WO2021179354A1 - 原代肝癌细胞培养基、原代肝癌细胞培养方法及其应用 - Google Patents

原代肝癌细胞培养基、原代肝癌细胞培养方法及其应用 Download PDF

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WO2021179354A1
WO2021179354A1 PCT/CN2020/081402 CN2020081402W WO2021179354A1 WO 2021179354 A1 WO2021179354 A1 WO 2021179354A1 CN 2020081402 W CN2020081402 W CN 2020081402W WO 2021179354 A1 WO2021179354 A1 WO 2021179354A1
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liver cancer
cells
primary liver
medium
culture medium
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刘青松
赵明
陈程
王文超
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 present invention belongs to the field of biotechnology. Specifically, it relates to a primary liver cancer cell culture medium for culturing or amplifying primary liver cancer cells in vitro and a method for culturing primary liver cancer cells, and also relates to the evaluation of the therapeutic effect of the cultured cells in medicines. And screening methods and applications.
  • Primary culture is also called primary culture, which refers to the first culture of tumor cells or tissues taken directly from the body.
  • the most commonly used primary cultures are tissue block culture and dispersed cell culture.
  • Tissue block culture is to directly transplant the shredded tissue block on the wall of the culture bottle, add the culture medium and carry out the culture.
  • Dispersed cell culture is the use of mechanical or chemical methods to disperse the cells.
  • proteolytic enzymes such as trypsin and collagenase
  • metal ion chelating agents such as EDTA
  • liver cancer drug susceptibility test system like a bacterial drug susceptibility test
  • the in vitro drug susceptibility results of liver cancer will correspond to the clinical in vivo response, and precise chemotherapy cannot be achieved. Therefore, research on the development of sensitive drugs for liver cancer is the key to treatment. .
  • High-throughput drug sensitivity test technology is a new type of drug sensitivity detection method, which has the advantages of wide coverage of detection drugs, high efficiency, and strong individualization. It has made excellent progress in the field of leukemia. Compared with other types of drug susceptibility experimental models (PDO, PDX), it also has the advantages of economy, rapid detection, and easy promotion. At present, the bottleneck encountered by this technology in the field of liver cancer is mainly the lack of effective, rapid and stable primary liver cancer cell culture and expansion technology.
  • conditional reprogramming culture using trophoblast cells can quickly and stably establish primary tumor cell lines (Xuefeng Liu, Ewa Krawczyk, et al. Conditional reprogramming and long-term expansion of normal). and tumor cells from human biospecimens. Nature Protocols, VOl.12,NO.2,2017,439-451).
  • stable cell lines selected in vitro using the conditioned medium (F Meduim) described in this document have a long culture cycle, which cannot meet the needs of rapid clinical testing. Therefore, it is necessary to develop a fast and stable culture medium and culture method suitable for primary liver cancer cells, so that high-throughput drug screening can be carried out within two weeks, and effectively guide the clinical rational use of drugs.
  • the present invention aims to provide a primary liver cancer cell culture medium and a primary liver cancer cell culture method for quickly and stably culturing primary liver cancer cells in vitro to overcome the deficiencies of the prior art.
  • the primary liver cancer cell culture medium and the primary liver cancer cell culture method of the present invention are used to construct a primary liver cancer cell model, primary liver cancer cells with the biological characteristics of liver cancer patients can be obtained, and can be used for new drug screening and in vitro drug sensitivity Sexual testing.
  • the invention provides a primary liver cancer cell culture medium for culturing primary liver cancer cells.
  • the primary liver cancer cell culture medium of the present invention contains an initial medium, glutamine (Glu), non-essential amino acids (NEAA), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and IL-6 , Epidermal growth factor, insulin, Y27632, N2 additives, B27 additives, Primocin, penicillin and streptomycin, fetal bovine serum, and optional hydrocortisone, optional R-spondin1, where the initial medium can be, for example, It is DMEM/F12, DMEM, F12 or RPMI-1640, preferably DMEM/F12.
  • the content of glutamine is 62.5ng/mL ⁇ 1000ng/mL, preferably 125ng/mL ⁇ 1000ng/mL;
  • the non-essential amino acid is selected from glycine, alanine, asparagine, aspartame One or more of acid, glutamic acid, proline and serine, the total content of the non-essential amino acids is 0.25 ⁇ M-4 ⁇ M, preferably 0.5 ⁇ M-4 ⁇ M;
  • the basic fibroblast growth factor The content is 5ng/ml ⁇ 80ng/ml, preferably 20ng/ml ⁇ 80ng/ml;
  • the content of the hepatocyte growth factor is 5ng/ml ⁇ 80ng/ml, preferably 20ng/ml ⁇ 80ng/ml;
  • the IL The content of -6 is 1.25ng/ml-20ng/ml, preferably 5ng/ml-20ng/ml;
  • the content of epidermal growth factor is 2.5ng/ml-40ng/ml, preferably 10
  • the content of the Primocin is 40 ⁇ g/ml ⁇ 80 ⁇ g/ml; the content of the penicillin is 80U/ml ⁇ 200U/ml; the content of the streptomycin is 50 ⁇ g/ml ⁇ 150 ⁇ g/ml; the fetus
  • the volume ratio of bovine serum to the culture medium is 1:5 ⁇ 1:20; the content of hydrocortisone is 0.4 ⁇ g/ml ⁇ 1.6 ⁇ g/ml; the content of R-spondin1 is 62.5ng/mL ⁇ 1000 ng/mL, preferably 250 ng/mL to 1000 ng/mL.
  • the invention also provides a method for culturing primary liver cancer cells.
  • the primary liver cancer culture medium of the present invention is used to culture the primary liver cancer cells.
  • the irradiated trophoblast cells are added at a cell density of 5 ⁇ 10 4 to 1.5 ⁇ 10 5 cells/cm 2.
  • the irradiated trophoblast cells are mouse fibroblasts, preferably NIH-3T3 or J2-3T3.
  • the irradiated trophoblast cells are irradiated with gamma rays or X-rays, preferably gamma rays.
  • the radiation dose is 10-50Gy.
  • the above-mentioned primary liver cancer cells can be obtained, for example, by the following methods: obtained by a liver cancer resection method; obtained by a puncture method; obtained from the ascites of a liver cancer patient; or obtained from the urine of a liver cancer patient.
  • a liver cancer tissue sample is collected within half an hour after the solid tumor patient is surgically removed or punctured.
  • the collection method is: in a sterile environment, cut a liver cancer tissue sample of more than 1 cm 3 from a non-necrotic site, place it in a pre-cooled 15 mL primary liver cancer cell culture medium of the present invention, and place it in a normal cell culture Stand by under conditions, or transport as soon as possible in an ice bag.
  • the method for culturing primary liver cancer cells of the present invention includes the step of separating and obtaining primary liver cancer cells from the above-mentioned tissue samples.
  • a biological safety cabinet transfer the tissue sample to a cell culture dish, and culture it with a medium containing a penicillin/streptomycin solution (referred to as a double antibody solution for short) (for example, DMEM/F12 containing a double antibody solution) Base, more preferably, it is added with glutamine, non-essential amino acids, basic fibroblast growth factor, hepatocyte growth factor, IL-6, hydrocortisone, epidermal growth factor, insulin, Y27632, N2 additives, One or more of B27 additive, R-spondin1 and Primocin, the concentration of the above-mentioned additive components can adopt the concentration in the primary liver cancer cell culture medium of the present invention, and this medium will be referred to as cell separation medium below) Three times, the blood cells and sloughed necrotic cells are washed away.
  • a double antibody solution for example, DMEM/F12 containing a double antibody solution
  • Base more preferably, it is added with glutamine, non-essen
  • the treated tissue sample to a new petri dish, drop an appropriate amount of the above-mentioned cell separation medium, and use a sterile scalpel blade, surgical scissors and surgical forceps to divide the tumor tissue into small pieces with a diameter of less than 1 mm.
  • the minced tumor tissue is transferred to a centrifuge tube for centrifugation, for example, at 1,000 to 1,800 revolutions per minute for 3 to 10 minutes.
  • centrifuge tube carefully remove the supernatant in the centrifuge tube, then mix and resuspend with tissue digestion solution, and place it on a constant temperature shaker for shaking (rotation speed can be 230 ⁇ 350rpm/min) for digestion for at least 3 hours (digestion time depends on the sample size) ; If the sample is larger than 1g, the digestion time will increase to 4-6 hours); then centrifuge, for example, centrifuge at 1000-1800 rpm for 3-10 minutes, discard the supernatant, and separate the digested tissue cells with an appropriate amount of the above-mentioned cells Resuspend in culture medium and sieving.
  • rotation speed can be 230 ⁇ 350rpm/min
  • digestion time depends on the sample size
  • centrifuge for example, centrifuge at 1000-1800 rpm for 3-10 minutes, discard the supernatant, and separate the digested tissue cells with an appropriate amount of the above-mentioned cells Resuspend in culture medium and sieving.
