeding, passage and drug sensitivity testing are cumbersome and time-consuming compared with 2D culture operations, and the technology formed
  • the size of organoids is not easy to control, and some organoids tend to grow too large and cause internal necrosis. Therefore, compared with 2D culture technology, organoid technology is less operable and applicable, requires professional technicians to operate, and is not suitable for large-scale and extensive clinical in vitro drug sensitivity testing (Nick Barker, Nat Cell Biol, 18(3 ): 246-54, 2016).
  • the cultured gastric cancer tumor cells can represent the biological characteristics of gastric cancer patients themselves.
  • the present invention provides a culture medium and culture method for rapidly expanding gastric cancer primary cells in vitro and its application.
  • One aspect of the present invention is to provide a culture medium for gastric cancer primary cells, the culture medium comprising MST1/2 kinase inhibitor; ROCK kinase selected from at least one of Y27632, Fasudil, and H-1152 Inhibitor; at least one of B27 Supplement and N2 Supplement; Basic Fibroblast Growth Factor; CHIR99021; Epidermal Growth Factor; ITS Cell Culture Supplement; SB202190; Dexamethasone; Fibroblast Growth Factor 10; N- Acetyl-L-cysteine; and Gastrin.
  • the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,
  • R 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by 1-2 independent R (such as phenyl and naphthyl, etc.), aryl C1-C6 alkyl (such as benzyl, etc.) and heteroaryl (such as thienyl, etc.);
  • R 2 and R 3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
  • R 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyran base, etc.);
  • R is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoro methyl).
  • halogen preferably fluorine and chlorine, more preferably fluorine
  • C1-C6 alkyl preferably methyl
  • C1-C6 alkoxy preferably methoxy
  • C1-C6 haloalkyl preferably trifluoro methyl
  • the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,
  • R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R6 , thienyl optionally substituted by 1-2 independently R6 , and optionally substituted by 1 -2 independently R6 substituted benzyl, R1 is more preferably optionally 1-2 independently R6 substituted phenyl;
  • R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R 5 is more preferably hydrogen;
  • R 6 is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R 6 is more preferably fluorine, methyl or trifluoromethyl.
  • the MST1/2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.
  • the MST1/2 kinase inhibitor of the present invention is Compound 1.
  • the content of each component in the culture medium of the present invention satisfies any one or more or all of the following:
  • the concentration of the MST1/2 kinase inhibitor is 2.5-20 ⁇ M
  • the volume ratio of the B27 or N2 cell culture additive to the medium is 1:25 to 1:400;
  • the concentration of the basic fibroblast growth factor is 1-30 ng/mL
  • the volume ratio of the ITS cell culture additive to the medium is 1:25 to 1:400;
  • the concentration of the ROCK kinase inhibitor is 2.5-40 ⁇ M
  • the concentration of the dexamethasone is 25-400nM
  • the concentration of the epidermal growth factor is 2.5-20 ng/mL
  • the concentration of the fibroblast growth factor 10 is 50-800 ng/mL
  • the concentration of the SB202190 is 50-800nM
  • 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 were used as the basal medium.
  • the streptomycin concentration ranges from 25 to 400 ⁇ g/mL, and the penicillin concentration ranges from 25 to 400 U/mL; when the antibiotic is selected from amphotericin B, The concentration range is 0.25-4 ⁇ g/mL, and when the antibiotic is selected from Primocin, the concentration range is 25-400 ⁇ g/mL.
  • the invention also provides a method for culturing primary gastric cancer cells.
  • primary gastric cancer cells are cultured using the culture medium for primary gastric cancer cells of the present invention.
  • the primary gastric cancer cell culture method of the present invention comprises the following steps.
  • the extracellular matrix gel is a low growth factor extracellular matrix gel, for example, commercially available Matrigel (purchased from Corning) or BME (purchased from Trevigen) can be used. More specifically, the extracellular matrix gel is diluted with a serum-free medium, which can be DMEM/F12 (purchased from Corning Incorporated). The dilution ratio of the extracellular matrix gel is 1:50-1:400, preferably 1:100-1:200.
  • the coating method is to add the diluted extracellular matrix gel into the culture vessel so that it completely covers the bottom of the culture vessel, and let it stand for more than 30 minutes for coating, preferably at 37°C for standing for coating, and the preferred coating time is 30 minutes. ⁇ 60 minutes. After coating, the excess extracellular matrix gel dilution was discarded, and the culture vessel was set aside.
  • Primary gastric cancer cells can be derived, for example, from gastric cancer surgical samples and biopsy endoscopic samples.
  • Gastric cancer surgical samples are, for example, derived from surgically resected cancer tissue samples of patients with gastric cancer tumors who have explained and obtained consent, and endoscopic samples are collected from gastric lesions through endoscopic guidance. The above tissue samples were collected within half an hour of the patient's surgical resection or biopsy. Taking surgical samples as an example, in a sterile environment, cut tissue samples from non-necrotic parts with a volume of more than 5mm3 , place them in pre-cooled 10-15mL DMEM/F12 medium or commercial preservation solution, and store them in a sterile environment. Transport to the laboratory on ice in plastic sterile capped centrifuge tubes.
  • tissue sample In a biological safety cabinet, transfer the tissue sample to a cell culture dish, rinse the tissue sample with the basal medium as described above, and wash away the blood cells on the surface of the tissue sample. Transfer the rinsed tissue sample to another new Petri dish, add 1-3 mL of basal medium, and use a sterile scalpel blade and forceps to divide the tissue sample into tissue fragments with a volume less than 3 mm 3 .
  • tissue digestion solution is: mix 1 ⁇ 2mg/mL collagenase II, 1 ⁇ 2mg/mL collagenase IV, 50 ⁇ 100U/mL deoxyribonucleic acid, 0.5 ⁇ 1mg/mL hyaluronidase, 1 ⁇ 5mM chloride Calcium chloride, 5-10 mg/mL bovine serum albumin dissolved in 1640 medium), mark the sample number, seal it with parafilm, and digest it with a constant temperature shaker (ZQLY-180N) at 37°C and 200-300 rpm.
  • ZQLY-180N constant temperature shaker
  • blood cells If there are blood cells, add 3-8mL blood cell lysate (purchased from Sigma Company), mix well, lyse at 4°C for 10-20 minutes, shake and mix once every 5 minutes, and the lysis is complete. Then take it out and centrifuge at 1000-3000rpm for 3-5 minutes.
  • 3-8mL blood cell lysate purchased from Sigma Company
  • step (3) Inoculate the primary gastric cancer cells isolated in step (3) into the coated culture vessel, and culture them with the primary cell culture medium in step (1).
  • the cell density reaches about 80%-90% of the bottom area of the multi-well plate, it is digested and passaged.
  • This inoculation step does not require the use of feeder cells, and compared with the cell conditional reprogramming technology, the operation steps of culturing and irradiating feeder cells are eliminated. Compared with the organoid technique, this step does not need to mix the primary cells and Matrigel on ice to form gel droplets, and wait for the gel droplets to solidify before adding the culture medium.
