WO2023060820A1 - 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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WO2023060820A1
WO2023060820A1 PCT/CN2022/078774 CN2022078774W WO2023060820A1 WO 2023060820 A1 WO2023060820 A1 WO 2023060820A1 CN 2022078774 W CN2022078774 W CN 2022078774W WO 2023060820 A1 WO2023060820 A1 WO 2023060820A1
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concentration
alkyl
medium
culture
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 carcinoma is a malignant tumor originating from the gastric mucosal epithelium. Its incidence rate ranks first among various malignant tumors in my country. 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. The vast majority of gastric cancer is adenocarcinoma.
  • 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with the results of drug susceptibility testing of patient primary cells; however, if the murine feeder cells are withdrawn, the patient's own primary cells detach In addition to the reprogramming environment, the cell proliferation rate and intracellular signaling pathways will be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), which greatly affects the response of the patient's own primary cells to the drug.
  • 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 a culture method for rapid expansion of gastric cancer primary cells in vitro.
  • 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; N-acetyl-L-cysteine acid; gastrin; A8301; oncostatin M; and cholera toxin.
  • 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 1.25-20 ⁇ M, more preferably 2.5-10 ⁇ M;
  • the volume ratio of the B27 or N2 cell culture supplement to the medium is 1:25 to 1:400, more preferably 1:50 to 1:200;
  • the concentration of the basic fibroblast cytokine is 2.5-40 ng/mL, more preferably 5-20 ng/mL;
  • the volume ratio of the ITS cell culture supplement to the medium is 1:25 to 1:400, more preferably 1:50 to 1:200;
  • the concentration of the ROCK kinase inhibitor is 2.5-40 ⁇ M, more preferably 5-20 ⁇ M;
  • the concentration of the dexamethasone is 25-400nM, more preferably 50-200nM;
  • the concentration of CHIR99021 is 1.25-20 ⁇ M, more preferably 2.5-10 ⁇ M;
  • the concentration of the epidermal growth factor is 2.5-40 ng/mL, more preferably 2.5-20 ng/mL;
  • the concentration of the cholera toxin is 1.25-20 ng/mL, more preferably 2.5-10 ng/mL;
  • the concentration of gastrin is 1.25-20nM, more preferably 5-20nM;
  • the concentration of the SB202190 is 50-800nM, more preferably 50-200nM;
  • the concentration of N-acetyl-L-cysteine is 0.25-4mM, more preferably 1-4mM;
  • the concentration of Oncostatin M is 2.5-40 ng/mL, more preferably 2.5-10 ng/mL;
  • the concentration of A8301 is 1.25-20 nM, more preferably 2.5-20 nM.
  • the medium also contains an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one selected from streptomycin/penicillin, amphotericin B and Primocin one or more antibiotics.
  • the streptomycin concentration ranges from 25 to 400 ⁇ g/mL, and the penicillin concentration ranges from 25 to 400 U/mL; when the antibiotic is selected from amphotericin B, The concentration range is 0.25-4 ⁇ g/mL, and when the antibiotic is selected from Primocin, the concentration range is 25-400 ⁇ g/mL.
  • the invention also provides a method for culturing 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 low growth factor extracellular matrix gel, for example, commercially available Matrigel (purchased from BD Biotechnology Company) or BME (purchased from Trevigen Company) 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 tissue samples for example, come 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 guided by an endoscope. The above tissue samples were collected within half an hour of the patient's surgical resection or biopsy. More specifically, under a sterile environment, tissue samples from non-necrotic sites with a volume of more than 5 mm 3 were excised and transported to the laboratory on ice.
