Culture medium, culture method and their use for primary gastrointestinal stromal tumor cells

Abstract

The present invention provides a cell culture medium for culturing primary gastrointestinal stromal tumor cells, comprising gastrin, N2, insulin, a receptor tyrosine kinase ligand, and a Rock kinase inhibitor.The present invention further provides a method for culturing gastrointestinal stromal tumor cells by using the cell culture medium, and the use of the expanded cell population obtained by using the method in efficacy evaluation or screening.

Description

[Technical field]

[0001] The present invention relates to the technical field of medicine, in particular to a culture medium and method for culturing or growing gastrointestinal stromal tumor cells in vitro, as well as to a method and use of the cultured cells in evaluating and screening the efficacy of drugs. [Background technology]

[0002] Gastrointestinal stromal tumor (GIST) is a type of tumor originating from mesenchymal tissue in the gastrointestinal tract, accounting for the majority of gastrointestinal mesenchymal tumors, mainly occurring in the stomach (50%-70%). Due to its insensitivity to conventional radiotherapy, surgical resection is an effective method to treat GIST. However, for patients who miss the opportunity for surgery due to tumor progression, metastasis, or postoperative recurrence, the median survival time of GIST is only 6-18 months, and the 5-year survival rate is less than 10%. Over the past decade, the widespread application of molecular biology techniques in the clinical diagnosis and treatment of GIST, especially the emergence and popularization of molecular targeted drugs represented by imatinib, has greatly improved the prognosis of GIST patients. At present, the remarkable curative effect of imatinib in the treatment of advanced GIST and postoperative adjuvant therapy of GIST has been widely recognized, but at the same time, researchers have also raised concerns about the first- and second-line drug resistance of imatinib. Studies have shown that 40%-50% of patients develop drug resistance within 2 years after receiving imatinib treatment, and patients with imatinib resistance only have a median progression-free survival of 6.8 months when given sunitinib as a second-line treatment, while the same patients only have a median progression-free survival of 4.8 months when given regorafenib as a third-line treatment. Therefore, it is urgent to conduct further research on the pathogenesis of gastrointestinal stromal tumors in clinical practice and develop new drugs to treat gastrointestinal stromal tumors.

[0003] However, currently there is a lack of suitable research models for gastrointestinal stromal tumors. This is mainly due to the differences between gastrointestinal stromal tumors and other common tumors. Common tumors such as lung cancer and breast cancer are of epithelial origin. Currently, methods for culturing epithelial-derived tumor cells in vitro have been developed relatively rapidly, but few methods for culturing non-epithelial tissue-derived tumor cells in vitro have been reported (Non-Patent Document 1). In addition, the number of established models of gastrointestinal stromal tumor cell lines is also very limited, with only a handful of gastrointestinal stromal tumor cell lines being commercially available from international commercial cell companies, which has caused many obstacles to basic research and new drug development for gastrointestinal stromal tumors.

[0004] The technique of developing in vitro culture of primary gastrointestinal stromal tumor cells using fresh tumor samples from patients with gastrointestinal stromal tumors is a good way to develop a research model for gastrointestinal stromal tumors. However, currently, no effective method has been recognized for culturing primary gastrointestinal stromal tumor cells from patients in vitro. Most of the reported methods add serum to the culture medium (Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5), and generally suffer from low success rate of culture and interference from non-gastrointestinal stromal tumor cells such as fibroblasts.

[0005] Therefore, to meet the demands of basic research and new drug development in the field of gastrointestinal stromal tumors, it is urgent to develop an effective method for culturing primary patient-derived gastrointestinal stromal tumor cells in vitro. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Xu et al., Journal of Hematology & Oncology, 11: 116, 2018 [Non-Patent Document 2] Liu et al., Am J Pathol, 183(6): 1862-1870, 2013 [Non-Patent Document 3] Liu et al., Ther Adv Med Oncol, 11: 1-15, 2019 [Non-Patent Document 4] Fukuda, Oncology Reports, 30: 71-78, 2013 [Non-Patent Document 5] Zhu et al., Journal of Central South University, 35(11): 1138-1144, 2010 Summary of the Invention