  • the pre-coated culture vessel can be directly used for primary Cell seeding. In addition, only a small amount of diluted extracellular matrix gel is needed to coat the culture vessel, which saves the use of expensive extracellular matrix gel and simplifies the operation steps compared with organoid technology.
  • the inoculated primary gastric cancer cells are cultured for 8 to 16 days, when the cell clones formed in the culture container reach 80% of the bottom area, the supernatant is discarded, and 0.5 to 2 mL of 0.05% trypsin (purchased from Thermo Fisher Company) for cell digestion, and incubated at room temperature for 5-20 minutes; then use DMEM/F12 culture solution containing, for example, 5% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin for 1-20 minutes.
  • trypsin purchased from Thermo Fisher Company
  • the amplified primary gastric cancer cells grow in 2D, which avoids the uneven size of organoids and internal necrosis of overgrown organoids caused by the expansion of organoid technology.
  • the present invention also provides a method for evaluating or screening a drug for the treatment of gastric cancer, comprising the following steps:
  • the cultured primary gastric cancer epithelial cells are not interfered by fibroblasts, and purified gastric cancer epithelial cells can be obtained;
  • the culture medium does not contain serum, so it is not affected by the quality and quantity of different batches of serum;
  • the amplification efficiency is high, and primary gastric cancer cells can be rapidly cultured, and the amplified primary gastric cancer cells can also be continuously passaged;
  • the passage step does not require operation on ice and dissociation of Matrigel, and the digestion and passage of cells can be completed within 10-15 minutes;
  • the culture cost is controllable, and there is no need to add expensive Wnt agonists, R-spondin family proteins, Noggin proteins, BMP inhibitors and other factors to the medium;
  • Figures 1A-1L are graphs showing the effects of different concentrations of factors added to the primary gastric cancer cell culture medium of the present invention on the proliferation of primary gastric cancer cells.
  • 2A-2D are photographs of gastric cancer primary cells cultured using the gastric cancer primary cell culture medium of the present invention observed under a microscope.
  • 3A and 3B are diagrams showing pathological and immunohistochemical identification results of gastric cancer primary tissue samples and gastric cancer primary cells obtained by culturing the primary gastric cancer cell culture medium using the gastric cancer primary cell culture medium of the present invention, respectively.
  • Fig. 4 is a cell growth curve of gastric cancer primary cells obtained by culturing gastric cancer primary tissue samples using the gastric cancer primary cell culture medium of the present invention.
  • 5A and 5B are graphs showing comparison results of culture of gastric cancer primary cells using the gastric cancer primary cell culture medium of the present invention and two existing media, respectively.
  • Fig. 6 is a graph showing the results of drug susceptibility testing of gastric cancer cells of different passages cultured using the gastric cancer primary 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 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 ).
  • Gastric cancer solid tumor tissue samples (intraoperative) were obtained from patients by professional medical staff from professional medical institutions, and all patients signed informed consent. Intraoperative samples of 0.25 cm 3 were stored and transported in commercial tissue preservation solution (manufacturer: Miltenyi Biotec).
  • Tissue digestion solution formula 1640 medium (Corning, 10-040-CVR), collagenase II (2mg/mL), collagenase IV (2mg/mL), DNase (50U/mL), hyaluronidase (0.75 mg/mL), calcium chloride (3.3mM), bovine serum albumin BSA (10mg/mL).
  • Collagenase II, collagenase IV, DNase, and hyaluronidase mentioned above were all purchased from Sigma; calcium chloride was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; BSA was purchased from Biofroxx.
  • the extracellular matrix gel (manufactured by BD Biotechnology Co., Ltd.) was diluted with serum-free DMEM/F12 medium at a ratio of 1:100 to prepare an extracellular matrix dilution, and 500 ⁇ l/well of the extracellular matrix dilution was added to a 48-well culture plate to completely cover the culture Bottom of plate well. Place in a 37°C incubator for 1 hour. After 1 hour, the extracellular matrix dilution was removed to obtain a Matrigel-coated culture plate.
  • the primary gastric cancer cells obtained in the above steps were resuspended in pre-cooled DMEM/F12 and counted.
  • the medium with different components (Table 1) was added to the 48-well plate coated with extracellular matrix gel (Matrigel) at a volume of 500 ⁇ l/well.
  • the counted gastric cancer primary cells (No. GQ-001) were inoculated into Matrigel-coated 48-well culture plates at a cell density of 2 ⁇ 10 4 cells/cm 2 , and placed at 37°C and 5% CO after surface disinfection. 2 incubator (purchased from Thermo Fisher), the same number of freshly isolated gastric cancer tumor cells (number GQ-001) were cultured under different medium formulation conditions.
  • the medium was replaced every 4 days after the start of the culture. After 12 days of culture, the cells were counted, and the promotion effects of each factor on the proliferation of primary gastric cancer cells were compared. Among them, as an experimental control, a basal medium without any additives was used, and the experimental results are shown in Table 1.
  • “+” means that compared with the basal medium, the medium added with this additive has the effect of promoting the proliferation of at least two cases of gastric cancer primary cells isolated from gastric cancer tissue; “-” means that the culture medium added with this additive
  • the base has an inhibitory effect on at least one case of primary gastric cancer cells isolated from gastric cancer tissue; Proliferation was not significantly affected.
  • gastric cancer primary cells were obtained from intraoperative tissue samples (numbered GQ-002, GQ-003), and the primary cell culture was performed using the medium formula in Table 2 below.
  • Y27632 prepared on the basis of formula 2 is added to the 48-well plate inoculated with primary cells, and the final concentrations of Y27632 are 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M, and 20 ⁇ M, respectively. , 40 ⁇ M; and set up control wells (BC) using the medium of formula 2.
  • bFGF prepared on the basis of formula 4 were added to the 48-well plate inoculated with primary cells, and the final concentrations of bFGF were 1 ng/mL, 3 ng/mL, 10 ng/mL, 30 ng/mL, 100 ng/mL; and set up control wells (BC) using the medium of recipe 4.
  • CHIR99021 prepared on the basis of formula 5 was added to the 48-well plate inoculated with primary cells, and the final concentrations of CHIR99021 were 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M; and the medium of formula 5 was used to set up control wells (BC).
  • EGF EGF prepared on the basis of formula 6
  • the final concentrations of EGF are 2.5 ng/mL and 5 ng/mL respectively.
  • 10ng/mL, 20ng/mL, 40ng/mL and use the medium of formula 6 to set up control wells (BC).
  • FGF10 prepared on the basis of formula 10 was added to the 48-well plate inoculated with primary cells, and the final concentrations of FGF10 were 50 ng/mL, 100 ng/mL, 200ng/mL, 400ng/mL, 800ng/mL; and set up control wells (BC) using the medium of Formulation 10.
