WO2023060681A1 - 一种用于宫颈癌原代细胞的培养基和培养方法 - Google Patents

一种用于宫颈癌原代细胞的培养基和培养方法 Download PDF

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WO2023060681A1
WO2023060681A1 PCT/CN2021/130057 CN2021130057W WO2023060681A1 WO 2023060681 A1 WO2023060681 A1 WO 2023060681A1 CN 2021130057 W CN2021130057 W CN 2021130057W WO 2023060681 A1 WO2023060681 A1 WO 2023060681A1
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cervical cancer
cells
culture medium
alkyl
culture
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French (fr)
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刘青松
胡洁
侯雪婷
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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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • the invention belongs to the technical field of medicine, and in particular relates to a culture medium and a culture method for culturing or expanding primary cervical cancer epithelial cells in vitro.
  • cervical cancer both rank fourth among female malignant tumors in the world, and it is the second largest malignant tumor after breast cancer in developing countries.
  • surgery, radiotherapy and chemotherapy for cervical cancer have become mature treatment methods. But for some patients with locally advanced cervical cancer, satisfactory results have not been achieved.
  • new targeted therapy drugs continue to emerge, bringing new hope to patients.
  • targeted therapy drugs that have definite curative effects on cervical cancer, and their long-term effects and side effects need further study.
  • Functional testing refers to the method of testing the sensitivity of antineoplastic drugs on cancer patient cells in vitro.
  • the key to applying this approach lies in the development of tumor cell models with short growth cycles that represent the biology of cervical cancer patients themselves.
  • the cell model should be easy to operate, and can quickly and efficiently predict the curative effect of clinical medication, so as to give precise medication guidance to cancer patients in a timely manner.
  • the success rate of primary tumor cells from cancer patients to establish cell models in vitro is often very low, the growth cycle is long, and there are problems such as excessive proliferation of mesenchymal cells such as fibroblasts, which restrict the development of this field.
  • there are two technologies for culturing primary epithelial cells/stem cells that are relatively mature in the field of functional testing of tumor cells.
  • irradiated feeder cells and ROCK kinase inhibitor Y27632 to promote the growth of primary epithelial cells to investigate the drug sensitivity of individual patients, that is, cell conditional reprogramming technology (Liu et al., Am J Pathol, 180: 599-607, 2012).
  • Another technique is to culture adult stem cells in vitro in 3D to obtain organoids similar to tissues and organs (Hans Clevers et al., Cell, 11, 172(1-2): 373-386, 2018).
  • 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 cervical cancer tumor cells can represent the biological characteristics of cervical cancer patients themselves.
  • the present invention aims at the deficiencies of the prior art, and provides a culture medium for culturing primary cervical cancer epithelial cells and a culture method for primary cervical cancer epithelial cells using the culture medium.
  • Adopting the primary cervical cancer epithelial cell culture medium and the culture method of the present invention for cell culture can achieve the purpose of short in vitro culture period, controllable cost, convenient operation and no interference from exogenous cells.
  • this technology is applied to the construction of primary cervical cancer tumor cell models, primary cervical cancer tumor cells with biological characteristics of cervical cancer patients can be obtained, and can be applied to new drug screening and in vitro drug sensitivity testing.
  • One aspect of the present invention is to provide a primary cell culture medium for culturing primary cervical cancer epithelial cells, which contains an MST1/2 kinase inhibitor; at least one selected from Y27632, Fasudil, and H-1152 A ROCK kinase inhibitor; fibroblast growth factor 7 (FGF7); at least one of B27 supplement and N2 supplement; hepatocyte growth factor (HGF); insulin-like growth factor 1 (IGF-1); CHIR99021 and a TGF ⁇ type I receptor inhibitor selected from at least one of A83-01, SB431542, Repsox, SB505124, SB525334, SD208, LY36494, and SJN2511, said MST1/2 kinase inhibitor comprising 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 the MST1/2 kinase inhibitor in the culture medium is usually 2 ⁇ M-20 ⁇ M, preferably 5 ⁇ M-20 ⁇ M.
  • the ROCK kinase inhibitor is preferably Y27632.
  • the content of the ROCK kinase inhibitor in the culture medium is usually 2 ⁇ M to 20 ⁇ M, preferably 10 ⁇ M.
  • the content of the fibroblast growth factor 7 is 2ng/ml ⁇ 40ng/ml, more preferably 10ng/ml ⁇ 40ng/ml; the volume of the B27 additive or N2 additive in the culture medium The concentration is 1:25 ⁇ 1:100, more preferably 1:50 ⁇ 1:100; the content of the hepatocyte growth factor is 2ng/ml ⁇ 40ng/ml, more preferably 10ng/ml ⁇ 40ng/ml; The content of the insulin-like growth factor 1 is 2ng/ml-40ng/ml, more preferably 10ng/ml; the content of the CHIR99021 is 1 ⁇ M-10 ⁇ M, more preferably 1 ⁇ M-3 ⁇ M; the TGF ⁇ type I receptor inhibitor is preferably A83-01, and the content of the TGF ⁇ type I receptor inhibitor is 50nM-500nM, more preferably 100nM-500nM.
  • the culture medium formula of the present invention also contains the initial culture medium that is selected from DMEM/F12, DMEM, F12 or RPMI-1640; And is selected from one or more antibiotics in streptomycin/penicillin, amphotericin B and Primocin .
  • the initial medium is preferably DMEM/F12, and the antibiotic is preferably Primocin.
  • the content of Primocin in the culture medium is 25-400 ⁇ g/mL, preferably 50-200 ⁇ g/mL.
  • the composition of the medium formula of the present invention is added with MST1/2 kinase inhibitor, and does not contain uncertain components such as serum and bovine pituitary extract, It also does not contain niche factors necessary for organoid culture such as Wnt agonists, R-spondin family proteins, and BMP inhibitors, and does not contain nicotinamide (Nicotinamide) and N-acetylcysteine (N-Acetylcysteine), thereby greatly The cost of the medium is reduced, the operation process of preparing the medium is simplified, and the in vitro culture of primary cervical cancer epithelial cells with controllable cost and convenient operation is realized.
  • primary cervical cancer epithelial cells can be cervical cancer tumor cells, normal cervical cancer epithelial cells, and cervical cancer epithelial stem cells.
  • One aspect of the present invention is to provide a method for culturing primary cervical cancer epithelial cells, which 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 cervical cancer epithelial cells can be derived from cervical cancer surgical samples or biopsy samples, for example.
  • Cervical cancer surgical samples are, for example, derived from surgically resected cancer tissue samples from patients with cervical cancer tumors who have explained and obtained consent. The above tissue samples were collected within half an hour of the patient's surgical resection or biopsy sampling. More specifically, in a sterile environment, cut tissue samples from non-necrotic parts with a volume of more than 0.5 cm 3 , and place them in pre-cooled 10-15 mL DMEM/F12 medium, which is contained in a plastic non-destructive medium.
  • the DMEM/F12 medium contains the MST1/2 kinase inhibitor of the present invention (such as compound 1) and 0.2-0.4% by volume Primocin (hereinafter referred to as tissue transport solution) .
  • the concentration range is 2 ⁇ M to 20 ⁇ M, preferably 3 ⁇ M to 5 ⁇ M; when using Primocin, the concentration range is 25 to 400 ⁇ g/mL, preferably 50 to 200 ⁇ g/mL, more preferably 100 ⁇ g/mL.
  • the tissue sample is transferred to a cell culture dish, and the tissue sample is rinsed with tissue transport solution to wash away the blood cells on the surface of the tissue sample. Transfer the rinsed tissue sample to another new Petri dish, add 1-3mL of tissue transport solution, 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, centrifuge at 1000-3000 rpm for 3-5 minutes with a desktop centrifuge (Sigma 3-18K); discard the supernatant, add tissue transport solution and tissue digestion at a ratio of 1:1 (About 5mL tissue digestion solution per 10mg tissue, the preparation method of tissue digestion solution is: mix 1-2mg/mL collagenase II, 1-2mg/mL collagenase IV, 50-100U/mL deoxyribose Nucleic acid I, 0.5 ⁇ 1mg/mL hyaluronidase, 0.1 ⁇ 0.5mg/mL calcium chloride, 5 ⁇ 10mg/mL bovine serum albumin dissolved in HBSS and RPMI-1640 with a volume ratio of 1:1), labeled samples Serial number, seal with parafilm, digest with a constant temperature shaker (ZQLY-180N) at 37°C, 200-300 rpm, and observe whether the digestion is complete every 1 hour; if no
  • tissue agglomerates are filtered out by a cell strainer (the pore size of the cell sieve is, for example, 70 ⁇ m).
