WO2022016642A1 - Culture medium for lung cancer epithelial cell, culture method and use thereof - Google Patents

Culture medium for lung cancer epithelial cell, culture method and use thereof Download PDF

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WO2022016642A1
WO2022016642A1 PCT/CN2020/109740 CN2020109740W WO2022016642A1 WO 2022016642 A1 WO2022016642 A1 WO 2022016642A1 CN 2020109740 W CN2020109740 W CN 2020109740W WO 2022016642 A1 WO2022016642 A1 WO 2022016642A1
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lung cancer
cells
alkyl
culture medium
medium
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刘青松
胡洁
王文超
陈程
任涛
王黎
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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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Definitions

  • the invention belongs to the technical field of medicine, in particular to a culture medium and a culture method for culturing or amplifying primary lung cancer epithelial cells in vitro, and also relates to a method and application of the cultured cells in the efficacy evaluation and screening of drugs .
  • Lung cancer remains one of the most common and deadly cancers, with 1.6 million cancer-related deaths worldwide each year. Lung cancer is a diverse, complex, and treatment-challenging disease. Over the past decade, with the advent of personalized medicine, there has been a greater understanding of the underlying biology and molecular mechanisms of lung cancer. Lung cancer is no longer a single disease entity, but is now subdivided into molecular subtypes, each corresponding to specialized targeted chemotherapy strategies.
  • Functional testing refers to the detection of the in vitro sensitivity of antitumor drugs on cells of cancer patients.
  • the key to applying this approach is to develop tumor cell models that have a short growth cycle and can represent the own biological characteristics of lung cancer patients.
  • the cell model should be easy to operate and be able to quickly and efficiently predict the efficacy of clinical medication, so as to give cancer patients precise medication guidance in a timely manner.
  • the success rate of establishing cell models in vitro from primary tumor cells from cancer patients is often 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.
  • organoid technology is a technology of embedding patient's primary epithelial cells in extracellular matrix for in vitro three-dimensional culture, but the medium of this technology needs to add a variety of specific growth factors (such as Wnt protein and R-spondin) family proteins), the cost is high, and it is not suitable for widespread clinical application on a large scale.
  • the organoids need to be embedded in extracellular matrix gel during the entire culture process, and the plating steps of cell seeding, passage and drug sensitivity test are cumbersome and time-consuming compared with 2D culture operations, and the technology formed by The size and size of organoids are not well controlled, and some organoids may grow too large and cause internal necrosis.
  • Cell reprogramming technology is a technology of co-culturing patients' autologous primary epithelial cells with mouse-derived feeder cells.
  • the culture medium used for in vitro expansion of primary lung cancer cells contains serum components. It is clear that there are large differences between different batches, which may easily interfere with the experimental results.
  • a primary lung cancer epithelial cell culture technology which has a short culture period, controllable cost, and convenient operation.
  • this technology is applied to construct primary lung cancer tumor cell models, the cultured The lung cancer tumor cells can represent the biological characteristics of lung cancer patients themselves.
  • the response rate of clinical antitumor drugs can be improved, and the pain caused by inappropriate drugs to patients and the waste of medical resources can be reduced.
  • the present invention aims at providing a culture medium for culturing primary lung cancer epithelial cells and a method for culturing primary lung cancer epithelial cells using the culture medium in view of the deficiencies of the prior art.
  • the primary lung cancer epithelial cell culture medium and culture method of the present invention can achieve the goals of short in vitro culture period, controllable cost and convenient operation.
  • the technology is applied to construct a primary lung cancer tumor cell model, primary lung cancer tumor cells with the own biological characteristics of lung cancer patients can be obtained, and can be applied to new drug screening and in vitro drug sensitivity detection.
  • One aspect of the present invention is to provide a primary cell culture medium for culturing primary lung cancer epithelial cells, which contains MST1/2 kinase inhibitor; insulin-like growth factor 1 (IGF-1); epidermal growth factor (EGF) ); hepatocyte growth factor (HGF); neuregulin-1; an additive selected from at least one of insulin-transferrin-selenium complex (ITS), B27 additive and N2 additive; selected from Y27632, Fasu A ROCK kinase inhibitor of at least one of dil, and H-1152, the MST1/2 kinase inhibitor comprising a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof.
  • MST1/2 kinase inhibitor insulin-like growth factor 1
  • EGF epidermal growth factor
  • HGF hepatocyte growth factor
  • ITS insulin-transferrin-selenium complex
  • B27 additive selected from Y27632
  • R 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl optionally substituted with 1-2 independently R 6 (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 alkylhydroxy, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl group is selected from, for example, piperidinyl, tetrahydropyran base, etc.);
  • R 6 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 1 is selected C1-C6 alkyl, optionally substituted with 1-2 R 6 independently substituted phenyl, optionally substituted with 1-2 R 6 independently substituted thienyl, and optionally substituted with 1 - 2 independently R 6 substituted benzyl, R 1 more preferably phenyl optionally substituted with 1-2 independently R 6;
  • 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, more preferably R 6 is fluoro, 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 medium is usually 0.625 ⁇ M to 20 ⁇ M, preferably 0.625 ⁇ M to 10 ⁇ M, and more preferably 2.5 ⁇ M.
  • the content of the insulin-like growth factor 1 is preferably 1.25-80 ng/ml, more preferably 5-80 ng/ml; the content of the hepatocyte growth factor is preferably 5-80 ng/ml, more preferably It is preferably 20-80ng/ml; the content of the epidermal growth factor is preferably 10-80ng/ml, more preferably 10-40ng/ml; the content of the neuregulin-1 is preferably 10-80ng/ml, More preferably 20-80ng/ml; the content of the additive is preferably 1:200-1:25 by volume, more preferably 1:50-1:25 by volume, and the additive is preferably an insulin-transferrin-selenium complex wherein the respective contents of insulin/transferrin/sodium selenite are preferably 2.5-20 ⁇ g/ml-1.25-10 ⁇ g/ml-1.25-10ng/ml, more preferably 10-20 ⁇ g/ml-5-10 ⁇ g, respectively /ml-5-10ng/
  • the composition of this medium is supplemented with MST1/2 kinase inhibitors, but does not contain uncertain components such as serum and bovine pituitary extract, nor does it contain Wnt agonists , R-spondin family proteins, BMP inhibitors and other necessary niche factors for organoid culture, and does not contain nicotinamide and N-acetylcysteine, which greatly reduces the cost of the medium and simplifies the operation of preparing the medium
  • the process realizes the in vitro culture of primary lung cancer epithelial cells with controllable cost and convenient operation.
  • the primary lung cancer epithelial cells can be lung cancer tumor cells, normal lung cancer epithelial cells, and lung cancer epithelial stem cells.
  • One aspect of the present invention is to provide a method for culturing primary lung cancer epithelial cells, comprising the following steps:
  • the primary cell culture medium of the present invention is prepared according to the above formula.
  • the trophoblasts can be, for example, irradiated NIH-3T3 cells
  • the irradiation source is X-rays or ⁇ -rays, preferably ⁇ -rays
  • the irradiation dose is 30-50 Gy, preferably 35 Gy.
  • the irradiated NIH-3T3 cells are seeded in a culture vessel such as a 48-well plate, a 24-well plate, a 12-well plate, a 6-well plate or a T25 cell culture flask at 2 ⁇ 10 4 cells/cm 2 . Reserve after sticking to the wall.
  • Primary lung cancer epithelial cells can be derived, for example, from lung cancer tissue samples and lung cancer biopsy or lung bronchoscopy samples.
  • Lung cancer tissue samples such as tumor tissue samples from surgically resected lung cancer tumor patients who have been described and obtained consent, lung cancer puncture or bronchoscopy samples, such as those obtained from lung cancer tumor patients who have been described and obtained consent for biopsy Puncture or bronchoscopy samples.
  • the above-mentioned tissue samples were collected within half an hour after the patient's surgical excision or biopsy.
  • tissue transport fluid 1-2 vol% penicillin/streptomycin, and/or 0.2-0.4 vol% Primocin (hereinafter referred to as tissue transport fluid).
  • the streptomycin concentration is in the range of 25-400 ⁇ g/mL, preferably 50-200 ⁇ g/mL, more preferably 200 ⁇ g/mL
  • the penicillin concentration is in the range of 25-400 U/mL, preferably 50- 200 U/mL, more preferably 200 U/mL
  • the concentration range is 25-400 ⁇ g/mL, preferably 50-200 ⁇ g/mL, more preferably 100 ⁇ g/mL.
  • tissue sample In the biological safety cabinet, transfer the tissue sample to a cell culture dish, rinse the tissue sample with the transport fluid, and wash away the blood cells on the surface of the tissue sample. Transfer the rinsed tissue sample to another new petri dish, add 1-3 mL of transport solution, and use a sterile scalpel blade and forceps to divide the tissue sample into tissue fragments with a volume of less than 3 mm 3 .
  • tissue sample fragments Transfer the tissue sample fragments to a centrifuge tube, and centrifuge with a desktop centrifuge (Sigma 3-18K) at 1000-3000 rpm for 3-5 minutes; discard the supernatant and add tissue transport solution and tissue digestion at a ratio of 1:1 (The usage amount is about 5mL tissue digestion solution per 10mg tissue, wherein the preparation method of tissue digestion solution is: 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 in a volume ratio of 1:1), labeled samples Numbered, sealed with parafilm, digested at 37°C, 200-300 rpm constant temperature shaker (Zhichu Instrument ZQLY-180N), and observed
  • tissue clumps are filtered out with a cell strainer (cell sieve pore size is, for example, 70 ⁇ m), the tissue clumps on the strainer are rinsed with tissue transport solution, and the remaining cells are flushed into a centrifuge tube, and the clumps are washed with a table top 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 1-5 mL of blood cell lysis solution (purchased from Sigma), mix well, lyse at 4°C for 10-20 minutes, shake and mix once in 5 minutes, and the lysis is over.
  • blood cell lysis solution purchased from Sigma
  • step (3) inoculating the primary lung cancer epithelial cells isolated in step (3) in a culture vessel pre-seeded with trophoblasts, and using the primary cell culture medium in step (1) for culturing.
  • a hole of a multi-well plate is pre- inoculated at a density of 2 ⁇ 10 4 to 4 ⁇ 10 4 /cm 2 (for example, 2 ⁇ 10 4 /cm 2 ) with an irradiation dose of 35 Gy after ⁇ -ray irradiation.
  • NIH-3H3 cells of 2 ⁇ 10 4 to 8 ⁇ 10 4 cells/cm 2 (for example, 4 ⁇ 10 4 cells/cm 2 ) were inoculated with primary lung cancer tumor cells after the cells adhered to the wall.
  • ⁇ 2mL of primary epithelial cell culture medium for example, at 37°C, 5% CO 2 in a cell incubator for 8-16 days, changing to fresh primary cell culture medium every 4 days during this period.
  • the cells were digested and passaged when the cells grew to a cell density of about 80% to 90% of the bottom area of the multi-well plate.
  • this step does not need to mix primary cells and Matrigel on ice to form gel droplets, and wait for the gel droplets to solidify before adding culture medium.
  • the usage of expensive extracellular matrix glue is saved, and the operation steps are simplified.
  • the inoculated primary lung cancer epithelial cells are cultured for 8-16 days, when the confluence of the cell clones formed in the culture vessel reaches 80% of the bottom area, the supernatant is discarded, and 1-2 mL of 0.25% trypsin (purchased) is added.
  • the expanded lung cancer epithelial cells grow in 2D, avoiding the non-uniform size of organoids and internal necrosis of overgrown organoids that occur with organoid technology expansion.
  • lung cancer epithelial cells especially lung cancer tumor cells, cultured by the method for culturing primary lung cancer epithelial cells of the present invention can be used for drug efficacy evaluation and screening, and the method includes the following steps:
  • Obtaining primary lung cancer epithelial cells particularly preferably, obtaining cancer tissue samples or biopsy cancer tissue samples derived from lung cancer patients, isolating primary lung cancer epithelial cells, culturing and Primary lung cancer epithelial cells (especially primary lung cancer tumor cells) are expanded to a cell number of the order of at least 10 5 , preferably of the order of at least 10 6 .
  • the drug takes its maximum plasma concentration Cmax as reference, and takes 2-5 times Cmax as the initial concentration, and dilutes multiple different drug concentration gradients, such as 5-10, preferably 6-8 drug concentration gradients.
  • step (1) Digest the lung cancer epithelial cells cultured in step (1) into a single cell suspension, count with a flow image counter, dilute the single cell suspension with the primary cell culture medium of the present invention, press each well At a density of 2000-4000 cells, the diluted cell suspension is evenly added to the multi-well plate, eg, 50 ⁇ L of cell dilution per well, and allowed to adhere overnight.
  • This step avoids the problem of cell reprogramming techniques that interfere with primary cell counts and subsequent primary cell viability assays due to the presence of feeder cells, and eliminates the need to mix cell suspensions with Matrigel on ice as in organoid techniques.
  • the tedious steps of hybrid embedding and re-plating greatly simplify the operation process and enhance the operability and practicality of the technology. Since the seeded cells are single-cell suspensions rather than organoid-like 3D structures, this technique, compared with organoid techniques, results in a more uniform number of plated cells and less variation in the number of cells between wells produced by plating, making it more suitable for Subsequent high-throughput drug screening operations.
  • step (4) Using a high-throughput automated workstation, add the selected traditional chemotherapeutic drugs, targeted drugs, antibody drugs or several drug combinations and other candidate drugs after gradient dilution to the adherent cells obtained in step (4), and carry out deal with.
  • the interaction time with the drug is also shorter than the drug detection time of the organoid technology (the average administration time of the organoid technology is 6 days).
  • the medium composition does not contain serum, so it is not affected by the quality and quantity of different batches of serum;
  • the efficiency of amplifying lung cancer epithelial cells is high. As long as there are 10 4- level cells, 10 6 -level lung cancer epithelial cells can be successfully expanded in about two weeks, and the expanded lung cancer epithelial cells can also be continuously passaged ;
  • Controllable culture cost The primary lung cancer culture medium does not need to add expensive Wnt agonists, R-spondin family proteins, BMP inhibitors and other factors, saving the cost of cell culture;
  • the lung cancer epithelial cells cultured by the technique have a large number and a high degree of homogeneity, which are suitable for high-throughput screening of new candidate compounds, and provide high-throughput in vitro drug sensitivity functional tests for patients.
  • lung cancer epithelial cells derived from humans or other mammals can be cultured, including lung cancer tumor cells, normal lung cancer epithelial cells, lung cancer epithelial stem cells, or cells comprising at least any of these cells. organization.
  • the culture medium of the present invention can also be used to develop a kit for expanding and culturing primary lung cancer cells in vitro.
  • the cells obtained by the culture method of this embodiment can be applied to regenerative medicine, basic medical research of lung cancer epithelial cells, screening of drug responses, and development of new drugs derived from lung cancer diseases.
  • 1A-1F are graphs showing the effect of different factors in the culture medium on the proliferation of primary lung cancer cells.
  • Figure 2 is a graph showing the effect of increasing the various factors in the culture medium on the proliferation of primary lung cancer cells.
  • 3A-3G are graphs showing the effect of the concentration of each additive factor on the proliferation of primary lung cancer cells.
  • Figures 4A and 4B are photographs taken under an inverted microscope of lung cancer tumor cells cultured on day 5 and day 10, respectively, with cells isolated from a lung cancer clinical tissue sample (No. OB0003) using the medium LM of the present invention.
  • Figure 5 is a summary graph of the primary culture cycle and the resulting cell number for 16 samples.
  • Figure 6 is a graph showing the growth curves of cells isolated from 8 lung cancer puncture samples (numbered 0B0002, 0B0004, 0B0006, 0B0009, 0B0010, 0B0011, 0B0012, and OB0013) continuously cultured under the condition of LM medium.
  • Fig. 7 is a graph showing the total number of cells obtained by culturing cells isolated from a clinical tissue sample of lung cancer (No. 0B0004) under 5 different medium conditions.
  • Figure 8 is a comparison chart of cell growth curves obtained by culturing cells isolated from a case of lung cancer clinical tissue sample (number 0B0004) under 5 different culture medium conditions.
  • Figure 9A is a picture of cells isolated from a lung cancer surgical resection sample (No. 0B0015) obtained by culturing the lung cancer tumor cells with the medium LM of the present invention, and then using the non-specific fluorescent dye DAPI to stain the cell nuclei
  • Figure 9B is a picture of the above
  • the obtained lung cancer tumor cells were stained with the lung adenocarcinoma-specific antibody NapsinA
  • FIG. 9C is the image obtained by combining FIG. 9A and FIG. 9B .
  • Figures 10A-10D are comparison charts of immunohistochemical results of primary tissue cells from a lung cancer surgical resection sample (No. 0B0004) and lung cancer tumor cells cultured with the cells in the medium LM of the present invention.
  • Figure 11 shows the dose-response curves of lung cancer tumor cells cultured to the first and fifth passages using the culture medium LM of the present invention on the cancer tissue samples (No. 0B0011) obtained from the puncture of the same lung cancer patient on different targeted 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 differentiation into epithelial cells, and refer to stem cells derived from epithelial tissue.
  • epithelial tissues include cornea, oral mucosa, skin, conjunctiva, bladder, renal tubule, kidney, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), and large intestine (including colon)) , liver, pancreas, breast, salivary gland, lacrimal gland, prostate, hair root, trachea, lung, etc.
  • the cell culture medium of the present embodiment is preferably a culture medium for lung cancer epithelial cells.
  • epithelial tumor cell refers to a cell derived from the above-described epithelial tissue-derived cell tumorigenic.
  • organoid refers to a three-dimensional, organ-like cellular organoid formed by spontaneously organizing and aggregating cells at a high density in a controlled space.
  • MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signaling.