  • the cell sieve can be 100 ⁇ M, for example, and then centrifuge again, for example, at 1000 to 1800 rpm for 3 to 10 minutes, discard the supernatant, and observe whether there are blood cells, such as blood cells, add Appropriate amount of blood cell lysate, mix and lyse, and then centrifuge again, for example, at 1000 to 1800 rpm for 3 to 10 minutes, discard the supernatant, add an appropriate amount of the primary liver cancer cell culture medium of the present invention to resuspend and collect the cells , Used for adhering to the wall in the culture flask to expand the culture.
  • the present invention also provides a method for evaluating the efficacy of a drug for treating liver cancer disease, which includes the following steps:
  • Hepatocarcinoma cells are cultured according to the primary hepatocarcinoma cell culture method of the present invention.
  • step (1) Digest the hepatocarcinoma cells cultured in step (1) into a single cell suspension, and dilute the single cell suspension with the primary hepatocarcinoma cell culture medium of the present invention to 2 ⁇ 10 4 cells/cm 2 ⁇ 4 ⁇ 10 Add the diluted cell suspension to the multi-well plate at a seeding density of 4 cells/cm 2 to adhere to the wall overnight, and add the drug after serial dilutions to the adherent cells;
  • a cell viability detection reagent is added to each well, and after shaking evenly, the chemiluminescence intensity value of each well is measured with a fluorescence microplate reader, a drug dose-effect curve is drawn based on the measured value, and each drug pair is calculated Inhibition strength of cell proliferation.
  • the cultured primary liver cancer cells are not interfered by interstitial cells such as fibroblasts and adipocytes, and purified liver cancer cells can be obtained;
  • the amplified hepatoma cells can also be serially passaged;
  • the culture cost is controllable: The primary liver cancer culture medium does not need to add expensive Wnt agonists, BMP inhibitors, FGF10 and other factors, which is an improvement to the existing conditional reprogramming medium.
  • the culture medium and the culture system have a large number of hepatocarcinoma cells cultured with a high degree of homogeneity, which is suitable for high-throughput screening of new candidate compounds and providing patients with high-throughput drug in vitro sensitivity functional testing.
  • hepatocarcinoma cells derived from humans can be cultured, including hepatocellular carcinoma cells, intrahepatic cholangiocarcinoma cells, and mixed hepatocellular carcinoma cells.
  • the cells obtained by the culture method of this embodiment can be used for basic medical research of primary liver cancer cells, screening of drug responses, and development of new drugs for liver cancer.
  • Figure 1 is an inverted phase contrast microscope photograph of primary liver cancer cells obtained by culturing primary liver cancer cells isolated from liver cancer tissue samples using the primary cell culture medium of the present invention.
  • Figure 2 shows the immunohistochemical staining results of liver cancer cells obtained by culturing primary liver cancer cells isolated from a liver cancer tissue sample using the primary cell culture medium of the present invention and the original tissue section immunohistochemical staining of the tissue sample itself Comparative Results.
  • Figure 3 is a graph showing the effect of different concentrations of different factors in the culture medium of primary liver cancer cells on the proliferation of primary liver cancer cells.
  • Figure 4 is an inverted phase-contrast microscope photograph of cells obtained by culturing primary liver cancer cells isolated from liver cancer tissue samples using existing conditioned medium (FM) and the primary liver cancer cell culture medium (HCCM) of the present invention .
  • FM conditioned medium
  • HCCM primary liver cancer cell culture medium
  • Figure 5 is a statistics of multiple passages of cells obtained by culturing primary liver cancer cells isolated from liver cancer tissue samples using the existing conditioned medium (FM) and the primary liver cancer cell culture medium (HCCM) of the present invention Effect picture.
  • FM existing conditioned medium
  • HCCM primary liver cancer cell culture medium
  • Fig. 6 shows the tumor formation of liver cancer cells in mice obtained by culturing primary liver cancer cells isolated from liver cancer tissue samples using the primary cell culture medium of the present invention.
  • Fig. 7 shows the dose-effect curve of different chemotherapeutic drugs and targeted drugs of liver cancer cells obtained by culturing the primary liver cancer cell culture medium of the present invention and the calculated half inhibition rate.
  • Example 1 Acquisition of primary liver cancer cells and treatment of trophoblast cells
  • Liver cancer tissue samples are collected within half an hour after surgical resection or puncture of liver cancer patients who have been explained and approved. In a sterile environment, cut a liver cancer tissue sample of more than 1 cm 3 from the non-necrotic site, place it in 15 mL DMEM/F12 medium pre-cooled in a refrigerator at 4° C., and label it.
  • NIH-3T3 purchased from ATCC was cultured in DMEM medium (manufacturer: Corning) supplemented with 10% FBS and 1% bi-antibody solution at 37°C and 5% CO 2 ;
  • the cells were resuspended in DMEM supplemented with 10% FBS and 1% bi-antibody solution, plated at a ratio of one to three, and passaged every three days.
  • the irradiation dose is 25Gy;
  • cytokines were added according to the concentrations in Table 1 below to obtain the single factor-added medium of Example 2.
  • primary liver cancer cells were obtained from the surgically removed liver cancer tissue samples of 4 liver cancer patients (patient 2, patient 3, patient 7, and patient 8) who had been explained and obtained consent.
  • the primary liver cancer cells obtained above were seeded in a 12-well plate (45,000 cells per well) at a viable cell density of 1 ⁇ 10 4 cells/cm 2 .
  • the culture was carried out to the 8th day, the 12-well plate was taken out, and 200 ⁇ l 0.25% trypsin (purchased from Gibco) was used to rinse for 1 minute. After aspiration, 500 ⁇ l 0.05% trypsin (purchased from Gibco) was added to each well. ), placed in a 37°C, 5% CO 2 incubator to react for 10 minutes, until the cells have been completely digested can be observed under the microscope (Invitrogen EVOS M500), and the digestion is terminated. After centrifugation at 1500 rpm for 4 minutes, discard the supernatant and add 1 Resuspend one milliliter of basal medium, and use a flow image counter (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL) for counting to obtain the total number of cells.
  • a flow image counter Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL
  • Glu (glutamine), NEAA (non-essential amino acid), bFGF (basic fibroblast growth factor), HGF (hepatocyte growth factor), IL-6, hydrocortisone, EGF ( Epidermal growth factor), Insulin (insulin), Y27632, N2 additive, B27 additive, R-spondin1 all have a strong proliferation effect on primary liver cancer cells.
  • the primary hepatoma cell culture medium (HCCM) of Example 3 was prepared according to the formula in Table 2 below.
  • primary liver cancer cells were obtained from a liver cancer tissue sample of a liver cancer patient (patient 1) who had been explained and obtained consent.
  • patient 1 Using the primary liver cancer cell culture medium of Example 3, the primary liver cancer cells obtained above were seeded in a 12-well plate (45,000 cells per well) at a viable cell density of 1 ⁇ 10 4 cells/cm 2 according to the cell density 5 ⁇ 10 4 cells/cm 2 were added to NIH-3T3 cells irradiated with gamma rays (irradiation dose 25Gy), and mixed. After the surface is disinfected, it is placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for cultivation.
  • Figure 1(A) is a photo taken under the microscope (Invitrogen EVOS M500, 10x objective lens) from the third day of cultivation
  • Figure 1(B) is a photo taken under the microscope from the seventh day of cultivation (Invitrogen EVOS M500, 10 Objective lens).
  • the circles in (A) and (B) of Figure 1 are the cell clones formed.
  • the cultured primary liver cancer cells can form a typical cell clone in 3 days.
  • Figure 1(B) cell clones have been proliferated rapidly.
  • immunohistochemical analysis was performed on the hepatocarcinoma cells obtained in the culture.
  • the method of immunohistochemistry is as follows.
  • the cultured primary liver cancer cells were digested and centrifuged, washed with PBS (purchased from Shanghai Shenggong) for 1 to 2 times, and then fixed in 95% ethanol (purchased from Shanghai Shenggong). The fixed cells were centrifuged and removed. After cleaning, wrap the cell pellet with embedding paper (purchased from Shanghai Shenggong) and soak it in 95% ethanol again to make cell wax blocks. The samples were prepared for cell wax blocks, and then analyzed for liver cancer immunohistochemical labeling (Ki67 and CK7 were purchased from Cell Signaling Technology).
  • Fig. 2(A) is the immunohistochemical result of the cells cultured using the primary liver cancer cell culture medium of this example
  • Fig. 2(B) is the immunohistochemical result of the liver cancer tissue of the same patient.