[0007] The present invention aims to provide a cell culture medium for culturing primary gastrointestinal stromal tumor cells, and a culture method for culturing primary gastrointestinal stromal tumor cells using the medium. The cell culture medium and culture method of the present invention can achieve the goals of high success rate, simple operation, and no interference from non-gastrointestinal stromal tumor cells such as fibroblasts. By applying the cell culture medium and culture method of the present invention to construct a primary gastrointestinal stromal tumor cell model, gastrointestinal stromal tumor cells with the biological characteristics of the gastrointestinal stromal tumor patient themselves can be obtained, which can be used for efficacy evaluation or screening of drugs.

[0008] One aspect of the present invention is to provide a cell culture medium for culturing primary gastrointestinal stromal tumor cells, the cell culture medium comprising gastrin, N2, insulin, a receptor tyrosine kinase ligand, and a Rock kinase inhibitor, the gastrin content being preferably 0.3 nM to 10 nM, more preferably 0.3 nM to 3 nM, and even more preferably 0.3 nM to 1 nM.

[0009] The volume concentration of N2 in the cell culture medium is in the range of 1:25 to 1:200, and preferably, the volume concentration of N2 is in the range of 1:50 to 1:100.

[0010] The insulin content is 2μg / ml to 20μg / ml, preferably 5μg / ml to 10μg / ml. For example, a commercially available insulin solution (10mg / mL, manufactured by Sigma) is added to the culture medium at a dilution ratio of 1:500 to 1:5000, preferably at a dilution ratio of 1:1000 to 1:2000, or alternatively, a commercially available insulin-containing culture additive, insulin-transferrin-selenium-ethanolamine (containing 1mg / mL insulin, manufactured by Gibco), is added to the culture medium at a dilution ratio of 1:50 to 1:500, preferably at a dilution ratio of 1:100 to 1:200.

[0011] The receptor tyrosine kinase ligand is at least one selected from the group consisting of platelet-derived growth factor AA, platelet-derived growth factor BB, platelet-derived growth factor CC, stem cell factor, insulin growth factor 1, basic fibroblast growth factor, and fibroblast growth factor 10, and the receptor tyrosine kinase ligand is preferably one or more selected from the group consisting of platelet-derived growth factor AA, stem cell factor, and basic fibroblast growth factor. When one or more receptor tyrosine kinase ligands are contained, the content of each receptor tyrosine kinase ligand is 5 ng / ml to 500 ng / ml, preferably 20 ng / ml to 100 ng / ml.

[0012] The Rock kinase inhibitor is at least one selected from the group consisting of Y27632, fasudil, and H-1152. A preferred Rock inhibitor is Y27632. The content of the Rock inhibitor is 2 μM to 50 μM, preferably 5 μM to 10 μM.

[0013] In addition, the cell culture medium of the present invention does not contain serum components required in reported culture media for primary gastrointestinal stromal tumor cells (Non-Patent Document 4, Liu et al., Nat Protoc. 2017, 12(2): 439-451), thereby avoiding interference from excessive proliferation of fibroblasts.

[0014] A second aspect of the present invention is a method for culturing primary gastrointestinal stromal tumor cells, comprising the steps of: (1) preparing the cell culture medium of the present invention; (2) coating the culture vessel with collagen; (3) inoculating primary gastrointestinal stromal tumor cells in the coated culture vessel, culturing the cells under an oxygen concentration of 0.1% to 25% by using the cell culture medium of the present invention, and digesting and subculturing the cells when the primary gastrointestinal stromal tumor cells grow to a cell density occupying about 80% to 90% of the bottom area of ​​the culture vessel; The present invention provides a culture method comprising the steps of:

[0015] Here, the collagen used in the culture method is rat tail type I collagen, and for example, commercially available collagen I (manufactured by Corning) can be used. More specifically, collagen is diluted with ultrapure water at a dilution ratio of 1:5 to 1:100, preferably 1:10 to 1:50.

[0016] The coating method involves adding diluted collagen to a culture vessel to completely cover the bottom of the vessel and leaving it for 30 minutes or more.