  • MST1/2 kinase inhibitors for example, bind to MST1/2 kinase and reduce its activity. Due to the structural similarity of MST1 and MST2, MST1/2 kinase inhibitors can also be, for example, compounds that bind to MST1 or MST2 and reduce their activity.
  • Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): In a round bottom flask, add 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) Methanol (30 mL) was then added, followed by dropwise addition of thionyl chloride (1.2 mL) in an ice bath. The reaction system was reacted at 85°C overnight. After the reaction, the solvent was evaporated to dryness in the system under reduced pressure, and the obtained white solid was directly used in the next step.
  • 2-amino-2-(2,6-difluorophenyl)acetic acid 2.0 g
  • Methanol 30 mL
  • thionyl chloride 1.2 mL
  • MST1/2 inhibitor compounds of the present invention were synthesized according to the method similar to compound 1, and their structures and mass spectrometry data are shown in the following table.
  • Lung cancer tissue samples were obtained from three patients with lung cancer tumors who had been described and obtained consent for surgical resection of cancer tissue samples, which were sample numbers 0B0010, 0B0011, and 0B0012, respectively. One of the samples (No. 0B0010) will be described below.
  • tissue samples were collected within half an hour after the patient's surgical excision or biopsy. More specifically, in a sterile environment, take a tissue sample from a non-necrotic site, the volume of which is more than 0.5 cm 3 , place it in a pre-cooled 4 mL tissue transport fluid (see Table 1 for specific preparation), and the transport fluid contains In a 5mL plastic sterile cryopreservation tube with a lid (purchased from Guangzhou Jiete Biotechnology), it was transported to the laboratory in a cold chain (0-10°C).
  • tissue sample No. 0B0010
  • tissue transport fluid transfer the tissue sample to a 100mm cell culture dish (purchased from NEST)
  • tissue transport fluid wash away the residual blood on the surface of the tissue sample, and remove the tissue Excess tissue such as fat on the surface of the sample.
  • transfer the rinsed tissue sample to another new 100 mm petri dish, add 2 mL of transport solution, and use a sterile scalpel blade and forceps to divide the tissue sample into tissue fragments with a volume of less than 3 mm 3 .
  • tissue sample fragments were transferred to a 15mL centrifuge tube, and centrifuged at 1500 rpm for 4 minutes with a desktop centrifuge (Sigma company 3-18K); discard the supernatant, and add tissue transport solution and tissue digestion solution (using The amount is about 5 mL of tissue digestion solution per 10 mg of tissue, the specific preparation is shown in Table 2), the sample number is marked, sealed with parafilm, and digested at 37 ° C, 300 rpm constant temperature shaker (Zhichu Instrument ZQLY-180N), every 1 hour Observe if digestion is complete.
  • tissue clumps were filtered out with a 70 ⁇ m filter, the tissue clumps on the filter were washed with tissue transport solution, and the residual cells were flushed into a centrifuge tube and centrifuged at 1500 rpm for 4 minutes.
  • the other two cases of lung cancer tumor tissue samples were separated according to the same method as above, and the total number of cells obtained were 100,000 (0B0011) and 80,000 (0B0012), respectively.
  • Cultured NIH-3T3 cells (purchased from ATCC, cultured in DMEM medium containing 10% fetal bovine serum) were digested with 0.25% trypsin (purchased from Thermo Fisher Company), and then cultured with 5% (v/v) Fetal bovine serum (purchased from Ekosai), 100 U/mL penicillin and 100 ⁇ g/mL streptomycin in DMEM (purchased from Corning) terminated digestion, and collected into a 15 mL centrifuge tube, centrifuged at 1500 rpm for 4 minutes, and discarded. supernatant.
  • BM basal medium
  • Primocin purchased from Invivogen Company, concentration of 50 mg/mL
  • DMEM/F-12 medium 0.2 vol% Primocin (purchased from Invivogen Company, concentration of 50 mg/mL) was added to the commercially available DMEM/F-12 medium to obtain a final concentration of 100 ⁇ g/mL. Get BM.
  • the medium of different composition was added to the 48-well culture plate pre-plated with NIH-3T3 cells after ⁇ -ray irradiation at a volume of 500 ⁇ l/well.
  • the lung cancer tumor cells (No. 0B0014) isolated from lung cancer tissue according to the same method in Example 1 were seeded in the above-mentioned 48 wells pre-plated with ⁇ -ray irradiated NIH-3T3 cells at a cell number of 4 ⁇ 10 4 cells/well.
  • the surface was sterilized and placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher Scientific), so that the same number of freshly isolated lung cancer tumor cells (number 0B0014) were cultured under different medium formulation conditions .
  • the vertical axis in the figure represents the ratio of the number of cells obtained after culturing in different media to the number of cells obtained after culturing in the basal medium BM.
  • adding different concentrations of different factors in Table 3 on the basis of BM had different effects on cell proliferation.
  • B27 additive, N2 additive, insulin-transferrin-selenium complex, hepatocyte growth factor, insulin sample growth factor 1, fibroblast growth factor 7, neuregulin-1, compound 1 And Y27632 has a certain promoting effect on cell proliferation.
  • the medium of different composition was added into the 48-well culture plate pre-plated with NIH-3T3 cells after ⁇ -ray irradiation at a volume of 500 ⁇ l/well, and the BM medium was used as the experimental control.
  • the lung cancer tumor cells (number 0B0016) isolated from lung cancer tissue according to the method of Example 1 were seeded in the above-mentioned 48 wells pre-plated with NIH-3T3 cells after ⁇ -ray irradiation at the number of 4 ⁇ 10 4 cells/well.
  • NO.7 was determined to be the most preferred medium for the patent for culturing and expanding primary lung cancer cells (hereinafter abbreviated as LM).
  • lung cancer epithelial cells derived from cancer tissues were isolated and obtained from cancer tissues (sample number 0B0002) of lung cancer patients.
  • the cancer tissue-derived lung cancer tumor cells were counted with a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.) to obtain the total number of cells.
  • 4 ⁇ 10 4 cells/cm 2 were seeded into 12-well plates pre-plated with ⁇ -irradiated NIH-3T3 cells.
  • Formula 2 Y27632 is not included in the LM component of the medium
  • Insulin-like growth factor 1 is not contained in the LM component of the medium;
  • Formula 4 the LM component of the medium does not contain hepatocyte growth factor
  • Insulin-transferrin-selenium complex is not included in the LM component of the medium;
  • Formulation 6 no epidermal cell growth factor is contained in the LM component of the medium;
  • Formulation 7 Neuregulin-1 is not included in the LM component of the medium.
  • the above-mentioned recipes 1-7 were used to dilute the above digested cell suspension, and seeded in a 48-well plate pre-plated with ⁇ -ray irradiated NIH-3T3 cells at a volume of 10,000 cells per well, with a volume of 250 microliters.
  • Y27632 When using the medium of formula 2, add 250 microliters of prepared Y27632 to each well of the 48-well plate seeded with primary cells.
  • the final concentrations of Y27632 are 40 ⁇ M, 20 ⁇ M, 10 ⁇ M, 5 ⁇ M, 2.5 ⁇ M, and 1.25 ⁇ M, respectively.
  • ⁇ M, 0.625 ⁇ M; and control wells (BC) were set up using the medium of Recipe 2.
  • hepatocyte growth factor When using the medium of formula 4, add 250 microliters of prepared hepatocyte growth factor to each well of the 48-well plate seeded with primary cells, and the final concentrations of hepatocyte growth factor are 80ng/ml and 40ng/ml, respectively. ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml; and control wells (BC) were set up using the medium of formula 4.
  • the ratio was calculated with reference to the number of cells in the control well (BC), and the results were shown in Figures 3A to 3G, respectively.
  • the ratio is the ratio of the number of cells obtained by culturing each medium for one generation to the number of cells obtained by culturing one generation of the corresponding control wells. If the ratio is greater than 1, it means that the prepared medium containing different concentrations of factors or small molecule compounds has a better effect of promoting proliferation than the control well medium; if the ratio is less than 1, it means that the prepared medium containing different concentrations of factors or small molecule compounds can promote proliferation The effect was weaker than that of the control well medium in promoting proliferation.
  • the content of compound 1 is preferably 0.625 ⁇ M to 20 ⁇ M, more preferably 0.625 ⁇ M to 10 ⁇ M; the content of Y27632 is preferably 1.25 ⁇ M to 20 ⁇ M, more preferably 2.5 ⁇ M to 10 ⁇ M; insulin-like growth factor 1
  • the content of hepatocyte growth factor is preferably 1.25ng/ml ⁇ 80ng/ml, more preferably 5ng/ml ⁇ 80ng/ml; the content of hepatocyte growth factor is preferably 5ng/ml ⁇ 80ng/ml, more preferably 20ng/ml ⁇ 80ng/ml
  • the volume concentration of the insulin-transferrin-selenium complex is preferably 1:25-1:200 (the respective contents of insulin/transferrin/sodium selenite are 2.5-20 ⁇ g/ml-1.25-10 ⁇ g/ml- 1.25 ⁇ 10ng/ml), more preferably 1:25 ⁇ 1:50 (the respective contents
  • lung cancer epithelial cells derived from cancer tissues were isolated and obtained from cancer tissues of lung cancer patients (sample number 0B0003).
  • the cancer tissue-derived lung cancer tumor cells were counted with a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.) to obtain the total number of cells.
  • 4 ⁇ 10 4 cells/cm 2 were seeded into 12-well plates pre-plated with ⁇ -irradiated NIH-3T3 cells.
  • 2 mL of the prepared primary lung cancer epithelial cell medium LM was added to the 12-well plate, and cultured in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher Scientific).
  • Figure 4A is a microscopic photo of the 12-well plate pre-plated with NIH-3T3 cells after ⁇ -ray irradiation at a density of 4 ⁇ 10 4 cells/cm 2 in this example, and cultured from the start of inoculation to the 5th day (100 photographed with an inverted phase contrast microscope). Microscopic observation showed that the cultured primary lung cancer tumor cells derived from cancer tissue had formed larger clones.
  • FIG. 4B is a microscopic photo (photographed by a 100-fold inverted phase contrast microscope) of the 10th day of culture after inoculation in this example, and the cells in the field of view have been overgrown.
  • the lung cancer puncture tissue samples (sample numbers 0B0001 to 0B0016) were digested according to the method of Example 1 to obtain primary lung cancer cells.
  • the cells were seeded in a 12-well plate at a viable cell density of 4 ⁇ 10 4 cells/cm 2 and cultured. After the cells were expanded to 85%, they were digested and counted. At the same time, the number of days of culture up to the time of digestion was recorded, and the number of days of culture until the time of digestion was regarded as one culture cycle.
  • the average number of cells in the 16 lung cancer samples at the beginning of the culture was 72,000, and the average number of cells obtained after the first expansion was 1.212 million, and the average culture period required was 12.6 days.
  • the samples numbered 0B0002, 0B0004, 0B0006, 0B0009, 0B0010, 0B0011, 0B0012, and 0B0013 were continuously cultured for a total of 8 samples, and the amplified cells were amplified in different passages, and each passage was digested and counted and recorded.
  • the primary lung cancer epithelial cell medium LM and the basal medium BM as a control were prepared in the same manner as in Example 2.
  • the medium FM used in the cell conditional reprogramming technical literature was prepared as another control example.
  • the medium formula is shown in Table 5.
  • lung cancer primary cell culture medium LM1 was prepared, and the formula was to replace insulin-transferrin-selenium complex with B27 additive in a volume ratio of 1:50 on the basis of LM.
  • the commercialized medium EpiCult TM Plus Medium (hereinafter also referred to as "Epi medium”) was purchased from STEMCELL Company, and the medium formula is shown in Table 6.
  • the technology of the present invention The technology of the present invention:
  • the primary lung cancer tumor cells were inoculated into a 24-well plate pre-plated with ⁇ -ray irradiated NIH-3T3 cells (purchased from ATCC Company) at an inoculation density of 4 ⁇ 10 4 cells/cm 2 .
  • the primary lung cancer tumor cells were seeded on the NIH-3T3 cells (purchased from ATCC) pre-plated with ⁇ -ray irradiation at a seeding density of 4 ⁇ 10 4 cells/cm 2 , using 1 mL
  • the cell conditional reprogramming technology medium FM was cultured in a 24-well plate (for specific steps, see (Liu et al., Nat.Protoc., 12(2):439-451, 2017);
  • the primary lung cancer tumor cells were inoculated into a 24-well plate pre-plated with ⁇ -irradiated NIH-3T3 cells (purchased from ATCC) at a seeding density of 4 ⁇ 10 4 cells/cm 2 , using 1 mL of culture Base LM1 was cultured in 24-well plates;
  • the primary lung cancer tumor cells were seeded into a 24-well plate at a seeding density of 4 ⁇ 10 4 cells/cm 2 , and cultured in a 24-well plate with 1 mL of the commercial medium Epi.
  • the primary lung cancer tumor cells were seeded into a 24-well plate pre-plated with ⁇ -irradiated NIH-3T3 cells (purchased from ATCC) at a seeding density of 4 ⁇ 10 4 cells/cm 2 , using 1 mL of basal Medium BM was cultured in 24-well plates.
  • the medium of the cells cultured under the five kinds of culture conditions was changed every 5 days.
  • the colony formation and cell proliferation status of the cells in each medium in the 24-well plate were observed, and the cell growth status was recorded by taking pictures with a microscope (EVOS M500 from Invitrogen).
  • Figure 7 is a graph of the total number of cells expanded under different conditions for cells numbered OB0006.
  • the primary lung cancer epithelial cell medium LM, and medium FM, LM1, Epi and BM as controls were obtained by the same method as in this Example (1).
  • the cultured cells are collected and counted by digesting again according to the above operation method. It was also seeded at a density of 4 ⁇ 10 4 /well and cultured continuously.
  • PD Population Doubling
  • Figure 8 shows the growth curves of cell OB0004 under five different culture conditions drawn by Graphpad Prism software.
  • the abscissa represents the days of cell culture, and the ordinate is the cumulative cell multiplication factor, which represents the multiplication factor of the cell in the culture cycle. The higher the number, the slope represents the rate of cell expansion. From the figure, it can be confirmed that the proliferation rate of lung cancer epithelial cells cultured in the medium LM and LM1 of the present invention is better than that of the other three culture conditions, and it can be confirmed that the technology of the present invention can continuously culture primary lung cancer epithelial cells.
  • lung cancer epithelial cells derived from cancer tissues were isolated and obtained from cancer tissues of lung cancer patients (sample number 0B0015).
  • the cancer tissue-derived lung cancer tumor cells were counted with a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.) to obtain the total number of cells.
  • JIMBIO FIL Jiangsu Zhuo Microorganism Technology Co., Ltd.
  • it was seeded into a 24-well plate pre-plated with ⁇ -ray-irradiated NIH-3T3 cells, and round cell fragments for immunofluorescence staining were pre-placed in the 24-well plate ( purchased from Thermo Fisher Scientific).
  • 1 mL of the prepared primary lung cancer epithelial cell culture medium LM was added to the 24-well plate, and cultured in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher Scientific).
  • the non-specific fluorescent dye DAPI (purchased from Sigma) was diluted with PBS at a volume ratio of 1:1000, stained at room temperature for 5 minutes, and washed with PBS for 5 minutes x 3 times. The images were imaged under a microscope (EVOS M500 from Invitrogen Company), and photographed and recorded.
  • Figures 9A-C are pictures taken under different conditions under a 10x objective lens, in which Figure 9A is a picture of nuclei stained with a non-specific fluorescent dye DAPI, and Figure 9B is a picture of a lung adenocarcinoma-specific antibody NapsinA (localized in the cytoplasm) stained picture of.
  • Fig. 9C is a picture obtained by combining Figs. 9A and 9B.
  • the nuclei marked in Figure 9A and the cytoplasm in Figure 9B can be marked by specific antibodies, indicating that the cultured cells are lung adenocarcinoma cells, which is consistent with the clinicopathological diagnosis.
  • a cancer tissue about the size of a mung bean grain (sample number 0B0004) was taken from a clinical surgical resection sample of a patient with lung cancer, and was immersed in 1 mL of 4% paraformaldehyde for fixation.
  • Lung cancer epithelial cells (sample number 0B0004) were obtained from the remaining cancer tissue using the same method as in Example 1.
  • the sample OB0004 was continuously cultured to the 5th passage using the medium LM of the present invention.
  • Immunohistochemical method was used to detect the expression of important biomarkers related to lung cancer in the original tissue of sample OB0004 and the primary cells that were continuously cultured to the third passage.
  • the tissue fixed with 4% paraformaldehyde was embedded in paraffin and cut into 4 ⁇ m thick tissue sections with a microtome. Routine immunohistochemical detection was then performed (for specific steps, see Li et al., Nature Conmunication, (2016) 9:2983).
  • the primary antibodies used were thyroid transcription factor 1 (TTF-1) (purchased from CST company) and ki-67 antibody (purchased from R&D company).
  • FIGS. 10A-10D are comparison diagrams of the immunohistochemical results of the original tissue cells and the lung cancer tumor cells obtained by culturing the cells with the medium LM of the present invention.
  • Fig. 10A and Fig. 10B are pictures of lung cancer tissue and cell marker thyroid transcription factor 1 antibody obtained after expansion and culture, respectively
  • Fig. 10C and Fig. 10D are pictures of lung cancer tissue and cell marker ki-67 antibody obtained after expansion and culture, respectively . From this, it can be confirmed that when the lung cancer tumor cells (sample number 0B0004) cultured by the technology of the present invention are cultured to the fifth passage, the expression of biomarkers related to lung cancer on the cells and the expression of markers in the original tissue sections derived from the cells Basically the same. It shows that the cells cultured by the technology of the present invention maintain the original pathological characteristics of the cancer tissue of lung cancer patients.
  • Lung cancer tumor cells (number 0B0003) were isolated from the cancer tissue of a patient with a pathological diagnosis of lung cancer using the same method as in Example 1, and the culture medium LM of the present invention was used to culture OB0003 according to the method in Example 3. When the number of cells reached 1 ⁇ 10 7 , lung cancer tumor cells were digested by the method of Example 5 and collected.