  • the tissue sample is transferred to a cell culture dish, and the blood cells on the surface of the tissue sample are washed away. Transfer the rinsed tissue sample to another new Petri dish, 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 sample fragments Transfer the tissue sample fragments to a centrifuge tube, and centrifuge at 1000-3000 rpm for 3-5 minutes with a desktop centrifuge (Sigma 3-18K); discard the supernatant, and add basal medium (such as DMEM /F12 medium containing 100 ⁇ g/mL Primocin) and tissue digestion solution (the preparation method of tissue digestion solution is: mix 1-2mg/mL collagenase II, 1-2mg/mL collagenase IV, 50-100U/mL deoxyribose Nuclease, 0.5 ⁇ 1mg/mL hyaluronidase, 1 ⁇ 5mM calcium chloride, 5 ⁇ 10mg/mL bovine serum albumin (dissolved in 1640 medium), mark the sample number, seal with parafilm, at 37°C, 200 ⁇ 300 rpm constant temperature shaker (ZQLY-180N) digestion, every half hour or 1 hour to observe whether the digestion is complete; if no obvious tissue blocks can be terminated, otherwise continue to
  • tissue clumps After the digestion is complete, filter out undigested tissue clumps with a cell strainer (cell sieve with a pore size of, for example, 70-100 ⁇ m), wash the tissue clumps on the filter with basal medium, wash the residual cells into a centrifuge tube, and centrifuge Centrifuge at 1000-3000 rpm for 3-5 minutes. Discard the supernatant and observe whether the remaining cell mass contains blood cells. If there are blood cells, add blood cell lysate (purchased from Sigma Company), mix well, lyse at 4°C for 10-20 minutes, shake and mix once every 5 minutes, take out after the lysis is completed, Centrifuge at 1000-3000 rpm for 3-5 minutes.
  • a cell strainer cell sieve with a pore size of, for example, 70-100 ⁇ m
  • 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).
  • 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 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;
  • 1A-1N 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.
  • 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.
  • extracellular matrix gel (manufactured by BD Biotechnology Co., Ltd.) Use serum-free DMEM/F12 medium at a ratio of 1:100 to prepare an extracellular matrix dilution, add 500 ⁇ l/well of the extracellular matrix dilution to a 48-well culture plate to completely cover it Bottom of culture plate wells. 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.
  • Counted primary gastric cancer cells were inoculated into Matrigel-coated 48-well culture plates at a cell density of 2 ⁇ 104 / cm2 , and placed in a 37°C, 5% CO2 incubator after surface disinfection (purchased from Thermo Fisher), the same number of freshly isolated gastric cancer tumor cells (No. GC-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.
  • MST1/2 kinase inhibitor compound 1 ROCK kinase inhibitor Y27632
  • B27 additive and N2 additive basic fibroblast growth factor
  • CHIR99021 basic fibroblast growth factor
  • epidermal growth factor ITS cell Culture additives
  • SB202190 dexamethasone
  • N-acetyl-L-cysteine gastrin
  • A8301 oncostatin M
  • cholera toxin cholera toxin and other factors
  • gastric cancer primary cells were obtained from intraoperative tissue samples (numbered GC-002, GC-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.
  • 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).
  • cholera toxin When the medium of formula 10 is used, 200 ⁇ L of prepared cholera toxin is added to each well of the 48-well plate inoculated with primary cells on the basis of formula 10, and the final concentrations of cholera toxin are 1.25 ng/mL, 2.5 ng/mL, respectively. ng/mL, 5ng/mL, 10ng/mL, 20ng/mL; and set up control wells (BC) using the medium of Formulation 10.
  • Oncostatin M prepared on the basis of Formula 13 to the 48-well plate inoculated with primary cells, and the final concentration of Oncostatin M is 2.5 ng /mL, 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL; and set up control wells (BC) using the medium of formula 13.