[0017] Primary gastrointestinal stromal tumor cells can be derived from gastrointestinal stromal tumor tissue from a patient.

[0018] For example, collection of the above-mentioned tissue samples is performed within 30 minutes after surgical resection or biopsy from the patient. More specifically, in a sterile environment, tissue samples from non-necrotic sites are collected in 0.5 cm 3 After cutting out the above volume, place it in pre-cooled 10 mL to 50 mL of DMEM / F12 medium, place it in a plastic sterile centrifuge tube with a lid, and transport it to the laboratory on ice. Here, the DMEM / F12 medium contains 50 U / mL to 200 U / mL (e.g., 100 U / mL) of penicillin and 50 μg / mL to 200 μg / mL (e.g., 100 μg / mL) of streptomycin (hereinafter referred to as "transport solution").

[0019] In a biosafety cabinet, the tissue sample is transferred to a cell culture dish, which is then rinsed with transport solution to wash away blood cells on the surface of the tissue sample and to remove unwanted tissues such as skin and fascia on the surface of the tissue sample.

[0020] Transfer the rinsed tissue sample to another new culture dish, add 5 mL to 25 mL of transport solution, and use a sterile scalpel blade and forceps to cut the tissue sample into pieces 1 mm in diameter. 3 Divide into smaller tissue pieces.

[0021] The tissue sample pieces are transferred to a centrifuge tube and centrifuged in a tabletop centrifuge at 1000 rpm or more for 3 to 10 minutes. The supernatant is carefully removed from the centrifuge tube with a pipette, and the resulting material is resuspended in 5 mL to 25 mL of serum-free DMEM / F12 medium containing collagenase II (0.5 mg / mL to 5 mg / mL, for example, 1 mg / mL) and collagenase IV (0.5 mg / mL to 5 mg / mL, for example, 1 mg / mL), and shaken at a constant temperature of 37°C. The digestion is carried out in a car with shaking for at least 30 minutes (digestion time depends on the sample size; if the sample is larger than 1 g, the digestion time is increased to 1.5 to 2 hours), then centrifuged in a tabletop centrifuge at 300 g / min or more for 3 to 10 minutes, and the supernatant is discarded. The digested tissue cells are then resuspended in, for example, 5 mL to 25 mL of DMEM / F12 medium containing 10% fetal bovine serum, then disrupted and sieved, for example, through a cell sieve with 100 μm openings, and the sieved cell suspension is collected in a centrifuge tube and the cells are counted with a hemocytometer.

[0022] The cell suspension is then centrifuged in a centrifuge at 300 g / min or more for 3 to 10 minutes, the supernatant is discarded, and the resultant is resuspended in the cell culture medium of the present invention and then cultured at 1 × 10 3 Cells / mL ~ 1 x 10 5 Inoculate and culture at a density of 10 cells / mL.

[0023] In the above culture method, in the step of culturing primary gastrointestinal stromal tumor cells, the cells are preferably cultured under an oxygen concentration of 0.1% to 4%.

[0024] A third aspect of the present invention provides a method for evaluating the efficacy of gastrointestinal stromal tumor drugs or a method for screening gastrointestinal stromal tumor drugs, comprising obtaining expanded cell progeny of gastrointestinal stromal tumor cells using the culture medium and culture method of the present invention, and applying the same in methods and uses for drug efficacy evaluation and screening, in particular in methods and uses for in vitro efficacy evaluation and screening of anti-tumor drugs.

[0025] Preferably, the method for evaluating the efficacy of a gastrointestinal stromal tumor drug or the method for screening a gastrointestinal stromal tumor drug comprises the following steps: (1) obtaining primary gastrointestinal stromal tumor cells and culturing the cells using the culture medium and culture method of the present invention to obtain gastrointestinal stromal tumor cells; (2) preparing the drug to be tested in the required concentration gradient; (3) adding the drug at various concentrations prepared in step (2) to the gastrointestinal stromal tumor cells cultured in step (1); (4) detecting cell viability; Includes.