  • lung cancer tumor cell culture medium LM of the present invention and (Purchased from BD Biotechnology Co., Ltd.) mixed at a ratio of 1:1, sucked 100 ⁇ L of the medium mixed with Matrigel to resuspend 5 ⁇ 10 6 lung cancer tumor cells, and injected them into 6-week-old females with high immunodeficiency Mouse (NCG) mice (purchased from Nanjing Institute of Model Animals) were used to observe the volume and growth rate of lung cancer tumor cells in the lung cancer fat pad and right forelimb axilla every three days.
  • NCG immunodeficiency Mouse
  • mice On the 15th day after tumor cell inoculation, tumor formation could be observed in two tumor cell inoculation sites in mice. From the 15th day to the 36th day, the tumor proliferation in the mice was obvious. This indicates that the lung cancer tumor cells derived from cancer tissue cultured by the culture method of the present invention have tumorigenicity in mice.
  • the following takes a puncture sample from a lung cancer patient as an example to illustrate that lung cancer tumor cells cultured from a patient-derived lung cancer tumor sample can be used to detect the sensitivity of the patient tumor cells to different drugs.
  • Plating of primary lung cancer tumor cells The isolated lung cancer tumor cell (No. 0B0011) cell suspension obtained according to the method in Example 1 was inoculated at a density of 4 ⁇ 10 4 cells/cm 2 to pre-plated ⁇ -ray radiation Illuminated NIH-3T3 cells within a 6-well plate. 2 mL of the prepared primary lung cancer epithelial cell culture medium LM was added to the 6-well plate, and cultured in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher Scientific).
  • the cell pellets after centrifugation were resuspended in LM medium, and counted using a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.) to obtain a total of 1.6 million first-generation cells.
  • the cells were seeded in 384-well plates at a density of 2000-4000 cells/well, and the cells were allowed to adhere overnight.
  • the remaining cells were continuously seeded at a density of 4 ⁇ 10 4 cells/cm 2 into a 6-well plate pre-plated with ⁇ -ray-irradiated NIH-3T3 cells for 4 consecutive passages, and digested and terminated according to the above method. After the cells were resuspended, the cells were counted using a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.), and the total number of cells of the fifth generation was 1.8 million. The cells were seeded in 384-well plates at a density of 2000-4000 cells/well, and the cells were allowed to adhere overnight.
  • a flow image counter JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.
  • the drugs to be tested are afatinib (purchased from MCE company), gefitinib (purchased from MCE company), osimertinib (purchased from MCE company) and crizotinib (purchased from MCE company) company).
  • afatinib purchased from MCE company
  • gefitinib purchased from MCE company
  • osimertinib purchased from MCE company
  • crizotinib purchased from MCE company
  • Detection of cell activity 72 hours after administration, use Cell Titer-Glo detection reagent (purchased from Promega) to detect the chemiluminescence value of the cells after drug addition and culture.
  • the size of the chemiluminescence value reflects cell viability and drug-induced cell viability 10 ⁇ L of the prepared Cell Titer-Glo detection solution was added to each well, and the chemiluminescence value was detected using a microplate reader (Envision, Perkin Elmer Company) after mixing.
  • cell viability (%) chemiluminescence value of drug-added well/chemiluminescence value of control well*100%, calculate the cell viability of cells treated with different drugs, and use Graphpad Prism software to draw the graph And calculate the half inhibition rate IC 50 , and calculate the cell survival rate of different drugs corresponding to the maximum blood concentration Cmax in the human body.
  • Figure 11 shows that the first-generation lung cancer tumor cells and the fifth-generation lung cancer tumor cells cultured from the surgically resected cancer tissue sample (No. 0B0011) from the same lung cancer patient showed that the targeted drugs afatinib, gefitinib, Sensitivity to osimertinib and crizotinib.
  • the concentration corresponding to the marked line on the abscissa in the figure is the maximum blood concentration Cmax of these four drugs in the human body.
  • the results show that the cells of the same patient have different sensitivities to different drugs at the maximum blood concentration in the human body, and the cells of different generations have basically the same sensitivity to the same drug. According to the results, the effectiveness of the drug in clinical use of the drug can be judged for lung cancer patients, and it can be shown that the sensitivity of tumor cells of different generations obtained according to the patented culture method to the drug is stable.
  • the invention provides a culture medium and a culture method for culturing or expanding primary lung cancer epithelial cells in vitro, and the cultured cells can be applied to the evaluation and screening of the curative effect of drugs.
  • the present invention is suitable for industrial applications.

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Abstract

Provided is a primary cell culture medium for culturing lung cancer epithelial cells, containing an MST1/2 kinase inhibitor; an insulin-like growth factor 1; epidermal cell growth factor; hepatocyte growth factor; neuregulin-1; an additive selected from at least one of an insulin-transferrin-selenium complex, a B27 additive, and an N2 additive; an ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152. Further provided are a culture method using the primary cell culture medium, and a method and the use of the culture medium and the culture method in the therapeutic effect evaluation and screening of a drug.

Description

一种用于肺癌上皮细胞的培养基、培养方法及其应用A kind of medium, culture method and application for lung cancer epithelial cells 技术领域technical field
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肺癌上皮细胞的培养基及培养方法，还涉及培养得到的细胞在药物的疗效评估和筛选中的方法和应用。The invention belongs to the technical field of medicine, in particular to a culture medium and a culture method for culturing or amplifying primary lung cancer epithelial cells in vitro, and also relates to a method and application of the cultured cells in the efficacy evaluation and screening of drugs .
背景技术Background technique
肺癌目前仍是最常见和致死性最高的肿瘤之一，全世界每年有160万例与肿瘤相关的死亡。肺癌是一种多样的、复杂的、治疗具有挑战性的疾病。在过去的十年里，随着个性化医疗的出现，对肺癌的基础生物学和分子机制有了进一步的理解。肺癌不再是一种单一的疾病实体，现在被细分为分子亚型，分别对应于专门的针对性化疗策略。Lung cancer remains one of the most common and deadly cancers, with 1.6 million cancer-related deaths worldwide each year. Lung cancer is a diverse, complex, and treatment-challenging disease. Over the past decade, with the advent of personalized medicine, there has been a greater understanding of the underlying biology and molecular mechanisms of lung cancer. Lung cancer is no longer a single disease entity, but is now subdivided into molecular subtypes, each corresponding to specialized targeted chemotherapy strategies.
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肺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且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成功率往往很低，生长周期长，并存在成纤维细胞等***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滋养细胞和ROCK激酶抑制剂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)。Functional testing refers to the detection of the in vitro sensitivity of antitumor drugs on cells of cancer patients. The key to applying this approach is to develop tumor cell models that have a short growth cycle and can represent the own biological characteristics of lung cancer patients. In addition, the cell model should be easy to operate and be able to quickly and efficiently predict the efficacy of clinical medication, so as to give cancer patients precise medication guidance in a timely manner. However, the success rate of establishing cell models in vitro from primary tumor cells from cancer patients is often 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 currently two techniques for culturing primary epithelial cells/stem cells that are relatively mature in the field of tumor cell functional testing applications. One is the use of irradiated trophoblast cells and ROCK kinase inhibitors to promote the growth of primary epithelial cells to investigate Techniques for drug sensitivity of individual patients, ie, cell conditional reprogramming techniques (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 tissue organs (Hans Clevers et al., Cell, 11, 172(1-2):373-386, 2018).
然而，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，但是该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)。However, organoid technology is a technology of embedding patient's primary epithelial cells in extracellular matrix for in vitro three-dimensional culture, but the medium of this technology needs to add a variety of specific growth factors (such as Wnt protein and R-spondin) family proteins), the cost is high, and it is not suitable for widespread clinical application on a large scale. In addition, the organoids need to be embedded in extracellular matrix gel during the entire culture process, and the plating steps of cell seeding, passage and drug sensitivity test are cumbersome and time-consuming compared with 2D culture operations, and the technology formed by The size and size of organoids are not well controlled, and some organoids may grow too large and cause internal necrosis. Therefore, compared with 2D culture technology, the operability and applicability of organoid technology is not strong, requiring professional and technical personnel to operate, and it is not suitable for large-scale and widely used in clinical in vitro drug sensitivity detection (Nick Barker, Nat. Cell Biol., 18 (3): 246-54, 2016).
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，现有文献中用于体外扩增肺癌原代细胞的培养基中含血清成分，由于血清成分不明确，不同批次之间差异大，容易对实验结果产生一定干扰。Cell reprogramming technology is a technology of co-culturing patients' autologous primary epithelial cells with mouse-derived feeder cells. In the existing literature, the culture medium used for in vitro expansion of primary lung cancer cells contains serum components. It is clear that there are large differences between different batches, which may easily interfere with the experimental results.
鉴于以上技术的局限性，临床上需要开发一种原代肺癌上皮细胞培养技术，其培养周期短，成本可控，操作便捷，在将该技术应用于构建原代肺癌肿瘤细胞模型时，所培养的肺癌肿瘤细胞能代表肺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。In view of the limitations of the above technologies, it is clinically necessary to develop a primary lung cancer epithelial cell culture technology, which has a short culture period, controllable cost, and convenient operation. When this technology is applied to construct primary lung cancer tumor cell models, the cultured The lung cancer tumor cells can represent the biological characteristics of lung cancer patients themselves. By evaluating the sensitivity of antitumor drugs in cell models derived from different cancer patients in vitro, the response rate of clinical antitumor drugs can be improved, and the pain caused by inappropriate drugs to patients and the waste of medical resources can be reduced.
发明内容SUMMARY OF THE INVENTION
本发明旨在针对现有技术的不足，提供一种用于培养原代肺癌上皮细胞的培养基以及使用该培养基的原代肺癌上皮细胞的培养方法。采用本发明的原代肺癌上皮细胞培养基和培养方法，能够达到体外培养周期短、成本可控、操作便捷的目的。在该技术应用于构建原代肺癌肿瘤细胞模型时，能够获得具有肺癌患者自身生物学特性的原代肺癌肿瘤细胞，并能够应用于新药筛选和体外药物敏感性检测。The present invention aims at providing a culture medium for culturing primary lung cancer epithelial cells and a method for culturing primary lung cancer epithelial cells using the culture medium in view of the deficiencies of the prior art. The primary lung cancer epithelial cell culture medium and culture method of the present invention can achieve the goals of short in vitro culture period, controllable cost and convenient operation. When the technology is applied to construct a primary lung cancer tumor cell model, primary lung cancer tumor cells with the own biological characteristics of lung cancer patients can be obtained, and can be applied to new drug screening and in vitro drug sensitivity detection.
本发明的一个方面在于提供一种用于培养原代肺癌上皮细胞的原代细胞培养基，其含有MST1/2激酶抑制剂；***1(IGF-1)；表皮细胞生长因子(EGF)；肝细胞生长因子(HGF)；神经调节蛋白-1；选自胰岛素-转铁蛋白-硒复合物(ITS)、B27添加剂和N2添加剂中的至少一种的添加剂；选自Y27632、法舒地尔、和H-1152中的至少一种的ROCK激酶抑制剂，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。One aspect of the present invention is to provide a primary cell culture medium for culturing primary lung cancer epithelial cells, which contains MST1/2 kinase inhibitor; insulin-like growth factor 1 (IGF-1); epidermal growth factor (EGF) ); hepatocyte growth factor (HGF); neuregulin-1; an additive selected from at least one of insulin-transferrin-selenium complex (ITS), B27 additive and N2 additive; selected from Y27632, Fasu A ROCK kinase inhibitor of at least one of dil, and H-1152, the MST1/2 kinase inhibitor comprising a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof.
Figure PCTCN2020109740-appb-000001
Figure PCTCN2020109740-appb-000001
其中，in,
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 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl optionally substituted with 1-2 independently R 6 (such as phenyl and naphthyl, etc.), aryl C1-C6 alkyl (such as benzyl, etc.) and heteroaryl (such as thienyl, etc.);
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 2 and R 3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
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 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl group is selected from, for example, piperidinyl, tetrahydropyran base, etc.);
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)。 R 6 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).
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，In a preferred embodiment, the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,
Figure PCTCN2020109740-appb-000002
Figure PCTCN2020109740-appb-000002
其中，in,
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 1 is selected C1-C6 alkyl, optionally substituted with 1-2 R 6 independently substituted phenyl, optionally substituted with 1-2 R 6 independently substituted thienyl, and optionally substituted with 1 - 2 independently R 6 substituted benzyl, R 1 more preferably phenyl optionally substituted with 1-2 independently R 6;
R 5选自氢、C1-C6烷基、和C3-C6环烷基，R 5更优选为氢； R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R 5 is more preferably hydrogen;
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。 R 6 is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, more preferably R 6 is fluoro, methyl or trifluoromethyl.
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。Preferably, the MST1/2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts, or solvates thereof.
Figure PCTCN2020109740-appb-000003
Figure PCTCN2020109740-appb-000003
Figure PCTCN2020109740-appb-000004
Figure PCTCN2020109740-appb-000004
Figure PCTCN2020109740-appb-000005
Figure PCTCN2020109740-appb-000005
Figure PCTCN2020109740-appb-000006
Figure PCTCN2020109740-appb-000006
Figure PCTCN2020109740-appb-000007
Figure PCTCN2020109740-appb-000007
最优选地，本发明的MST1/2激酶抑制剂为化合物1。Most preferably, the MST1/2 kinase inhibitor of the present invention is Compound 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0.625μM～20μM，优选为0.625μM～10μM，更优选为2.5μM。In an embodiment of the present invention, the content of the MST1/2 kinase inhibitor in the medium is usually 0.625 μM to 20 μM, preferably 0.625 μM to 10 μM, and more preferably 2.5 μM.
在优选的实施方式中，所述***1的含量优选为1.25～80ng/ml，更优选为5～80ng/ml；所述肝细胞生长因子的含量优选为5～80ng/ml，更优选为20～80ng/ml；所述表皮细胞生长因子的含量优选为10～80ng/ml，更优选为10～40ng/ml；所述神经调节蛋白-1的含量优选为10～80ng/ml，更优选20～80ng/ml；所述添加剂的含量优选体积比1:200～1:25，更优选体积比1:50～1:25，且所述添加剂优选为胰岛素-转铁蛋白-硒复合物，其中胰岛素/转铁蛋白/***钠各自的含量优选分别为2.5～20μg/ml-1.25～10μg/ml-1.25～10ng/ml，更优选分别为10～20μg/ml-5～10μg/ml-5～10ng/ml；所述ROCK激酶抑制剂的含量优选为1.25～20μM，更优选为2.5～10μM，所述ROCK激酶抑制剂优选为Y27632。In a preferred embodiment, the content of the insulin-like growth factor 1 is preferably 1.25-80 ng/ml, more preferably 5-80 ng/ml; the content of the hepatocyte growth factor is preferably 5-80 ng/ml, more preferably It is preferably 20-80ng/ml; the content of the epidermal growth factor is preferably 10-80ng/ml, more preferably 10-40ng/ml; the content of the neuregulin-1 is preferably 10-80ng/ml, More preferably 20-80ng/ml; the content of the additive is preferably 1:200-1:25 by volume, more preferably 1:50-1:25 by volume, and the additive is preferably an insulin-transferrin-selenium complex wherein the respective contents of insulin/transferrin/sodium selenite are preferably 2.5-20μg/ml-1.25-10μg/ml-1.25-10ng/ml, more preferably 10-20μg/ml-5-10μg, respectively /ml-5-10ng/ml; the content of the ROCK kinase inhibitor is preferably 1.25-20 μM, more preferably 2.5-10 μM, and the ROCK kinase inhibitor is preferably Y27632.
该培养基配方成分与细胞条件重编程培养基和类器官培养基成分相比，添加了MST1/2激酶抑制剂，但不包含血清、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，并且不包含烟酰胺和N-乙酰半胱氨酸， 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肺癌上皮细胞的体外培养。Compared with the composition of cell-conditioned reprogramming medium and organoid medium, the composition of this medium is supplemented with MST1/2 kinase inhibitors, but does not contain uncertain components such as serum and bovine pituitary extract, nor does it contain Wnt agonists , R-spondin family proteins, BMP inhibitors and other necessary niche factors for organoid culture, and does not contain nicotinamide and N-acetylcysteine, which greatly reduces the cost of the medium and simplifies the operation of preparing the medium The process realizes the in vitro culture of primary lung cancer epithelial cells with controllable cost and convenient operation.
本发明中，原代肺癌上皮细胞可以为肺癌肿瘤细胞、正常肺癌上皮细胞、肺癌上皮干细胞。In the present invention, the primary lung cancer epithelial cells can be lung cancer tumor cells, normal lung cancer epithelial cells, and lung cancer epithelial stem cells.
本发明的一个方面在于提供一种原代肺癌上皮细胞的培养方法，其包括以下步骤：One aspect of the present invention is to provide a method for culturing primary lung cancer epithelial cells, comprising the following steps:
(1)按上述配方配制本发明的原代细胞培养基。(1) The primary cell culture medium of the present invention is prepared according to the above formula.
(2)使用辐照后的滋养细胞预铺培养容器。(2) Use the irradiated trophoblast to pre-plate the culture vessel.
具体地，所述的滋养细胞例如可以为辐照后的NIH-3T3细胞，辐照源为X射线或者γ射线，优选为γ射线，辐照剂量为30～50Gy，优选为35Gy。具体的，将辐照后的NIH-3T3细胞按照2×10 4个/cm 2接种在培养容器例如48孔板、24孔板、12孔板、6孔板或者T25细胞培养瓶中，待细胞贴壁后备用。 Specifically, the trophoblasts can be, for example, irradiated NIH-3T3 cells, the irradiation source is X-rays or γ-rays, preferably γ-rays, and the irradiation dose is 30-50 Gy, preferably 35 Gy. Specifically, the irradiated NIH-3T3 cells are seeded in a culture vessel such as a 48-well plate, a 24-well plate, a 12-well plate, a 6-well plate or a T25 cell culture flask at 2×10 4 cells/cm 2 . Reserve after sticking to the wall.