  • the tissue agglomerates on the strainer are washed with tissue transport solution, and the residual cells are washed into a centrifuge tube, and then washed with a benchtop centrifuge to 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 3-5mL 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.
  • 3-5mL blood cell lysate purchased from Sigma Company
  • step (3) Inoculate the primary cervical cancer epithelial cells isolated in step (3) into the coated culture vessel, and culture with the primary cell culture medium in step (1).
  • the cancer epithelial cells are digested and passaged when the cell density reaches about 80%-90% of the bottom area of the multi-well plate.
  • 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 cervical cancer epithelial cells are cultured for 8 to 16 days, when the cell clones formed in the culture vessel are confluent and 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 incubate at room temperature for 5-20 minutes; ⁇ 4mL resuspended digested cells, centrifuged at 1000 ⁇ 3000 rpm for 3 ⁇ 5 minutes, used the primary cell culture medium of the present invention to resuspend the digested single cells, and put the obtained cell suspension into Continue to expand the culture in the T25 cell culture flask coated with extracellular matrix gel.
  • the coating operation of the T25 cell culture flask is the same as step (2).
  • the expanded cervical cancer epithelial 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 cervical cancer, comprising the following steps:
  • the cultured primary cervical cancer epithelial cells are not interfered by fibroblasts, and purified cervical 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;
  • cervical cancer epithelial cells The efficiency of expanding cervical cancer epithelial cells is high. As long as there are 10 4 cells, cervical cancer epithelial cells of the order of 10 6 can be successfully amplified in about two weeks. Can be passed down continuously;
  • 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 cost of culture is controllable.
  • the primary cervical cancer cell culture medium does not need to add expensive Wnt agonists, R-spondin family proteins, BMP inhibitors and other factors.
  • the cell inoculation does not need to use a higher concentration of extracellular matrix to mix with the primary cells to form gel droplets, but only needs to use a dilution prepared by a small amount of extracellular matrix gel, which saves the costly extracellular matrix dosage;
  • the operation is convenient. Compared with the conditional reprogramming technology, this technology does not need to cultivate feeder cells and irradiate the feeder cells, avoiding the problem that the quality and quantity of different batches of feeder cells affect the efficiency of primary cell culture, and the plating of drug screening
  • the object of detection and detection is only the primary cervical cancer epithelial cells, and is not disturbed by the feeder cells in the co-culture system required by the cell conditional reprogramming technology;
  • the coating method of extracellular matrix gel used in the present invention the culture vessel can be prepared in advance, no need to embed the cells in the Matrigel like the organoid technique, and the operation steps of the technique are simple and easy;
  • cervical cancer epithelial cells derived from humans or other mammals can be cultured, including cervical cancer tumor cells, normal cervical epithelial cells, cervical cancer epithelial stem cells, or at least any of these cells A kind of organization.
  • the culture medium of the technology of the present invention can also be used to develop a kit for expanding and culturing primary cervical cancer cells in vitro.
  • the cells obtained by the culture method of this embodiment can be applied to regenerative medicine, basic medical research of cervical cancer epithelial cells, screening of drug response, and research and development of new drugs derived from cervical cancer diseases.
  • 1A-1H are graphs showing the effect of the concentration of each added factor on the proliferation of cervical cancer primary cells.
  • Figures 2A and 2B are photographs taken under an inverted microscope of cervical cancer tumor cells cultured on the 4th and 12th day of cells isolated from a clinical tissue sample of cervical cancer using the medium FCM of the present invention.
  • 3A-3C are photos taken under an inverted microscope after cells isolated from a surgical resection sample of a case of cervical cancer were cultured in three different medium conditions for 15 days.
  • Fig. 4 is a comparison chart of the cell proliferation effect obtained after the cells isolated from surgical resection samples of 9 cases of cervical cancer were cultured under three different medium conditions for 16 days.
  • Fig. 5 is a comparison chart of cell growth curves obtained by culturing cells isolated from a clinical tissue sample of cervical cancer using three different medium conditions.
  • Fig. 6 is a comparison chart of immunohistochemical results of cervical cancer tumor cells cultured with the culture medium FCM of the present invention from cells isolated from a surgical resection sample of cervical cancer.
  • Figure 7 shows the cell activity curves of cervical cancer epithelial cells obtained by culturing cells isolated from 3 cases of cervical cancer surgical resection samples according to the method of the present invention under the influence of 8 different drugs.
  • epithelial cells include differentiated epithelial cells and epithelial stem cells obtained from epithelial tissues.
  • Epithelial stem cells refer to cells with long-term self-renewal ability and epithelial cell differentiation, and refer to stem cells derived from epithelial tissue.
  • epithelial tissues include cervix, cornea, oral mucosa, skin, conjunctiva, bladder, renal tubules, kidneys, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), large intestine (including colon )), liver, pancreas, mammary gland, salivary gland, lacrimal gland, prostate gland, hair root, trachea, lung, etc.
  • the cell culture medium of the present embodiment is preferably a culture medium for culturing cervical epithelial cells.
  • epithelial tumor cell means the cell which the cell derived from the above-mentioned epithelial tissue was tumorized.
  • organoid refers to a three-dimensional, organ-like cell organoid formed by spontaneously organizing and aggregating cells at high density in a controlled space.
  • 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.
  • Cervical cancer tissue samples were obtained from surgically resected cancer tissue samples from patients with cervical cancer tumors who had explained and obtained consent. One of the samples (No. CCa2) will be described below.
  • tissue samples were collected within half an hour of the patient's surgical resection or biopsy. More specifically, in a sterile environment, cut tissue samples from non-necrotic parts with a volume of more than 0.5 cm 3 , and place them in 4 mL of pre-cooled tissue transport solution (see Table 1 for specific preparation). In 5mL plastic sterile cryopreservation tubes with lids (purchased from Guangzhou Jiete Biology), cold chain (0-10°C) transport to the laboratory.
  • Tissue Transport Fluid Components supplier Final concentration DMEM/F12 Corning 99.8% by volume Primocin Invivogen 0.2% by volume (commercially available product concentration 50mg/ml) Compound 1 self made 3 ⁇ M
  • tissue sample code CCa2
  • tissue transport solution a 100mm cell culture dish (purchased from NEST Company)
  • tissue transport solution a 100mm cell culture dish
  • tissue transport solution a 100mm cell culture dish
  • tissue transport solution a 100mm cell culture dish
  • tissue transport solution a 100mm cell culture dish
  • tissue transport solution a sterile scalpel blade and forceps to divide the tissue sample into tissue fragments with a volume less than 3mm 3 .
  • tissue sample fragments Transfer the tissue sample fragments to a 15mL centrifuge tube, and centrifuge at 1500 rpm for 4 minutes with a desktop centrifuge (Sigma 3-18K); discard the supernatant, and add tissue transport solution and tissue digestion solution at a ratio of 1:1 (using The amount is about 5mL of tissue digestion solution per 10mg of tissue, see Table 2 for specific preparation), mark the sample number, seal with parafilm, and digest with a constant temperature shaker (ZQLY-180N) at 37°C and 300 rpm, with an interval of 1 hour Watch to see if digestion is complete.
  • a constant temperature shaker ZQLY-180N
  • tissue agglomerates are filtered out with a 70 ⁇ m filter, and the tissue agglomerates on the filter are washed with tissue transport solution, and the residual cells are washed into a centrifuge tube, and centrifuged at 1500 rpm for 4 minutes.
  • 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.
  • BM basal medium
  • Primocin purchased from Invivogen, the concentration is 50 mg/mL
  • DMEM/F-12 medium DMEM/F-12 medium
  • Cervical cancer tumor cells (coded CCa5) isolated from cervical cancer tissue according to the same method as in Example 1 were inoculated in a 384-well culture plate at a cell density of 3000 cells/well, and 50 ⁇ l/well of medium with different components were added. After disinfection, they were placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher), and the same number of freshly isolated cervical cancer tumor cells (code CCa5) were cultured under different medium formulation conditions.
  • the ratio refers to the ratio of the absorbance value detected by adding CCK8 after culturing in different media to the absorbance value detected after culturing in basal medium BM. As shown in the table, adding different factors in Table 3 on the basis of BM produced different effects on cell proliferation.
  • B27 additive in a specific concentration range, B27 additive, N2 additive, fibroblast growth factor 7, CHIR99021, hepatocyte growth factor, insulin-like growth factor 1, compound 1, Y27632 and A83-01 all had better promoting effects on cell proliferation. is significant.
  • 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 200 ⁇ 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.
  • FGF7 fibroblast growth factor 7
  • HGF Hepatocyte growth factor
  • IGF-1 insulin-like growth factor 1
  • B27 additive at a final concentration of 1:50 volume ratio
  • compounds at a final concentration of 5 ⁇ M 1.