  • the cells were expanded to about 85% of the 48 wells, they were digested and counted, and the proliferation multiples were calculated with reference to the number of cells in the control well (BC).
  • the data collected from the two samples were summarized in Figures 1A-1L.
  • the ratios are the ratios 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 wells 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.
  • the content of MST1/2 kinase inhibitor Compound 1 is preferably 2.5-20 ⁇ M, more preferably 5-10 ⁇ M;
  • the volume concentration of B27 is preferably 1:25-1:400, more preferably 1: 50 ⁇ 1:400;
  • the concentration of basic fibroblast growth factor bFGF is preferably 1 ⁇ 30ng/mL, more preferably 10 ⁇ 30ng/mL;
  • the volume concentration of ITS cell culture supplement relative to the medium is preferably 1:25 ⁇ 1:400, more preferably 1:50 ⁇ 1:200;
  • the concentration of Y27632 is preferably 2.5 ⁇ 40 ⁇ M, more preferably 5 ⁇ 20 ⁇ M;
  • the concentration of dexamethasone is preferably 25 ⁇ 400nM, more preferably 50 ⁇ 400nM; CHIR99021
  • the concentration of is preferably 1.25 ⁇ 10 ⁇ M, more preferably 2.5 ⁇ 10 ⁇ M;
  • the concentration of epidermal growth factor EGF is preferably 2.5 ⁇ 20ng/mL, more preferably 5 ⁇ 10ng/mL
  • gastric cancer primary cells from intraoperative tissue samples (numbered GQ-004, GQ-007, GQ-009, GQ-0010) according to the method of step (2) of 3 of Example 1, and use the method in Example 2 GC-2.1 medium for cultivation.
  • the obtained primary gastric cancer cells were seeded in a 6-well plate pre-coated with Matrigel (100,000 cells per well) at a living cell density of 1 ⁇ 10 4 cells/cm 2 , and mixed evenly. After surface disinfection, they were cultured in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher).
  • Figures 2A-2D are samples GQ-004, GQ-007, GQ-009, and GQ taken under a 10x objective lens, respectively.
  • Example 2 Obtain an intraoperative tissue sample (number GQ-008) according to the method of step (2) of Example 1, and use the GC-2.1 medium in Example 2 to cultivate the sample GC-008 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 it in a 37°C, 5% CO2 incubator for 2 to 10 minutes until Digestion is terminated when the cells have been digested completely. After centrifugation at 1500rpm for 4 minutes, the supernatant was discarded, and 500 ⁇ L GC-2.1 medium was added to resuspend. Phase II Building H4) for pathological and immunohistochemical identification.
  • trypsin purchased from Gibco
  • Figure 3A is the result of pathological and immunohistochemical identification of the original gastric cancer tissue sample GC-008, and Figure 3B is the pathological examination of the gastric cancer primary cells obtained after in vitro culture of the sample GC-008 using the GC-2.1 medium of the present invention and the results of immunohistochemical identification are pictures taken under a 20x objective lens.
  • both the cultured primary cells and original tissue samples expressed CDX-2, CK7, VILLIN, and Ki67, suggesting that the cultured primary cells were gastric cancer cells, and the GC-2.1 of the present invention
  • the primary cells cultured in the culture medium were consistent with the diagnostic results of gastric cancer tissue.
  • Example 4 Primary gastric cancer primary culture cycle and cell number statistics and Population Doubling (PD) value calculation
  • gastric cancer primary cells were obtained from 3 gastric cancer tissue samples (numbered GQ-001, GQ-002, and GQ-003).
  • gastric cancer primary cells use the GC-2.1 medium in Example 2 to culture, and inoculate the cells in a T25 flask according to the living cell density of 2 ⁇ 104 cells/ cm2 and culture them until the cells are expanded to After 95% of the cells were digested and counted, the number of days of culture until digestion was recorded at the same time, and the number of days of culture at the time of digestion was regarded as a culture cycle.
  • the Graphpad Prism software is used to draw the growth curves of 3 examples of primary cells cultured using the gastric cancer primary cells of the present invention
  • the abscissa represents the days of cell culture
  • the ordinate is the cumulative cell proliferation multiple, which represents the cell growth rate.
  • the multiple of 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, and the slope represents the rate of cell expansion. It can be confirmed from Figure 4 that the primary gastric cancer cells cultured in the GC-2.1 medium of the present invention can be continuously cultured and expanded, and the cell expansion rate remains basically unchanged for at least 50 days, and still has the ability to continue to expand.
  • Embodiment 5 and the comparison of existing culture medium effect
  • the culture medium used in the preparation literature (Xuefeng Liu et al., Nat Protoc.2017,12(2):439-451), its formula is DMEM/F12 medium+250ng/ml amphotericin B (purchased from Selleck company)+ 10 ⁇ g/ml gentamicin (purchased from MCE company)+0.1nM cholera toxin (purchased from MCE company)+0.125ng/ml EGF+25ng/ml hydrocortisone (purchased from Sigma)+10 ⁇ M Y27632+10%FBS (available from Excell).
  • LXF medium for short.
  • Gastric cancer primary cells were obtained from intraoperative tissue samples (GQ-001, GQ-002) according to the method of step (2)-3 of Example 1, and cultured in GC-2.1, LXF and A1 medium respectively.
  • the 48-well plate was taken out, the medium was discarded, and 100 ⁇ L of 0.05% trypsin (purchased from Gibco) was used to rinse once, and then 200 ⁇ L of 0.05% trypsin was added to each well.
  • 0.05% trypsin purchased from Gibco
  • GC-2.1 medium can significantly promote the expansion of gastric cancer primary cells, and its effect is better than that of LXF medium and A1 medium used in the prior art.
  • Example 6 Using the medium of the present invention to amplify primary gastric cancer cells for drug screening
  • Gastric cancer primary cells were isolated from the obtained intraoperative gastric cancer sample (GQ-003) in the same manner as in Example 1, and cultured in GC-2.1 medium. After the cells were amplified to 85%, they were digested and passaged as the first generation. The cultured 1st, 2nd, 3rd, 4th, and 5th passage cells were used for drug screening.
  • the cells were digested and counted.
  • the cells were mixed thoroughly in the sample tank (purchased from Corning Corporation) according to the viable cell density of 5.76 ⁇ 104 cells/mL, and then the cells were mixed at 384 Wells of an opaque white cell culture plate (purchased from Corning Corporation) were cultured, the volume of each well was 50 ⁇ L, and the number of cells was 3000/well.
  • the surface was sterilized with 75% alcohol (purchased from Lierkang), cultured in a 37°C, 5% CO 2 incubator, and the drug was added after 24 hours.
  • cell inhibition rate (%) 100%-chemiluminescence value of drug-dosing well/chemiluminescence value of control well*100%, calculate the cell inhibition rate after different drugs act on cells, and use graphpad prism software to calculate the effect of drugs on cells Half inhibition rate (IC 50 ). The results are shown in FIG. 6 .