(3)从肺癌组织分离得到原代肺癌上皮细胞。(3) Primary lung cancer epithelial cells were isolated from lung cancer tissue.
原代肺癌上皮细胞例如可以来源于肺癌组织样本和肺癌穿刺或者肺支气管内镜样本。肺癌组织样本例如来源于进行过说明并获得同意的肺癌肿瘤患者手术切除的癌组织样本，肺癌穿刺或者支气管内镜样本例如来源于进行过说明并获得同意的肺癌肿瘤患者进行活体检查时所取的穿刺或者支气管内镜样本。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，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3-5mL 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1-2体积％青霉素/链霉素、和/或0.2-0.4体积％Primocin(以下简称组织运输液)。当使用链霉素/青霉素时，链霉素浓度范围为25～400μg/mL，优选为50～200μg/mL，更优选为200μg/mL，青霉素浓度范围为25～400U/mL，优选为50～200U/mL，更优选为200U/mL；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。 Primary lung cancer epithelial cells can be derived, for example, from lung cancer tissue samples and lung cancer biopsy or lung bronchoscopy samples. Lung cancer tissue samples, such as tumor tissue samples from surgically resected lung cancer tumor patients who have been described and obtained consent, lung cancer puncture or bronchoscopy samples, such as those obtained from lung cancer tumor patients who have been described and obtained consent for biopsy Puncture or bronchoscopy samples. The above-mentioned tissue samples were collected within half an hour after the patient's surgical excision or biopsy. More specifically, in a sterile environment, take a tissue sample from a non-necrotic site with a volume of more than 0.5 cm 3 and place it in a pre-cooled 3-5 mL DMEM/F12 medium, which is placed in a plastic-free medium. The bacteria were placed in a capped centrifuge tube and transported to the laboratory on ice; DMEM/F12 medium contained 1-2 vol% penicillin/streptomycin, and/or 0.2-0.4 vol% Primocin (hereinafter referred to as tissue transport fluid). When streptomycin/penicillin is used, the streptomycin concentration is in the range of 25-400 μg/mL, preferably 50-200 μg/mL, more preferably 200 μg/mL, and the penicillin concentration is in the range of 25-400 U/mL, preferably 50- 200 U/mL, more preferably 200 U/mL; when Primocin is used, the concentration range is 25-400 μg/mL, preferably 50-200 μg/mL, more preferably 100 μg/mL.
在生物安全柜内，将组织样本转移至细胞培养皿内，用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。 In the biological safety cabinet, transfer the tissue sample to a cell culture dish, rinse the tissue sample with the transport fluid, and wash away the blood cells on the surface of the tissue sample. Transfer the rinsed tissue sample to another new petri dish, add 1-3 mL of transport solution, and use a sterile scalpel blade and forceps to divide the tissue sample into tissue fragments with a volume of less than 3 mm 3 .
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1～5mL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。Transfer the tissue sample fragments to a centrifuge tube, and centrifuge with a desktop centrifuge (Sigma 3-18K) at 1000-3000 rpm for 3-5 minutes; discard the supernatant and add tissue transport solution and tissue digestion at a ratio of 1:1 (The usage amount is about 5mL tissue digestion solution per 10mg tissue, wherein the preparation method of tissue digestion solution is: 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 in a volume ratio of 1:1), labeled samples Numbered, sealed with parafilm, digested at 37°C, 200-300 rpm constant temperature shaker (Zhichu Instrument ZQLY-180N), and observed every 1 hour to see if the digestion is complete; if no obvious tissue block is seen, the digestion can be terminated, otherwise continue to digest , until the digestion is sufficient, and the digestion time ranges from 4 to 8 hours. After digestion is complete, undigested tissue clumps are filtered out with a cell strainer (cell sieve pore size is, for example, 70 μm), the tissue clumps on the strainer are rinsed with tissue transport solution, and the remaining cells are flushed into a centrifuge tube, and the clumps are washed with a table top 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 1-5 mL of blood cell lysis solution (purchased from Sigma), mix well, lyse at 4°C for 10-20 minutes, shake and mix once in 5 minutes, and the lysis is over. Then take it out and centrifuge at 1000-3000 rpm for 3-5 minutes. The supernatant was discarded, the primary cell culture medium of the present invention was added to resuspend, and a flow image counter (Jiangsu Zhuo Microorganism Technology Co., Ltd. JIMBIO FIL) was used to count to obtain the total number of cells.
(4)在预接种有滋养细胞的培养器皿内接种步骤(3)中分离得到的原代肺癌上皮细胞，并采用步骤(1)中的原代细胞培养基进行培养。(4) inoculating the primary lung cancer epithelial cells isolated in step (3) in a culture vessel pre-seeded with trophoblasts, and using the primary cell culture medium in step (1) for culturing.
更具体而言，预先在多孔板的一个孔中按2×10 4～4×10 4个/cm 2(例如2×10 4个/cm 2)的密度接种35Gy辐照剂量γ射线辐照后的NIH-3H3细胞，待细胞贴壁后，按2×10 4～8×10 4个/cm 2(例如4×10 4个/cm 2)的密度接种原代肺癌肿瘤细胞，每孔加入0.5～2mL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，在原代肺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。 More specifically, a hole of a multi-well plate is pre- inoculated at a density of 2 × 10 4 to 4 × 10 4 /cm 2 (for example, 2 × 10 4 /cm 2 ) with an irradiation dose of 35 Gy after γ-ray irradiation. NIH-3H3 cells of 2 × 10 4 to 8 × 10 4 cells/cm 2 (for example, 4 × 10 4 cells/cm 2 ) were inoculated with primary lung cancer tumor cells after the cells adhered to the wall. ~2mL of primary epithelial cell culture medium, for example, at 37°C, 5% CO 2 in a cell incubator for 8-16 days, changing to fresh primary cell culture medium every 4 days during this period. The cells were digested and passaged when the cells grew to a cell density of about 80% to 90% of the bottom area of the multi-well plate.
该步骤相比类器官技术，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。此外，还节约了价格昂贵 的细胞外基质胶的使用量，简化了操作步骤。Compared with organoid technology, this step does not need to mix primary cells and Matrigel on ice to form gel droplets, and wait for the gel droplets to solidify before adding culture medium. In addition, the usage of expensive extracellular matrix glue is saved, and the operation steps are simplified.
任选地，接种后的原代肺癌上皮细胞在培养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1～2mL0.25％胰酶(购自Thermo Fisher公司)消化1分钟，随后吸出0.25％胰酶，再加入1～2mL 0.05％胰酶进行细胞消化，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链霉素的培养液2～4mL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预铺有滋养细胞的T25细胞培养瓶中继续扩大培养。T25细胞培养瓶的预处理操作同步骤(2)。Optionally, after the inoculated primary lung cancer epithelial cells are cultured for 8-16 days, when the confluence of the cell clones formed in the culture vessel reaches 80% of the bottom area, the supernatant is discarded, and 1-2 mL of 0.25% trypsin (purchased) is added. Digest from Thermo Fisher for 1 minute, then aspirate 0.25% trypsin, add 1-2 mL of 0.05% trypsin for cell digestion, and incubate at room temperature for 5-20 minutes; Resuspend the digested cells in 2 to 4 mL of a culture medium of serum, 100 U/mL penicillin and 100 μg/mL streptomycin, centrifuge at 1000 to 3000 rpm for 3 to 5 minutes, and use the primary cell culture medium of the present invention. The digested single cells were resuspended, and the obtained cell suspension was placed in a T25 cell culture flask pre-plated with feeder cells to continue expanding the culture. The pretreatment operation of the T25 cell culture flask is the same as step (2).
扩增的肺癌上皮细胞呈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。The expanded lung cancer epithelial cells grow in 2D, avoiding the non-uniform size of organoids and internal necrosis of overgrown organoids that occur with organoid technology expansion.
另一方面，由本发明的原代肺癌上皮细胞的培养方法培养得到的肺癌上皮细胞、特别是肺癌肿瘤细胞，能够用于药物的疗效评估和筛选，所述方法包括以下步骤：On the other hand, lung cancer epithelial cells, especially lung cancer tumor cells, cultured by the method for culturing primary lung cancer epithelial cells of the present invention can be used for drug efficacy evaluation and screening, and the method includes the following steps:
(1)获取原代肺癌上皮细胞，特别优选获取源自肺癌患者的癌组织样本或活检癌组织样本，分离得到原代肺癌上皮细胞，根据如上所述的原代肺癌上皮细胞的培养方法培养并扩增原代肺癌上皮细胞(特别是原代肺癌肿瘤细胞)达至少10 5数量级、优选至少10 6数量级的细胞数目。 (1) Obtaining primary lung cancer epithelial cells, particularly preferably, obtaining cancer tissue samples or biopsy cancer tissue samples derived from lung cancer patients, isolating primary lung cancer epithelial cells, culturing and Primary lung cancer epithelial cells (especially primary lung cancer tumor cells) are expanded to a cell number of the order of at least 10 5 , preferably of the order of at least 10 6 .
(2)选定需要检测的药物。(2) Select the drugs to be tested.
(3)药物以其最大血浆浓度C max为参考，以2-5倍C max为起始浓度，稀释多个不同的药物浓度梯度，例如5-10个、优选6-8个药物浓度梯度。 (3) The drug takes its maximum plasma concentration Cmax as reference, and takes 2-5 times Cmax as the initial concentration, and dilutes multiple different drug concentration gradients, such as 5-10, preferably 6-8 drug concentration gradients.
(4)将步骤(1)中培养得到的肺癌上皮细胞消化成单细胞悬液，用流式图像计数仪进行计数，用本发明的原代细胞培养基将单细胞悬液稀释，按每孔2000-4000个的密度将稀释后的细胞悬液均匀地加入到多孔板内，例如每孔50μL细胞稀释液，并进行过夜贴壁。(4) Digest the lung cancer epithelial cells cultured in step (1) into a single cell suspension, count with a flow image counter, dilute the single cell suspension with the primary cell culture medium of the present invention, press each well At a density of 2000-4000 cells, the diluted cell suspension is evenly added to the multi-well plate, eg, 50 μL of cell dilution per well, and allowed to adhere overnight.
该步骤避免了细胞重编程技术出现的由于饲养细胞的存在而干扰原代细胞计数和后续的原代细胞活力检测的问题，也无需像类器官技术一样，在冰上将细胞悬液与基质胶混合包埋再铺板的繁琐步骤，从 而大大简化了操作流程，增强了技术的可操作性和实用性。由于接种的细胞为单细胞悬液而不是像类器官一样的3D结构，所以该技术与类器官技术相比，铺板的细胞数更加均一，铺板产生的孔间细胞数差异小，也更适合进行后续的高通量药物筛选操作。This step avoids the problem of cell reprogramming techniques that interfere with primary cell counts and subsequent primary cell viability assays due to the presence of feeder cells, and eliminates the need to mix cell suspensions with Matrigel on ice as in organoid techniques. The tedious steps of hybrid embedding and re-plating greatly simplify the operation process and enhance the operability and practicality of the technology. Since the seeded cells are single-cell suspensions rather than organoid-like 3D structures, this technique, compared with organoid techniques, results in a more uniform number of plated cells and less variation in the number of cells between wells produced by plating, making it more suitable for Subsequent high-throughput drug screening operations.
(5)采用高通量自动化工作站，对步骤(4)中得到的贴壁细胞添加梯度稀释后的所选定的传统化疗药物、靶向药物、抗体药物或几种药物组合等候选药物，进行处理。(5) Using a high-throughput automated workstation, add the selected traditional chemotherapeutic drugs, targeted drugs, antibody drugs or several drug combinations and other candidate drugs after gradient dilution to the adherent cells obtained in step (4), and carry out deal with.
(6)加药处理数小时后，例如72小时后，采用Cell-Titer Glo发光法细胞活力检测试剂盒(购自Promega公司)检测肺癌上皮细胞的存活率，进行药物活性筛选。(6) After a few hours of drug addition, for example, 72 hours later, use the Cell-Titer Glo luminescence cell viability detection kit (purchased from Promega) to detect the survival rate of lung cancer epithelial cells to screen for drug activity.
具体而言，向每孔加入例如10μL Cell Titer-Glo试剂(购自Promega公司)，均匀震荡后，用荧光酶标仪测量各孔的化学发光强度，根据测得的数值，以药物浓度为横坐标，荧光强度为纵坐标，应用GraphPad Prism软件绘制药物量-效曲线，计算各个药物对所测试细胞的增殖的抑制强度。Specifically, add, for example, 10 μL of Cell Titer-Glo reagent (purchased from Promega) to each well, and after uniform shaking, measure the chemiluminescence intensity of each well with a fluorescence microplate reader, and according to the measured values, take the drug concentration as the horizontal Coordinates, the fluorescence intensity is the ordinate, the drug dose-effect curve was drawn by GraphPad Prism software, and the inhibitory intensity of each drug on the proliferation of the tested cells was calculated.
在本发明的原代肺癌肿瘤细胞在药物筛选和体外药物敏感性检测的应用中。由于细胞呈2D生长，与药物的作用时间也比类器官技术的药物检测时间短(类器官技术的平均给药时间为6天)。In the application of the primary lung cancer tumor cells of the present invention in drug screening and in vitro drug sensitivity detection. Due to the 2D growth of the cells, the interaction time with the drug is also shorter than the drug detection time of the organoid technology (the average administration time of the organoid technology is 6 days).
本发明的有益效果还包括：The beneficial effects of the present invention also include:
(1)提高原代肺癌上皮细胞培养的成功率，成功率达到85％以上；(1) Improve the success rate of primary lung cancer epithelial cell culture, with a success rate of over 85%;
(2)保证体外原代培养的肺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；(2) To ensure that the primary cultured lung cancer epithelial cells in vitro can maintain the pathological phenotype and heterogeneity of the primary cell-derived patient;
(3)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；(3) The medium composition does not contain serum, so it is not affected by the quality and quantity of different batches of serum;
(4)扩增肺癌上皮细胞效率高，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数量级的肺癌上皮细胞，扩增出的肺癌上皮细胞还可以连续传代； (4) The efficiency of amplifying lung cancer epithelial cells is high. As long as there are 10 4- level cells, 10 6 -level lung cancer epithelial cells can be successfully expanded in about two weeks, and the expanded lung cancer epithelial cells can also be continuously passaged ;
(5)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；(5) There is no need to operate on ice and dissociate Matrigel in the passage step, and the digestion and passage of cells can be completed within 10-15 minutes;
(6)培养成本可控：原代肺癌癌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，节约了细胞培养的 成本；(6) Controllable culture cost: The primary lung cancer culture medium does not need to add expensive Wnt agonists, R-spondin family proteins, BMP inhibitors and other factors, saving the cost of cell culture;
(7)操作便捷，该技术相比类器官技术，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；(7) The operation is convenient. Compared with the organoid technology, this technology does not need to embed cells in Matrigel like the organoid technology, and the operation steps of the technology are simple and easy;
(9)所述技术培养获得的肺癌上皮细胞数量大，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。(9) The lung cancer epithelial cells cultured by the technique have a large number and a high degree of homogeneity, which are suitable for high-throughput screening of new candidate compounds, and provide high-throughput in vitro drug sensitivity functional tests for patients.
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肺癌上皮细胞，包括肺癌肿瘤细胞、正常肺癌上皮细胞、肺癌上皮干细胞、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肺癌原代细胞扩增培养的试剂盒。Using the cell culture medium of this embodiment, lung cancer epithelial cells derived from humans or other mammals can be cultured, including lung cancer tumor cells, normal lung cancer epithelial cells, lung cancer epithelial stem cells, or cells comprising at least any of these cells. organization. At the same time, the culture medium of the present invention can also be used to develop a kit for expanding and culturing primary lung cancer cells in vitro.
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、肺癌上皮细胞的基础医学研究、药物应答的筛选、以及来源于肺癌疾病的新药研发等。In addition, the cells obtained by the culture method of this embodiment can be applied to regenerative medicine, basic medical research of lung cancer epithelial cells, screening of drug responses, and development of new drugs derived from lung cancer diseases.
附图说明Description of drawings
图1A-1F为显示培养基中不同因子对肺癌原代细胞增殖的影响的图。1A-1F are graphs showing the effect of different factors in the culture medium on the proliferation of primary lung cancer cells.
图2是显示培养基中不同因子递增对肺癌原代细胞增殖的影响的图。Figure 2 is a graph showing the effect of increasing the various factors in the culture medium on the proliferation of primary lung cancer cells.
图3A-3G是显示各添加因子的浓度对肺癌原代细胞增殖的影响的图。3A-3G are graphs showing the effect of the concentration of each additive factor on the proliferation of primary lung cancer cells.
图4A和4B是将从1例肺癌临床组织样本(编号0B0003)分离得到的细胞采用本发明的培养基LM分别培养至第5天和第10天的肺癌肿瘤细胞在倒置显微镜下拍摄的照片。Figures 4A and 4B are photographs taken under an inverted microscope of lung cancer tumor cells cultured on day 5 and day 10, respectively, with cells isolated from a lung cancer clinical tissue sample (No. OB0003) using the medium LM of the present invention.
图5是16例样本初次培养周期和所得细胞数的汇总图。Figure 5 is a summary graph of the primary culture cycle and the resulting cell number for 16 samples.
图6是从8例肺癌穿刺样本(编号0B0002、0B0004、0B0006、0B0009、0B0010、0B0011、0B0012、0B0013)分离得到的细胞在LM培养基条件下持续培养得到的生长曲线图。Figure 6 is a graph showing the growth curves of cells isolated from 8 lung cancer puncture samples (numbered 0B0002, 0B0004, 0B0006, 0B0009, 0B0010, 0B0011, 0B0012, and OB0013) continuously cultured under the condition of LM medium.