  • Add Y27632 at a final concentration of 10 ⁇ M
  • add TGF ⁇ 1 inhibitor A83-01 at a final concentration of 500 nM
  • CHIR99021 at a final concentration of 10 ⁇ M to prepare primary cervical cancer epithelial cell culture medium.
  • cervical cancer epithelial cells derived from cancer tissue were isolated from cancer tissue (coded CCa8) of a cervical cancer patient.
  • the cervical cancer epithelial cells derived from the cancer tissue were counted with a flow image counter (Jimbio FIL, Jiangsu Zhuo Microbial Technology Co., Ltd.) to obtain the total number of cells.
  • the basal medium BM was used to resuspend the centrifuged cell pellet, and the flow image counter (Jiangsu Zhuo Microbe Technology Co., Ltd. JIMBIO FIL) was used for counting to obtain the total number of cells.
  • the obtained cells were used in the following culture experiments.
  • Formula 1 the above medium components do not contain B27 additive
  • Formula 2 the above medium components do not contain fibroblast growth factor 7;
  • Insulin-like growth factor 1 is not included in the above medium components;
  • Formula 4 the above medium components do not contain hepatocyte growth factor
  • the above-mentioned recipes 1-8 were respectively used to dilute the above-mentioned digested cell suspension, and seeded into a 48-well plate with 10,000 cells per well and a volume of 250 microliters.
  • 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 proliferation-promoting effect than the culture medium of the control well, and 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 concentration of the B27 additive in the medium is preferably 1:25 to 1:100, more preferably 1:50 to 1:100;
  • the content of fibroblast growth factor 7 is preferably 2ng/ml ⁇ 40ng/ml, more preferably 10ng/ml ⁇ 40ng/ml;
  • the content of insulin-like growth factor 1 is preferably 2ng/ml ⁇ 40ng/ml, more preferably 10ng/ml ⁇ 40ng/ml;
  • the content of hepatocyte growth factor is preferably 2ng/ml ⁇ 40ng/ml, more preferably 10ng/ml ⁇ 40ng/ml;
  • the content of Y27632 is preferably 2 ⁇ M ⁇ 20 ⁇ M, more preferably 2 ⁇ M ⁇ 10 ⁇ M;
  • the content of compound 1 is preferably 2 ⁇ M ⁇ 20 ⁇ M, more preferably 5 ⁇ M ⁇ 20 ⁇ M;
  • the content of A83-01 is preferably 50 nM-500 nM, more preferably 100 nM-500 nM;
  • the preferred medium formula FCM of the present invention is prepared, which comprises: basal medium (BM), 10ng/ml fibroblast growth factor 7 (FGF7), 10ng/ml hepatocyte growth factor (HGF), 10 ng/ml insulin-like growth factor 1 (IGF-1), 1:50 volume ratio of B27 supplement, 5 ⁇ M compound 1, 10 ⁇ M Y27632, 500 nM A83-01, and 3 ⁇ M CHIR99021.
  • BM basal medium
  • FGF7 fibroblast growth factor 7
  • HGF hepatocyte growth factor
  • IGF-1 insulin-like growth factor 1
  • cervical cancer epithelial cells derived from cancer tissue were isolated from cancer tissue of a patient with cervical cancer (sample number CCa15).
  • the cervical cancer epithelial cells derived from the cancer tissue were counted with a flow image counter (Jimbio FIL, Jiangsu Zhuo Microbial Technology Co., Ltd.) to obtain the total number of cells.
  • inoculate at a density of 4 ⁇ 10 4 /cm 2 to (purchased from BD Biotechnology Company) wrapped in a treated 12-well plate.
  • Add 2 mL of the prepared primary cervical cancer epithelial cell culture medium FCM to the 12-well plate, and place it in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher) for culture.
  • Fig. 2A is a photomicrograph (photographed by a 10-fold inverted phase-contrast microscope) of this example inoculated onto a Matrigel-coated 6-well plate at a density of 4 ⁇ 10 4 cells/cm 2 and cultured from the beginning of inoculation to the 4th day. Observation under the microscope shows that the primary cervical cancer tumor cells derived from the cultured cancer tissue are of high purity and do not contain fibroblasts.
  • Fig. 2B is a microscopic photograph (photographed with a 40-fold inverted phase-contrast microscope) of this example from the 12th day after inoculation.
  • Example 2 The same method as in Example 2 was used to prepare the primary cervical cancer epithelial cell medium FCM and the basal medium BM as a control.
  • literature medium RM was prepared as another control example.
  • the preparation steps see (Ma Liping et al., Jilin Medicine, 42(6):1289-1293, 2021), and the medium formula is shown in Table 4.
  • Example 2 The same method as in Example 1 was used to obtain primary cervical cancer tumor cells (number CCa10) derived from cervical cancer tissue. Next, they were cultured under the following three culture conditions at the same density (4 ⁇ 10 4 cells/cm 2 ):
  • the technology of the present invention inoculate the primary cervical cancer tumor cells into the ((BD Biotechnology Co., Ltd.)) wrapped in a treated 24-well plate, using 2 mL of the primary cervical cancer epithelial cell culture medium FCM of the present invention to cultivate;
  • the medium was changed every 4 days for the cells cultured under the three culture conditions.
  • EVOS M500 from Invitrogen Company
  • the primary cervical cancer tumor cells (numbered CCa10) cultivated by the technology of the present invention, when the cell growth in the culture plate reaches about 80% of the bottom area, discard the supernatant of the culture medium in the 24-well plate, and add 500 ⁇ L of 0.05% Digest the cells with trypsin (purchased from GIBCO), and incubate at 37°C for 10 minutes until it can be observed under a microscope (EVOS M500 from Invitrogen that the cells have been completely digested), that is, with 5% (v/v) fetal bovine serum 1 mL of DMEM/F12 culture medium containing 100 U/mL penicillin and 100 ⁇ g/mL streptomycin was used to stop the digestion, and collected into a 15 mL centrifuge tube, centrifuged at 1500 rpm for 4 minutes, and discarded the supernatant.
  • trypsin purchased from GIBCO
  • the culture medium of the present invention to resuspend the centrifuged cell pellet, use a flow image counter (Jiangsu Zhuo Microbial Technology Co., Ltd. JIMBIO FIL) to count, and the total number of cells is 190,000.
  • the cells cultured under the other two culture conditions were digested and counted in the same manner as above, and the total number of cells cultured with medium FM and CM were 45,000 and 46,000, respectively.
  • Figure 3A-3C are microscopic photographs (under a 10-fold inverted phase-contrast microscope) of samples numbered CCa10 cultured to the 15th day under three different culture conditions: where Figure 3A is that CCa10 was cultured to the 15th day using basal medium BM Microscopic photos on the 15th day; Figure 3B is a microscopic photo of CCa10 cultured to the 15th day using the patent medium FCM; Figure 3C is a microscopic photo of CCa10 cultured to the 15th day using the literature medium RM.
  • Figure 4 is a comparison chart of the cell proliferation effect obtained after 16 days of primary cervical cancer cells obtained from 9 cases of cervical cancer patient samples according to the method of Example 1 and cultured in the above three different medium conditions, where ⁇ represents general cloning formation Ability and proliferation-promoting effect, ⁇ means that it has obvious clone formation ability and proliferation-promoting effect, ⁇ means it has strong clone-forming ability and proliferation-promoting effect, and ⁇ means it cannot form clones. From Figure 6, it can be confirmed that the culture medium of the present invention has obvious advantages over the other two culture conditions in terms of colony formation ability, cell proliferation promoting effect, and culture success rate when culturing primary cells obtained from cervical cancer tissue .
  • the primary cervical cancer epithelial cell culture medium FCM, and culture medium BM and RM as controls were obtained by the same method as in this example (1).
  • the primary cervical cancer tumor cells (coded CCa5) derived from cervical cancer tissue were cultured in three culture medium conditions, digested, passaged and counted.
  • the cultured cells were digested and collected according to the above operation method again and counted. Also inoculate at a density of 4 ⁇ 10 4 cells/well and continue culturing.
  • PD Population Doubling
  • Figure 5 is the growth curve of CCa5 cells drawn under three different culture conditions using Graphpad Prism software.
  • the abscissa indicates the number of days of cell culture, and the ordinate indicates the cumulative cell proliferation multiple, which indicates the multiple of cell expansion during the culture period. The larger the value, the more times the cells are expanded within a certain period, that is, the expanded cells The more the number, the slope represents the rate of cell expansion. It can be confirmed from the figure that the proliferation rate of the cervical cancer epithelial cells cultured in the medium FCM of the present invention is better than the other two culture conditions.