  • the gastric cancer cells cultured in the gastric cancer primary cell culture medium of the present invention are used for drug screening, and the inhibitory effect of the same drug on the cultured cells of different generations is basically consistent (the inhibition curves are basically consistent).
  • 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, the effectiveness of the drug in clinical use can be judged for patients with gastric cancer, 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 this patent is stable to the drug.
  • the invention provides a medium and a culture method for culturing gastric cancer primary cells, and the cultured gastric cancer primary 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

一种用于培养原代胃癌细胞的培养基，其含有MST1/2激酶抑制剂、ROCK激酶抑制剂、B27添加剂和N2添加剂中的至少一种添加剂、碱性成纤维细胞生长因子、CHIR99021、表皮细胞生长因子、ITS细胞培养添加剂、SB202190、地塞米松、成纤维细胞生长因子10、N-乙酰-L-半胱氨酸、和胃泌素。以及使用该原代细胞培养基的培养方法，该培养方法使用上述培养基在包被有细胞外基质胶的培养器皿上培养原代细胞，使得原代细胞快速增殖。

Description

胃癌原代细胞的培养基和培养方法 技术领域
本发明属于生物技术领域，具体涉及一种用于胃癌原代细胞的培养基、和使用该培养基培养胃癌原代细胞的方法。
背景技术
胃癌(gastric carcinoma)是起源于胃黏膜上皮的恶性肿瘤，在我国各种恶性肿瘤中发病率居首位。胃癌发病有明显的地域性差别，在我国的西北与东部沿海地区胃癌发病率比南方地区明显为高。好发年龄在50岁以上，男女发病率之比为2：1。由于饮食结构的改变、工作压力增大以及幽门螺杆菌的感染等原因，使得胃癌呈现年轻化倾向。胃癌可发生于胃的任何部位，其中半数以上发生于胃窦部，胃大弯、胃小弯及前后壁均可受累。绝大多数胃癌属于腺癌，早期无明显症状，或出现上腹不适、嗳气等非特异性症状，常与胃炎、胃溃疡等胃慢性疾病症状相似，易被忽略。因此，目前我国胃癌的早期诊断率仍较低。目前全国早期胃癌的诊断率仍然低于20％，胃癌患者5年生存率仅为27.4％。
近几年，在分子生物学的兴起与发展下，肿瘤药物治疗呈现多样化趋势，其中分子靶向药物因其针对性强、安全性高等优势成为胃癌临床治疗中研究的热点。但是临床上针对众多治疗方案，怎么选择适合病人的方案就尤为重要。尽管有基因检测作为指标，但是有些病人没有基因突变，或者有些病人即使有某种突变，但是针对该突变有多种靶向药物，这时确定治疗方案在临床上有一定的难度。除了基因测序，体外对胃癌病人样本进行原代细胞培养已经成为未来体外预测疗效和指导临床用药的重要手段，但是体外快速获得胃癌原代细胞一直是亟待解决的技术问题。
目前主要有两种培养原代细胞的技术发展得相对成熟。一种是使用经辐射的饲养细胞和ROCK激酶抑制剂Y27632来促进原代上皮细胞的生长，即细胞条件重编程技术(Liu等，Am J Pathol，180：599-607，2012)。另一种技术是体外3D培养成体干细胞从而获得类似于组织器 官的类器官技术(Hans Clevers等，Cell，11，172(1-2)：373-386，2018)。
然而，这两种技术都存在一定的局限性。细胞重编程技术是一种将患者自体原代上皮细胞与鼠源性饲养细胞共培养的技术。在对患者原代细胞进行药物敏感性测试时，这些鼠源性细胞的存在会干扰患者自体原代细胞的药物敏感性检测结果；但如果撤除鼠源性饲养细胞，病人自体原代细胞就脱离了重编程环境，细胞的增殖速率和细胞内信号通路会发生明显的改变(Liu等，Am J Pathol，183(6)：1862-1870，2013；Liu等，Cell Death Dis.，9(7)：750，2018)，从而使患者自体原代细胞对药物的响应结果受到较大影响。类器官技术是将患者自体原代上皮细胞包埋在细胞外基质内进行体外三维立体培养的技术，该技术无需饲养细胞，因此不存在鼠源性饲养细胞的干扰问题。但是类器官技术的培养基内需添加多种特定的生长因子(如Wnt蛋白和R-spondin家族蛋白)，成本昂贵，不适于普及到临床进行大规模应用。另外，类器官在整个培养过程中均需将细胞包埋在细胞外基质胶中，其细胞接种、传代和药物敏感性测试的铺板步骤相较于2D培养操作繁琐费时，且该技术所形成的类器官大小尺寸不好控制，易出现部分类器官生长过大而导致内部发生坏死的情况。因此，类器官技术相较于2D培养技术可操作性和适用性不强，需要专业技术人员操作，不适合大规模广泛应用于临床体外药物敏感性检测(Nick Barker，Nat Cell Biol，18(3)：246-54，2016)。
鉴于以上技术的局限性，临床上需要开发一种原代胃癌细胞培养技术，其培养周期短，成本可控，操作便捷，不受外源性细胞干扰。在将该技术应用于构建原代胃癌肿瘤细胞模型时，所培养的胃癌肿瘤细胞能代表胃癌患者自身的生物学特性。通过体外评估抗肿瘤药物在不同癌症患者个体所衍生的细胞模型上的敏感性，来提高临床上抗肿瘤药物的响应率，减少不合适的药物给患者造成的痛苦及医疗资源的浪费。
发明内容
为了解决上述技术问题，本发明提供了一种用于在体外快速扩增 胃癌原代细胞的培养基和培养方法及其应用。
本发明的一个方面在于提供一种胃癌原代细胞的培养基，所述培养基包含MST1/2激酶抑制剂；选自Y27632、法舒地尔、和H-1152中的至少一种的ROCK激酶抑制剂；B27添加剂和N2添加剂中的至少一种添加剂；碱性成纤维细胞生长因子；CHIR99021；表皮细胞生长因子；ITS细胞培养添加剂；SB202190；地塞米松；成纤维细胞生长因子10；N-乙酰-L-半胱氨酸；和胃泌素。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 PCTCN2021140029-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 PCTCN2021140029-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 PCTCN2021140029-appb-000003
Figure PCTCN2021140029-appb-000004
Figure PCTCN2021140029-appb-000005
Figure PCTCN2021140029-appb-000006
Figure PCTCN2021140029-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)所述MST1/2激酶抑制剂的浓度为2.5～20μM；
(2)所述B27或N2细胞培养添加剂相对于培养基的体积比为1:25～1:400；
(3)所述碱性成纤维细胞生长因子的浓度为1～30ng/mL；
(4)所述ITS细胞培养添加剂相对于培养基的体积比为1:25～1:400；
(5)所述ROCK激酶抑制剂的浓度为2.5～40μM；
(6)所述地塞米松的浓度为25～400nM；
(7)所述CHIR99021的浓度为1.25～10μM；
(8)所述表皮细胞生长因子的浓度为2.5～20ng/mL；
(9)所述成纤维细胞生长因子10的浓度为50～800ng/mL；
(10)所述胃泌素的浓度为1.25～20nM；
(11)所述SB202190的浓度为50～800nM；
(12)所述N-乙酰-L-半胱氨酸的浓度为0.25～4mM。