图7是将从1例肺癌临床组织样本(编号0B0004)分离得到的细胞分别采用5种不同培养基条件下培养所获得的细胞总数图。Fig. 7 is a graph showing the total number of cells obtained by culturing cells isolated from a clinical tissue sample of lung cancer (No. 0B0004) under 5 different medium conditions.
图8是将从1例肺癌临床组织样本(编号0B0004)分离得到的细 胞分别采用5种不同培养基条件下培养所获得的细胞生长曲线对比图。Figure 8 is a comparison chart of cell growth curves obtained by culturing cells isolated from a case of lung cancer clinical tissue sample (number 0B0004) under 5 different culture medium conditions.
图9A是从1例肺癌手术切除样本(编号0B0015)分离得到的细胞采用本发明的培养基LM培养获得肺癌肿瘤细胞后，使用非特异性荧光染料DAPI染细胞核的图片，图9B是将如上所述获得的肺癌肿瘤细胞使用肺腺癌特异性抗体NapsinA染色的图片，图9C是图9A和图9B合并后得到的图片。Figure 9A is a picture of cells isolated from a lung cancer surgical resection sample (No. 0B0015) obtained by culturing the lung cancer tumor cells with the medium LM of the present invention, and then using the non-specific fluorescent dye DAPI to stain the cell nuclei, and Figure 9B is a picture of the above The obtained lung cancer tumor cells were stained with the lung adenocarcinoma-specific antibody NapsinA, and FIG. 9C is the image obtained by combining FIG. 9A and FIG. 9B .
图10A-10D是从1例肺癌手术切除样本(编号0B0004)的原始组织细胞和采用该细胞以本发明的培养基LM培养而获得的肺癌肿瘤细胞的免疫组化结果对比图。Figures 10A-10D are comparison charts of immunohistochemical results of primary tissue cells from a lung cancer surgical resection sample (No. 0B0004) and lung cancer tumor cells cultured with the cells in the medium LM of the present invention.
图11表示从同一例肺癌患者的穿刺得到的癌组织样本(编号0B0011)采用本发明的培养基LM培养至第一代和第五代的肺癌肿瘤细胞对不同靶向药物的剂量-效应曲线图。Figure 11 shows the dose-response curves of lung cancer tumor cells cultured to the first and fifth passages using the culture medium LM of the present invention on the cancer tissue samples (No. 0B0011) obtained from the puncture of the same lung cancer patient on different targeted drugs .
具体实施方式detailed description
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、***、毛根、气管、肺等。其中，本实施方式的细胞培养基较好是用于来源于肺癌上皮细胞的培养基。In the present specification, 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 differentiation into epithelial cells, and refer to stem cells derived from epithelial tissue. Examples of epithelial tissues include cornea, oral mucosa, skin, conjunctiva, bladder, renal tubule, kidney, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), and large intestine (including colon)) , liver, pancreas, breast, salivary gland, lacrimal gland, prostate, hair root, trachea, lung, etc. Among them, the cell culture medium of the present embodiment is preferably a culture medium for lung cancer epithelial cells.
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。In addition, in this specification, "epithelial tumor cell" refers to a cell derived from the above-described epithelial tissue-derived cell tumorigenic.
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。In the present specification, "organoid" refers to a three-dimensional, organ-like cellular organoid formed by spontaneously organizing and aggregating cells at a high density in a controlled space.
[MST1/2激酶抑制剂的制备实施例][Preparation Example of MST1/2 Kinase Inhibitor]
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。In the present specification, MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signaling. In general, MST1/2 kinase inhibitors, for example, bind to MST1/2 kinase and reduce its activity. Due to the structural similarity of MST1 and MST2, MST1/2 kinase inhibitors can also be, for example, compounds that bind to MST1 or MST2 and reduce their activity.
1.MST1/2激酶抑制剂化合物1的制备1. Preparation of MST1/2 Kinase Inhibitor Compound 1
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4-((7-(2,6-Difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene 磺酰胺1Sulfonamide 1
Figure PCTCN2020109740-appb-000008
Figure PCTCN2020109740-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。Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): In a round bottom flask, add 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) Methanol (30 mL) was then added, followed by dropwise addition of thionyl chloride (1.2 mL) in an ice bath. The reaction system was reacted at 85°C overnight. After the reaction, the solvent was evaporated to dryness in the system under reduced pressure, and the obtained white solid was directly used in the next step.
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。Methyl 2-((2-Chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (A3): In a round bottom flask, add 2-amino- After methyl 2-(2,6-difluorophenyl)acetate (2 g) was added acetone (30 mL) and potassium carbonate (2.2 g), the system was cooled to -10°C with an ice-salt bath, and then slowly added 2,4-Dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction system was stirred at room temperature overnight. After the reaction is completed, filter the filtrate, remove the solvent under reduced pressure, and purify the residue by pressurized silica gel column chromatography to obtain compound 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***洗涤后得化合物A4。LC/MS：M+H 297.0。2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridine-6(5H)-one (A4): In a round bottom flask, add 2-((2-chloro- Methyl 5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g) was added followed by acetic acid (50 mL) and iron powder (3.9 g). The reaction system was stirred at 60°C for two hours. After the reaction, the solvent was evaporated to dryness in the system under reduced pressure, and the resultant was neutralized with saturated sodium bicarbonate until basic. It was extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain the crude product. The crude product was washed with ether to obtain compound 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***洗涤后得化合物A5。LC/MS：M+H 325.0。2-Chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridine-6(5H)-one (A5): in a round bottom flask 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridine-6(5H)-one (2 g) and N,N-dimethylacetamide (10 mL) , cooled to -35°C, methyl iodide (0.9 mL) was added, followed by sodium hydride (615 mg), and the reaction system continued to stir for two hours. After the reaction was completed, it was quenched by adding water, extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain the crude product. The crude product was washed with ether to obtain compound 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***洗涤得化合物1。LC/MS：M+H 461.1。4-((7-(2,6-Difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide (1): In a round bottom flask was added 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridine-6(5H) - ketone (100 mg), sulfonamide (53 mg), p-toluenesulfonic acid (53 mg) and sec-butanol (5 mL). The reaction system was stirred at 120°C overnight. After the reaction, filter, wash with methanol and ether to obtain compound 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2. Preparation of other MST1/2 inhibitor compounds of the present invention
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。Other MST1/2 inhibitor compounds of the present invention were synthesized according to the method similar to compound 1, and their structures and mass spectrometry data are shown in the following table.
Figure PCTCN2020109740-appb-000009
Figure PCTCN2020109740-appb-000009
Figure PCTCN2020109740-appb-000010
Figure PCTCN2020109740-appb-000010
Figure PCTCN2020109740-appb-000011
Figure PCTCN2020109740-appb-000011
Figure PCTCN2020109740-appb-000012
Figure PCTCN2020109740-appb-000012
Figure PCTCN2020109740-appb-000013
Figure PCTCN2020109740-appb-000013
[实施例1][Example 1]
人原代肺癌上皮细胞的分离Isolation of Human Primary Lung Cancer Epithelial Cells
肺癌组织样本来源于三例进行过说明并获得同意的肺癌肿瘤患者手术切除癌组织样本，它们分别是样本编号0B0010、0B0011、0B0012。下面以其中一例样本(编号0B0010)进行说明。Lung cancer tissue samples were obtained from three patients with lung cancer tumors who had been described and obtained consent for surgical resection of cancer tissue samples, which were sample numbers 0B0010, 0B0011, and 0B0012, respectively. One of the samples (No. 0B0010) will be described below.
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。 The above-mentioned tissue samples were collected within half an hour after the patient's surgical excision or biopsy. More specifically, in a sterile environment, take a tissue sample from a non-necrotic site, the volume of which is more than 0.5 cm 3 , place it in a pre-cooled 4 mL tissue transport fluid (see Table 1 for specific preparation), and the transport fluid contains In a 5mL plastic sterile cryopreservation tube with a lid (purchased from Guangzhou Jiete Biotechnology), it was transported to the laboratory in a cold chain (0-10°C).
表1 组织运输液配方Table 1 Formulation of tissue transport solution
Figure PCTCN2020109740-appb-000014
Figure PCTCN2020109740-appb-000014
表2 组织消化液配方Table 2 Formula of tissue digestive juice
组织消化液成分Tissue Digestive Fluid Components 供应商supplier 终浓度Final concentration
HBSSHBSS
GibcoGibco 50％(体积)50% (volume)
RPMI-1640RPMI-1640 康宁 Corning 50％(体积)50% (volume)
胶原酶ⅡCollagenase II SigmaSigma 2mg/mL2mg/mL
胶原酶ⅣCollagenase IV SigmaSigma 2mg/mL2mg/mL
脱氧核糖核酸ⅠDNA I SigmaSigma 50U/mL50U/mL
透明质酸酶Hyaluronidase SigmaSigma 0.5mg/mL0.5mg/mL
氯化钙calcium chloride 上海生工Shanghai Shenggong 0.33mg/mL0.33mg/mL
牛血清白蛋白bovine serum albumin 上海生工Shanghai Shenggong 10mg/mL10mg/mL
在生物安全柜内，将组织样本(编号0B0010)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。 In the biological safety cabinet, transfer the tissue sample (No. 0B0010) to a 100mm cell culture dish (purchased from NEST), rinse the tissue sample with tissue transport fluid, wash away the residual blood on the surface of the tissue sample, and remove the tissue Excess tissue such as fat on the surface of the sample. Transfer the rinsed tissue sample to another new 100 mm petri dish, add 2 mL of transport solution, and use a sterile scalpel blade and forceps to divide the tissue sample into tissue fragments with a volume of less than 3 mm 3 .
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(使用量约为每10mg组织使用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。The tissue sample fragments were transferred to a 15mL centrifuge tube, and centrifuged at 1500 rpm for 4 minutes with a desktop centrifuge (Sigma company 3-18K); discard the supernatant, and add tissue transport solution and tissue digestion solution (using The amount is about 5 mL of tissue digestion solution per 10 mg of tissue, the specific preparation is shown in Table 2), the sample number is marked, sealed with parafilm, and digested at 37 ° C, 300 rpm constant temperature shaker (Zhichu Instrument ZQLY-180N), every 1 hour Observe if digestion is complete.
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。After the digestion was completed, undigested tissue clumps were filtered out with a 70 μm filter, the tissue clumps on the filter were washed with tissue transport solution, and the residual cells were flushed into a centrifuge tube and centrifuged at 1500 rpm for 4 minutes.
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，得到消化分离后的肺癌原代细胞，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10万。Discard the supernatant and observe whether the remaining cell mass contains blood cells. If there are blood cells, add 3 mL of blood cell lysis buffer (purchased from Sigma), mix well, lyse at 4°C for 15 minutes, shake and mix once for 5 minutes, take out after the lysis is complete, and use Centrifuge at 1500 rpm for 4 minutes. The supernatant was discarded to obtain primary lung cancer cells after digestion and separation, and basal medium (BM) was added to resuspend, wherein the basal medium was a commercially available DMEM/F-12 medium with 0.2 vol% Primocin (purchased from BM). Invivogen, 50 mg/mL) to obtain a final concentration of 100 μg/mL. Using a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.), the total number of cells was 100,000.
另外两例肺癌肿瘤组织样本按照以上同样的方法进行分离，得到的细胞总数分别为10万(0B0011)和8万(0B0012)。The other two cases of lung cancer tumor tissue samples were separated according to the same method as above, and the total number of cells obtained were 100,000 (0B0011) and 80,000 (0B0012), respectively.
[实施例2][Example 2]
原代肺癌上皮细胞培养基的优化Optimization of culture medium for primary lung cancer epithelial cells
(1)不同因子的作用(1) The role of different factors
以0.25％胰酶(购自Thermo Fisher公司)将培养的NIH-3T3细胞(购自ATCC，使用含10％的胎牛血清的DMEM培养液进行培养)消化，用含有5％(v/v)胎牛血清(购自依科赛公司)、100U/mL青霉素和100μg/mL链霉素的DMEM培养液(购自康宁公司)终止消化，并收集至15mL离心管内，1500rpm离心4分钟后，弃上清。使用上述含10％的胎牛血清的DMEM培养液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，用35Gy辐照剂量γ射线进行辐照，随后按2×10 4个/cm 2的密度接种至培养器皿中。在37℃培养箱内培养，待细胞贴壁。接种原代细胞前，移除培养器皿中培养基。 Cultured NIH-3T3 cells (purchased from ATCC, cultured in DMEM medium containing 10% fetal bovine serum) were digested with 0.25% trypsin (purchased from Thermo Fisher Company), and then cultured with 5% (v/v) Fetal bovine serum (purchased from Ekosai), 100 U/mL penicillin and 100 μg/mL streptomycin in DMEM (purchased from Corning) terminated digestion, and collected into a 15 mL centrifuge tube, centrifuged at 1500 rpm for 4 minutes, and discarded. supernatant. Use the above-mentioned DMEM medium containing 10% fetal bovine serum to resuspend the cell pellet after centrifugation, use a flow image counter (Jiangsu Zhuo Microorganism Technology Co., Ltd. JIMBIO FIL) to count, and irradiate with 35Gy irradiation dose of gamma rays , and then seeded into culture vessels at a density of 2×10 4 /cm 2 . Culture in a 37°C incubator until cells adhere. Before seeding the primary cells, remove the medium from the culture vessel.
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。Preparation of basal medium (abbreviated as BM): 0.2 vol% Primocin (purchased from Invivogen Company, concentration of 50 mg/mL) was added to the commercially available DMEM/F-12 medium to obtain a final concentration of 100 μg/mL. Get 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肺癌上皮细胞培养基。Next, different types and concentrations of additive factors (Table 3) were added to the basal medium (BM) to prepare a lung cancer epithelial cell culture medium containing different additive components.
表3 不同组分培养基的配制(浓度为终浓度)Table 3 Preparation of different components of culture medium (concentration is final concentration)
Figure PCTCN2020109740-appb-000015
Figure PCTCN2020109740-appb-000015
Figure PCTCN2020109740-appb-000016
Figure PCTCN2020109740-appb-000016
将不同成分的培养基按500μl/孔体积加入预铺有γ射线辐照过后的NIH-3T3细胞的48孔培养板内。将按照实施例1相同方法从肺癌组织分离得到的肺癌肿瘤细胞(编号0B0014)以4×10 4个/孔的细胞数量接种在上述预铺有γ射线辐照过后的NIH-3T3细胞的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肺癌肿瘤细胞(编号0B0014)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和滋养细胞的补充。培养7天后，进行细胞计数。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)。将结果示于图1A-F。 The medium of different composition was added to the 48-well culture plate pre-plated with NIH-3T3 cells after γ-ray irradiation at a volume of 500 μl/well. The lung cancer tumor cells (No. 0B0014) isolated from lung cancer tissue according to the same method in Example 1 were seeded in the above-mentioned 48 wells pre-plated with γ-ray irradiated NIH-3T3 cells at a cell number of 4×10 4 cells/well. Inside the culture plate, the surface was sterilized and placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher Scientific), so that the same number of freshly isolated lung cancer tumor cells (number 0B0014) were cultured under different medium formulation conditions . Medium change and feeder cell supplementation were performed every 4 days after the start of culture. After 7 days of culture, cell counts were performed. Among them, as an experimental control, a basal medium (BM) without any additives was used. The results are shown in Figures 1A-F.
图中纵坐标表示不同培养基培养之后得到的细胞数与基础培养基BM培养之后得到细胞数的比值。如图所示，在BM基础上加入表3中不同浓度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、胰岛素-转铁蛋白-硒复合物、肝细胞生长因子、胰岛素样本生长因子1、成纤维细胞生长因子7、神经调节蛋白-1、化合物1和Y27632对细胞增殖有一定的促进作用。The vertical axis in the figure represents the ratio of the number of cells obtained after culturing in different media to the number of cells obtained after culturing in the basal medium BM. As shown in the figure, adding different concentrations of different factors in Table 3 on the basis of BM had different effects on cell proliferation. Among them, in a specific concentration range, B27 additive, N2 additive, insulin-transferrin-selenium complex, hepatocyte growth factor, insulin sample growth factor 1, fibroblast growth factor 7, neuregulin-1, compound 1 And Y27632 has a certain promoting effect on cell proliferation.
(2)培养基中不同因子递增对本专利方法获得的肺癌原代细胞增殖的影响(2) The effect of different factors in the culture medium on the proliferation of primary lung cancer cells obtained by the patented method
向基础培养基BM中分别依次递加不同小分子、添加剂以及生长因子(表4)，配制成含有不同添加成分的肺癌上皮细胞培养基。Different small molecules, additives and growth factors (Table 4) were successively added to the basal medium BM to prepare a lung cancer epithelial cell medium containing different supplementary components.
表4 不同组分培养基的配制(浓度为终浓度)Table 4 Preparation of different components of culture medium (concentration is final concentration)
Figure PCTCN2020109740-appb-000017
Figure PCTCN2020109740-appb-000017
将不同成分的培养基按500μl/孔体积加入预铺有γ射线辐照过后的NIH-3T3细胞的48孔培养板内，同时将BM培养基作为实验对照。将按照实施例1的方法从肺癌组织分离得到的肺癌肿瘤细胞(编号0B0016)以4×10 4个/孔的细胞数量接种在上述预铺有γ射线辐照过后的NIH-3T3细胞的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肺癌肿瘤细胞(编号0B0016)在不同的培养基配方条件下进行培养。培养7天后，进行细胞计数。将实验结果示于图2。 The medium of different composition was added into the 48-well culture plate pre-plated with NIH-3T3 cells after γ-ray irradiation at a volume of 500 μl/well, and the BM medium was used as the experimental control. The lung cancer tumor cells (number 0B0016) isolated from lung cancer tissue according to the method of Example 1 were seeded in the above-mentioned 48 wells pre-plated with NIH-3T3 cells after γ-ray irradiation at the number of 4×10 4 cells/well. Inside the culture plate, the surface was sterilized and placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher Scientific), so that the same number of freshly isolated lung cancer tumor cells (number 0B0016) were cultured under different medium formulation conditions . After 7 days of culture, cell counts were performed. The experimental results are shown in FIG. 2 .