  • a cancer tissue about the size of a mung bean (sample number CCa14) was taken from a clinical surgical resection sample of a patient with cervical cancer, soaked in 1 mL of 4% paraformaldehyde and fixed. The remaining cancer tissue was obtained using the same method as in Example 1 to obtain cervical cancer epithelial cells (sample number CCa14). Using the method of Example 3, the sample CCa14 was continuously cultured to the third generation using the medium FCM of the present invention.
  • Immunohistochemical method was used to detect the expression of important biomarkers related to cervical cancer in original CCa14 tissue and primary cells obtained from continuous culture to the third passage. Tissues fixed with 4% paraformaldehyde were embedded in paraffin and cut into 4 ⁇ m thick tissue sections with a microtome. Followinged by routine immunohistochemical detection (see Li et al., Nature Communication, (2016) 9:2983 for specific steps).
  • the primary antibodies used were P16 antibody (purchased from Affinit Company), P63 antibody (purchased from CST Company) and Ki67 antibody (purchased from CST Company).
  • the following takes surgical resection samples from patients with cervical cancer as an example to illustrate that cervical cancer tumor cells cultured from patient-derived cervical cancer tumor samples can be used to detect the sensitivity of patient tumor cells to different drugs.
  • Plating of primary cervical cancer tumor cells the suspension of isolated cervical cancer tumor cells (numbered CCa5, numbered CCa6 and numbered CCa9) obtained according to the method in Example 1 was inoculated at a density of 4 ⁇ 10 cells/cm 2 into a 12-well plate. Add 2 mL of the prepared primary cervical cancer epithelial cell culture medium FCM to the 12-well plate, and place it in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher) for culture.
  • the cell pellets after centrifugation were resuspended in FCM medium, and counted using a flow image counter (Jiangsu Zhuo Microbial Technology Co., Ltd. JIMBIO FIL), and the total number of cells was 530,000, 780,000, and 630,000, respectively.
  • the cells were seeded in 384-well plates at a density of 1000-2000 cells/well, and the cells were allowed to adhere to the wall overnight.
  • the drug storage plate is prepared by the method of concentration gradient dilution: draw 40 ⁇ L of 10 ⁇ M mother solution of the drug to be tested as the highest concentration, and then draw 10 ⁇ L from it and add it to a 0.5mL EP tube containing 20 ⁇ L of DMSO, and then draw from the above EP Pipette 10 ⁇ L into the second 0.5mL EP tube filled with 20 ⁇ L of DMSO, that is, dilute the drug 1:3. Repeat the above method, dilute in turn, and finally obtain the 7 concentrations required for dosing. Drugs at different concentrations were added to 384-well drug storage plates. An equal volume of DMSO was added to each well of the solvent control group as a control.
  • the drugs to be tested are cisplatin (purchased from MCE Company), paclitaxel (purchased from MCE Company), 5-fluorouracil (5-F, purchased from MCE Company), topotecan (purchased from MCE Company), Bortezomib (purchased from MCE Company), anlotinib (purchased from MCE Company), pazopanib (purchased from MCE Company), and apatinib (purchased from MCE Company) and.
  • Detection of cell activity 72 hours after administration, use the Cell Titer-Glo detection reagent (purchased from Promega) to detect the chemiluminescence value of the cells after drug-added culture.
  • the chemiluminescence value reflects the cell viability and the drug's effect on the cell 10 ⁇ L of the prepared Cell Titer-Glo detection solution was added to each well, and after mixing, the chemiluminescence value was detected with a microplate reader (Envision of Perkin Elmer Company).
  • cell survival rate (%) chemiluminescence value of drug-dosing well/chemiluminescence value of control well*100%, calculate the cell survival rate after different drugs act on the cells, use Graphpad Prism software to plot and calculate the half inhibitory rate IC50 .
  • Fig. 7 respectively shows that the cervical cancer tumor cells obtained from three different cervical cancer patients' surgical resection cancer tissue samples (numbering CCa5, numbering CCa6 and numbering CCa9) have different effects on four chemotherapeutic drugs cisplatin, paclitaxel and 5-fluorouracil. and topotecan drug sensitivity and sensitivity to four targeted drugs bortezomib, anlotinib, pazopanib and apatinib.