  • (A) and (B) of Figure 2 both show CK7(-) and Ki67(+), that is to say, the hepatocarcinoma cells cultured with the primary hepatocarcinoma cell culture medium of this example can maintain the pathology of the hepatocarcinoma tissue characteristic.
  • Example 3 In the same manner as in Example 3, using the culture medium prepared in Example 3, primary liver cancer cells were obtained from 9 liver cancer patients (1-9) who had been explained and agreed to undergo surgical resection of liver cancer tissue samples. Example 3 was used The primary hepatocarcinoma cell culture medium was cultured in the same manner as in Example 3. When the cells were expanded to 85%, they were digested and counted. At the same time, the number of culture days until digestion was recorded as a culture cycle. The results are shown in Table 3 below.
  • Sample information Training days Number of implanted cells (*10 4 ) Number of collected cells (*10 4 ) Patient 1 18 6 100 Patient 2 13 10 180 Patient 3 13 10 160 Patient 4 13 10 164 Patient 5 10 10 440 Patient 6 14 10 264 Patient 7 twenty two 8 280 Patient 8 12 10 178 Patient 9 11 10 276 average value 12.6 8.4 204
  • the present invention hepatoma cell primary cultures of liver cancer cell obtained average realized of the order of 104 to the order of 12.6 days, the proliferation of 106.
  • Example 5 The effect of the concentration of added factors on the proliferation of liver cancer cells
  • liver cancer cells were obtained from the surgical resection of liver cancer tissue samples of 4 liver cancer patients (patient 4, patient 8, patient 9, and patient 10) who were explained and obtained consent, and used the examples
  • the primary hepatocellular carcinoma cell culture medium in 3 was used to obtain hepatocellular carcinoma cells.
  • the obtained hepatocellular carcinoma cells were seeded in a 12-well plate (45,000 cells per well) at a living cell density of 1 ⁇ 10 4 cells/cm 2 according to the cell density. 5 ⁇ 10 4 cells/cm 2 were added to NIH-3T3 cells irradiated with gamma rays (irradiation dose 25Gy), and mixed.
  • the surface After the surface is disinfected, it is placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for cultivation. After the cells are expanded to 85%, take out the 12-well plate, rinse with 200 microliters of 0.25% trypsin (purchased from Gibco) for 1 minute, and then add 500 microliters of 0.05% trypsin (purchased from Gibco) to each well. Company), placed in a 37°C, 5% CO 2 incubator for 10 minutes until the cells have been completely digested can be observed under the microscope (Invitrogen’s EVOS M500), and the digestion is terminated.
  • a 37°C, 5% CO 2 incubator purchased from Thermo Fisher
  • Formulation 1 The primary liver cancer cell culture medium components in Example 3 do not contain N2 additives;
  • Formulation 2 The primary liver cancer cell culture medium components in Example 3 do not contain non-essential amino acids;
  • Formulation 3 The primary liver cancer cell culture medium components in Example 3 do not contain glutamine;
  • Formulation 4 The primary liver cancer cell culture medium components in Example 3 do not contain insulin;
  • Formulation 5 The primary liver cancer cell culture medium components in Example 3 do not contain B27 additives;
  • Formulation 6 The primary liver cancer cell culture medium components in Example 3 do not contain HGF;
  • Formulation 7 The primary liver cancer cell culture medium component in Example 3 does not contain Y27632;
  • Formulation 8 The primary liver cancer cell culture medium components in Example 3 do not contain hydrocortisone;
  • Formulation 9 The primary liver cancer cell culture medium component in Example 3 does not contain EGF;
  • Formulation 10 The primary liver cancer cell culture medium components in Example 3 do not contain bFGF;
  • Formulation 11 The primary liver cancer cell culture medium components in Example 3 do not contain R-spondin1;
  • Formulation 12 The primary liver cancer cell culture medium components in Example 3 do not contain IL-6;
  • the primary liver cancer cell culture medium in the above formulas 1-12 and Example 3 were used to dilute the digested cell suspension, and 20,000 cells per well, 500 microliters of volume were seeded into a 24-well plate.
  • the ratio is the ratio of the number of cells obtained by culturing for one generation using each medium to the number of cells obtained by culturing for one generation in the corresponding control wells.
  • a ratio greater than 1 indicates that the proliferation-promoting effect of the formulated medium containing different concentrations of factors or small molecule compounds is better than that of the control well.
  • the ratio is less than 1, indicating that the formulated medium containing different concentrations of factors or small molecule compounds has a better proliferation effect.
  • the medium of the control wells had a weak proliferation promotion effect.
  • the concentration range of N2 additive is 1:400 ⁇ 1:25 in terms of volume ratio, and the cell proliferation effect is the most obvious when added at 1:100; the concentration range of non-essential amino acids
  • the cell proliferation effect is most obvious when the concentration is 0.25 ⁇ 4 ⁇ M, and the concentration is 1 ⁇ M
  • the concentration range of glutamine is 62.5 ⁇ 1000ng/mL, and the cell proliferation effect is the most obvious when the concentration is 250ng/mL
  • the concentration range of insulin is 1.25 ⁇ 20ng/mL
  • the cell proliferation effect is the most obvious when the concentration is 5-20ng/mL
  • the concentration range of B27 additive is 1:12.5 ⁇ 1:200 by volume ratio, and the cell proliferation effect is the most obvious when added at 1:12.5 ⁇ 1:50
  • HGF The concentration range is 5 ⁇ 80ng/mL, the cell proliferation effect is most obvious when the concentration is 20 ⁇ 80ng/mL; the Y27632 concentration range is 5 ⁇ 40 ⁇ M, and the cell proliferation effect is the most obvious when the concentration is 10 ⁇ M;
  • the FM medium was prepared according to the formula in Table 4 below.
  • Example 3 Using the FM medium and the primary hepatocarcinoma cell culture medium in Example 3 (denoted as HCCM in the figure), respectively, the surgical removal of liver cancer tissue samples from liver cancer patients (patients 3 and 4) who have explained and obtained consent Primary liver cancer cells were cultured in the same manner as in Example 3.
  • Figure 4 shows the culture results of primary liver cancer cells from patient 3.
  • Figure 4(A) is a primary liver cancer cell cultured using FM medium
  • Figure 4(B) is a primary liver cancer cell cultured using the primary liver cancer cell culture medium of Example 3, as shown in Figure 4
  • the culture was continued, with the number of days of culture as the abscissa and the cell population doubling number as the ordinate, the Graphpad Prism7.0 software was used to draw the growth curve of two cases of primary hepatocellular carcinoma cells cultured with two different media.
  • the results of primary hepatocarcinoma cell culture of patient 4 are shown in Fig. 5(A) and Fig. 5(B), respectively. It can be confirmed from (A) and (B) of FIG. 5 that when the primary liver cancer cell culture medium of Example 3 is used, the proliferation rate of primary liver cancer cells is better than that of FM culture medium.
  • Example 7 Xenotransplantation tumor formation experiment of primary liver cancer cells cultured in the primary liver cancer cell culture medium of the present invention in mice
  • the primary liver cancer cells were obtained from the liver cancer tissue samples of the liver cancer patient (patient 4) who had explained and obtained consent.
  • the primary liver cancer culture medium of Example 3 was used for culture in the same manner as in Example 3, and the number of liver cancer tumor cells was determined When it reaches 1 ⁇ 10 7 cells, the digestion method in Example 2 is used to digest, collect and count the liver cancer cells. Use the primary liver cancer cell culture medium of Example 3 to mix well, and draw 100 ⁇ L to remove 5 ⁇ 10 6 cells.
  • NCG 6-week-old female high immunodeficiency mice (purchased from Nanjing Model Animal Research Institute), and the tumor volume formed by liver cancer cells in the mice was observed every three days And growth rate, and record.
  • Fig. 6 is a record of the tumor volume of liver cancer cells cultured from primary liver cancer cells from patient 4 in mice.
  • liver cancer cells cultured from a patient's liver cancer sample can be used to detect the sensitivity of the patient's liver cancer cells to different drugs.
  • Plating primary liver cancer cells According to the method described in Example 1, primary liver cancer cells will be obtained from the surgical resection of liver cancer tissue samples from 4 liver cancer patients (patients 1 to 4) who have been explained and obtained consent.
  • the primary liver cancer culture medium of Example 3 was used for culture in the same manner as in Example 3.
  • the digestion method in Example 2 was used to digest, collect and count the liver cancer cells. Inoculate in a 384-well plate at a density of 3000-5000 cells/well to allow the cells to adhere overnight.
  • (1) Prepare the drug storage plate by the method of concentration gradient dilution: draw 10 ⁇ L of the drug mother solution to be tested (the concentration of the drug mother solution is prepared by 2 times the maximum blood drug concentration C max of the drug in the human body), and add it to the 20 ⁇ L In the 0.5 mL EP tube of DMSO, draw 10 ⁇ L from the above EP tube to the second 0.5 mL EP tube that already contains 20 ⁇ L of DMSO, that is, dilute the medicine according to 1:3. Repeat the above method, dilute in sequence, and finally obtain the 7 concentrations required for dosing. Add different concentrations of drugs to the 384-well drug storage plate. In the solvent control group, an equal volume of DMSO was added to each well as a control.