如图所示，确定NO.7为本专利最优选的培养基用于培养扩增肺癌原代细胞(下文缩写为LM)。As shown in the figure, NO.7 was determined to be the most preferred medium for the patent for culturing and expanding primary lung cancer cells (hereinafter abbreviated as LM).
(3)所添加因子的不同浓度对本专利获得的肺癌原代细胞的增殖作用(3) The proliferation effect of the different concentrations of the added factors on the primary lung cancer cells obtained by this patent
使用与实施例1同样的方法从肺癌患者的癌组织(样本编号0B0002)中分离获得癌组织来源的肺癌上皮细胞。接着，将癌组织来源的肺癌肿瘤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密度接种至预铺有γ射线辐照过后的NIH-3T3细胞的12孔板内。向12孔板中添加2mL制备好的上述原代肺癌上皮细胞培养基NO.7(LM)，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0.5mL0.25％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，室温下孵育5～20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。 Using the same method as in Example 1, lung cancer epithelial cells derived from cancer tissues were isolated and obtained from cancer tissues (sample number 0B0002) of lung cancer patients. Next, the cancer tissue-derived lung cancer tumor cells were counted with a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.) to obtain the total number of cells. Then, 4×10 4 cells/cm 2 were seeded into 12-well plates pre-plated with γ-irradiated NIH-3T3 cells. 2 mL of the prepared primary lung cancer epithelial cell culture medium NO.7 (LM) was added to the 12-well plate, and cultured in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher Scientific). When the cell growth in the culture plate reaches about 80% of the bottom area, discard the medium supernatant in the 12-well plate, add 0.5 mL of 0.25% trypsin (purchased from Thermo Fisher) to digest for 1 minute, and then aspirate 0.25% Trypsin, then add 0.5 mL of 0.05% trypsin for cell digestion, and incubate at room temperature for 5 to 20 minutes, until the cells can be observed under the microscope (EVOS M500 from Invitrogen) to be completely digested. 1 mL of DMEM/F12 medium containing fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin was used to terminate the digestion, and then collected into a 15 mL centrifuge tube. After centrifugation at 1500 rpm for 4 minutes, the supernatant was discarded. The cell pellet after centrifugation was resuspended in basal medium BM, and counted using a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.) to obtain the total number of cells. The resulting cells were used for the following culture experiments.
接着，配制以下7种配方培养基进行实验：Next, prepare the following 7 kinds of formula medium for experiment:
配方1：培养基LM组分中不含化合物1；Formula 1: Compound 1 is not contained in the LM component of the medium;
配方2：培养基LM组分中不含Y27632；Formula 2: Y27632 is not included in the LM component of the medium;
配方3：培养基LM组分中不含***1；Formulation 3: Insulin-like growth factor 1 is not contained in the LM component of the medium;
配方4：培养基LM组分中不含肝细胞生长因子；Formula 4: the LM component of the medium does not contain hepatocyte growth factor;
配方5：培养基LM组分中不含胰岛素-转铁蛋白-硒复合物；Formulation 5: Insulin-transferrin-selenium complex is not included in the LM component of the medium;
配方6：培养基LM组分中不含表皮细胞生长因子；Formulation 6: no epidermal cell growth factor is contained in the LM component of the medium;
配方7：培养基LM组分中不含神经调节蛋白-1。Formulation 7: Neuregulin-1 is not included in the LM component of the medium.
分别使用上述配方1～7来稀释上述消化后的细胞悬液，按照每孔1万细胞，250微升体积种入预铺有γ射线辐照过后的NIH-3T3细胞的48孔板中。The above-mentioned recipes 1-7 were used to dilute the above digested cell suspension, and seeded in a 48-well plate pre-plated with γ-ray irradiated NIH-3T3 cells at a volume of 10,000 cells per well, with a volume of 250 microliters.
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化合物1每孔250微升，化合物1的终浓度分别为40μM、20μM、10μM、5μM、2.5μM、1.25μM、0.625μM；并使用配方1的培养基设置对照孔(BC)。When using the medium of formula 1, add 250 microliters of compound 1 to each well of the 48-well plate seeded with primary cells. The final concentrations of compound 1 are 40 μM, 20 μM, 10 μM, 5 μM, and 2.5 μM, respectively. , 1.25 μM, 0.625 μM; and control wells (BC) were set up using the medium of Recipe 1.
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Y27632每孔250微升，Y27632的终浓度分别为40μM、20μM、10μM、5μM、2.5μM、1.25μM、0.625μM；并使用配方2的培养基设置对照孔(BC)。When using the medium of formula 2, add 250 microliters of prepared Y27632 to each well of the 48-well plate seeded with primary cells. The final concentrations of Y27632 are 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, respectively. μM, 0.625 μM; and control wells (BC) were set up using the medium of Recipe 2.
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***1每孔250微升，***1的终浓度分别为80ng/ml、40ng/ml、20ng/ml、10ng/ml、5ng/ml、2.5ng/ml、1.25ng/ml；并使用配方3的培养基设置对照孔(BC)。When using the medium of formula 3, 250 microliters of prepared insulin-like growth factor 1 were added to each well of the 48-well plate seeded with primary cells, and the final concentrations of insulin-like growth factor 1 were 80 ng/ml, 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml; and control wells (BC) were set up using the medium of formula 3.
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80ng/ml、40ng/ml、20ng/ml、10ng/ml、5ng/ml、2.5ng/ml、1.25ng/ml；并使用配方4的培养基设置对照孔(BC)。When using the medium of formula 4, add 250 microliters of prepared hepatocyte growth factor to each well of the 48-well plate seeded with primary cells, and the final concentrations of hepatocyte growth factor are 80ng/ml and 40ng/ml, respectively. ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml; and control wells (BC) were set up using the medium of formula 4.
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胰岛素-转铁蛋白-硒复合物每孔250微升，胰岛素-转铁蛋白-硒复合物的终浓度分别为1:1600、1:800、1:400、1:200、1:100、1:50、1:25；并使用配方5的培养基设置对照孔(BC)。When using the medium of formula 5, 250 microliters of the prepared insulin-transferrin-selenium complex was added to each well of the 48-well plate seeded with primary cells. The final concentrations were 1:1600, 1:800, 1:400, 1:200, 1:100, 1:50, 1:25; and control wells (BC) were set up using the medium of formula 5.
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表皮细胞生长因子每孔250微升，表皮细胞生长因子的终浓度分别为80ng/ml、40ng/ml、20ng/ml、10ng/ml、5ng/ml、2.5ng/ml、1.25ng/ml；并使用配方6的培养基设置对照孔(BC)。When using the medium of formula 6, 250 microliters of prepared epidermal growth factor was added to each well of the 48-well plate seeded with primary cells, and the final concentrations of epidermal growth factor were 80ng/ml and 40ng/ml, respectively. ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml; and control wells (BC) were set up using the medium of formula 6.
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神经调节蛋白-1每孔250微升，神经调节蛋白-1的终浓度分别为80ng/ml、40ng/ml、20ng/ml、10ng/ml、5ng/ml、2.5ng/ml、1.25ng/ml；并使用配方7的培养基设置对照孔(BC)。When using the medium of formula 7, 250 microliters of prepared neuregulin-1 were added to each well of the 48-well plate seeded with primary cells, and the final concentrations of neuregulin-1 were 80ng/ml, 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml; and control wells (BC) were set up using the medium of formula 7.
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3A～3G。图3A～图3G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。After the cells were expanded to about 85% of the 48-well digestion count, the ratio was calculated with reference to the number of cells in the control well (BC), and the results were shown in Figures 3A to 3G, respectively. In FIGS. 3A to 3G , the ratio is the ratio of the number of cells obtained by culturing each medium for one generation to the number of cells obtained by culturing one generation of the corresponding control wells. If the ratio is greater than 1, it means that the prepared medium containing different concentrations of factors or small molecule compounds has a better effect of promoting proliferation than the control well medium; if the ratio is less than 1, it means that the prepared medium containing different concentrations of factors or small molecule compounds can promote proliferation The effect was weaker than that of the control well medium in promoting proliferation.
根据图3A～3G的结果，化合物1的含量优选为0.625μM～20μM，更优选为0.625μM～10μM；Y27632的含量优选为1.25μM～20μM，更优选为2.5μM～10μM；***1的含量优选为1.25ng/ml～80ng/ml，更优选为5ng/ml～80ng/ml；肝细胞生长因子的含量优选为5ng/ml～80ng/ml，更优选为20ng/ml～80ng/ml；胰岛素-转铁蛋白-硒复合物的体积浓度优选为1:25～1:200(胰岛素/转铁蛋白/***钠各自的含量分别为2.5～20μg/ml-1.25～10μg/ml-1.25～10ng/ml)，更优选1:25～1:50(胰岛素/转铁蛋白/***钠各自的含量分别为10～20μg/ml-5～10μg/ml-5～10ng/ml)；表皮细胞生长因子的含量优选为10ng/ml～80ng/ml，更优选为10ng/ml～40ng/ml；神经调节蛋白 -1在培养基中的含量优选为10～80ng/ml，更优选20～80ng/ml。According to the results of Figures 3A to 3G, the content of compound 1 is preferably 0.625 μM to 20 μM, more preferably 0.625 μM to 10 μM; the content of Y27632 is preferably 1.25 μM to 20 μM, more preferably 2.5 μM to 10 μM; insulin-like growth factor 1 The content of hepatocyte growth factor is preferably 1.25ng/ml～80ng/ml, more preferably 5ng/ml～80ng/ml; the content of hepatocyte growth factor is preferably 5ng/ml～80ng/ml, more preferably 20ng/ml～80ng/ml The volume concentration of the insulin-transferrin-selenium complex is preferably 1:25-1:200 (the respective contents of insulin/transferrin/sodium selenite are 2.5-20 μg/ml-1.25-10 μg/ml- 1.25～10ng/ml), more preferably 1:25～1:50 (the respective contents of insulin/transferrin/sodium selenite are 10～20μg/ml-5～10μg/ml-5～10ng/ml) The content of epidermal growth factor is preferably 10ng/ml～80ng/ml, more preferably 10ng/ml～40ng/ml; The content of neuregulin-1 in the medium is preferably 10～80ng/ml, more preferably 20 ~80ng/ml.
[实施例3][Example 3]
人肺癌组织来源的原代肺癌肿瘤细胞的培养Culture of primary lung cancer tumor cells derived from human lung cancer tissue
使用与实施例1同样的方法从肺癌患者的癌组织(样本编号0B0003)中分离获得癌组织来源的肺癌上皮细胞。接着，将癌组织来源的肺癌肿瘤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密度接种至预铺有γ射线辐照过后的NIH-3T3细胞的12孔板内。向12孔板中添加2mL制备好的原代肺癌上皮细胞培养基LM，置于37℃、5％CO 2培养箱(购自赛默飞)中进行培养。 Using the same method as in Example 1, lung cancer epithelial cells derived from cancer tissues were isolated and obtained from cancer tissues of lung cancer patients (sample number 0B0003). Next, the cancer tissue-derived lung cancer tumor cells were counted with a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.) to obtain the total number of cells. Then, 4×10 4 cells/cm 2 were seeded into 12-well plates pre-plated with γ-irradiated NIH-3T3 cells. 2 mL of the prepared primary lung cancer epithelial cell medium LM was added to the 12-well plate, and cultured in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher Scientific).
图4A是本实施例按4×10 4个/cm 2密度接种预铺有γ射线辐照过后的NIH-3T3细胞的12孔板，自接种开始后培养至第5天的镜下照片(100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肺癌肿瘤细胞已经形成较大克隆。图4B是本实施例自接种后培养第10天的镜下照片(100倍倒置相差显微镜拍照)，视野中细胞已经长满。从图4A和4B两张图可以看出，分离后得到肺癌原代细胞在体外培养5天在镜下就可以看到明显的克隆形成，并且经过10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肺癌上皮细胞的技术。 Figure 4A is a microscopic photo of the 12-well plate pre-plated with NIH-3T3 cells after γ-ray irradiation at a density of 4 × 10 4 cells/cm 2 in this example, and cultured from the start of inoculation to the 5th day (100 photographed with an inverted phase contrast microscope). Microscopic observation showed that the cultured primary lung cancer tumor cells derived from cancer tissue had formed larger clones. FIG. 4B is a microscopic photo (photographed by a 100-fold inverted phase contrast microscope) of the 10th day of culture after inoculation in this example, and the cells in the field of view have been overgrown. It can be seen from Figure 4A and Figure 4B that the primary lung cancer cells obtained after separation were cultured in vitro for 5 days, and obvious clone formation could be seen under the microscope, and after 10 days of expansion, the number of cells was significantly expanded. increase, suggesting that the technology of the present invention is an efficient technology for expanding lung cancer epithelial cells in vitro.
[实施例4][Example 4]
肺癌初次培养周期和细胞数统计及Population Doubling(PD)值计算Statistics of primary culture cycle and cell number of lung cancer and calculation of Population Doubling (PD) value
按照实施例1的方法消化肺癌穿刺组织样本(样本编号0B0001～0B0016号)获得肺癌原代细胞。对于所获得的肺癌原代细胞，使用LM培养基，按照活细胞密度4×10 4个/cm 2将细胞接种在12孔板中并进行培养，待细胞扩增至85％后消化并计数，同时记录直至消化时培养的天数，将该直至消化时培养的天数作为一个培养周期。如图5所示，16例肺癌样本培养起始时的平均细胞数是7.2万，首次扩增后得到的平均细胞数是121.2万，所需平均培养周期是12.6天。 The lung cancer puncture tissue samples (sample numbers 0B0001 to 0B0016) were digested according to the method of Example 1 to obtain primary lung cancer cells. For the obtained primary lung cancer cells, using LM medium, the cells were seeded in a 12-well plate at a viable cell density of 4×10 4 cells/cm 2 and cultured. After the cells were expanded to 85%, they were digested and counted. At the same time, the number of days of culture up to the time of digestion was recorded, and the number of days of culture until the time of digestion was regarded as one culture cycle. As shown in Figure 5, the average number of cells in the 16 lung cancer samples at the beginning of the culture was 72,000, and the average number of cells obtained after the first expansion was 1.212 million, and the average culture period required was 12.6 days.
在该实验条件下持续培养样本编号0B0002、0B0004、0B0006、0B0009、0B0010、0B0011、0B0012、0B0013总共8例样本，将扩增所得的细胞进行不同代数扩增，每一代进行消化后计数并记录相应培养的周期，根据公式Population Doubling(PD)＝3.32*log10(消化后细胞总数/初始种入细胞数)计算PD，如图6所示，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。Under the experimental conditions, the samples numbered 0B0002, 0B0004, 0B0006, 0B0009, 0B0010, 0B0011, 0B0012, and 0B0013 were continuously cultured for a total of 8 samples, and the amplified cells were amplified in different passages, and each passage was digested and counted and recorded. The culture period was calculated according to the formula Population Doubling(PD)=3.32*log10 (the total number of cells after digestion/the number of initial seeded cells), as shown in Figure 6, the abscissa represents the days of cell culture, and the ordinate is the accumulated cells Proliferation fold, indicating the fold of cell expansion in the culture cycle, the larger the value, the more times the cell is expanded in a certain cycle, that is, the more cells are amplified, and the slope represents the cell expansion. rate.
从图6可以看出，使用本发明的肺癌原代细胞培养基对上述8例样本进行培养时，扩增58天后的细胞扩增速率基本保持不变，仍具有继续扩增的能力。It can be seen from FIG. 6 that when the above-mentioned 8 samples were cultured with the primary lung cancer cell culture medium of the present invention, the cell expansion rate after 58 days of expansion remained basically unchanged, and the cells still had the ability to continue expansion.
[实施例5][Example 5]
不同培养基对肺癌组织来源的原代肺癌肿瘤细胞的促增殖效果Proliferation effect of different media on primary lung cancer tumor cells derived from lung cancer tissue
(1)不同培养基对初代原代细胞增殖效果的比较(1) Comparison of the effects of different media on the proliferation of primary cells
使用与实施例2同样的方法制备原代肺癌上皮细胞培养基LM，和作为对照的基础培养基BM。另外制备细胞条件重编程技术文献中所用培养基FM作为另一对照例，配制步骤参见(Liu等，Nat.Protoc.，12(2):439-451，2017)，培养基配方见表5。同时，作为另外对照例，制备肺癌原代细胞培养基LM1，配方是在LM基础上使用1:50体积比的B27添加剂替换胰岛素-转铁蛋白-硒复合物。此外，作为另一对照例，自STEMCELL公司购买商品化培养基EpiCult TM Plus Medium，以下也称“Epi培养基”)，培养基配方见表6。 The primary lung cancer epithelial cell medium LM and the basal medium BM as a control were prepared in the same manner as in Example 2. In addition, the medium FM used in the cell conditional reprogramming technical literature was prepared as another control example. For the preparation steps, see (Liu et al., Nat. Protoc., 12(2): 439-451, 2017), and the medium formula is shown in Table 5. Meanwhile, as another control example, lung cancer primary cell culture medium LM1 was prepared, and the formula was to replace insulin-transferrin-selenium complex with B27 additive in a volume ratio of 1:50 on the basis of LM. In addition, as another control example, the commercialized medium EpiCult ™ Plus Medium (hereinafter also referred to as "Epi medium") was purchased from STEMCELL Company, and the medium formula is shown in Table 6.