  • the results show that the cells of the same patient have different sensitivities to different concentrations of drugs, and the cells of different patients have different sensitivities to the same drug. According to the results, the effectiveness of the drug in cervical cancer patients can be judged. .
  • the invention provides a culture medium and a culture method for culturing primary cervical cancer epithelial cells, and the cultured cells can be applied to the curative effect evaluation and screening of drugs.
  • the present invention is suitable for industrial applications.

Abstract

一种用于培养原代宫颈癌上皮细胞的培养基，其含有MST1/2激酶抑制剂、ROCK激酶抑制剂、成纤维细胞生长因子7、B27添加剂和N2添加剂中的至少一种添加剂、肝细胞生长因子、胰岛素样生长因子1、CHIR99021、和TGFβI型受体抑制剂。还涉及使用该原代细胞培养基的培养方法及其应用。该培养方法使用上述原代细胞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，培养获得的细胞可用于评估或筛选用于治疗宫颈癌的药物。

Description

一种用于宫颈癌原代细胞的培养基和培养方法 技术领域
本发明属于医药技术领域，具体而言，涉及用于在体外培养或扩增原代宫颈癌上皮细胞的培养基及培养方法。
背景技术
宫颈癌的发病率和病死率均居全球女性恶性肿瘤的第四位，而在发展中国家中是仅次于乳腺癌的第二大恶性肿瘤。全世界每年发病50万人，我国每年13万人发病，且近来年发病年龄趋于年轻化。目前宫颈癌的手术、放射治疗及化学治疗，已经成为成熟的治疗方法。但对于某些局部晚期宫颈癌患者并未取得令人满意的疗效。随着针对宫颈癌分子靶点的研究深入，新的靶向治疗药物不断涌现，给患者带来了新的希望。但是，目前对宫颈癌有确切疗效的靶向治疗药物还很有限，其远期效果及毒副作用尚待进一步研究。除了基因检测选取靶向药物，体外对宫颈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，但是体外快速获得宫颈癌原代细胞一直是亟待解决的技术问题。
功能性测试是指在体外对抗肿瘤药物在癌症患者细胞上的敏感性进行检测的方法。应用这一方法的关键在于开发生长周期短且能够代表宫颈癌患者自身生物学特性的肿瘤细胞模型。另外，所述细胞模型应操作便捷，能快速高效地预测临床用药的疗效，从而及时给予癌症患者精准用药指导。然而，取自癌症患者的原代肿瘤细胞在体外建立细胞模型的成功率往往很低，生长周期长，并存在成纤维细胞等间质细胞过度增殖等问题，制约着这一领域的发展。目前有两种培养原代上皮细胞/干细胞的技术在肿瘤细胞功能性测试应用领域发展得相对成熟。一种是使用经辐射的饲养细胞和ROCK激酶抑制剂Y27632来促进原代上皮细胞的生长以考察个体患者的药物敏感性的技术，即细胞条件重编程技术(Liu等，Am J Pathol，180：599-607，2012)。另一种技术是体外3D培养成体干细胞从而获得类似于组织器官的类器官技术(Hans Clevers等，Cell，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宫颈癌上皮细胞培养技术，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宫颈癌肿瘤细胞模型时，所培养的宫颈癌肿瘤细胞能代表宫颈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
本发明旨在针对现有技术的不足，提供一种用于培养原代宫颈癌上皮细胞的培养基以及使用该培养基的原代宫颈癌上皮细胞的培养方法。采用本发明的原代宫颈癌上皮细胞培养基和培养方法进行细胞培 养，能够实现体外培养周期短、成本可控、操作便捷并不受外源性细胞干扰的目的。在该技术应用于构建原代宫颈癌肿瘤细胞模型时，能够获得具有宫颈癌患者自身生物学特性的原代宫颈癌肿瘤细胞，并能够应用于新药筛选和体外药物敏感性检测。
本发明的一个方面在于提供一种用于培养原代宫颈癌上皮细胞的原代细胞培养基，其含有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；成纤维细胞生长因子7(FGF7)；B27添加剂和N2添加剂中的至少一种添加剂；肝细胞生长因子(HGF)；胰岛素样生长因子1(IGF-1)；CHIR99021；和选自A83-01、SB431542、Repsox、SB505124、SB525334、SD208、LY36494、和SJN2511中的至少一种的TGFβI型受体抑制剂，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 PCTCN2021130057-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 PCTCN2021130057-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 PCTCN2021130057-appb-000003
Figure PCTCN2021130057-appb-000004
Figure PCTCN2021130057-appb-000005
Figure PCTCN2021130057-appb-000006
Figure PCTCN2021130057-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，MST1/2激酶抑制剂在培养基中的含量通常为2μM～20μM，优选为5μM～20μM。
在又一实施方式中，ROCK激酶抑制剂优选地为Y27632。另外优选的，ROCK激酶抑制剂在培养基中的含量通常为2μM～20μM，优选为10μM。
在优选的实施方式中，所述成纤维细胞生长因子7的含量为2ng/ml～40ng/ml，更优选为10ng/ml～40ng/ml；所述B27添加剂或N2添加剂在培养基中的体积浓度为1:25～1:100，更优选为1:50～1:100；所述肝细胞生长因子的含量为2ng/ml～40ng/ml，更优选为10ng/ml～40ng/ml；所述胰岛素样生长因子1的含量为2ng/ml～40ng/ml，更优选为10ng/ml；所述CHIR99021的含量为1μM～10μM，更优选为1μM～3μM；所述TGFβI型受体抑制剂优选A83-01，且TGFβI型受体抑制剂的含量为50nM～500nM，更优选为100nM～500nM。
本发明的培养基配方还含有选自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。在一些的实施方式中，初始培养基优选为DMEM/F12，抗生素优选为Primocin。在进一步优选的实施方式中，Primocin在培养基中的含量为25～400μg/mL，优选为50～200μg/mL。
本发明的培养基配方成分与细胞条件重编程培养基和宫颈癌上皮细胞类器官培养基成分相比，添加了MST1/2激酶抑制剂，且不包含血清、牛垂体提取物等不确定成分，也不包含Wnt激动剂、R-spondin家族蛋白、BMP抑制剂等类器官培养所必须的龛因子，并且不包含烟酰胺(Nicotinamide)和N-乙酰半胱氨酸(N-Acetylcysteine)，从而大大降低了培养基的成本，简化了配制培养基的操作流程，实现了成本可控和操作便捷的原代宫颈癌上皮细胞的体外培养。
本发明中，原代宫颈癌上皮细胞可以为宫颈癌肿瘤细胞、正常宫颈癌上皮细胞、宫颈癌上皮干细胞。
本发明的一个方面在于提供一种原代宫颈癌上皮细胞的培养方法，其包括以下步骤：
(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)从宫颈癌组织分离得到原代宫颈癌上皮细胞。
原代宫颈癌上皮细胞例如可以来源于宫颈癌手术样本或者活检样本。宫颈癌手术样本例如来源于进行过说明并获得同意的宫颈癌肿瘤患者手术切除的癌组织样本。在患者手术切除后或者活检取样后的半小时内进行上述组织样本的收集。更具体而言，在无菌环境下，切取非坏死部位的组织样本，其体积在0.5cm 3以上，将其置于预冷的10-15mL DMEM/F12培养基中，培养基盛在塑料无菌带盖离心管内，冰上运输至实验室；其中，DMEM/F12培养基中含有本发明的MST1/2激酶抑制剂(例如化合物1)和0.2-0.4体积％Primocin(以下简称组织运输液)。当使用本发明的MST1/2激酶抑制剂时，浓度范围为2μM～20μM，优选为3μM～5μM；当使用Primocin时，浓度范围为25～400μg/mL，优选为50～200μg/mL，更优选为100μg/mL。
在生物安全柜内，将组织样本转移至细胞培养皿内，用组织运输液润洗组织样本，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组织运输液，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1:1比例加入组织运输液和组织消化液(使用量约为每10mg组织使用5mL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Ⅰ、0.5～1mg/mL透明质酸酶、0.1～0.5mg/mL氯化钙、5～10mg/mL牛血清白蛋白溶于体积比1:1的HBSS和RPMI-1640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70μm)过滤掉未消化的组织团块，滤网上的组织团块用组织运输液冲洗，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3～5mL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。弃上清，加入本发明的原代细胞培养基重悬，使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数。