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作为基础培养基。
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。
本发明还提供一种胃癌原代细胞的培养方法。在本发明的胃癌原代细胞的培养方法中，使用本发明的胃癌原代细胞培养基对胃癌原代细胞进行培养。
本发明的胃癌原代细胞培养方法包括以下步骤。
(1)按上述配方配制本发明的原代细胞培养基。
(2)用细胞外基质胶稀释液包被培养器皿。
具体地，该细胞外基质胶使用低生长因子型细胞外基质胶，例如，可采用市售的Matrigel(购自康宁公司)或BME(购自Trevigen公司)。更具体而言，用无血清的培养基稀释细胞外基质胶，培养基可以是DMEM/F12(购自康宁公司)。细胞外基质胶的稀释比例为1:50-1:400，优选为1:100-1:200。包被方法为将稀释后的细胞外基质胶加入培养器皿内，使其完全覆盖培养器皿底部，静置包被30分钟以上，优选在37℃条件下静置包被，优选包被时间为30～60分钟。包被结束后吸弃多余的细胞外基质胶稀释液，培养器皿备用。
(3)从胃癌实体瘤组织分离样本，获得胃癌原代细胞。
原代胃癌细胞例如可以来源于胃癌手术样本和活检内镜样本。胃癌手术样本例如来源于进行过说明并获得同意的胃癌肿瘤患者手术切除癌组织样本，内镜样本经由内镜引导采集自胃内病灶。在患者手术切除或活检后的半小时内进行上述组织样本的收集。以手术样本为例，在无菌环境下，切取非坏死部位的组织样本，其体积在5mm 3以上，将其置于预冷的10-15mL DMEM/F12培养基或商品化保存液中，盛在塑料无菌带盖离心管内，冰上运输至实验室。
在生物安全柜内，将组织样本转移至细胞培养皿内，用如上文所述的基础培养基润洗组织样本，将组织样本表面的血细胞清洗掉。将润洗后的组织样本转移至另一个新的培养皿内，加入1-3mL基础培养基，用无菌手术刀片和手术镊将组织样本分割为体积小于3mm 3的组织碎块。
将组织样本碎块转移至离心管内，用台式离心机(Sigma公司3-18K)以1000～3000rpm离心3～5分钟；弃上清，按1:3比例加入基础培养基和组织消化液(其中组织消化液的配制方法为：将1～2mg/mL胶原酶Ⅱ、1～2mg/mL胶原酶Ⅳ、50～100U/mL脱氧核糖核酸、0.5～1mg/mL透明质酸酶、1～5mM氯化钙、5～10mg/mL牛血清白蛋白溶于1640培养基中)，标记样本编号，封口膜密封，以37℃、200～300rpm恒温摇床(知楚仪器ZQLY-180N)消化，每间隔半小时或1小时观察消化是否完成；若未见明显组织块即可终止消化，否则继续消化，直至消化充分，消化时间范围为4～8小时。消化完成后，细胞滤网(细胞筛孔径为例如70-100μm)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，将残留细胞冲入离心管中，用台式离心机以1000～3000rpm离心3～5分钟。弃上清，观察剩余细胞团是否含有血细胞，若有血细胞，加3～8mL血细胞裂解液(购自Sigma公司)，混匀，4℃裂解10～20分钟，5分钟摇晃混匀一次，裂解结束后取出，以1000～3000rpm离心3～5分钟。
(4)在包被好的培养器皿内接种步骤(3)中分离得到的原代胃癌细胞，并采用步骤(1)中的原代细胞培养基进行培养。
更具体而言，在多孔板的一个孔中按1×10 4～8×10 4个/cm 2(例如 4×10 4个/cm 2)的密度接种原代胃癌肿瘤细胞，加入适量如2-3mL原代胃癌细胞培养基，在例如37℃、5％CO 2的条件下于细胞培养箱中培养8-16天，期间每4天换成新鲜的原代细胞培养基，在原代胃癌细胞长至占多孔板底面积80％～90％左右的细胞密度时进行消化传代。
该接种步骤无需使用饲养细胞，相比细胞条件重编程技术，免去了培养和辐照饲养细胞的操作步骤。该步骤相比类器官技术，也无需在冰上将原代细胞和基质胶混匀后形成胶滴，并等待胶滴凝固后加入培养基，预先包被好的培养器皿可直接用于原代细胞接种。此外，包被培养器皿仅需少量稀释后的细胞外基质胶，相比类器官技术，节约了价格昂贵的细胞外基质胶的使用量，也简化了操作步骤。
任选地，接种后的原代胃癌细胞在培养8～16天后，当培养容器内形成的细胞克隆汇合达到底面积80％，弃去上清，加入0.5～2mL0.05％胰酶(购自Thermo Fisher公司)进行细胞消化，室温下孵育5～20分钟；然后用含有例如5％(v/v)胎牛血清、100U/mL青霉素和100μg/mL链霉素的DMEM/F12培养液1～4mL重悬消化处理后的细胞，以1000～3000rpm离心3～5分钟；使用本发明的原代细胞培养基将消化后的单细胞重悬，将所得到的细胞悬液置入包被有细胞外基质胶的T25细胞培养瓶中继续扩大培养。T25细胞培养瓶的包被操作同步骤(2)。
扩增的胃癌原代细胞呈2D生长，避免了类器官技术扩增出现的类器官大小不均一和生长过大的类器官出现内部坏死等情况。
本发明还提供一种用于评估或筛选治疗胃癌疾病的药物的方法，其包括以下步骤：
(1)使用本发明的胃癌原代细胞的培养方法培养胃癌原代细胞；
(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)中培养得到的细胞添加稀释后的所述药物；
(4)进行细胞活性测试。
本发明的有益效果包括：
(1)提高胃癌原代细胞培养的成功率，成功率达到90％以上；
(2)保证体外原代培养的胃癌原代细胞能够保持病人的病理特性；
(3)所培养的原代胃癌上皮细胞不受成纤维细胞干扰，能得到纯化的胃癌上皮细胞；
(4)培养基成分不含血清，所以不受不同批次血清质量和数量的影响；
(5)扩增效率高，能快速培养出胃癌原代细胞，扩增出的胃癌原代细胞还可以连续传代；
(6)传代步骤无需冰上操作和解离基质胶，10-15分钟内即可完成细胞的消化传代；
(7)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、Noggin蛋白、BMP抑制剂等因子；
(8)所述技术培养获得的胃癌原代细胞数量大，适合高通量筛选候选化合物和为病人提供高通量药物体外敏感性功能测试。
附图说明
图1A-1L为显示本发明的胃癌原代细胞培养基所添加因子的不同浓度对胃癌原代细胞增殖的影响的图。
图2A-2D为利用显微镜观察使用本发明的胃癌原代细胞培养基培养得到的胃癌原代细胞的照片。
图3A和3B分别为显示对胃癌原始组织样本和使用本发明的胃癌原代细胞培养基对该原始组织样本进行培养而得到的胃癌原代细胞进行病理和免疫组化鉴定的结果的图。
图4为使用本发明的胃癌原代细胞培养基对胃癌原代组织样本进行培养得到的胃癌原代细胞的细胞生长曲线图。
图5A和5B为显示分别使用本发明的胃癌原代细胞培养基和两种现有培养基对胃癌原代细胞进行培养的比较结果的图。
图6为显示使用本发明的胃癌原代细胞培养基培养得到的不同代数的胃癌细胞用于药物敏感性测试的结果的图。
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 PCTCN2021140029-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 PCTCN2021140029-appb-000009
Figure PCTCN2021140029-appb-000010
Figure PCTCN2021140029-appb-000011
Figure PCTCN2021140029-appb-000012
Figure PCTCN2021140029-appb-000013
实施例1胃癌原代细胞培养基中各添加因子对胃癌原代细胞增殖的影响
(1)胃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胃癌原代细胞培养基。
(2)胃癌原代细胞的分离和处理
1样品选择
胃癌实体瘤组织样品(术中)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，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。
组织消化液配方：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)、牛血清白蛋白BSA(10mg/mL)。