  • the drugs to be tested are sorafenib (manufactured by MCE), regorafenib (manufactured by MCE), 5-fluorouracil (manufactured by MCE), and doxorubicin (manufactured by MCE).
  • Cell viability detection 72 hours after administration, use Cell Titer-Glo detection reagent (manufactured by Promega) to detect the chemiluminescence value of the cells after the drug is added.
  • the value of the chemiluminescence value reflects the cell viability and the effect of the drug on the cell viability Influence, add the prepared Cell Titer-Glo detection solution to each well, and use the microplate reader to detect the chemiluminescence value after mixing.
  • Figure 7 (A) ⁇ (D) respectively show the susceptibility of liver cancer tumor cells cultured from four different liver cancer patients with surgically removed cancer tissue samples to two chemotherapeutic drugs, doxorubicin and 5-fluorouracil, Sensitivity to targeted drugs soratinib and regorafenib.
  • the results show that the cells of the same patient have different sensitivities to different drugs, and the cells of different patients have different sensitivities to the same drug.
  • liver cancer cells derived from cancer tissues of liver cancer patients cultured by the present invention to chemotherapy drugs and targeted drugs are consistent with the patient's clinical medication remission rate, such as chemotherapy drugs (doxorubicin) It exhibits extensive drug resistance, and sorafenib, the first-line clinical targeted drug for liver cancer, is only effective in some patients, suggesting that the liver cancer cells cultivated by the technology of the present invention have application potential in predicting the clinical efficacy of liver cancer patients.
  • chemotherapy drugs doxorubicin
  • the present invention it is possible to provide a primary liver cancer cell culture medium and a primary liver cancer cell culture method for quickly and stably culturing primary liver cancer cells.
  • the cell model obtained from the primary liver cancer cell culture medium of the present invention and the primary liver cancer cell culture method of the present invention can be used for drug curative effect evaluation and screening.

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Abstract

提供一种用于快速、稳定地培养原代肝癌细胞的原代肝癌细胞培养基和原代肝癌细胞培养方法。上述原代肝癌细胞培养基含有谷氨酰胺、非必需氨基酸、碱性成纤维细胞生长因子、肝细胞生长因子、IL-6、表皮生长因子、胰岛素、Y27632、N2添加剂、B27添加剂、Primocin、青霉素和链霉素、胎牛血清、以及任选的氢化可的松、任选的R-spondin1。由所述原代肝癌细胞培养基以及原代肝癌细胞培养方法得到的细胞模型，能够用于药物的疗效评估和筛选。

Description

原代肝癌细胞培养基、原代肝癌细胞培养方法及其应用 技术领域
本发明属于生物技术领域，具体而言，涉及用于在体外培养或扩增原代肝癌细胞的原代肝癌细胞培养基和原代肝癌细胞培养方法，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。
背景技术
原代培养(primary culture)也叫初代培养，指直接从体内取出的肿瘤细胞或组织进行的第一次培养。最常用的原代培养有组织块培养和分散细胞培养。组织块培养是将剪碎的组织块直接移植在培养瓶壁上，加入培养基后进行培养。分散细胞培养则是将组织块用机械法或化学法使细胞分散。如欲从临床活检或切除的肿瘤组织分离到活性最好的游离细胞，经典的方法是用蛋白水解酶(如胰蛋白酶和胶原酶)消化细胞间的结合物，或用金属离子螯合剂(如EDTA)除去细胞互相粘着所依赖的Ca 2+，再经机械轻度振荡，使之成为单细胞。
近几年来，肝癌术后辅助化疗作为一种新型辅助治疗方式已逐渐得到临床医生的重视与认可，包括术后TACE治疗，口服药物治疗等，但由于缺乏规范化疗方案，常规凭经验化疗，忽略个体差异，带有一定的盲目性，因此效果一直不佳，单药及联合用药的有效率低于20％(Jindal A,Thadi A,Shailubhai K.Hepatocellular Carcinoma:Etiology and Current and Future Drugs[J].J Clin Exp Hepatol,2019,9(2):221-232)。新兴的靶向药物虽然在一定程度上降低了毒副作用，但是数量太少，治疗费用昂贵，且有效率随个体差异，难以满足大多数患者的治疗需求。主要是由于缺乏有效的肝癌药敏试验体系，如同细菌药敏试验一般，将把肝癌体外药敏结果与临床体内反应相对应，无法做到精准化疗，因此研究对肝癌敏感药物的开发就是治疗关键。
高通量药敏实验技术(High-throuphput Drug Sensitivity)是一种新型药敏检测方法，具有检测药物覆盖率广，有效率高，个体化强等优点，已在白血病领域取得优异进展。对比其他类型药敏实验模型(PDO、PDX)同时也具有经济，快速检测，易于推广等优点。而目前这一技 术在肝癌领域所遇到的瓶颈主要是缺乏有效的、快速的、稳定的原代肝癌细胞培养扩增技术。
现已发现，使用滋养层细胞的条件性重编程培养法(conditional reprogramming culture，CRC)可以快速稳定建立原代肿瘤细胞系(Xuefeng Liu,Ewa Krawczyk,et al.Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens.Nature Protocols,VOl.12,NO.2,2017,439-451)。然而使用该文献中记载的条件培养基(F Meduim)经过体外筛选的稳定细胞株培养周期较长，不能满足临床快速检测的需求。因此，需要开发一种适用于原代肝癌细胞的快速稳定的培养基及培养方法，使得能够在两周以内进行高通量药物筛选，有效指导临床合理用药。
发明内容