(4)在包被好的培养器皿内接种步骤(3)中分离得到的原代宫颈癌上皮细胞，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)的密度接种原代宫颈癌肿瘤细胞，加入适量如2-3mL原代上皮细胞培养基，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，在原代宫颈癌上皮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，接种后的原代宫颈癌上皮细胞在培养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0.5～2mL 0.05％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)。
扩增的宫颈癌上皮细胞呈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宫颈癌疾病的药物的方法，其包括以下步骤：
(1)使用本发明的原代宫颈癌上皮细胞的培养方法培养宫颈癌上皮细胞；
(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)中培养得到的宫颈癌上皮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)提高原代宫颈癌上皮细胞培养的成功率，成功率达到80％以上；
(2)保证体外原代培养的宫颈癌上皮细胞能够保持原代细胞来源病人的病理表型和异质性；
(3)所培养的原代宫颈癌上皮细胞不受成纤维细胞干扰，能得到纯化的宫颈癌上皮细胞；
(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)扩增宫颈癌上皮细胞效率高，只要有10 4级别的细胞数量就可在两周左右时间内成功扩增出10 6数量级的宫颈癌上皮细胞，扩增出的宫颈癌上皮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，原代宫颈癌细胞培养基无需加入价格昂贵的Wnt激动剂、R-spondin家族蛋白、BMP抑制剂等因子，是对已有原代宫颈癌上皮细胞或类器官培养基的简化和改进，细胞接种也无需使用浓度较高的细胞外基质与原代细胞混合形成胶滴，而只需使用少量细 胞外基质胶制备的稀释液，节约了成本较高的细胞外基质的用量；
(8)操作便捷，该技术相比条件重编程技术，无需培养饲养细胞并对饲养细胞进行辐射，避免了不同批次饲养细胞的质量和数量影响原代细胞培养效率的问题，药物筛选的铺板和检测的对象只有原代宫颈癌上皮细胞，而不受细胞条件重编程技术所需的共培养体系中饲养细胞的干扰；相比类器官技术，本发明采用的细胞外基质胶的包被方法，培养器皿可预先准备，无需像类器官技术一样将细胞包埋于基质胶内，所述技术操作步骤简便易行；
(9)所述技术培养获得的宫颈癌上皮细胞数量大，均一化程度高，适合高通量筛选新候选化合物，并为病人提供高通量药物体外敏感性功能测试。
采用本实施方式的细胞培养基，可培养来源于包括人的或其他哺乳动物的宫颈癌上皮细胞，包括宫颈癌肿瘤细胞、正常宫颈上皮细胞、宫颈癌上皮干细胞、或者包含这些细胞中的至少任一种的组织。同时本发明技术的培养基还可以用于开发体外宫颈癌原代细胞扩增培养的试剂盒。
另外，通过本实施方式的培养方法获得的细胞可应用于再生医疗、宫颈癌上皮细胞的基础医学研究、药物应答的筛选、以及来源于宫颈癌疾病的新药研发等。
附图说明
图1A-1H是显示各添加因子的浓度对宫颈癌原代细胞增殖的影响的图。
图2A和2B是将从1例宫颈癌临床组织样本分离得到的细胞采用本发明的培养基FCM分别培养至第4天和第12天的宫颈癌肿瘤细胞在倒置显微镜下拍摄的照片。
图3A-3C是从1例宫颈癌手术切除样本分离得到的细胞采用三种不同培养基条件下培养15天后在倒置显微镜下拍摄的照片。
图4是从9例宫颈癌手术切除样本分离得到的细胞在三种不同培养基条件下培养16天后得到细胞增殖效果的比较图。
图5是将从1例宫颈癌临床组织样本分离得到的细胞分别采用三 种不同培养基条件培养所获得的细胞生长曲线对比图。
图6是从1例宫颈癌手术切除样本分离得到的细胞采用本发明的培养基FCM培养获得的宫颈癌肿瘤细胞的免疫组化结果对比图。
图7显示从3例宫颈癌手术切除样本分离得到的细胞按照本发明的方法培养所获得的宫颈癌上皮细胞在8种不同药物影响下的细胞活性曲线。
具体实施方式
本说明书中，上皮细胞包括从上皮组织获取的已分化的上皮细胞及上皮干细胞。“上皮干细胞”是指具有长期的自我更新能力和向上皮细胞分化的细胞，是指来源于上皮组织的干细胞。作为上皮组织，可例举例如宫颈、角膜、口腔粘膜、皮肤、结膜、膀胱、肾小管、肾脏、消化器官(食道、胃、十二指肠、小肠(包括空肠及回肠)、大肠(包括结肠))、肝脏、胰脏、乳腺、唾液腺、泪腺、前列腺、毛根、气管、肺等。其中，本实施方式的细胞培养基较好是用于培养来源于宫颈上皮细胞的培养基。
此外，本说明书中，“上皮肿瘤细胞”是指来源于上述的上皮组织的细胞肿瘤化而得的细胞。
本说明书中，“类器官”是指通过使细胞在受控的空间内高密度地自发组织和聚集而成的三维立体的、类似于器官的细胞组织体。
[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 PCTCN2021130057-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 PCTCN2021130057-appb-000009
Figure PCTCN2021130057-appb-000010
Figure PCTCN2021130057-appb-000011
Figure PCTCN2021130057-appb-000012
Figure PCTCN2021130057-appb-000013
[实施例1]
人原代宫颈癌上皮细胞的分离
宫颈癌组织样本来源于进行过说明并获得同意的宫颈癌肿瘤患者手术切除癌组织样本。下面以其中一例样本(编号CCa2)进行说明。
在患者手术切除或活检后的半小时内进行上述组织样本的收集。更具体而言，在无菌环境下，切取非坏死部位的组织样本，其体积在0.5cm 3以上，将其置于预冷的4mL组织运输液(具体配制见表1)中，运输液盛在5mL塑料无菌带盖冻存管(购自广州洁特生物)内，冷链(0-10℃)运输至实验室。
表1组织运输液配方
组织运输液成分 供应商 终浓度
DMEM/F12 康宁 99.8体积％
Primocin Invivogen 0.2体积％(市售产品浓度50mg/ml)
化合物1 自制 3μM
表2组织消化液配方
组织消化液成分 供应商 终浓度
HBSS Gibco 50％(体积)
RPMI-1640 康宁 50％(体积)
胶原酶Ⅱ Sigma 2mg/mL
胶原酶Ⅳ Sigma 2mg/mL
脱氧核糖核酸Ⅰ Sigma 50U/mL
透明质酸酶 Sigma 0.5mg/mL
氯化钙 上海生工 0.33mg/mL
牛血清白蛋白 上海生工 10mg/mL
在生物安全柜内，将组织样本(编号CCa2)转移至100mm细胞培养皿(购自NEST公司)内，用组织运输液润洗组织样本，将组织样本表面的残留的血液清洗掉，并剔除组织样本表面的脂肪等多余的组织。将润洗后的组织样本转移至另一个新的100mm培养皿内，加入2mL运输液，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15mL离心管内，用台式离心机(Sigma公司3-18K)以1500转/分钟离心4分钟；弃上清，按1:1比例加入组织运输液和组织消化液(使用量为每10mg组织使用约5mL组织消化液，具体配制见表2)，标记样本编号，封口膜密封，以37℃、300转恒温摇床(知楚仪器ZQLY-180N)消化，每间隔1小时观察消化是否完成。
消化完成后，70μm滤网过滤掉未消化的组织团块，滤网上的组织团块用组织运输液冲洗，将残留细胞冲入离心管中，1500转/分钟离心4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3mL血细胞裂解液(购自Sigma公司)，混匀，4℃裂解15分钟，5分钟摇晃混匀一次，裂解结束后取出，以1500转/分钟离心4分钟。弃上清， 得到消化分离后的宫颈癌原代细胞，加入基础培养基(BM)重悬，其中，基础培养基是由市售的DMEM/F-12培养基中加入0.2体积％Primocin(购自Invivogen公司，浓度为50mg/mL)，以得到100μg/mL的最终浓度。使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数为162万。
[实施例2]
原代宫颈癌上皮细胞培养基的优化
(1)不同因子的作用
将细胞外基质胶
Figure PCTCN2021130057-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。
配制基础培养基(缩写为BM)：向市售的DMEM/F-12培养基中加入0.2体积％Primocin(购自Invivogen公司，浓度为50mg/mL)，以得到100μg/mL的最终浓度，配制得到BM。
接着，在基础培养基(BM)中分别加入不同种类和不同浓度的添加剂因子(表3)，配制成含有不同添加成分的宫颈癌上皮细胞培养基。表3不同组分培养基的配制(浓度为终浓度)
Figure PCTCN2021130057-appb-000015
Figure PCTCN2021130057-appb-000016
将按照实施例1相同方法从宫颈癌组织分离得到的宫颈癌肿瘤细胞(编号CCa5)以3000个/well的细胞密度接种在384孔培养板内，按50μl/孔加入不同成分的培养基，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宫颈癌肿瘤细胞(编号CCa5)在不同的培养基配方条件下进行培养。培养6天后，按5μl/孔加入CCK8检测试剂(购自MCE公司)，放入培养箱中孵育2-4小时，使用多功能酶标仪(购自PE公司)测定在OD450处的吸光值。其中，作为实验对照，使用未添加任何添加剂的基础培养基(BM)。将结果示于表3。比值是指不同培养基培养之后加入CCK8检测得到的吸光值与基础培养基BM培养之后检测得到的吸光值的比值。如表所示，在BM基础上加入表3中不同因子对细胞增殖产生不同的作用。其中，在特定浓度范围下，B27添加剂、N2添加剂、成纤维细胞生长因子7、CHIR99021、肝细胞生长因子、胰岛素样生长因子1、化合物1、Y27632和A83-01均对细胞增殖的促进作用更为显著。
(2)所添加因子的不同浓度对本专利获得的宫颈癌原代细胞的增殖作用
将细胞外基质胶(
Figure PCTCN2021130057-appb-000017
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200μl/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。