以上提及的胶原酶Ⅱ、胶原酶Ⅳ、DNA酶、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样品分离
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，加入1:3比例的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；
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(生产厂家：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(生产厂家：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)胃癌原代细胞的培养
将细胞外基质胶(
Figure PCTCN2021140029-appb-000014
BD生物科技公司制)使用无血清DMEM/F12培养基按1:100比例稀释，配制成细胞外基质稀释液，在48孔培养板内加入500μl/孔的细胞外基质稀释液使其完全覆盖培养板孔的底部。在37℃培养箱内静置1小时。1小时后，移除细胞外基质稀释液，得到包被有Matrigel的培养板。
将上述步骤中获得的胃癌原代细胞用预冷的DMEM/F12重悬并计数。将不同成分的培养基(表1)按500μl/孔体积加入至包被有细胞外基质胶(Matrigel)的48孔板内。将计数好的胃癌原代细胞(编号GQ-001)以2×10 4个/cm 2的细胞密度接种在Matrigel包被过的48孔培养板内，表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)，使相同数量的新鲜分离的胃癌肿瘤细胞(编号GQ-001)在不同的培养基配方条件下进行培养。培养开始后每4天进行一次培养基的更换。培养12天后，进行细胞计数，比较各因子对胃癌原代细胞增殖的促进作用。其中，作为实验对照，使用未添加任何添加剂的基础培养基，将实验结果示于表1。
表1培养基中的添加成分及促类器官增殖效果
序号 培养基添加剂种类 供应商 终浓度 促增殖程度分级
1 N2 Gibco 1:50 +
2 表皮细胞生长因子EGF R&D 10ng/mL +
3 R-spondin1 R&D 20ng/mL ○
4 前列腺素E2 Tocris 0.5μM ○
5 胰岛素 Peprotech 1.5μg/mL ○
6 B27 Gibco 1:50 +
7 SB202190 MCE 200nM +
8 碱性成纤维细胞生长因子bFGF R&D 10ng/mL +
9 氢化可的松 Sigma 10ng/mL ○
10 Noggin R&D 30ng/mL ○
11 胎牛血清FBS Excell 5％ +
12 胰岛素样生长因子-1 IGF-1 R&D 45ng/mL ○
13 角化细胞生长因子KGF R&D 5ng/mL ○
14 GlutaMAX Gibco 1:100 ○
15 非必需氨基酸 Corning 100μM ○
16 地塞米松 MCE 100nM +
17 神经调节蛋白1 NRG1 sino biological 5ng/mL -
18 Y27632 MCE 10μM +
19 ITS细胞培养添加剂 Gibco 1:100 +
20 化合物1 制备例 5μM +
21 CHIR99021 MCE 5μM +
22 肝细胞生长因子HGF R&D 5ng/mL -
23 成纤维细胞生长因子10 FGF10 R&D 100ng/mL +
24 胃泌素 MCE 5nM +
25 N-乙酰-L-半胱氨酸NAC MCE 1mM +
其中，“+”表示与基础培养基相比，加入该添加剂的培养基对从胃癌组织分离出的胃癌原代细胞中的至少两例有促进增殖的作用；“－”表示添加该添加剂的培养基对从胃癌组织分离出的胃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胃癌组织分离出的胃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，拟选择化合物1、Y27632、B27、碱性成纤维细胞生长因子(bFGF)、CHIR99021、表皮细胞生长因子(EGF)、ITS细胞培养添加剂、SB202190、地塞米松、成纤维细胞生长因子10(FGF10)、N-乙酰-L-半胱氨酸(NAC)、胃泌素等因子进行进一步培养实验。
实施例2培养基添加因子的不同浓度对胃癌原代细胞的增殖作用
按照实施例1之(2)的方法从术中组织样本(编号为GQ-002、GQ-003)获得胃癌原代细胞，并使用下表2中的培养基配方进行原代细胞培养。
表2培养基配方(浓度为终浓度)
Figure PCTCN2021140029-appb-000015
在使用配方1的培养基时，在接种有原代细胞的48孔板中在配方1的基础上分别添加配制好的化合物1每孔200μL，化合物1的终浓度分别为1.25μM、2.5μM、5μM、10μM、20μM；并使用配方1的培养基设置对照孔(BC)。该系列的培养基中其他添加因子的终浓度与GC-2.1培养基相同。以下配方1-12的实验也以同样的方式进行，不再赘述。
在使用配方2的培养基时，在接种有原代细胞的48孔板中在配方2的基础上分别添加配制好的Y27632每孔200μL，Y27632的终浓度分别为2.5μM、5μM、10μM、20μM、40μM；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孔板中在配方 3的基础上分别添加配制好的B27每孔200μL，B27的终浓度分别为1:25、1:50、1:100、1:200、1:400；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孔板中在配方4的基础上分别添加配制好的bFGF每孔200μL，bFGF的终浓度分别为1ng/mL、3ng/mL、10ng/mL、30ng/mL、100ng/mL；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孔板中在配方5的基础上分别添加配制好的CHIR99021每孔200μL，CHIR99021的终浓度分别为1.25μM、2.5μM、5μM、10μM、20μM；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孔板中在配方6的基础上分别添加配制好的EGF每孔200μL，EGF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48孔板中在配方7的基础上分别添加配制好的ITS细胞培养添加剂每孔200μL，ITS细胞培养添加剂的终浓度分别为1:25、1:50、1:100、1:200、1:400；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孔板中在配方8的基础上分别添加配制好的SB202190每孔200μL，SB202190的终浓度分别为50nM、100nM、200nM、400nM、800nM；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孔板中在配方9的基础上分别添加配制好的地塞米松每孔200μL，地塞米松的终浓度分别为25nM、50nM、100nM、200nM、400nM；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孔板中在配方10的基础上分别添加配制好的FGF10每孔200μL，FGF10的终浓度分别为50ng/mL、100ng/mL、200ng/mL、400ng/mL、800ng/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48孔板中在配方11的基础上分别添加配制好的NAC每孔200μL，NAC的终浓度分别为0.25mM、0.5mM、1mM、2mM、4mM；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48孔板中在配方12的基础上分别添加配制好的胃泌素每孔200μL，胃泌素的终浓度分别为1.25nM、2.5nM、5nM、10nM、20nM；并使用配方12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2例样本收集的数据汇总示于图1A～1L。