本发明旨在针对现有技术的不足，提供一种用于在体外快速、稳定地培养原代肝癌细胞的原代肝癌细胞培养基和原代肝癌细胞培养方法。采用本发明的原代肝癌细胞培养基、原代肝癌细胞培养方法构建原代肝癌细胞模型时，能够获得具有肝癌患者自身生物学特性的原代肝癌细胞，并能够应用于新药筛选和体外药物敏感性检测。
本发明提供一种用于培养原代肝癌细胞的原代肝癌细胞培养基。本发明的原代肝癌细胞培养基含有初始培养基、谷氨酰胺(Glu)、非必需氨基酸(NEAA)、碱性成纤维细胞生长因子(bFGF)、肝细胞生长因子(HGF)、IL-6、表皮生长因子、胰岛素、Y27632、N2添加剂、B27添加剂、Primocin、青霉素和链霉素、胎牛血清、以及任选的氢化可的松、任选的R-spondin1，其中，初始培养基例如可以为DMEM/F12、DMEM、F12或RPMI-1640，优选为DMEM/F12。
其中，所述谷氨酰胺的含量为62.5ng/mL～1000ng/mL，优选为125ng/mL～1000ng/mL；所述非必需氨基酸为选自甘氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、脯氨酸和丝氨酸中的一种或多种，所述非必需氨基酸的总含量为0.25μM～4μM，优选为0.5μM～4μM；所述碱性成纤维细胞生长因子的含量为5ng/ml～80ng/ml，优选为20ng/ml～80ng/ml；所述肝细胞生长因子的含量为5ng/ml～80ng/ml，优 选为20ng/ml～80ng/ml；所述IL-6的含量为1.25ng/ml～20ng/ml，优选为5ng/ml～20ng/ml；所述表皮生长因子的含量为2.5ng/ml～40ng/ml，优选为10ng/ml～40ng/ml；所述胰岛素的含量为1.25ng/ml～20ng/ml，优选为2.5ng/ml～20ng/ml；所述Y27632的含量为5μM～40μM，优选为10μM～40μM；所述N2添加剂相对于培养基的体积比为1:25～1:400，优选为1:25～1:100；所述B27添加剂相对于培养基的体积比为1:12.5～1:200，优选为1:12.5～1:50；所述Primocin的含量为40μg/ml～80μg/ml；所述青霉素的含量为80U/ml～200U/ml；所述链霉素的含量为50μg/ml～150μg/ml；所述胎牛血清相对于培养基的体积比为1:5～1:20；所述氢化可的松的含量为0.4μg/ml～1.6μg/ml；所述R-spondin1的含量为62.5ng/mL～1000ng/mL，优选为250ng/mL～1000ng/mL。
本发明还提供一种原代肝癌细胞的培养方法。本发明的原代肝癌细胞培养方法中，使用本发明的原代肝癌培养基对原代肝癌细胞进行培养。
本发明的原代肝癌细胞培养方法中，以5×10 4～1.5×10 5个/cm 2的细胞密度加入经辐照的滋养层细胞。所述经辐照的滋养层细胞为小鼠的成纤维细胞，优选为NIH-3T3或者J2-3T3。所述经辐照的滋养层细胞经γ射线或X射线照射，优选经γ射线照射。辐照剂量为10～50Gy。
其中，上述原代肝癌细胞例如可以通过以下方法获得：通过肝癌切除方法获得；通过穿刺方法获得；从肝癌患者的腹水中获得；或者从肝癌患者的尿液中获得。
更具体而言，在通过肝癌切除方法或穿刺方法获得时，肝癌组织样本在实体瘤患者手术切除或穿刺后的半小时内进行收集。收集方法为：在无菌环境下，切取非坏死部位的肝癌组织样本lcm 3以上，将其置于盛有提前预冷的15mL本发明的原代肝癌细胞培养基中，置于通常的细胞培养条件下待用，或用冰袋尽快运输。
本发明的原代肝癌细胞的培养方法包括从上述组织样本分离获得原代肝癌细胞的步骤。
具体而言，在生物安全柜内，将组织样本转移至细胞培养皿内， 用含有青霉素/链霉素溶液(简称为双抗溶液)的培养基(例如为含有双抗溶液的DMEM/F12培养基，更优选还添加有选自谷氨酰胺、非必需氨基酸、碱性成纤维细胞生长因子、肝细胞生长因子、IL-6、氢化可的松、表皮生长因子、胰岛素、Y27632、N2添加剂、B27添加剂、R-spondin1和Primocin的一种或多种，上述添加组分的浓度可以采用本发明的原代肝癌细胞培养基中的浓度，以下将该培养基称为细胞分离用培养基)冲洗三次，将血细胞及脱落的坏死细胞清洗掉。将处理后的组织样本转移至新的培养皿内，滴加适量的上述细胞分离用培养基，用无菌手术刀片，手术剪和手术镊将肿瘤组织分割为直径小于lmm的小碎块。将切碎的肿瘤组织转移至离心管中进行离心，例如以1000～1800转/分钟离心3～10分钟。然后小心移除离心管内上清，再用组织消化液混匀重悬，置于恒温摇床上进行振荡(转速可以为230～350rpm/分钟)消化，时间为至少3小时(消化时间取决于样本大小；如果样本大于lg则消化时间增至4～6小时)；之后进行离心，例如以1000～1800转/分钟离心3～10分钟，弃去上清液，消化后的组织细胞适量的上述细胞分离用培养基重悬，过筛，细胞筛例如可以为100μM，之后再次离心，例如以1000～1800转/分钟离心3～10分钟，弃去上清液，观察是否有血细胞，例如有血细胞，加适量血细胞裂解液，混匀裂解，之后再次离心，例如以1000～1800转/分钟离心3～10分钟，弃去上清液，加入适量的本发明的原代肝癌细胞培养基重悬并收集细胞，用于培养瓶中贴壁扩大培养。
待细胞铺满75％以上瓶壁时，进行细胞常规传代培养或药敏实验。
本发明还提供一种评估用于治疗肝癌疾病的药物的疗效的方法，其包括以下步骤：
(1)根据本发明的原代肝癌细胞培养方法培养得到肝癌细胞；
(2)选定需要检测的药物，所述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，稀释成不同的药物浓度梯度；
(3)将步骤(1)培养得到的肝癌细胞消化成单细胞悬液，并用本发明的原代肝癌细胞培养基将单细胞悬液稀释，按2×10 4个/cm 2～4×10 4个/cm 2的接种密度将稀释后的细胞悬液加入多孔板内，进行过夜贴壁，对贴壁细胞添加梯度稀释后的所述药物；
(4)进行细胞活力检测。
所述细胞活力检测中，向每孔加入细胞活力检测试剂，震荡均匀后，用荧光酶标仪测量各孔的化学发光强度值，根据测得的数值绘制药物量-效曲线，计算各个药物对细胞的增殖的抑制强度。
本发明的有益效果包括：
(1)提高原代肝癌细胞培养的成功率，成功率达到90％以上；
(2)保证体外原代培养的肝癌细胞能够再现原代细胞来源病人的病理表型和异质性；
(3)所培养的原代肝癌细胞不受成纤维细胞、脂肪细胞等间质细胞的干扰，能得到纯化的肝癌细胞；
(4)扩增原代肝癌细胞效率高，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数量级的肝癌细胞，扩增出的肝癌细胞还可以连续传代；
(5)培养成本可控：原代肝癌培养基无需加入价格昂贵的Wnt激动剂、BMP抑制剂、FGF10等因子，是对已有条件重编程培养基的改进。
(6)所述培养基及培养体系培养获得的肝癌细胞数量多，均一化程度高，适合高通量筛选新候选化合物和为病人提供高通量药物体外敏感性功能测试。
采用本实施方式的原代肝癌细胞培养基，可培养来源于包括人的肝癌细胞，包括肝细胞癌细胞、肝内胆管癌细胞及混合型肝癌细胞。
另外，通过本实施方式的培养方法获得的细胞可应用于原代肝癌细胞的基础医学研究、药物应答的筛选、以及肝癌的新药研发等。
附图说明
图1为对从肝癌组织样本分离得到的原代肝癌细胞使用本发明的原代细胞培养基进行培养所获得的原代肝癌细胞的倒置相差显微镜下照片。
图2是对从肝癌组织样本分离得到的原代肝癌细胞使用本发明的原代细胞培养基进行培养所获得的肝癌细胞的免疫组化染色结果与该组织样本自身原始的组织切片免疫组化染色结果对比。
图3是表示原代肝癌细胞培养基中不同因子的不同浓度对于原代肝癌细胞增殖的影响的图。
图4是对从肝癌组织样本分离得到的原代肝癌细胞分别使用现有的条件培养基(FM)和本发明的原代肝癌细胞培养基(HCCM)进行培养得到的细胞的倒置相差显微镜下照片。
图5是将从肝癌组织样本分离得到的原代肝癌细胞分别使用现有的条件培养基(FM)和本发明的原代肝癌细胞培养基(HCCM)进行培养得到的细胞进行多次传代的统计效果图。
图6是对从肝癌组织样本分离得到的原代肝癌细胞使用本发明的原代细胞培养基进行培养所获得的肝癌细胞在小鼠体内的成瘤情况。
图7是表示采用本发明的原代肝癌细胞培养基培养所获得的肝癌细胞对不同化疗药物和靶向药物的剂量-效应曲线及经计算得到的半数抑制率。
具体实施方式
以下通过具体实施方式的描述并结合附图对本发明作进一步说明，这些实施例仅用于说明本发明，本发明的范围不限定于这些实施例。
实施例1原代肝癌细胞的获取以及滋养层细胞的处理
1.肝癌组织样本的收集
肝癌组织样本在进行过说明并获得同意的肝癌患者的手术切除或穿刺后的半小时内进行收集。在无菌环境下，切取非坏死部位的肝癌组织样本lcm 3以上，将其置于盛有提前在4℃冰箱中预冷的15mL DMEM/F12培养基中，做好标记。
2.肝癌组织样本的处理