  • 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 1.25-20 ⁇ M, more preferably 2.5-10 ⁇ M; the concentration of Y27632 is preferably 2.5-40 ⁇ M, more preferably 5-20 ⁇ M; the volume of B27 The concentration is preferably 1:25-1:400, more preferably 1:50-1:200; the concentration of basic fibroblast cytokine is preferably 2.5-40 ng/mL, more preferably 5-20 ng/mL; the concentration of CHIR99021 Preferably 1.25-20 ⁇ M, more preferably 2.5-10 ⁇ M; the concentration of epidermal growth factor is preferably 2.5-40 ng/mL, more preferably 2.5-20 ng/mL; the volume concentration of ITS cell culture additive relative to the medium is preferably 1 :25 ⁇ 1:400, more preferably 1:50 ⁇ 1:200; the concentration of SB202190 is preferably 50 ⁇ 800nM, more preferably 50 ⁇ 200nM; the concentration of dexamethas
  • 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、N-乙酰-L-半胱氨酸、胃泌素、A8301、抑瘤素M、和霍乱毒素。使用该原代细胞培养基的培养方法，其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胃癌原代细胞，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培养的培养基、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，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及培养方法。
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；N-乙酰-L-半胱氨酸；胃泌素；A8301；抑瘤素M；和霍乱毒素。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 PCTCN2022078774-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 PCTCN2022078774-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 PCTCN2022078774-appb-000003
Figure PCTCN2022078774-appb-000004
Figure PCTCN2022078774-appb-000005
Figure PCTCN2022078774-appb-000006
Figure PCTCN2022078774-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1.25～20μM，更优选为2.5～10μM；
(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，更优选为1:50～1:200；
(3)所述碱性成纤维细胞因子的浓度为2.5～40ng/mL，更优选为5～20ng/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，更优选为1:50～1:200；
(5)所述ROCK激酶抑制剂的浓度为2.5～40μM，更优选为5～20μM；
(6)所述地塞米松的浓度为25～400nM，更优选为50～200nM；
(7)所述CHIR99021的浓度为1.25～20μM，更优选为2.5～10μM；
(8)所述表皮细胞生长因子的浓度为2.5～40ng/mL，更优选为2.5～20ng/mL；
(9)所述霍乱毒素的浓度为1.25～20ng/mL，更优选为2.5～10ng/mL；
(10)所述胃泌素的浓度为1.25～20nM，更优选为5～20nM；
(11)所述SB202190的浓度为50～800nM，更优选为50～200nM；
(12)所述N-乙酰-L-半胱氨酸的浓度为0.25～4mM，更优选为1～4mM；
(13)所述抑瘤素M的浓度为2.5～40ng/mL，更优选为2.5～10ng/mL；
(14)所述A8301的浓度为1.25～20nM，更优选为2.5～20nM。
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。
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BD生物科技公司)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。胃癌组织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。更具体而言，在无菌环境下，切取非坏死部位的组织样本，其体积在5mm 3以上，将其冰上运输至实验室。
在生物安全柜内，将组织样本转移至细胞培养皿内，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，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1000～3000转/分钟离心3～5分钟；弃上清，按1:3比例加入基础培养基(例如DMEM/F12培养基含100μg/mL Primocin)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脱氧核糖核酸酶、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℃、200～300转恒温摇床(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1000～3000转/分钟离心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1000～3000转/分钟离心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2×10 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1000～3000转/分钟离心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-1N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。
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 PCTCN2022078774-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 PCTCN2022078774-appb-000009
Figure PCTCN2022078774-appb-000010
Figure PCTCN2022078774-appb-000011
Figure PCTCN2022078774-appb-000012