图1A～1L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1A～1L的结果，MST1/2激酶抑制剂化合物1的含量优选为2.5～20μM，更优选为5～10μM；B27的体积浓度优选为1:25～1:400，更优选为1:50～1:400；碱性成纤维细胞生长因子bFGF的浓度优选为1～30ng/mL，更优选为10～30ng/mL；ITS细胞培养添加剂相对于培养基的体积浓度优选为1:25～1:400，更优选为1:50～1:200；Y27632的浓度优选为2.5～40μM，更优选为5～20μM；地塞米松的浓度优选为25～400nM，更优选为50～400nM；CHIR99021的浓度优选为1.25～10μM，更优选为2.5～10μM；表皮细胞生长因子EGF的浓度优选为2.5～20ng/mL，更优选为5～10ng/mL；成纤维细胞生长因子10FGF10的浓度优选为50～800ng/mL，更优选为100～400ng/mL；胃泌素的浓度优选为1.25～20nM，更优选为1.25～10nM；SB202190的浓度优选为50～800nM，更优选为100～400nM；N-乙酰-L-半胱氨酸NAC的浓度优选为0.25～4mM，更优选为0.5～2mM。
实施例3胃癌原代细胞培养及鉴定
按照实施例1的步骤(2)之3的方法从术中组织样本(编号为 GQ-004、GQ-007、GQ-009、GQ-0010)获得胃癌原代细胞，并使用实施例2中的GC-2.1培养基进行培养。所获得的胃癌原代细胞，按照活细胞密度1×10 4个/cm 2接种于预包被基质胶的6孔板中(每孔10万细胞数)，混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。
在第3-7天，使用显微镜(Invitrogen公司EVOS M500)观察培养得到的胃癌原代细胞，图2A-2D分别是10倍物镜下拍摄由样本GQ-004、GQ-007、GQ-009、GQ-0010培养得到的原代细胞的照片，细胞在镜下呈紧密排列，形态略不规则。
按照实施例1的步骤(2)之3的方法获得术中组织样本(编号为GQ-008)，并使用实施例2中的GC-2.1培养基培养样本GC-008直至细胞长至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，加入500μL GC-2.1培养基重悬，对培养得到的胃癌原代细胞由合肥金域医学检验实验室有限公司(合肥市高新区创新大道2800号创新产业园二期H4号楼)进行病理和免疫组化鉴定。
图3A为对胃癌原始组织样本GC-008进行病理和免疫组化鉴定的结果，图3B为对使用本发明的GC-2.1培养基对样本GC-008体外培养后得到的胃癌原代细胞进行病理和免疫组化鉴定的结果，分别为20倍物镜下拍照的图片。如图3A和3B所示，培养后的原代细胞和原始组织样本均有CDX-2、CK7、VILLIN、Ki67表达，提示培养后的原代细胞为胃癌细胞，且使用本发明的GC-2.1培养基培养的原代细胞与胃癌组织的诊断结果一致。
实施例4胃癌原代细胞初次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3例胃癌组织样本(编号为GQ-001、GQ-002、GQ-003)获得胃癌原代细胞。对于所获得的胃癌原代细胞，使用实施例2中的GC-2.1培养基培养，按照活细胞密度2× 10 4个/cm 2将细胞接种在T25瓶中并进行培养，待细胞扩增至95％后消化并计数，同时记录直至消化时培养的天数，将消化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，每一代进行消化后计数并记录相应培养的周期，根据公式Population Doubling(PD)＝3.32*log 10(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)。
如图4所示，采用Graphpad Prism软件绘制使用本发明的胃癌原代细胞培养的3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，其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4中可以确认，本发明的GC-2.1培养基培养的胃癌原代细胞可进行持续培养扩增，且至少50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5与现有培养基培养效果的比较
(1)对照培养基的配制
配制文献(Xuefeng Liu等，Nat Protoc.2017,12(2):439-451)中使用的培养基，其配方为DMEM/F12培养基+250ng/ml两性霉素B(购自Selleck公司)+10μg/ml庆大霉素(购自MCE公司)+0.1nM霍乱毒素(购自MCE公司)+0.125ng/ml EGF+25ng/ml氢化可的松(购自Sigma)+10μM Y27632+10％FBS(购自Excell)。以下简称为LXF培养基。
配置另一文献(Jigui Peng等，Cancer Cell Int.(2020)20:437)中使用的培养基，其配方为DF12(购自Corning公司)+2％FBS(购自Excell公司)+100U/ml青霉素(购自corning公司)+100μg/ml链霉素(购自corning公司)+0.1ng/ml EGF+0.1ng/ml bFGF+25μg/ml氢化可的松(购自Sigma)。以下简称为A1培养基。
(2)胃癌原代细胞的获取和培养
按照实施例1的步骤(2)之3的方法从术中组织样本(GQ-001、GQ-002)获得胃癌原代细胞，分别利用GC-2.1、LXF和A1培养基进 行培养。
在培养第7天，取出48孔板，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％血清DF12终止消化，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，将计数结果示于图5A和5B。
根据图5的结果可知，与LXF培养基和A1培养基相比，GC-2.1培养基能显著促进胃癌原代细胞扩增，其效果优于现有技术采用的LXF培养基和A1培养基。
实施例6使用本发明培养基扩增得到的胃癌原代细胞用于药物筛选
1、细胞培养和铺板
从得到的胃癌术中样本(GQ-003)与实施例1同样地分离得到胃癌原代细胞，并使用GC-2.1培养基进行培养，待细胞扩增至85％，进行消化传代，作为一代。分别取培养第1代、第2代、第3代、第4代、第5代细胞进行药物筛选。
按照实施例1中步骤将细胞消化计数，使用GC-2.1培养基，将细胞按照活细胞密度5.76×10 4个/mL细胞于加样槽(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)进行培养，每孔体积50μL，细胞数目为3000个/孔。从孔板边缘加入GC-2.1培养基封板，板上标注样品名称及CellTiter-Glo(购自Promega公司)检测时间。表面75％酒精(购自利尔康)消毒，置37℃、5％CO 2培养箱培养，24小时后加药。
2、筛选药物配制
按照下表配制7个浓度梯度的4种药物(阿糖胞苷、环磷酰胺、吉西他滨、苯达莫司汀；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阿糖胞苷、环磷酰胺、吉西他滨、苯达莫司汀添加液的配制
阿糖胞苷 苯达莫司汀 环磷酰胺 吉西他滨
最终浓度(uM) 最终浓度(uM) 最终浓度(uM) 最终浓度(uM)
5.83 163.41 893.37 273.42
1.94 54.47 297.79 91.14
0.65 18.16 99.26 30.38
0.22 6.05 33.09 10.13
0.07 2.02 11.03 3.38
0.02 0.67 3.68 1.13
0.01 0.22 1.23 0.38
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。对培养有口腔癌细胞的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继续培养，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6。