(1)在生物安全柜中取组织置于100mm培养皿(Corning，430167)中，去除带血液的组织，使用在DMEM/F12培养基(生产厂家：Corning)中添加有1％链霉素-青霉素溶液(以下简称为双抗溶液，购自美国Corning公司，其中，母液中链霉素浓度为10mg/ml，青霉素浓度为10000U/ml)得到的培养基(以下称为细胞分离用培养基)冲洗3次，转移至新的100mm培养皿中，加入10ml细胞分离用培养基，用无菌 手术刀、手术剪和镊子进行机械分离，使组织分割为直径小于lmm的小碎块；
(2)将切碎的组织转移至50mL离心管中，加入5mL细胞分离用培养基，混匀，1400rpm离心5分钟；
(3)用移液器小心移除离心管内上清，加入细胞分离用培养基和2X消化酶(含胶原酶II(1mg/mL)(生成厂家Sigma)和胶原酶IV(1mg/mL)(生成厂家Sigma))按照体积比1:1的比例配制好的组织消化液混匀重悬，所加入消化酶的量大约是1g肿瘤组织使用10mL消化酶，对样品标记好样品名称及编号，封口膜密封，37℃、300rpm摇床消化，期间每1h观察消化是否完成；
(4)消化完成后，以1400rpm离心5分钟，弃去上清液，采用等体积的含10％胎牛血清的细胞分离用培养基进行中和，然后100μm滤网(生产厂家：Biosharp)过滤掉未消化的组织团块，滤网上的组织团块用细胞分离用培养基冲洗入离心管中(减少细胞损失)，1400rpm离心5分钟；
(5)弃上清，观察是否有血细胞，若有血细胞，加10mL血细胞裂解液(生产厂家：Sigma)，混匀，4℃裂解20分钟，期间颠倒混匀一次，之后1400rpm离心5分钟；
(6)弃上清，加入10mL细胞分离用培养基重悬并收集细胞，用于扩大培养。
3.培养滋养层细胞
(1)将NIH-3T3(购自ATCC)用添加有10％FBS和1％双抗溶液的DMEM培养基(生产厂家：Corning)在37℃，5％CO 2条件下进行培养；
(2)传代时，吸掉培养基，用PBS(磷酸盐缓冲液(1×)，0.0067M(PO 4))洗一遍，加0.25％胰酶(购自美国Corning公司)于37℃消化3分钟，待细胞变圆并部分悬浮时，用同体积的添加有10％FBS(购自美国Gibco公司)和1％双抗溶液的DMEM培养基中和，得到细胞悬液；
(3)将细胞悬液1000rpm离心5分钟，弃上清；
(4)用添加有10％FBS和1％双抗溶液的DMEM将细胞重悬， 按一传三的比例进行铺板，每三天传代一次。
4.对滋养层细胞进行辐照
(1)待NIH-3T3长至80％左右的密度时，按照上述3(2)的方法对细胞进行消化，重悬于完全DMEM培养基中；
(2)使用γ射线进行辐照，辐照剂量为25Gy；
(3)辐照完成后的滋养层细胞用于后续培养实验中。
实施例2原代肝癌细胞培养基的成分优化
使用在DMEM/F12培养基中添加有1％双抗溶液(购自Corning公司，其中，母液中链霉素浓度为10mg/ml，青霉素浓度为10000U/ml)、10％胎牛血清(购自Gibco公司)和0.1％Primocin溶液(购自Invivogen公司，其中，母液中Primocin浓度为50mg/ml)得到的培养基作为基础培养基。
在基础培养基中，分别根据下述表1的浓度添加细胞因子，得到实施例2的单一因子添加培养基。
按照实施例1中记载的方法，从4例进行过说明并获得同意的肝癌患者(患者2、患者3、患者7、患者8)的手术切除肝癌组织样本获得原代肝癌细胞。分别使用实施例2的单一因子添加培养基以及基础培养基，将上述获得的原代肝癌细胞按照活细胞密度1×10 4个/cm 2接种于12孔板中(每孔4.5万细胞数)，按照细胞密度5×10 4个/cm 2加入经γ射线辐照(辐照剂量25Gy)的NIH-3T3细胞，混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。
培养进行至第8天，取出12孔板，使用200微升0.25％胰蛋白酶(购自Gibco公司)润洗1分钟，吸去后再每孔加入500微升0.05％胰蛋白酶(购自Gibco公司)，置于37℃、5％CO 2培养箱中反应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终止消化，1500rpm离心4分钟后，弃上清，加入1毫升基础培养基重悬，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。
在与使用基础培养基时相比，使用加入该添加剂的培养基时4例原代肝癌细胞中的至少三例有促进增殖的作用时，记为“+”；在与使 用基础培养基时相比，使用加入该添加剂的培养基时4例原代肝癌细胞中的至少两例显示有抑制增殖的作用时，记为“－”。
将实验结果一并示于表1。
【表1】
Figure PCTCN2020081402-appb-000001
如表1所示，Glu(谷氨酰胺)、NEAA(非必需氨基酸)、bFGF(碱性成纤维细胞生长因子)、HGF(肝细胞生长因子)、IL-6、氢化可的松、EGF(表皮生长因子)、Insulin(胰岛素)、Y27632、N2添加剂、B27添加剂、R-spondin1均对肝癌原代细胞具有较强的增殖作用。
实施例3原代肝癌细胞培养及鉴定
根据下述表2的配方配制实施例3的原代肝癌细胞培养基(HCCM)。
【表2】
序号 培养基添加剂种类 供应商 终浓度
1 N2 Gibico 1:100
2 表皮生长因子 R&D 10ng/ml
3 肝细胞生长因子 Sino Biological 20ng/ml
4 碱性成纤维细胞生长因子 Sino Biological 20ng/ml
5 R-spondin1 Sino Biological 250ng/ml
6 谷氨酰胺 Invitrogen 250ng/ml
7 非必需氨基酸 Corning 1μM
8 胰岛素 Sigma-Aldrich 5ng/ml
9 Y27632 MCE 10μM
10 IL-6 R&D 5ng/ml
11 DMEM/F12培养基 Corning 90％(体积％)
12 胎牛血清 Gibico 10％(体积％)
13 氢化可的松 Sigma-Aldrich 400ng/ml
14 链霉素-青霉素 Corning 1％(体积％)
15 Primocin Invivogen 0.1％(体积％)
16 B27 Gibco 1:50
按照实施例1中记载的方法，从1例进行过说明并获得同意的肝癌患者(患者1)的手术切除肝癌组织样本获得原代肝癌细胞。使用实施例3的原代肝癌细胞培养基，将上述获得的原代肝癌细胞按照活细胞密度1×10 4个/cm 2接种于12孔板中(每孔4.5万细胞数)，按照细胞密度5×10 4个/cm 2加入经γ射线辐照(辐照剂量25Gy)的NIH-3T3细胞，混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。
分别在培养第三天和第七天进行镜下观察。
图1的(A)为培养至第三天的镜下照片(Invitrogen公司EVOS M500，10倍物镜)，图1的(B)为培养至第七天的镜下照片(Invitrogen公司EVOS M500，10倍物镜)。图1的(A)和(B)的圆圈所圈出的是形成的细胞克隆，如图1的(A)所示，所培养的原代肝癌细胞能够在3天时间内形成典型的细胞克隆，如图1的(B)所示，细胞克隆得到了快速增殖。
在培养第28天，对培养获得的肝癌细胞进行免疫组化分析。免疫组化的方法如下所述。
将所培养的原代肝癌细胞消化离心，用PBS(购自上海生工)洗1～ 2次，之后固定于95％乙醇(购自上海生工)中，将固定好的细胞离心，去除上清后，将细胞沉淀用包埋纸(购自上海生工)包裹好，再次浸泡于95％乙醇中，用于制作细胞蜡块。样品进行细胞蜡块的制作，之后进行肝癌免疫组化标记(Ki67、CK7均购自Cell Signaling Technology)分析。
在通风橱中将石蜡切片依次置于二甲苯1(购自上海生工)10分钟、二甲苯2(购自上海生工)10分钟、100％乙醇1(购自上海生工)5分钟、100％乙醇2(购自上海生工)5分钟、95％乙醇5分钟、90％乙醇5分钟、80％乙醇5分钟、70％乙醇5分钟，之后，使用PBS洗3次，5分钟/次后置于透明液(PBS+0.3％H 2O 2+0.3％Triton X-100)(购自上海生工)中，摇床100rpm、室温避光孵育30分钟，PBS洗3次，5分钟/次。然后使用免疫组化笔勾圈出染色组织，滴入5％羊血清或驴血清(0.3％Triton X-100的PBS按照1:50稀释)，37℃水浴锅孵育30分钟(II抗若为羊抗则选羊血清封闭，若为驴抗，则选驴血清封闭)。去血清，使用0.3％triton X-100的PBS(购自上海生工)按照1:50稀释I抗，滴入切片，37℃水浴锅孵育1h，移入4℃冰箱孵育过夜。取出孵育I抗的组织切片，平衡至室温，PBS洗3次，5分钟/次。0.3％triton X-100的PBS按照1:50稀释I抗(生物素)，滴入组织切片，37℃水浴锅孵育1h，PBS洗3次，5分钟/次。滴入苏木素(购自上海生工)，室温染色5～10分钟，ddH 2O洗3次。加入0.5％盐酸乙醇(70％乙醇配制)，反应30s，0.5％氨水蓝化30s，ddH 2O洗3次，置于显微镜(Invitrogen公司EVOS M500，10倍物镜)拍照。