[0026] The beneficial effects of the present invention include the following: (1) Gastrointestinal stromal tumor cells cultured in vitro using the culture medium and culture method of the present invention can maintain the pathological phenotype and heterogeneity of the patient from which the cells were derived, and can be applied in the field of regenerative medicine. (2) The cultured gastrointestinal stromal tumor cells are not interfered with by cells such as fibroblasts, and purified gastrointestinal stromal tumor cells and their progeny can be obtained. (3) The composition of the culture medium does not contain uncertain components such as serum, and is therefore not affected by the quality and quantity of serum from different batches. (4) This technology can provide gastrointestinal stromal tumor cells with high uniformity, which has a manageable cost and is suitable for the fields of drug efficacy evaluation and screening, including high-throughput screening of new candidate compounds and in vitro high-throughput drug sensitivity functional tests for patients, as well as toxicity testing.

[0027] The cell culture medium of this embodiment can be used to culture gastrointestinal stromal tumor cells derived from humans or other mammals, or tissues containing at least one of these cells, to obtain the corresponding expanded progeny of the gastrointestinal stromal tumor cells.

[0028] Furthermore, the cells obtained by the culture method of this embodiment can be used for regenerative medicine, toxicity testing, basic medical research on gastrointestinal stromal tumor cells, screening of drug response, determination of the metabolic stability and metabolic spectrum of drugs in vitro, and development of new drugs targeting gastrointestinal stromal tumors. [Brief description of the drawings]

[0029] [Figure 1] 1A and 1B are graphs showing the effect of various concentrations of gastrin I and N2 in culture medium on the growth of primary gastrointestinal stromal tumor cells in vitro. [Diagram 2] 2A-2C are graphs showing the effect of various receptor tyrosine kinase ligands at various concentrations in culture medium on the growth of primary gastrointestinal stromal tumor cells in vitro. [Diagram 3] 3A-3F are graphs showing the effect of combinations of various factors in the culture medium on the growth of primary gastrointestinal stromal tumor cells in vitro. [Figure 4] FIG. 4 shows a photograph (100x magnification) taken under an inverted phase contrast microscope of primary gastrointestinal stromal tumor cells obtained by culturing primary gastrointestinal stromal tumor cells isolated from three tissue samples from clinical gastrointestinal stromal tumor patients for 14 days using the culture method for culturing gastrointestinal stromal tumor cells of the present invention. [Diagram 5] FIG. 5 shows photographs (100x magnification) of gastrointestinal stromal tumor cells obtained by culturing primary gastrointestinal stromal tumor cells isolated from the same tissue sample of a clinical gastrointestinal stromal tumor patient, the cultures being carried out in parallel for 8 days using the culture method for culturing gastrointestinal stromal tumor cells of the present invention and two culture methods reported for culturing primary gastrointestinal stromal tumor cells, in a collagen I-coated and non-coated format, respectively. [Figure 6] Figure 6A shows the results of immunofluorescence staining (under a microscope at 100x magnification) of specific biomarkers of gastrointestinal stromal tumor cells and fibroblasts performed on gastrointestinal stromal tumor cells obtained by culturing primary gastrointestinal stromal tumor cells separated from clinical tissue samples of gastrointestinal stromal tumors, and the culture was performed in parallel for 14 days using the culture method of culturing gastrointestinal stromal tumor cells of the present invention and two culture methods reported for culturing gastrointestinal stromal tumor cells, respectively. Here, GIST-T1 cells were used as a positive control. Figure 6B shows the results of statistical analysis of the number of CKIT-positive and CKIT-negative labeled cells after staining the cells obtained from the three different culture methods described above. [Figure 7] FIG. 7 shows a cell growth curve obtained by continuously culturing primary gastrointestinal stromal tumor cells isolated from clinical tissue samples of gastrointestinal stromal tumors for 30 days using the culture method for culturing gastrointestinal stromal tumor cells of the present invention. [Figure 8] FIG. 8 shows the results of gene mutation concordance analysis between three surgically resected samples of gastrointestinal stromal tumor and gastrointestinal stromal tumor cells obtained by culturing primary gastrointestinal stromal tumor cells derived from the corresponding samples using the culture method for culturing gastrointestinal stromal tumor cells of the present invention. [Figure 9] 9A and 9B are diagrams showing dose-response curves of various targeted drugs against gastrointestinal stromal tumor cells cultured using the culture medium for gastrointestinal stromal tumor cells of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0030] [Example 1] Isolation of human primary gastrointestinal stromal tumor cells and optimization of culture medium for gastrointestinal stromal tumor cells (1) Isolation of primary human gastrointestinal stromal tumor cells For transport and washing of samples, a commercially available penicillin-streptomycin double antibody solution (manufactured by Corning Inc., containing 10,000 U / ml penicillin and 10 mg / ml streptomycin) was added to DMEM / F12 medium (manufactured by Corning) at a volume ratio of 2%, which is hereinafter referred to as the "transport solution."