由图6可以确认，使用本发明的胃癌原代细胞培养基培养得到的胃癌细胞进行药物筛选，相同药物对于培养的不同代数细胞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。根据结果可以判断胃癌病人在临床使用该种药物时的有效性，同时可以说明根据本专利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胃癌原代细胞培养的培养基及培养方法，可将培养得到的胃癌原代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 (10)

  1. 一种用于胃癌原代细胞的培养基，其特征在于包括MST1/2激酶抑制剂；选自Y27632、法舒地尔、和H-1152中的至少一种的ROCK激酶抑制剂；B27添加剂和N2添加剂中的至少一种添加剂；碱性成纤维细胞生长因子；CHIR99021；表皮细胞生长因子；ITS细胞培养添加剂；SB202190；地塞米松；成纤维细胞生长因子10；N-乙酰-L-半胱氨酸；和胃泌素，
    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 PCTCN2021140029-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 PCTCN2021140029-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 PCTCN2021140029-appb-100003
    Figure PCTCN2021140029-appb-100004
    Figure PCTCN2021140029-appb-100005
    Figure PCTCN2021140029-appb-100006
    Figure PCTCN2021140029-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：
    所述MST1/2激酶抑制剂的浓度为2.5～20μM；
    所述B27或N2细胞培养添加剂相对于培养基的体积比为1:25～1:400；
    所述碱性成纤维细胞生长因子的浓度为1～30ng/mL；
    所述ITS细胞培养添加剂相对于培养基的体积比为1:25～1:400；
    所述ROCK激酶抑制剂的浓度为2.5～40μM；
    所述地塞米松的浓度为25～400nM；
    所述CHIR99021的浓度为1.25～10μM；
    所述表皮细胞生长因子的浓度为2.5～20ng/mL；
    所述成纤维细胞生长因子10的浓度为50～800ng/mL；
    所述胃泌素的浓度为1.25～20nM；
    所述SB202190的浓度为50～800nM；
    所述N-乙酰-L-半胱氨酸的浓度为0.25～4mM。
  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. 如权利要求1～7中任一项所述的培养基，其特征在于所述培养基不含Wnt激动剂、R-spondin家族蛋白、Noggin蛋白、或BMP抑制剂。
  9. 一种胃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1～8中任一项所述的培养基；
    (2)用细胞外基质胶稀释液包被培养器皿，所述细胞外基质胶选自Matrigel和BME中的至少一种；
    (3)在包被有细胞外基质胶的培养器皿内接种从胃癌组织分离得到原代胃癌细胞，使用步骤(1)中的所述培养基进行培养。
  10. 一种用于评估或筛选治疗胃癌疾病的药物的方法，其特征在于，包括以下步骤：
    (1)使用如权利要求9所述的胃癌原代细胞的培养方法培养胃癌原代细胞；
    (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)中培养得到的细胞添加稀释后的所述药物；
    (4)进行细胞活性测试。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116590235A (zh) * 2023-07-14 2023-08-15 北京嘉士腾医学检验实验室有限公司 一种高效的消化道肿瘤类器官培养方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108060119A (zh) * 2016-11-07 2018-05-22 云南济慈再生医学研究院有限公司 小分子化合物组合及利用该小分子化合物组合诱导分化的细胞制备血管平滑肌细胞的方法
CN111039944A (zh) * 2018-10-12 2020-04-21 中国科学院合肥物质科学研究院 Mst1激酶抑制剂及其用途
CN111808816A (zh) * 2019-04-11 2020-10-23 北京基石生命科技有限公司 一种用于培养胃癌实体瘤原代细胞的培养基
CN113403278A (zh) * 2020-03-16 2021-09-17 合肥中科普瑞昇生物医药科技有限公司 胃癌原代细胞的培养基及培养方法
CN113528425A (zh) * 2020-04-15 2021-10-22 合肥中科普瑞昇生物医药科技有限公司 一种用于乳腺上皮干细胞的培养基和培养方法
CN113528444A (zh) * 2020-04-15 2021-10-22 合肥中科普瑞昇生物医药科技有限公司 一种用于食管鳞癌上皮细胞的培养基、培养方法及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108060119A (zh) * 2016-11-07 2018-05-22 云南济慈再生医学研究院有限公司 小分子化合物组合及利用该小分子化合物组合诱导分化的细胞制备血管平滑肌细胞的方法
CN111039944A (zh) * 2018-10-12 2020-04-21 中国科学院合肥物质科学研究院 Mst1激酶抑制剂及其用途
CN111808816A (zh) * 2019-04-11 2020-10-23 北京基石生命科技有限公司 一种用于培养胃癌实体瘤原代细胞的培养基
CN113403278A (zh) * 2020-03-16 2021-09-17 合肥中科普瑞昇生物医药科技有限公司 胃癌原代细胞的培养基及培养方法
CN113528425A (zh) * 2020-04-15 2021-10-22 合肥中科普瑞昇生物医药科技有限公司 一种用于乳腺上皮干细胞的培养基和培养方法
CN113528444A (zh) * 2020-04-15 2021-10-22 合肥中科普瑞昇生物医药科技有限公司 一种用于食管鳞癌上皮细胞的培养基、培养方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG HAINA, WANG RUI, HUANG DAN, LI SIHAN, GAO BEIBEI, KANG ZHIJIE, TANG BO, XIE JIAJUN, YAN FANZHI, LIANG RUI, LI HUA, YAN JINSON: "Homoharringtonine Exerts Anti-tumor Effects in Hepatocellular Carcinoma Through Activation of the Hippo Pathway", FRONTIERS IN PHARMACOLOGY, vol. 12, XP093056583, DOI: 10.3389/fphar.2021.592071 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116590235A (zh) * 2023-07-14 2023-08-15 北京嘉士腾医学检验实验室有限公司 一种高效的消化道肿瘤类器官培养方法
CN116590235B (zh) * 2023-07-14 2023-11-21 北京嘉士腾医学检验实验室有限公司 一种高效的消化道肿瘤类器官培养方法

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