结果如图2所示。图2的(A)是使用本实施例的原代肝癌细胞培养基培养所得的细胞的免疫组化结果，图2的(B)是同一病人的肝癌组织的免疫组化结果。图2的(A)和(B)均显示为CK7(-)、Ki67(+)，也就是说，采用本实施例的原代肝癌细胞培养基培养所得的肝癌细胞能够保持该肝癌组织的病理特性。
实施例4原代肝癌细胞初次培养周期及细胞数统计
与实施例3同样地，使用实施例3中配制的培养基，从9例进行过说明并获得同意的肝癌患者(1～9)的手术切除肝癌组织样本获得 原代肝癌细胞，使用实施例3的原代肝癌细胞培养基与实施例3同样地进行培养。待细胞扩增至85％时进行消化并计数，同时记录直至消化时的培养天数，将其作为一个培养周期，结果如下述表3所示。
【表3】
样本信息 培养天数 种入细胞数(*10 4) 收集细胞数(*10 4)
患者1 18 6 100
患者2 13 10 180
患者3 13 10 160
患者4 13 10 164
患者5 10 10 440
患者6 14 10 264
患者7 22 8 280
患者8 12 10 178
患者9 11 10 276
平均值 12.6 8.4 204
如表3所示，使用本发明的原代肝癌细胞培养基培养得到的肝癌细胞平均在12.6天内实现10 4数量级到10 6数量级的增殖。
实施例5所添加的因子的浓度对肝癌细胞增殖的影响
按照实施例1中记载的方法，从4例进行过说明并获得同意的肝癌患者(患者4、患者8、患者9、患者10)的手术切除肝癌组织样本获得原代肝癌细胞，并使用实施例3中的原代肝癌细胞培养基培养获得肝癌细胞，将所获得的肝癌细胞按照活细胞密度1×10 4个/cm 2接种于12孔板中(每孔4.5万细胞数)，按照细胞密度5×10 4个/cm 2加入经γ射线辐照(辐照剂量25Gy)的NIH-3T3细胞，混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。待细胞扩增至85％，取出12孔板，使用200微升0.25％胰蛋白酶(购自Gibco公司)润洗1分钟，吸去后再每孔加入500微升0.05％胰蛋白酶(购自Gibco公司)，置于37℃、5％CO 2培养箱中反应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终止消化，1500rpm 离心4分钟后，弃上清，加入1毫升基础培养基重悬，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。所得细胞用于以下培养实验。
接着，配制以下12种配方培养基进行实验：
配方1：实施例3中的原代肝癌细胞培养基组分中不含N2添加剂；
配方2：实施例3中的原代肝癌细胞培养基组分中不含非必需氨基酸；
配方3：实施例3中的原代肝癌细胞培养基组分中不含谷氨酰胺；
配方4：实施例3中的原代肝癌细胞培养基组分中不含胰岛素；
配方5：实施例3中的原代肝癌细胞培养基组分中不含B27添加剂；
配方6：实施例3中的原代肝癌细胞培养基组分中不含HGF；
配方7：实施例3中的原代肝癌细胞培养基组分中不含Y27632；
配方8：实施例3中的原代肝癌细胞培养基组分中不含氢化可的松；
配方9：实施例3中的原代肝癌细胞培养基组分中不含EGF；
配方10：实施例3中的原代肝癌细胞培养基组分中不含bFGF；
配方11：实施例3中的原代肝癌细胞培养基组分中不含R-spondin1；
配方12：实施例3中的原代肝癌细胞培养基组分中不含IL-6；
分别使用上述配方1～12和实施例3中的原代肝癌细胞培养基来稀释上述消化后的细胞悬液，按照每孔2万细胞，500微升体积种入24孔板中。
在使用配方1的培养基时，按照终浓度分别为1:400、1:200、1:100、1:50、1:25配制5个浓度梯度的N2添加剂，在接种有原代细胞的24孔板中分别添加配制好的N2添加剂每孔500微升；并使用配方1的培养基设置对照孔(BC)。
在使用配方2的培养基时，按照终浓度分别为4μM、2μM、1μM、0.5μM、0.25μM配制5个浓度梯度的非必需氨基酸，在接种有原代细胞的24孔板中分别添加配制好的非必需氨基酸每孔500微升；并使用配方2的培养基设置对照孔(BC)。
在使用配方3的培养基时，按照终浓度分别为1000ng/mL、500ng/mL、250ng/mL、125ng/mL、62.5ng/mL配制5个浓度梯度的谷氨酰胺，在接种有原代细胞的24孔板中分别添加配制好的谷氨酰胺每孔500微升；并使用配方3的培养基设置对照孔(BC)。
在使用配方4的培养基时，按照终浓度分别为20ng/mL、10ng/mL、5ng/mL、2.5ng/mL、1.25ng/mL配制5个浓度梯度的胰岛素，在接种有原代细胞的24孔板中分别添加配制好的胰岛素每孔500微升；并使用配方4的培养基设置对照孔(BC)。
在使用配方5的培养基时，按照终浓度分别为1:200、1:100、1:50、1:25、1:12.5配制5个浓度梯度的B27添加剂，在接种有原代细胞的24孔板中分别添加配制好的B27添加剂每孔500微升；并使用配方5的培养基设置对照孔(BC)。
在使用配方6的培养基时，按照终浓度分别为80ng/mL、40ng/mL、20ng/mL、10ng/mL、5ng/mL配制5个浓度梯度的HGF，在接种有原代细胞的24孔板中分别添加配制好的HGF每孔500微升；并使用配方6的培养基设置对照孔(BC)。
在使用配方7的培养基时，按照终浓度分别为40μM、20μM、10μM、5μM、2.5μM配制5个浓度梯度的Y27632，在接种有原代细胞的24孔板中分别添加配制好的Y27632每孔500微升；并使用配方7的培养基设置对照孔(BC)。
在使用配方8的培养基时，按照终浓度分别为1.6μg/mL、0.8μg/mL、0.4μg/mL、0.2μg/mL、0.1μg/mL配制5个浓度梯度的氢化可的松，在接种有原代细胞的24孔板中分别添加配制好的氢化可的松每孔500微升；并使用配方8的培养基设置对照孔(BC)。
在使用配方9的培养基时，按照终浓度分别为40ng/mL、20ng/mL、10ng/mL、5ng/mL、2.5ng/mL配制5个浓度梯度的EGF，在接种有原代细胞的24孔板中分别添加配制好的EGF每孔500微升；并使用配方9的培养基设置对照孔(BC)。
在使用配方10的培养基时，按照终浓度分别为80ng/mL、40ng/mL、20ng/mL、10ng/mL、5ng/mL配制5个浓度梯度的bFGF，在接种有原代细胞的24孔板中分别添加配制好的bFGF每孔500微升；并使用配 方10的培养基设置对照孔(BC)。
在使用配方11的培养基时，按照终浓度分别为1000ng/mL、500ng/mL、250ng/mL、125ng/mL、62.5ng/mL配制5个浓度梯度的R-spondin1，在接种有原代细胞的24孔板中分别添加配制好的R-spondin1每孔500微升；并使用配方11的培养基设置对照孔(BC)。
在使用配方12的培养基时，按照终浓度分别为20ng/mL、10ng/mL、5ng/mL、2.5ng/mL、1.25ng/mL配制5个浓度梯度的IL-6，在接种有原代细胞的24孔板中分别添加配制好的IL-6每孔500微升；并使用配方12的培养基设置对照孔(BC)。
同时上述各孔加入1万个辐照后的NIH-3T3细胞作为滋养层细胞。
待细胞扩增至24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3的(A)～(L)。图3的(A)～(L)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。
如图3的(A)～(L)所示，N2添加剂的浓度范围以体积比计为1:400～1:25，以1:100加入时细胞增殖效果最明显；非必需氨基酸的浓度范围为0.25～4μM，浓度为1μM时细胞增殖效果最明显；谷氨酰胺的浓度范围为62.5～1000ng/mL，浓度为250ng/mL时细胞增殖效果最明显；胰岛素的浓度范围为1.25～20ng/mL，浓度为5～20ng/mL时细胞增殖效果最明显；B27添加剂的浓度范围以体积比计为1:12.5～1:200，以1:12.5～1:50加入时细胞增殖效果最明显；HGF浓度范围为5～80ng/mL，浓度为20～80ng/mL时细胞增殖效果最明显；Y27632浓度范围为5～40μM，浓度为10μM时细胞增殖效果最明显；氢化可的松浓度范围为0.4～1.6μg/mL，浓度为0.4μg/mL时细胞增殖效果最明显；EGF浓度范围为2.5～40ng/mL，浓度为10ng/mL时细胞增殖效果最明显；bFGF浓度范围为5～80ng/mL，浓度为20～80ng/mL时细胞增殖效果最明显；R-spondin1浓度范围为62.5～1000ng/mL，浓度为250ng/mL时细胞增殖效果最明显；IL-6浓度范围为1.25～20 ng/mL，浓度为5～20ng/mL时细胞增殖效果最明显。
实施例6与现有培养基的对比
根据下述表4的配方配制FM培养基。
【表4】
培养基成分 供应商 终浓度
DMEM培养基 Corning 65％(体积％)
胎牛血清 Gibico 10％(体积％)