表5 细胞条件重编程技术文献中所用培养基(FM)成分Table 5 Medium (FM) components used in the technical literature on cell conditional reprogramming
培养基成分Medium composition 供应商supplier 终浓度Final concentration
DMEM培养基DMEM medium CorningCorning 65体积％65% by volume
胎牛血清fetal bovine serum GibicoGibico 10体积％10% by volume
Ham’s F12营养液Ham's F12 Nutrient Solution GibicoGibico 25体积％25% by volume
氢化可的松Hydrocortisone Sigma-AldrichSigma-Aldrich 25ng/ml25ng/ml
表皮生长因子epidermal growth factor R&DR&D 0.125ng/ml0.125ng/ml
胰岛素insulin Sigma-AldrichSigma-Aldrich 5μg/ml5μg/ml
两性霉素Bamphotericin B Sigma-AldrichSigma-Aldrich 250ng/ml250ng/ml
庆大霉素Gentamicin Sigma-AldrichSigma-Aldrich 10μg/ml10μg/ml
霍乱毒素Cholera toxin Sigma-AldrichSigma-Aldrich 0.1nM0.1nM
Y27632Y27632 MCEMCE 10μM10μM
表6 商品化培养基EpiCult TM Plus Medium(Epi)成分 Table 6 Composition of commercial medium EpiCult TM Plus Medium (Epi)
培养基成分Medium composition 供应商supplier 终浓度Final concentration
EpiCult TM Plus Basal Medium EpiCult ™ Plus Basal Medium STEMCELLSTEMCELL 99体积％99% by volume
EpiCult TM Plus Supplement EpiCult ™ Plus Supplement STEMCELLSTEMCELL 1体积％1 vol%
使用与实施例1同样的方法获得肺癌组织来源的原代肺癌肿瘤细胞(编号0B0006)。接着，按照相同的密度(4×10 4个/cm 2)分别在以下5种培养条件下培养： Primary lung cancer tumor cells (No. 0B0006) derived from lung cancer tissue were obtained by the same method as in Example 1. Next, at the same density (4×10 4 cells/cm 2 ), the cells were cultured under the following five culture conditions:
A.本发明技术：按4×10 4个/cm 2接种密度将原代肺癌肿瘤细胞接种至预铺有γ射线辐照过后的NIH-3T3细胞(购自ATCC公司)的24孔板内，采用1mL的本发明的原代肺癌上皮细胞培养基LM进行培养； A. The technology of the present invention: The primary lung cancer tumor cells were inoculated into a 24-well plate pre-plated with γ-ray irradiated NIH-3T3 cells (purchased from ATCC Company) at an inoculation density of 4×10 4 cells/cm 2 . Use 1 mL of the primary lung cancer epithelial cell culture medium LM of the present invention for cultivation;
B.细胞条件重编程技术：按4×10 4个/cm 2接种密度将原代肺癌肿瘤细胞接种至预铺有γ射线辐照过后的NIH-3T3细胞(购自ATCC公司)上，采用1mL细胞条件重编程技术培养基FM在24孔板中进行培养(具体步骤参见(Liu等，Nat.Protoc.，12(2):439-451，2017)； B. Cell Conditional Reprogramming Technology: The primary lung cancer tumor cells were seeded on the NIH-3T3 cells (purchased from ATCC) pre-plated with γ-ray irradiation at a seeding density of 4×10 4 cells/cm 2 , using 1 mL The cell conditional reprogramming technology medium FM was cultured in a 24-well plate (for specific steps, see (Liu et al., Nat.Protoc., 12(2):439-451, 2017);
C.按4×10 4个/cm 2接种密度将原代肺癌肿瘤细胞接种至预铺有γ射线辐照过后的NIH-3T3细胞(购自ATCC公司)的24孔板内，采用1mL的培养基LM1在24孔板中进行培养； C. The primary lung cancer tumor cells were inoculated into a 24-well plate pre-plated with γ-irradiated NIH-3T3 cells (purchased from ATCC) at a seeding density of 4×10 4 cells/cm 2 , using 1 mL of culture Base LM1 was cultured in 24-well plates;
D.按4×10 4个/cm 2接种密度将原代肺癌肿瘤细胞接种至24孔板内，采用1mL的商品化培养基Epi在24孔板中进行培养。 D. The primary lung cancer tumor cells were seeded into a 24-well plate at a seeding density of 4×10 4 cells/cm 2 , and cultured in a 24-well plate with 1 mL of the commercial medium Epi.
E.按4×10 4个/cm 2接种密度将原代肺癌肿瘤细胞接种至预铺有γ射线辐照过后的NIH-3T3细胞(购自ATCC公司)的24孔板内，采用1mL的基础培养基BM在24孔板中进行培养。 E. The primary lung cancer tumor cells were seeded into a 24-well plate pre-plated with γ-irradiated NIH-3T3 cells (purchased from ATCC) at a seeding density of 4×10 4 cells/cm 2 , using 1 mL of basal Medium BM was cultured in 24-well plates.
上述五种培养中，每5天对五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。In the above-mentioned five kinds of culture, the medium of the cells cultured under the five kinds of culture conditions was changed every 5 days. At the same time, the colony formation and cell proliferation status of the cells in each medium in the 24-well plate were observed, and the cell growth status was recorded by taking pictures with a microscope (EVOS M500 from Invitrogen).
对于采用本发明技术培养的原代肺癌肿瘤细胞(编号0B0006)，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44.6万。另4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、LM1、Epi和BM培养得到的细胞总数分别是29.6万、39.6万、25万和6.9万。For the primary lung cancer tumor cells (No. 0B0006) cultured by the technology of the present invention, when the cells in the culture plate grow to about 80% of the bottom area, discard the medium supernatant in the 24-well plate, add 0.5 mL of 0.25% Trypsin (purchased from Thermo Fisher) for 1 minute, then aspirate 0.25% trypsin, add 0.5 mL of 0.05% trypsin for cell digestion, and incubate at 37°C for 10 minutes until it can be observed under a microscope (EVOS M500 from Invitrogen). When the cells have been digested completely, stop the digestion with 1 mL of DMEM/F12 medium containing 5% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, and collect them into a 15 mL centrifuge tube and centrifuge at 1500 rpm. After 4 minutes, discard the supernatant. Use the culture medium of the present invention to resuspend the cell pellet after centrifugation, and use a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.) to count, and the total number of cells obtained is 446,000. The cells cultured under the other four culture conditions were digested and counted in the same manner as above, and the total number of cells cultured with media FM, LM1, Epi and BM were 296,000, 396,000, 250,000, and 69,000, respectively.
图7是编号0B0006细胞在不同条件下扩增的到的细胞总数作图。Figure 7 is a graph of the total number of cells expanded under different conditions for cells numbered OB0006.
(2)不同培养基对原代肺癌肿瘤细胞的持续培养和生长曲线的绘制(2) Continuous culture and growth curve of primary lung cancer tumor cells in different media
使用与本实施例(1)中同样的方法获得原代肺癌上皮细胞培养基LM，以及作为对照的培养基FM、LM1、Epi和BM。The primary lung cancer epithelial cell medium LM, and medium FM, LM1, Epi and BM as controls were obtained by the same method as in this Example (1).
使用与本实施例(1)中同样的方法将肺癌组织来源的原代肺癌肿瘤细胞(编号0B0004)分别在五种培养基培养，并进行消化传代和计数。Primary lung cancer tumor cells (No. 0B0004) derived from lung cancer tissue were cultured in five culture media by the same method as in Example (1), digested, passaged and counted.
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。 When the passaged cells grow in the culture plate and reach about 80% of the bottom area of the plate again, the cultured cells are collected and counted by digesting again according to the above operation method. It was also seeded at a density of 4×10 4 /well and cultured continuously.
以下为原代肺癌上皮细胞在不同技术培养条件下细胞的扩增倍数(Population Doubling)的计算公式：The following is the formula for calculating the Population Doubling of primary lung cancer epithelial cells under different technical culture conditions:
Population Doubling(PD)＝3.32*log 10(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)。 Population Doubling (PD) = 3.32*log 10 (total number of cells after digestion/number of initially seeded cells), see formula (Chapman et al., Stem Cell Research & Therapy 2014, 5:60).
如图8所示是采用Graphpad Prism软件绘制的五种不同培养条件下的细胞0B0004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培养基LM 以及LM1培养的肺癌上皮细胞的增殖速率优于其他三种培养条件，同时可以确认本发明技术可以对原代肺癌上皮细胞进行持续培养。Figure 8 shows the growth curves of cell OB0004 under five different culture conditions drawn by Graphpad Prism software. The abscissa represents the days of cell culture, and the ordinate is the cumulative cell multiplication factor, which represents the multiplication factor of the cell in the culture cycle. The higher the number, the slope represents the rate of cell expansion. From the figure, it can be confirmed that the proliferation rate of lung cancer epithelial cells cultured in the medium LM and LM1 of the present invention is better than that of the other three culture conditions, and it can be confirmed that the technology of the present invention can continuously culture primary lung cancer epithelial cells.
[实施例6][Example 6]
癌组织来源的原代肺癌肿瘤细胞的鉴定Identification of primary lung cancer tumor cells derived from cancer tissue
(1)原代肺癌组织和传代培养后的肺癌细胞免疫荧光鉴定(1) Immunofluorescence identification of primary lung cancer tissue and subcultured lung cancer cells
使用与实施例1同样的方法从肺癌患者的癌组织(样本编号0B0015)中分离获得癌组织来源的肺癌上皮细胞。接着，将癌组织来源的肺癌肿瘤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密度接种至预铺有γ射线辐照过后的NIH-3T3细胞的24孔板内，同时24孔板中预先放置用于免疫荧光染色的圆形细胞破片(购自赛默飞公司)。向24孔板中添加1mL制备好的原代肺癌上皮细胞培养基LM，置于37℃、5％CO 2培养箱(购自赛默飞)中进行培养。 Using the same method as in Example 1, lung cancer epithelial cells derived from cancer tissues were isolated and obtained from cancer tissues of lung cancer patients (sample number 0B0015). Next, the cancer tissue-derived lung cancer tumor cells were counted with a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.) to obtain the total number of cells. Then, at a density of 4×10 4 cells/cm 2 , it was seeded into a 24-well plate pre-plated with γ-ray-irradiated NIH-3T3 cells, and round cell fragments for immunofluorescence staining were pre-placed in the 24-well plate ( purchased from Thermo Fisher Scientific). 1 mL of the prepared primary lung cancer epithelial cell culture medium LM was added to the 24-well plate, and cultured in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher Scientific).
待24孔板中细胞扩增80％底面积时，弃培养液，使用4％甲醛冰上固定细胞30分钟。PBS(购自上海生工)洗5分钟x 3次。弃PBS，加入通透液，避光下，摇床(100rpm左右)破膜30分钟，PBS洗5分钟x 3次。随后使用PBS+0.3％Triton X-100(购自上海生工)配制5％体积浓度的BSA(购自上海生工)溶液用于封闭，37℃封闭30分钟。When the cells in the 24-well plate expanded by 80% of the bottom area, the culture medium was discarded, and the cells were fixed on ice with 4% formaldehyde for 30 minutes. Washed with PBS (purchased from Shanghai Shenggong) for 5 minutes x 3 times. Discard the PBS, add permeabilization solution, and in the dark, shake the membrane (about 100 rpm) for 30 minutes, and wash with PBS for 5 minutes x 3 times. Then use PBS+0.3% Triton X-100 (purchased from Shanghai Sangong) to prepare a 5% volume concentration of BSA (purchased from Shanghai Sangong) solution for blocking, and block at 37°C for 30 minutes.
提前配制PBS+0.3％Triton X-100用于稀释抗体，按照1:50比例稀释肺腺癌特异性抗体NapsinA(购自CST公司)，弃封闭液，加入配制好的一抗稀释液，至4℃冰箱孵育过夜。4℃取出，平衡至室温，37℃继续孵育1小时，PBS洗5分钟x 3次。Prepare PBS+0.3% Triton X-100 in advance to dilute the antibody, dilute the lung adenocarcinoma-specific antibody NapsinA (purchased from CST) at a ratio of 1:50, discard the blocking solution, and add the prepared primary antibody dilution solution to 4 Incubate overnight in the refrigerator. Take out at 4°C, equilibrate to room temperature, continue to incubate at 37°C for 1 hour, and wash with PBS for 5 minutes x 3 times.
提前配制PBS+0.3％Triton X-100用于二抗稀释，按照1:1000比例稀释激发光为488nm且种属为鼠的荧光二抗(购自赛默飞公司)，常温避光孵育1小时，PBS洗5分钟x 3次。Prepare PBS+0.3% Triton X-100 in advance for secondary antibody dilution, dilute the fluorescent secondary antibody with excitation light of 488 nm and mouse species (purchased from Thermo Fisher Scientific) at a ratio of 1:1000, and incubate at room temperature for 1 hour in the dark , PBS washed 5 min x 3 times.
用PBS按照1:1000体积比稀释非特异性荧光染料DAPI(购自Sigma公司)，常温避光染色5分钟，PBS洗5分钟x 3次。显微镜(Invitrogen公司EVOS M500)下成像，拍照记录。The non-specific fluorescent dye DAPI (purchased from Sigma) was diluted with PBS at a volume ratio of 1:1000, stained at room temperature for 5 minutes, and washed with PBS for 5 minutes x 3 times. The images were imaged under a microscope (EVOS M500 from Invitrogen Company), and photographed and recorded.
图9A-C分别为10倍物镜下不同条件下拍照的图片，其中图9A 是使用非特异性荧光染料DAPI染细胞核的图片，图9B是使用肺腺癌特异性抗体NapsinA(定位在胞浆)染色的图片。图9C是将图9A和9B合并得到的图片。如图所示，在图9A中标记细胞核的位置在图9B中的胞浆均能被特异性抗体标记，表明培养后的细胞为肺腺癌细胞，与临床病理诊断一致。Figures 9A-C are pictures taken under different conditions under a 10x objective lens, in which Figure 9A is a picture of nuclei stained with a non-specific fluorescent dye DAPI, and Figure 9B is a picture of a lung adenocarcinoma-specific antibody NapsinA (localized in the cytoplasm) stained picture of. Fig. 9C is a picture obtained by combining Figs. 9A and 9B. As shown in the figure, the nuclei marked in Figure 9A and the cytoplasm in Figure 9B can be marked by specific antibodies, indicating that the cultured cells are lung adenocarcinoma cells, which is consistent with the clinicopathological diagnosis.
(2)原代肺癌组织和传代培养后的肺癌细胞免疫组化鉴定(2) Immunohistochemical identification of primary lung cancer tissues and subcultured lung cancer cells
从一例肺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0B0004)，浸泡在1mL 4％多聚甲醛中固定。剩余癌组织使用与实施例1相同的方法获得肺癌上皮细胞(样本编号0B0004)。使用实施例3的方法采用本发明的培养基LM将样本0B0004持续培养至第5代。A cancer tissue about the size of a mung bean grain (sample number 0B0004) was taken from a clinical surgical resection sample of a patient with lung cancer, and was immersed in 1 mL of 4% paraformaldehyde for fixation. Lung cancer epithelial cells (sample number 0B0004) were obtained from the remaining cancer tissue using the same method as in Example 1. Using the method of Example 3, the sample OB0004 was continuously cultured to the 5th passage using the medium LM of the present invention.
采用免疫组化法检测样本0B0004原始组织和持续培养至第3代得到的原代细胞中与肺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)。所使用的一抗为甲状腺转录因子1(TTF-1)(购自CST公司)和ki-67抗体(购自R&D公司)。Immunohistochemical method was used to detect the expression of important biomarkers related to lung cancer in the original tissue of sample OB0004 and the primary cells that were continuously cultured to the third passage. The tissue fixed with 4% paraformaldehyde was embedded in paraffin and cut into 4 μm thick tissue sections with a microtome. Routine immunohistochemical detection was then performed (for specific steps, see Li et al., Nature Conmunication, (2018) 9:2983). The primary antibodies used were thyroid transcription factor 1 (TTF-1) (purchased from CST company) and ki-67 antibody (purchased from R&D company).
图10A-10D是原始组织细胞和采用该细胞以本发明的培养基LM培养而获得的肺癌肿瘤细胞的免疫组化结果对比图。图10A和图10B分别是肺癌组织和扩增培养后得到的细胞标记甲状腺转录因子1抗体的图片，图10C和图10D分别是肺癌组织和扩增培养后得到的细胞标记ki-67抗体的图片。由此可以确认，采用本发明技术培养的肺癌肿瘤细胞(样本编号0B0004)培养至第5代时，细胞上与肺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。说明采用本发明技术所培养的细胞保持了肺癌病人癌组织的原始病理特性。10A-10D are comparison diagrams of the immunohistochemical results of the original tissue cells and the lung cancer tumor cells obtained by culturing the cells with the medium LM of the present invention. Fig. 10A and Fig. 10B are pictures of lung cancer tissue and cell marker thyroid transcription factor 1 antibody obtained after expansion and culture, respectively, and Fig. 10C and Fig. 10D are pictures of lung cancer tissue and cell marker ki-67 antibody obtained after expansion and culture, respectively . From this, it can be confirmed that when the lung cancer tumor cells (sample number 0B0004) cultured by the technology of the present invention are cultured to the fifth passage, the expression of biomarkers related to lung cancer on the cells and the expression of markers in the original tissue sections derived from the cells Basically the same. It shows that the cells cultured by the technology of the present invention maintain the original pathological characteristics of the cancer tissue of lung cancer patients.