[0031] Tissue samples of gastrointestinal stromal tumors, namely GIST-1, GIST-2, GIST-3, and GIST-4, were obtained from surgically removed cancer tissue samples from four patients with gastrointestinal stromal tumors who provided informed consent. One of the samples (GIST-1) is illustrated below. The above-mentioned tissue samples were collected within 30 minutes after surgical resection from the patients. More specifically, in a sterile environment, tissue samples from non-necrotic areas were cut into 0.5 cm 3 The above volumes were collected and placed in pre-chilled transport medium before being shipped to the laboratory on ice.

[0032] In a biosafety cabinet, the tissue sample (GIST-1) was transferred to a 100 mm cell culture dish and rinsed with transport solution. Blood cells on the surface of the tissue sample were washed away, and unwanted tissues such as skin and fascia on the surface of the tissue sample were removed.

[0033] Transfer the rinsed tissue sample to another new 100 mm culture dish, add 10 mL of transport solution, and cut the tissue sample into pieces with a diameter of 1 mm using a sterile scalpel blade and forceps. 3 The tissue was separated into smaller pieces.

[0034] The tissue sample pieces were transferred to a 50 mL centrifuge tube and centrifuged at 1200 rpm for 5 minutes using a tabletop centrifuge, the supernatant was carefully removed from the centrifuge tube with a pipette, and the resultant was resuspended in 10 mL of serum-free DMEM / F12 medium containing collagenase II (1 mg / mL) (Sigma) and collagenase IV (1 mg / mL) (Sigma), which was placed on a shaker with a constant temperature of 37°C and digested by shaking for 30 to 90 minutes, and then centrifuged at 350 g / min for 5 minutes in a tabletop centrifuge, and after discarding the supernatant, the digested tissue cells were resuspended in 10 mL of DMEM / F12 medium containing 10% fetal bovine serum (Gibco), and then the resultant was crushed and sieved through a cell sieve with a 100 μm opening, the sieved cell suspension was collected in a 50 mL centrifuge tube, and the cells were counted with a hemocytometer.

[0035] The cell suspension was then centrifuged in a centrifuge at 350 g / min for 5 minutes, and after discarding the supernatant, the resultant was resuspended in the culture medium described below for primary gastrointestinal stromal tumor cells.

[0036] The other three gastrointestinal stromal tumor tissue samples were isolated using the same method as above.

[0037] (2) Coating of cell culture plates Rat tail type I collagen (type I collagen from Corning Inc.) was diluted with ultrapure water at a ratio of 1:50 to prepare a collagen dilution solution. The collagen dilution solution was added to a 24-well culture plate at 500 μl per well to completely cover the bottom of the well of the culture plate. After being left in a 37° C. incubator for 1 hour, the collagen dilution solution was removed to obtain a collagen-coated culture plate.

[0038] (3) Screening of factors added to culture media for primary gastrointestinal stromal tumor cells First, a basal medium was prepared by adding GlutaMAX-I (Thermo Fisher SCIENTIFIC) at a dilution rate of 1:100, insulin-containing culture additive insulin-transferrin-selenium-ethanolamine (ITS-X, Gibco) at a dilution rate of 1:100, ROCK kinase inhibitor Y27632 (Sigma) at a final concentration of 10 μM, and commercial Primocin solution (InvivoGen) (commercial product at a concentration of 50 mg / ml) at a dilution rate of 1:500 to a commercially available DMEM / F-12 medium to obtain a basal medium.