Figure PCTCN2022078774-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，与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 PCTCN2022078774-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GC-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
Figure PCTCN2022078774-appb-000015
Figure PCTCN2022078774-appb-000016
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MST1/2激酶抑制剂化合物1、ROCK激酶抑制剂Y27632、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、N-乙酰-L-半胱氨酸、胃泌素、A8301、抑瘤素M、霍乱毒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GC-002、 GC-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 PCTCN2022078774-appb-000017
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-2.2培养基相同。以下配方1-14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碱性成纤维细胞因子每孔200μL，碱性成纤维细胞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表皮细胞生长因子每孔200μL，表皮细胞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霍乱毒素每孔200μL，霍乱毒素的终浓度分别为1.25ng/mL、2.5ng/mL、5ng/mL、10ng/mL、20ng/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N-乙酰-L-半胱氨酸每孔200μL，N-乙酰-L-半胱氨酸的终浓度分别为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)。
在使用配方13的培养基时，在接种有原代细胞的48孔板中在配方13的基础上分别添加配制好的抑瘤素M每孔200μL，抑瘤素M的终浓度分别为2.5ng/mL、5ng/mL、10ng/mL、20ng/mL、40ng/mL；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48孔板中在配方14的基础上分别添加配制好的A8301每孔200μL，A8301的终浓度分别为1.25nM、2.5nM、5nM、10nM、20nM；并使用配方14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～1N。图1A～1N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～1N的结果，MST1/2激酶抑制剂化合物1的含量优选为1.25～20μM，更优选为2.5～10μM；Y27632的浓度优选为2.5～40μM，更优选为5～20μM；B27的体积浓度优选为1:25～1:400，更优选为1:50～1:200；碱性成纤维细胞因子的浓度优选为2.5～40ng/mL，更优选为5～20ng/mL；CHIR99021的浓度优选为1.25～20μM，更优选为2.5～10μM；表皮细胞生长因子的浓度优选为2.5～40ng/mL，更优选为2.5～20ng/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；SB202190的浓度优选为50～800nM，更优选为50～200nM；地塞米松的浓度优选为25～400 nM，更优选为50～200nM；霍乱毒素的浓度优选为1.25～20ng/mL，更优选为2.5～10ng/mL；N-乙酰-L-半胱氨酸的浓度优选为0.25～4mM，更优选为1～4mM；胃泌素的浓度优选为1.25～20nM，更优选为5～20nM；抑瘤素M的浓度优选为2.5～40ng/mL，更优选为2.5～10ng/mL；A8301的浓度优选为1.25～20nM，更优选为2.5～20nM。
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；N-乙酰-L-半胱氨酸；胃泌素；A8301；抑瘤素M；和霍乱毒素，
    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 PCTCN2022078774-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 PCTCN2022078774-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 PCTCN2022078774-appb-100003
    Figure PCTCN2022078774-appb-100004
    Figure PCTCN2022078774-appb-100005
    Figure PCTCN2022078774-appb-100006
    Figure PCTCN2022078774-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1.25～20μM；
    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因子的浓度为2.5～40ng/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1.25～20μM；
    所述表皮细胞生长因子的浓度为2.5～40ng/mL；
    所述霍乱毒素的浓度为1.25～20ng/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；
    所述抑瘤素M的浓度为2.5～40ng/mL；
    所述A8301的浓度为1.25～20nM。
  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：
    所述MST1/2激酶抑制剂的浓度为2.5～10μM；
    所述B27或N2细胞培养添加剂相对于培养基的体积比为1:50～1:200；
    所述碱性成纤维细胞因子的浓度为5～20ng/mL；
    所述ITS细胞培养添加剂相对于培养基的体积比为1:50～1:200；
    ROCK激酶抑制剂的浓度为5～20μM；
    所述地塞米松的浓度为50～200nM；
    所述CHIR99021的浓度为2.5～10μM；
    所述表皮细胞生长因子的浓度为2.5～20ng/mL；
    所述霍乱毒素的浓度为2.5～10ng/mL；
    所述胃泌素的浓度为5～20nM；
    所述SB202190的浓度为为50～200nM；
    所述N-乙酰-L-半胱氨酸的浓度为1～4mM；
    所述抑瘤素M的浓度为2.5～10ng/mL；
    所述A8301的浓度为2.5～20nM。
  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. 如权利要求1～8中任一项所述的培养基，其特征在于所述培养基不含Wnt激动剂、R-spondin家族蛋白、Noggin蛋白、BMP抑制剂。
  10. 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1～9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上皮细胞，并使用步骤(1)中所述的培养基进行培养。
PCT/CN2022/078774 2021-10-15 2022-03-02 胃癌原代细胞的培养基和培养方法 WO2023060820A1 (zh)

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