配制本实施例的原代宫颈癌上皮细胞培养基：向基础培养基(BM)中以最终浓度40ng/ml的条件添加成纤维细胞生长因子7(FGF7)，以最终浓度40ng/ml的条件添加肝细胞生长因子(HGF)，以最终浓度40ng/ml的条件添加胰岛素样生长因子1(IGF-1)，以最终浓度1:50体积比的条件添加B27添加剂，以最终浓度5μM的条件添加化合物1，以最终浓度10μM的条件添加Y27632，以最终浓度500nM的条件添加TGFβ1抑制剂A83-01，以最终浓度10μM的条件添加CHIR99021，制备原代宫颈癌上皮细胞培养基。
使用与实施例1同样的方法从宫颈癌患者的癌组织(编号CCa8)中分离获得癌组织来源的宫颈癌上皮细胞。接着，将癌组织来源的宫颈癌上皮细胞用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数。然后按4×10 4个/cm 2密度接种至
Figure PCTCN2021130057-appb-000018
(购自BD生物科技公司)包被处理过的48孔板内。向48孔板中添加2mL制备好的原代宫颈癌上皮细胞培养基，置于37℃、5％CO 2培养箱(购自赛默飞)中进行培养。在培养板内细胞生长达到底面积的80％左右时，弃去48孔板内的培养基上清，加入500μL 0.05％胰酶(购自Gibco公司)对细胞进行消化，37℃下孵育10分钟，直至显微镜(Invitrogen公司EVOS M500)下能观察到细胞已经消化完全，即用含有5％(v/v)胎牛血清(购自依科赛公司)、100U/mL青霉素(购自康宁公司)和100μg/mL链霉素(购自康宁公司)的DMEM/F12培养液1mL终止消化，并收集至15mL离心管内，1500rpm离心4分钟后，弃上清。使用基础培养基BM重悬离心后的细胞沉淀，使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数。所得细胞用于以下培养实验。
接着，配制以下8种配方培养基进行实验：
配方1：上述培养基组分中不含B27添加剂；
配方2：上述培养基组分中不含成纤维细胞生长因子7；
配方3：上述培养基组分中不含胰岛素样生长因子1；
配方4：上述培养基组分中不含肝细胞生长因子；
配方5：上述培养基组分中不含Y27632；
配方6：上述培养基组分中不含化合物1；
配方7：上述培养基组分中不含A83-01；
配方8：上述培养基组分中不含CHIR99021。
分别使用上述配方1～8来稀释上述消化后的细胞悬液，按照每孔1万细胞，250微升体积种入48孔板中。
在使用配方1的培养基时，在接种有原代细胞的48孔板中分别添加配制好的B27添加剂每孔250微升，B27添加剂的终浓度分别为1:100、1:50、1:25；并使用配方1的培养基设置对照孔(BC)。
在使用配方2的培养基时，在接种有原代细胞的48孔板中分别添加配制好的成纤维细胞生长因子7每孔250微升，成纤维细胞生长因子7的终浓度分别为40ng/mL、10ng/mL、2ng/mL；并使用配方2的培养基设置对照孔(BC)。
在使用配方3的培养基时，在接种有原代细胞的48孔板中分别添加配制好的胰岛素样生长因子1每孔250微升，胰岛素样生长因子1的终浓度分别为40ng/mL、10ng/mL、2ng/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48孔板中分别添加配制好的肝细胞生长因子每孔250微升，肝细胞生长因子的终浓度分别为40ng/mL、10ng/mL、2ng/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孔板中分别添加配制好的Y27632每孔250微升，Y27632的终浓度分别为20μM、10μM、2μM；并使用配方5的培养基设置对照孔(BC)。
在使用配方6的培养基时，在接种有原代细胞的48孔板中分别添加配制好的化合物1每孔250微升，化合物1的终浓度分别为20μM、5μM、2μM；并使用配方6的培养基设置对照孔(BC)。
在使用配方7的培养基时，在接种有原代细胞的48孔板中分别添加配制好的A83-01每孔250微升，A83-01的终浓度分别为500nM、 100nM、50nM；并使用配方7的培养基设置对照孔(BC)。
在使用配方8的培养基时，在接种有原代细胞的48孔板中分别添加配制好的CHIR99021每孔250微升，CHIR99021的终浓度分别为10μM、3μM、1μM；并使用配方8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)细胞数计算比值，将结果分别示于图1A～1H。图1A～图1H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～1H的结果，B27添加剂在培养基中的体积浓度优选为1:25～1:100，更优选1:50～1:100；成纤维细胞生长因子7的含量优选为2ng/ml～40ng/ml，更优选10ng/ml～40ng/ml；胰岛素样生长因子1的含量优选为2ng/ml～40ng/ml，更优选为10ng/ml～40ng/ml；肝细胞生长因子的含量优选为2ng/ml～40ng/ml，更优选10ng/ml～40ng/ml；Y27632的含量优选为2μM～20μM，更优选为2μM～10μM；化合物1的含量优选为2μM～20μM，更优选为5μM～20μM；A83-01的含量优选为50nM～500nM，更优选为100nM～500nM；CHIR99021的含量优选为1μM～10μM，更优选为1μM～3μM。
根据以上个组分的优选浓度，配制本发明的优选培养基配方FCM，其包含：基础培养基(BM)、10ng/ml成纤维细胞生长因子7(FGF7)、10ng/ml肝细胞生长因子(HGF)、10ng/ml胰岛素样生长因子1(IGF-1)、1:50体积比的B27添加剂、5μM化合物1、10μM Y27632、500nM A83-01、和3μM CHIR99021。
[实施例3]
宫颈癌组织来源的原代宫颈癌肿瘤细胞的培养
使用与实施例1同样的方法从宫颈癌患者的癌组织(样本编号CCa15)中分离获得癌组织来源的宫颈癌上皮细胞。接着，将癌组织来源的宫颈癌上皮细胞用流式图像计数仪(江苏卓微生物科技有限公司 JIMBIO FIL)进行计数，得到细胞总数。然后按4×10 4个/cm 2密度接种至
Figure PCTCN2021130057-appb-000019
(购自BD生物科技公司)包被处理过的12孔板内。向12孔板中添加2mL制备好的原代宫颈癌上皮细胞培养基FCM，置于37℃、5％CO 2培养箱(购自赛默飞)中进行培养。
图2A是本实施例按4×10 4个/cm 2密度接种至Matrigel包被处理过的6孔板，自接种开始后培养至第4天的镜下照片(10倍倒置相差显微镜拍照)。镜下观察可见，所培养的癌组织来源的原代宫颈癌肿瘤细胞纯度较高，不含有成纤维细胞。图2B是本实施例自接种后培养第12天的镜下照片(40倍倒置相差显微镜拍照)。从图2A和2B两张图可以看出，分离后得到宫颈癌原代细胞在体外培养4天在镜下就可以看到明显的克隆形成，并且经过12天的扩增，细胞数目得到显著的扩增，提示本发明技术是一种高效的体外扩增宫颈癌上皮细胞的技术。
[实施例4]
不同培养基对宫颈癌组织来源的原代宫颈癌肿瘤细胞的促增殖效果
(1)不同培养基对初代原代细胞克隆形成的影响和增殖效果的比较
使用与实施例2同样的方法制备原代宫颈癌上皮细胞培养基FCM，和作为对照的基础培养基BM。另外制备文献培养基RM作为另一对照例，配制步骤参见(马丽萍等，吉林医学，42(6):1289-1293，2021)，培养基配方见表4。
表4文献培养基(RM)成分
培养基成分 供应商 终浓度
DMEM/F12培养基 Corning 100体积％
成纤维细胞生长因子 北京义翘 10ng/ml
表皮生长因子 北京义翘 20ng/ml
使用与实施例1同样的方法获得宫颈癌组织来源的原代宫颈癌肿瘤细胞(编号CCa10)。接着，按照相同的密度(4×10 4个/cm 2)分别在以下三种培养条件下培养：
A.本发明技术：按4×10 4个/cm 2接种密度将原代宫颈癌肿瘤细胞 接种至
Figure PCTCN2021130057-appb-000020
((BD生物科技公司制)包被处理过的24孔板内，采用2mL的本发明的原代宫颈癌上皮细胞培养基FCM进行培养；
B.按4×10 4个/cm 2接种密度将原代宫颈癌肿瘤细胞接种至
Figure PCTCN2021130057-appb-000021
((BD生物科技公司制)包被处理过的24孔板内，采用2mL的文献培养基RM在24孔板中进行培养。
C.按4×10 4个/cm 2接种密度将原代宫颈癌肿瘤细胞接种至
Figure PCTCN2021130057-appb-000022
((BD生物科技公司制)包被处理过的24孔板内，采用2mL的基础培养基BM在24孔板中进行培养。
上述三种培养中，每4天对三种培养条件下培养的细胞进行换液。同时观察24孔板中各培养基培养下细胞形成克隆和细胞增殖状态，使用显微镜(Invitrogen公司EVOS M500)进行拍照记录细胞生长状况。
对于采用本发明技术培养的原代宫颈癌肿瘤细胞(编号CCa10)，分别在培养板内细胞生长达到底面积的80％左右时，弃去24孔板内的培养基上清，加入500μL 0.05％胰酶(购自GIBCO公司)对细胞进行消化，37℃下孵育10分钟，直至显微镜(Invitrogen公司EVOS M500)下能观察到细胞已经消化完全，即用含有5％(v/v)胎牛血清、100U/mL青霉素和100μg/mL链霉素的DMEM/F12培养液1mL终止消化，并收集至15mL离心管内，1500rpm离心4分钟后，弃上清。使用本发明的培养基重悬离心后的细胞沉淀，使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数为19万。另二种培养条件下培养的细胞采用如上述同样的操作方式进行消化和计数，使用培养基FM和CM培养得到的细胞总数分别是4.5万和4.6万。
图3A-3C的细胞照片为编号CCa10的样本在三种不同的培养条件下培养至第15天时的镜下照片(10倍倒置相差显微镜下)：其中图3A为CCa10使用基础培养基BM培养至第15天时的镜下照片；图3B为CCa10使用本专利培养基FCM培养至第15天时的镜下照片；图3C为CCa10使用文献培养基RM培养至第15天时的镜下照片。从图中可以看出，样本CCa10使用基础培养基BM(图3A)培养15天不能形成细胞克隆；使用文献培养基RM(图3C)培养15天不能形成细胞克隆，且细胞状态不佳；使用本发明的培养基FCM(图3B)培养15天细胞形成克隆，且促增殖效果明显。
图4是9例宫颈癌病人样本按照实施例1的方法获得的宫颈癌原代细胞在以上三种不同培养基条件下培养16天后得到细胞增殖效果的比较图，其中√代表有一般的克隆形成能力和促增殖效果，√√代表有较明显的克隆形成能力和促增殖效果，√√√代表有较强的克隆形成能力和促增殖效果，×代表不能形成克隆。从图6中可以确认，本发明的培养基在对宫颈癌组织来源获得的原代细胞进行培养时，克隆形成能力和促细胞增殖效果、以及培养成功率都较其他两种培养条件有明显优势。