[实施例7][Example 7]
癌组织来源的原代肺癌肿瘤细胞在小鼠体内的异种移植成瘤实验Xenotransplantation experiment of primary lung cancer tumor cells derived from cancer tissue in mice
使用与实施例1同样的方法从一例病理诊断为肺癌患者的癌组织中分离获得肺癌肿瘤细胞(编号0B0003)，按照实施例3的方法采用 本发明的培养基LM对0B0003进行培养，待肺癌肿瘤细胞数量达到1×10 7个时，采用实施例5的方法对肺癌肿瘤细胞进行消化，并收集。采用本发明的肺癌肿瘤细胞培养基LM和
Figure PCTCN2020109740-appb-000018
(购自BD生物科技公司)按照1:1混匀，吸取100μL与Matrigel基质胶混匀后的培养基将5×10 6个肺癌肿瘤细胞重悬，分别注射入6周大的雌性高度免疫缺陷小鼠(NCG)小鼠(购自南京模式动物研究所)的肺癌脂肪垫和右前肢腋下部位，每三天观察一次肺癌肿瘤细胞在小鼠体内形成肿瘤的体积和生长速率。 Lung cancer tumor cells (number 0B0003) were isolated from the cancer tissue of a patient with a pathological diagnosis of lung cancer using the same method as in Example 1, and the culture medium LM of the present invention was used to culture OB0003 according to the method in Example 3. When the number of cells reached 1×10 7 , lung cancer tumor cells were digested by the method of Example 5 and collected. Using the lung cancer tumor cell culture medium LM of the present invention and
Figure PCTCN2020109740-appb-000018
(Purchased from BD Biotechnology Co., Ltd.) mixed at a ratio of 1:1, sucked 100 μL of the medium mixed with Matrigel to resuspend 5×10 6 lung cancer tumor cells, and injected them into 6-week-old females with high immunodeficiency Mouse (NCG) mice (purchased from Nanjing Institute of Model Animals) were used to observe the volume and growth rate of lung cancer tumor cells in the lung cancer fat pad and right forelimb axilla every three days.
在肿瘤细胞接种后的第15天可观察到小鼠的两处肿瘤细胞接种部位均有瘤体形成，自第15天起至第36天，小鼠体内肿瘤增殖明显。这说明采用本发明的培养方法所培养的癌组织来源的肺癌肿瘤细胞在小鼠体内具有成瘤性。On the 15th day after tumor cell inoculation, tumor formation could be observed in two tumor cell inoculation sites in mice. From the 15th day to the 36th day, the tumor proliferation in the mice was obvious. This indicates that the lung cancer tumor cells derived from cancer tissue cultured by the culture method of the present invention have tumorigenicity in mice.
[实施例8][Example 8]
癌组织来源的肺癌肿瘤细胞的药物敏感性功能测试Drug Sensitivity Functional Testing of Lung Cancer Cells Derived from Cancer Tissue
下面以肺癌患者穿刺样本为例，说明由病人来源的肺癌肿瘤样本培养得到的肺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。The following takes a puncture sample from a lung cancer patient as an example to illustrate that lung cancer tumor cells cultured from a patient-derived lung cancer tumor sample can be used to detect the sensitivity of the patient tumor cells to different drugs.
一、原代肺癌肿瘤细胞的铺板：按照实施例1中方法得到的分离后的肺癌肿瘤细胞(编号0B0011)细胞悬液，按照4×10 4个/cm 2密度接种至预铺有γ射线辐照过后的NIH-3T3细胞的6孔板内。向6孔板中添加2mL制备好的原代肺癌上皮细胞培养基LM，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6孔板内的培养基上清，加入1mL 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1mL 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2mL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LM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，得到第一代细胞总数为160万。按2000～4000个/孔密度接种于384孔板中，使细胞贴壁过夜。剩下的细胞继续按照4×10 4个/cm 2密度接种至预铺有γ射线辐照过后的NIH-3T3细胞的6孔板内进行持续传代4次，按照上述方法进行消化和终止消化，重悬细胞后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，得到第五代细胞总数为180万。按2000～4000个/孔密度接种于384孔板中，使细胞贴壁过夜。 1. Plating of primary lung cancer tumor cells: The isolated lung cancer tumor cell (No. 0B0011) cell suspension obtained according to the method in Example 1 was inoculated at a density of 4×10 4 cells/cm 2 to pre-plated γ-ray radiation Illuminated NIH-3T3 cells within a 6-well plate. 2 mL of the prepared primary lung cancer epithelial cell culture medium LM was added to the 6-well plate, and cultured in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher Scientific). When the cells in the plate grow to about 80% of the bottom area, discard the medium supernatant in the 6-well plate, add 1 mL of 0.25% trypsin (purchased from Thermo Fisher) to digest for 1 minute, and then aspirate 0.25% trypsin , and then add 1 mL of 0.05% trypsin to digest the cells, and incubate at 37°C for 10 minutes, until the cells can be observed under the microscope (EVOS M500 from Invitrogen) to be completely digested. 2 mL of DMEM/F12 medium of 100 U/mL penicillin and 100 μg/mL streptomycin was used to terminate the digestion, and collected into a 15 mL centrifuge tube. After centrifugation at 1500 rpm for 4 minutes, the supernatant was discarded. The cell pellets after centrifugation were resuspended in LM medium, and counted using a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.) to obtain a total of 1.6 million first-generation cells. The cells were seeded in 384-well plates at a density of 2000-4000 cells/well, and the cells were allowed to adhere overnight. The remaining cells were continuously seeded at a density of 4×10 4 cells/cm 2 into a 6-well plate pre-plated with γ-ray-irradiated NIH-3T3 cells for 4 consecutive passages, and digested and terminated according to the above method. After the cells were resuspended, the cells were counted using a flow image counter (JIMBIO FIL, Jiangsu Zhuo Microorganism Technology Co., Ltd.), and the total number of cells of the fifth generation was 1.8 million. The cells were seeded in 384-well plates at a density of 2000-4000 cells/well, and the cells were allowed to adhere overnight.
二、药物梯度实验：2. Drug gradient experiment:
(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，待测药物为阿法替尼(购自MCE公司)、吉非替尼(购自MCE公司)、奥希替尼(购自MCE公司)和克唑替尼(购自MCE公司)。(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肺癌肿瘤细胞的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。(1) Prepare the drug storage plate by concentration gradient dilution method: draw 40 μL of the 10 μM drug stock solution to be tested as the highest concentration, and then draw 10 μL from it and add it to a 0.5 mL EP tube containing 20 μL of DMSO. Pipette 10 μL from the tube into a second 0.5 mL EP tube containing 20 μL of DMSO, i.e. dilute the drug 1:3. Repeat the above method, dilute in turn, and finally obtain 7 concentrations required for dosing. Different concentrations of drugs 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. In this example, the drugs to be tested are afatinib (purchased from MCE company), gefitinib (purchased from MCE company), osimertinib (purchased from MCE company) and crizotinib (purchased from MCE company) company). (2) Using a high-throughput automated workstation (JANUS, Perkin Elmer Company), different concentrations of drugs and solvent controls in the 384-well drug storage plate were added to the 384-well cell culture plate plated with lung cancer tumor cells. Drug group and solvent control group Each has 3 duplicate holes. The volume of drug added to each well was 100 nL.
(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。(3) Detection of cell activity: 72 hours after administration, use Cell Titer-Glo detection reagent (purchased from Promega) to detect the chemiluminescence value of the cells after drug addition and culture. The size of the chemiluminescence value reflects cell viability and drug-induced cell viability 10 μL of the prepared Cell Titer-Glo detection solution was added to each well, and the chemiluminescence value was detected using a microplate reader (Envision, Perkin Elmer Company) after mixing.
(4)细胞活性检测：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，同时计算不同药物在人体内最大血药浓度Cmax对应下的细胞存活率。 (4) Cell viability detection: According to the formula, cell viability (%)=chemiluminescence value of drug-added well/chemiluminescence value of control well*100%, calculate the cell viability of cells treated with different drugs, and use Graphpad Prism software to draw the graph And calculate the half inhibition rate IC 50 , and calculate the cell survival rate of different drugs corresponding to the maximum blood concentration Cmax in the human body.
(5)药物敏感性测试结果如图11所示。(5) The results of the drug sensitivity test are shown in FIG. 11 .
图11表示从同一个肺癌患者的手术切除癌组织样本(编号0B0011) 所培养获得的第1代肺癌肿瘤细胞和第5代肺癌肿瘤细胞，对靶向药物阿法替尼、吉非替尼、奥希替尼和克唑替尼的敏感性。图中横坐标上标线对应的浓度为这四种药物在人体内最大血药浓度Cmax。结果显示，同一病人的细胞对不同药物在人体内最大血药浓度时的敏感性不同，不同代数的细胞对同一种药物的敏感性基本一致。根据结果可以判断肺癌病人在临床使用该种药物时的有效性，同时可以说明根据本专利培养方法得到不同代数的肿瘤细胞对药物的敏感性是稳定的。Figure 11 shows that the first-generation lung cancer tumor cells and the fifth-generation lung cancer tumor cells cultured from the surgically resected cancer tissue sample (No. 0B0011) from the same lung cancer patient showed that the targeted drugs afatinib, gefitinib, Sensitivity to osimertinib and crizotinib. The concentration corresponding to the marked line on the abscissa in the figure is the maximum blood concentration Cmax of these four drugs in the human body. The results show that the cells of the same patient have different sensitivities to different drugs at the maximum blood concentration in the human body, and the cells of different generations have basically the same sensitivity to the same drug. According to the results, the effectiveness of the drug in clinical use of the drug can be judged for lung cancer patients, and it can be shown that the sensitivity of tumor cells of different generations obtained according to the patented culture method to the drug is stable.
工业应用性Industrial applicability
本发明提供一种用于在体外培养或扩增原代肺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。The invention provides a culture medium and a culture method for culturing or expanding primary lung cancer epithelial cells in vitro, and the cultured cells can be applied to the evaluation and screening of the curative effect of drugs. Thus, the present invention is suitable for industrial applications.
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。Although the present invention is described in detail herein, the present invention is not limited thereto, and those skilled in the art can make modifications according to the principles of the present invention. Therefore, all modifications made according to the principles of the present invention should be understood as falling into the protection scope of the present invention.

Claims (13)

  1. 一种用于培养肺癌上皮细胞的原代细胞培养基，其特征在于：A primary cell culture medium for culturing lung cancer epithelial cells, characterized in that:
    含有MST1/2激酶抑制剂；***1；表皮细胞生长因子；肝细胞生长因子；神经调节蛋白-1；选自胰岛素-转铁蛋白-硒复合物、B27添加剂和N2添加剂中的至少一种的添加剂；选自Y27632、法舒地尔、和H-1152中的至少一种的ROCK激酶抑制剂，Contains MST1/2 kinase inhibitor; insulin-like growth factor 1; epidermal growth factor; hepatocyte growth factor; neuregulin-1; An additive; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152,
    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，The MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof,
    Figure PCTCN2020109740-appb-100001
    Figure PCTCN2020109740-appb-100001
    其中，in,
    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 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl optionally substituted with 1-2 independently R 6 base, aryl C1-C6 alkyl and heteroaryl;
    R 2和R 3各自独立地选自C1-C6烷基； R 2 and R 3 are each independently selected from C1-C6 alkyl;
    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 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl;
    R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。 R 6 is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
  2. 如权利要求1所述的原代细胞培养基，其中The primary cell culture medium of claim 1, wherein
    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 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and benzene optionally substituted with 1-2 independently R 6 base, naphthyl, benzyl and thienyl;
    R 2和R 3各自独立地选自C1-C3烷基； R 2 and R 3 are each independently selected from C1-C3 alkyl;
    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 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, piperidinyl C1-C6 alkyl, and tetrahydropyranyl C1-C6 alkyl;
    R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。 R 6 is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
  3. 如权利要求1所述的原代细胞培养基，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，The primary cell culture medium of claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,
    Figure PCTCN2020109740-appb-100002
    Figure PCTCN2020109740-appb-100002
    其中，in,
    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 is selected C1-C6 alkyl, optionally substituted with 1-2 R 6 independently substituted phenyl, optionally substituted with 1-2 R 6 independently substituted thienyl, and optionally substituted with 1 - 2 independently R 6 substituted benzyl groups;
    R 5选自氢、C1-C6烷基、和C3-C6环烷基； R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl;
    R 6各自独立地选自卤素、C1-C6烷基、和C1-C6卤代烷基。 Each R 6 is independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl.
  4. 如权利要求3所述的原代细胞培养基，其中The primary cell culture medium of claim 3, wherein
    R 1为任选地被1-2个独立地R 6取代的苯基； R 1 is phenyl optionally substituted with 1-2 independently R 6;
    R 5为氢； R 5 is hydrogen;
    R 6优选为氟、甲基或三氟甲基。 R 6 is preferably fluorine, methyl or trifluoromethyl.
  5. 如权利要求1所述的原代细胞培养基，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：The primary cell culture medium of claim 1, wherein the MST1/2 kinase inhibitor is selected from at least one of the following compounds or pharmaceutically acceptable salts thereof:
    Figure PCTCN2020109740-appb-100003
    Figure PCTCN2020109740-appb-100003
    Figure PCTCN2020109740-appb-100004
    Figure PCTCN2020109740-appb-100004
    Figure PCTCN2020109740-appb-100005
    Figure PCTCN2020109740-appb-100005
    Figure PCTCN2020109740-appb-100006
    Figure PCTCN2020109740-appb-100006
    Figure PCTCN2020109740-appb-100007
    Figure PCTCN2020109740-appb-100007
  6. 如权利要求1～5中任一项所述的原代细胞培养基，其特征在于：The primary cell culture medium according to any one of claims 1 to 5, characterized in that:
    所述MST1/2激酶抑制剂的含量为0.625～20μM，优选为0.625～10μM。The content of the MST1/2 kinase inhibitor is 0.625-20 μM, preferably 0.625-10 μM.
  7. 如权利要求1-5中任一项所述的原代细胞培养基，其特征在于：The primary cell culture medium of any one of claims 1-5, wherein:
    所述***1的含量为1.25～80ng/ml，更优选为5～80ng/ml；The content of the insulin-like growth factor 1 is 1.25-80 ng/ml, more preferably 5-80 ng/ml;
    所述表皮细胞生长因子的含量为10～80ng/ml，更优选为10～40ng/ml；The content of the epidermal growth factor is 10-80ng/ml, more preferably 10-40ng/ml;
    所述肝细胞生长因子为5～80ng/ml，更优选为20～80ng/ml；The hepatocyte growth factor is 5-80ng/ml, more preferably 20-80ng/ml;
    所述神经调节蛋白-1的含量为10～80ng/ml，更优选20～80ng/ml；The content of the neuregulin-1 is 10-80ng/ml, more preferably 20-80ng/ml;
    所述添加剂的含量优选为体积比1:200～1:25，更优选为体积比1:50～1:25；The content of the additive is preferably 1:200-1:25 by volume, more preferably 1:50-1:25 by volume;
    所述ROCK激酶抑制剂的含量优选为1.25～20μM，更优选为2.5～10μM。The content of the ROCK kinase inhibitor is preferably 1.25-20 μM, more preferably 2.5-10 μM.
  8. 如权利要求7所述的原代细胞培养基，其特征在于：The primary cell culture medium of claim 7, wherein:
    所述添加剂为胰岛素-转铁蛋白-硒复合物，其中胰岛素/转铁蛋白/***钠各自的含量优选分别为2.5～20μg/ml-1.25～10μg/ml-1.25～10ng/ml，更优选分别为10～20μg/ml-5～10μg/ml-5～10ng/ml。The additive is an insulin-transferrin-selenium complex, wherein the respective contents of insulin/transferrin/sodium selenite are preferably 2.5-20 μg/ml-1.25-10 μg/ml-1.25-10 ng/ml, and more Preferably, they are 10-20 μg/ml-5-10 μg/ml-5-10 ng/ml, respectively.
  9. 如权利要求7所述的原代细胞培养基，其特征在于：The primary cell culture medium of claim 7, wherein:
    所述ROCK激酶抑制剂为Y27632。The ROCK kinase inhibitor is Y27632.
  10. 如权利要求1～5中任一项所述的原代细胞培养基，其特征在于：The primary cell culture medium according to any one of claims 1 to 5, characterized in that:
    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。Free of serum, bovine pituitary extract, Wnt agonists, R-spondin family proteins, BMP inhibitors, nicotinamide and N-acetylcysteine.
  11. 如权利要求1～5中任一项所述的原代细胞培养基，其特征在于：The primary cell culture medium according to any one of claims 1 to 5, characterized in that:
    所述肺癌上皮细胞选自肺癌肿瘤细胞、正常肺癌上皮细胞、和肺癌上皮干细胞。The lung cancer epithelial cells are selected from lung cancer tumor cells, normal lung cancer epithelial cells, and lung cancer epithelial stem cells.
  12. 一种肺癌上皮细胞的培养方法，其特征在于，包括以下步骤：A method for culturing lung cancer epithelial cells, comprising the following steps:
    (1)配制如权利要求1～11中任一项所述的原代细胞培养基；(1) preparing the primary cell culture medium according to any one of claims 1 to 11;
    (2)用X射线或者γ射线辐照后的滋养细胞预铺培养器皿；(2) Pre-plating culture vessels with trophoblasts irradiated with X-rays or γ-rays;
    (3)在预铺有滋养细胞的培养器皿内接种从肺癌组织分离得到原代肺癌上皮细胞，使用步骤(1)中的所述原代细胞培养基进行培养。(3) Inoculating primary lung cancer epithelial cells isolated from lung cancer tissue in a culture vessel pre-plated with trophoblasts, and culturing using the primary cell culture medium in step (1).
  13. 一种评估或筛选用于治疗肺癌疾病的药物的方法，其特征在于，包括以下步骤：A method for evaluating or screening a drug for the treatment of lung cancer, comprising the steps of:
    (1)根据权利要求12所述的培养方法培养得到肺癌上皮细胞；(1) the culturing method according to claim 12 is cultivated to obtain lung cancer epithelial cells;
    (2)选定需要检测的药物，稀释成不同的药物浓度梯度；(2) Select the drugs to be detected and dilute them into different drug concentration gradients;
    (3)向步骤(1)培养得到的肺癌上皮细胞中添加梯度稀释后的所述药物，并进行细胞活力检测。(3) adding the drug after gradient dilution to the lung cancer epithelial cells cultured in step (1), and performing cell viability detection.
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