Ham’s F12营养液 Gibico 25％
氢化可的松 Sigma-Aldrich 25ng/ml
表皮生长因子 R&D 0.125ng/ml
胰岛素 Sigma-Aldrich 5μg/ml
两性霉素B Sigma-Aldrich 250ng/ml
庆大霉素 Gibico 10μg/ml
霍乱毒素 Sigma-Aldrich 0.1nM
Y27632 Enzo 10μM
分别使用FM培养基和实施例3中的原代肝癌细胞培养基(图中记为HCCM)，对从进行过说明并获得同意的肝癌患者(患者3、患者4)的手术切除肝癌组织样本的原代肝癌细胞，与实施例3同样地进行原代肝癌细胞培养。
在培养第3天，使用倒置显微镜(Invitrogen公司EVOS M500，10倍)进行观察，在图4中表示来自患者3的原代肝癌细胞培养结果。图4的(A)是使用FM培养基培养得到的原代肝癌细胞，图4的(B)是使用实施例3的原代肝癌细胞培养基培养得到的原代肝癌细胞，如图4所示，使用实施例3的原代肝癌细胞培养基时，在第3天已经形成了明显的细胞克隆，效果优于FM培养基。
持续进行培养，以培养天数为横坐标，细胞群体倍增数为纵坐标，采用Graphpad Prism7.0软件绘制使用两种不同培养基进行培养的两例原代肝癌细胞的生长曲线，将来自患者3和患者4的原代肝癌细胞培养结果分别示于图5的(A)和图5的(B)。由图5的(A)和(B)可以确认，在使用实施例3的原代肝癌细胞培养基时，原代肝癌细胞的增殖速度优于FM培养基。
实施例7使用本发明的原代肝癌细胞培养基培养得到的原代肝癌细胞在小鼠体内的异种移植成瘤实验
从进行过说明并获得同意的肝癌患者(患者4)的手术切除肝癌组织样本获得原代肝癌细胞，使用实施例3的原代肝癌培养基与实施例3同样地进行培养，待肝癌肿瘤细胞数量达到1×10 7个时，采用实施例2中的消化方法对肝癌细胞进行消化，并收集、计数，采用实施例3的原代肝癌细胞培养基混匀，并吸取100μL将5×10 6个肝癌肿瘤细胞重悬，分别注射入6周大的雌性高度免疫缺陷(NCG)小鼠(购自南京模式动物研究所)的皮下部位，每三天观察一次肝癌细胞在小鼠体内形成肿瘤的体积和生长速率，并记录。
图6是从来自患者4的原代肝癌细胞培养得到的肝癌细胞在小鼠体内的肿瘤体积的记录。
如图6所示，在肿瘤细胞接种后的第13天即可观察到小鼠的肿瘤细胞接种部位均有瘤体形成，自第13天起至第22天，小鼠体内肿瘤增殖明显，这说明采用实施例3的原代肝癌细胞培养基及培养方法所培养的肝癌组织来源的肝癌肿瘤细胞在小鼠体内具有成瘤性。
实施例8肝癌组织来源的肝癌细胞药物敏感性测试
下面以肝癌患者手术切除样本为例，说明由病人来源的肝癌样本培养得到的肝癌细胞可以用于检测病人肝癌细胞对不同药物的敏感性。
1、原代肝癌细胞的铺板：按照实施例1中记载的方法，将从4例进行过说明并获得同意的肝癌患者(患者1～患者4)的手术切除肝癌组织样本获得原代肝癌细胞，使用实施例3的原代肝癌培养基与实施例3同样地进行培养，待肝癌细胞数量达到1×10 7个时，采用实施例2中的消化方法对肝癌细胞进行消化，并收集、计数，按3000～5000个/孔密度接种于384孔板中，使细胞贴壁过夜。
2、药物梯度实验：
(1)采用浓度梯度稀释的方法配制药物贮存板：分别吸取10μL的待测药物母液(药物母液浓度按2倍于该药物在人体中的最大血药浓度C max配制)，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，待测药物为索拉非尼(MCE公司制)、瑞戈非尼(MCE公司制)、5-氟尿嘧啶(MCE公司制)和多柔比星(MCE公司制)。
(2)使用高通量自动化工作站(购自Perkin Elmer公司)将384孔药物贮存板内的不同浓度药物和溶剂对照加入到上述步骤1中获得的铺有肝癌细胞的384孔细胞培养板中，药物组和溶剂对照组都各设3个复孔。每孔加入药物体积为100nL。
(3)细胞活性检测：给药72小时后，用Cell Titer-Glo检测试剂(Promega公司制)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，每孔加入配制好的Cell Titer-Glo检测液，混匀后使用酶标仪检测化学发光数值。
使用Graphpad Prism 7.0软件作图并计算半数抑制率IC50。
(4)药物敏感性测试结果如图7所示。
图7的(A)～(D)分别表示从四例不同的肝癌患者的手术切除癌组织样本所培养获得的肝癌肿瘤细胞对两个化疗药物多柔比星和5-氟尿嘧啶的药物敏感性、对靶向药物索拉替尼、瑞戈非尼的敏感性。结果显示，同一病人的细胞对不同药物具有不同的敏感性，不同病人的细胞对同一药物的敏感性也不同。
根据图7可以确认，本发明技术所培养的肝癌病人癌组织来源的肝癌细胞对化疗药物和靶向药物的敏感性测试结果与病人的临床用药缓解率相符，如化疗药物(多柔比星)表现出广泛的耐药性，而肝癌临床一线靶向药物索拉非尼也仅在部分患者中具有有效性，提示本发明技术所培养的肝癌细胞在预测肝癌患者临床用药疗效方面具有应用潜力。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业上的可利用性
根据本发明，能够提供一种用于快速、稳定地培养原代肝癌细胞的原代肝癌细胞培养基和原代肝癌细胞培养方法。由本发明的原代肝癌细胞培养基以及本发明的原代肝癌细胞培养方法得到的细胞模型，能够用于药物的疗效评估和筛选。

Claims (9)

  1. 一种用于培养原代肝癌细胞的原代肝癌细胞培养基，其特征在于：
    在初始培养基中含有谷氨酰胺、非必需氨基酸、碱性成纤维细胞生长因子、肝细胞生长因子、IL-6、表皮生长因子、胰岛素、Y27632、N2添加剂、B27添加剂、Primocin、青霉素和链霉素、胎牛血清、以及任选的氢化可的松、任选的R-spondin1，
    所述初始培养基选自DMEM/F12、DMEM、F12或RPMI-1640。
  2. 如权利要求1的原代肝癌细胞培养基，其特征在于：
    所述谷氨酰胺的含量为62.5ng/mL～1000ng/mL；
    所述非必需氨基酸为选自甘氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、脯氨酸和丝氨酸中的一种或多种，所述非必需氨基酸的总含量为0.25μM～4μM；
    所述碱性成纤维细胞生长因子的含量为5ng/ml～80ng/ml；
    所述肝细胞生长因子的含量为5ng/ml～80ng/ml；
    所述IL-6的含量为1.25ng/ml～20ng/ml；
    所述表皮生长因子的含量为2.5ng/ml～40ng/ml；
    所述胰岛素的含量为1.25ng/ml～20ng/ml；
    所述Y27632的含量为5μM～40μM；
    所述N2添加剂相对于培养基的体积比为1:25～1:400；
    所述B27添加剂相对于培养基的体积比为1:12.5～1:200；
    所述Primocin的含量为40μg/ml～80μg/ml；
    所述青霉素的含量为80U/ml～200U/ml；
    所述链霉素的含量为50μg/ml～150μg/ml；
    所述胎牛血清相对于培养基的体积比为1:5～1:20；
    若含有所述氢化可的松，其含量为0.4μg/ml～1.6μg/ml；
    若含有所述R-spondin1，其含量为62.5ng/mL～1000ng/mL。
  3. 一种原代肝癌细胞的培养方法，其特征在于，包括以下步骤：
    使用权利要求1或2所述的原代肝癌细胞培养基对原代肝癌细胞进行培养。
  4. 如权利要求3所述的培养方法，其特征在于：
    在培养中，以5×10 4～1.5×10 5个/cm 2的细胞密度加入经辐照的滋养层细胞。
  5. 如权利要求4所述的培养方法，其特征在于：
    所述经辐照的滋养层细胞为小鼠的成纤维细胞，优选为NIH-3T3或者J2-3T3。
  6. 如权利要求4所述的培养方法，其特征在于：
    所述经辐照的滋养层细胞经γ射线或X射线照射，优选经γ射线照射。
  7. 如权利要求6所述的培养方法，其特征在于：
    辐照剂量为10～50Gy。
  8. 如权利要求3～7中任一项所述的培养方法，其特征在于：
    在原代肝癌细胞生长至占培养器皿底面积75％以上时进行消化传代。
  9. 一种评估用于治疗肝癌疾病的药物的疗效的方法，其特征在于，包括以下步骤：
    (1)根据权利要求3～8中任一项所述的培养方法培养得到肝癌细胞；
    (2)选定需要检测的药物，所述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，稀释成不同的药物浓度梯度；
    (3)将步骤(1)培养得到的肝癌细胞消化成单细胞悬液，并加入多孔板内进行贴壁，对所述贴壁细胞添加梯度稀释后的所述药物；
    (4)进行细胞活力检测。
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