(2)不同培养基对原代宫颈癌肿瘤细胞的持续培养和生长曲线的绘制
使用与本实施例(1)中同样的方法获得原代宫颈癌上皮细胞培养基FCM，以及作为对照的培养基BM和RM。
使用与本实施例(1)中同样的方法将宫颈癌组织来源的原代宫颈癌肿瘤细胞(编号CCa5)分别在三种培养基条件下培养，并进行消化传代和计数。
当传代后的细胞在培养板内生长再次达到约80％板底面积时，再次按上述操作方法消化收集所培养获得的细胞并计数。同样按4×10 4个/孔密度接种并持续培养。
以下为原代宫颈癌上皮细胞在不同培养条件下细胞的扩增倍数(Population Doubling)的计算公式：
Population Doubling(PD)＝3.32*log10(消化后细胞总数/初始种入细胞数)，公式参见(Chapman等.Stem Cell Research&Therapy 2014,5:60)。
图5是采用Graphpad Prism软件绘制的三种不同培养条件下的细胞CCa5的生长曲线。横坐标表示细胞培养的天数，纵坐标是累计的细胞增殖倍数，表示细胞在培养周期内扩增的倍数，数值越大表示细胞在一定周期内扩增的次数越多，即扩增得到的细胞数也就越多，斜率代表的是细胞扩增的速率。从图中可以确认本发明的培养基FCM培养的宫颈癌上皮细胞的增殖速率优于其他两种培养条件。
[实施例5]
原代宫颈癌组织和传代培养后的宫颈癌细胞免疫组化鉴定
从一例宫颈癌患者的临床手术切除样本取出约绿豆粒大小的癌组织(样本编号CCa14)，浸泡在1mL 4％多聚甲醛中固定。剩余癌组织使用与实施例1相同的方法获得宫颈癌上皮细胞(样本编号CCa14)。使用实施例3的方法采用本发明的培养基FCM将样本CCa14持续培养至第三代。
采用免疫组化法检测样本CCa14原始组织和持续培养至第三代得到的原代细胞中与宫颈癌相关的重要生物标记物的表达。4％多聚甲醛固定后的组织，经石蜡包埋，用切片机切成4μm厚的组织切片。随后进行常规的免疫组织化学检测(具体步骤参见Li等，Nature Conmunication，(2018)9:2983)。所使用的一抗为P16抗体(购自Affinit公司)、P63抗体(购自CST公司)和Ki67抗体(购自CST公司)。
由图6可以确认，采用本发明的培养基培养的宫颈癌肿瘤细胞(样本编号CCa14)培养至第3代时，细胞上与宫颈癌相关的生物标记物的表达情况与细胞来源的原始组织切片的标记物表达情况基本一致。说明采用本发明的培养基所培养的细胞保持了宫颈癌病人癌组织的原始病理特性。
[实施例6]
癌组织来源的宫颈癌肿瘤细胞的药物敏感性功能测试
下面以宫颈癌患者手术切除样本为例，说明由病人来源的宫颈癌肿瘤样本培养得到的宫颈癌肿瘤细胞可以用于检测病人肿瘤细胞对不同药物的敏感性。
一、原代宫颈癌肿瘤细胞的铺板：按照实施例1中方法得到的分离后的宫颈癌肿瘤细胞(编号CCa5、编号CCa6和编号CCa9)悬液，按照4×10 4个/cm 2密度接种至12孔板内。向12孔板中添加2mL制备好的原代宫颈癌上皮细胞培养基FCM，置于37℃、5％CO 2培养箱(购自赛默飞)中进行培养。在培养板内细胞生长达到底面积的80％左右时，弃去12孔板内的培养基上清，加入0.5mL 0.25％胰酶(购自Thermo Fisher公司)消化1分钟，随后吸出0.25％胰酶，再加入0.5mL 0.05％胰酶进行细胞消化，37℃下孵育10分钟，直至显微镜(Invitrogen公司EVOS M500)下能观察到细胞已经消化完全，即用含有5％(v/v) 胎牛血清、100U/mL青霉素和100μg/mL链霉素的DMEM/F12培养液1mL终止消化，并收集至15mL离心管内，1500rpm离心4分钟后，弃上清。使用FCM培养基重悬离心后的细胞沉淀，使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数分别为53万、78万和63万。按1000～2000个/孔密度接种于384孔板中，使细胞贴壁过夜。
二、药物梯度实验：
(1)采用浓度梯度稀释的方法配制药物贮存板：分别吸取40μL的10μM待测药物母液作为最高浓度，再分别从中吸取10μL加入到含20μL的DMSO的0.5mL的EP管中，再从上述EP管中吸取10μL到第二个已装有20μL的DMSO的0.5mL的EP管中，即按照1:3稀释药品。重复以上方法，依次稀释，最后得到加药所需的7种浓度。将不同浓度的药物加入384孔药物储存板中。溶剂对照组各孔加入等体积的DMSO作为对照。本实施例中，待测药物为顺铂(购自MCE公司)、紫杉醇(购自MCE公司)、5-氟尿嘧啶(5-F，购自MCE公司)、拓扑替康(购自MCE公司)、硼替佐米(购自MCE公司)、安罗替尼(购自MCE公司)、帕唑帕尼(购自MCE公司)、和阿帕替尼(购自MCE公司)和。
(2)使用高通量自动化工作站(Perkin Elmer公司JANUS)将384孔药物贮存板内的不同浓度药物和溶剂对照加入到铺有宫颈癌肿瘤细胞的384孔细胞培养板中，药物组和溶剂对照组都各设3个复孔。每孔加入药物体积为100nL。
(3)细胞活性检测：给药72小时后，用Cell Titer-Glo检测试剂(购自Promega公司)检测加药培养后细胞的化学发光数值，化学发光数值的大小反映细胞活力以及药物对细胞活力的影响，每孔加入10μL配制好的Cell Titer-Glo检测液，混匀后使用酶标仪(Perkin Elmer公司Envision)检测化学发光数值。按照公式细胞存活率(％)＝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存活率，使用Graphpad Prism软件作图并计算半数抑制率IC 50。
(4)药物敏感性测试结果如图7所示。
图7分别表示从三个不同的宫颈癌患者的手术切除癌组织样本(编号CCa5、编号CCa6和编号CCa9)所培养获得的宫颈癌肿瘤细胞，对四个化疗药物顺铂、紫杉醇、5-氟尿嘧啶和拓扑替康的药物敏感性和对四个靶向药物硼替佐米、安罗替尼、帕唑帕尼和阿帕替尼的敏感性。结果显示，同一病人的细胞对不同浓度药物作用时的敏感性不同，不同病人的细胞对在同一种药物的敏感性也不同，根据结果可以判断宫颈癌病人在临床使用该种药物时的有效性。
工业应用性
本发明提供一种用于培养原代宫颈癌上皮细胞的培养基及培养方法，可将培养得到的细胞应用于药物的疗效评估和筛选。因而，本发明适于工业应用。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本说明书基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (13)

  1. 一种用于培养原代宫颈癌上皮细胞的培养基，其特征在于：
    含有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；成纤维细胞生长因子7；B27添加剂和N2添加剂中的至少一种添加剂；肝细胞生长因子；胰岛素样生长因子1；CHIR99021；和选自A83-01、SB431542、Repsox、SB505124、SB525334、SD208、LY36494、和SJN2511中的至少一种的TGFβI型受体抑制剂，
    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 PCTCN2021130057-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 PCTCN2021130057-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 PCTCN2021130057-appb-100003
    Figure PCTCN2021130057-appb-100004
    Figure PCTCN2021130057-appb-100005
    Figure PCTCN2021130057-appb-100006
    Figure PCTCN2021130057-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MST1/2激酶抑制剂在培养基中的含量为2μM～20μM。
  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满足以下任意一项或多项或全部满足：
    所述ROCK激酶抑制剂在培养基中的含量为2μM～20μM；
    所述成纤维细胞生长因子7的含量为2ng/ml～40ng/ml；
    所述B27添加剂或N2添加剂在培养基中的体积浓度为1:25～1:100；
    所述肝细胞生长因子的含量为2ng/ml～40ng/ml；
    所述胰岛素样生长因子1的含量为2ng/ml～40ng/ml；
    所述CHIR99021的含量为1μM～10μM；
    所述TGFβI型受体抑制剂的含量为50nM～500nM。
  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激酶抑制剂为化合物1；
    所述ROCK激酶抑制剂为Y27632；
    所述TGFβI型受体抑制剂为A83-01。
  9. 如权利要求1～8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。
  10. 如权利要求1～9中任一项所述的培养基，其特征在于：
    不含血清、牛垂体提取物、Wnt激动剂、R-spondin家族蛋白、BMP抑制剂、烟酰胺和N-乙酰半胱氨酸。
  11. 如权利要求1～10中任一项所述的培养基，其特征在于：
    所述原代宫颈癌上皮细胞选自宫颈癌肿瘤细胞、正常宫颈癌上皮细胞、和宫颈癌上皮干细胞。
  12. 一种原代宫颈癌上皮细胞的培养方法，其特征在于，包括以下步骤：
    (1)配制如权利要求1～11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宫颈癌组织分离得到原代宫颈癌上皮细胞，使用步骤(1)中的培养基进行培养。
  13. 一种用于评估或筛选治疗宫颈癌疾病的药物的方法，其包括以下步骤：
    (1)使用如权利要求12所述的原代宫颈癌上皮细胞的培养方法培养宫颈癌上皮细胞；
    (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)中培养得到的宫颈癌上皮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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