[0039] Next, various kinds of additives (Table 1) were added to the base medium to prepare culture media for gastrointestinal stromal tumor cells containing various components. The culture media containing various components were added to a 24-well plate coated with collagen I at a volume of 500 μl per well, with three wells per medium formulation. GIST cells (GIST-1) isolated from GIST tissue in section (1) of this Example were cultured at 3 × 10 cells per well in a 24-well culture plate coated with collagen I. 4 Cells were seeded at a cell density of 1000× and incubated in various media formulations at 37°C with 5% CO 2 The cells were cultured at 1000 x g for 14 h at 37 °C and 2% oxygen concentration. The medium was changed every 3 days after the start of culture. After 14 days of culture, cell counts were performed. As an experimental control, basal medium without any of the additional factors shown in Table 1 was used.

[0040] Tumor cells isolated from the other three gastrointestinal stromal tumor tissue samples were cultured and counted in the same manner as above.

[0041] The statistical results are shown in Table 1.

[0042] [Table 1]

[0043] Here, "+" indicates that, compared with the basic medium, the medium to which the additive(s) has been added has the effect of promoting the proliferation of primary gastrointestinal stromal tumor cells isolated from gastrointestinal stromal tumor tissue in at least three cases, and the number of "+" indicates the degree of the promoting effect. "-" indicates that the medium to which the additive(s) has been added has the effect of inhibiting the proliferation of primary gastrointestinal stromal tumor cells isolated from gastrointestinal stromal tumor tissue in at least two cases. "○" indicates that the medium to which the additive(s) has been added has no significant effect on the proliferation of primary gastrointestinal stromal tumor cells isolated from gastrointestinal stromal tumor tissue in at least three cases. These results demonstrated that N2, platelet-derived growth factor AA-platelet-derived growth factor CC, stem cell factor, insulin growth factor 1, basic fibroblast growth factor, fibroblast growth factor 10, and gastrin were able to promote the proliferation of primary gastrointestinal stromal tumor cells isolated from gastrointestinal stromal tumor tissues in at least three cases, and among them, N2, platelet-derived growth factor AA, stem cell factor, basic fibroblast growth factor, and gastrin had more potent effects.

[0044] (4) Optimization of the concentrations of factors added to the culture medium for primary gastrointestinal stromal tumor cells Gastrin I (Anaspec) was added to the basal medium to prepare culture media for gastrointestinal stromal tumor cells containing gastrin I at final concentrations of 0.3 nM, 1 nM, 3 nM, and 10 nM, and these were added to a 24-well plate coated with collagen I in a volume of 500 μl per well in three wells for each concentration. Primary gastrointestinal stromal tumor cells (GIST-1) isolated from gastrointestinal stromal tumor tissue in section (1) of this Example were cultured at 3 × 10 per well in a 24-well culture plate coated with collagen I. 4 The cells were seeded at a cell density of 100 x 100 and incubated at 37 °C in 5% CO in a medium formulation containing various concentrations of gastrin I. 2The cells were cultured at 0.1% CO2 concentration and 2% oxygen concentration. The medium was changed every 3 days after the start of culture. After 14 days of culture, cell counts were performed. Base medium with 0 nM gastrin I concentration was used as an experimental control. The results are shown in Figure 1A.

[0045] Culture media for gastrointestinal stromal tumor cells with various concentrations of N2 were prepared by adding N2 additive (Thermo Fisher) to the base medium at dilution ratios of 1:200, 1:100, and 1:50. Various medium formulations were added to a collagen I-coated 24-well plate in triplicate wells at each concentration at a volume of 500 μl per well. Primary cell culture and counting were performed as described above. Base medium with an N2 concentration of 0 was used as the experimental control. The results are shown in Figure 1B.

[0046] Then, 1:100 diluted N2 and 1 nM gastrin were added to the basic medium to prepare basic medium 2. Stem cell factor (SCF, R&D) was added to basic medium 2 to prepare culture media for gastrointestinal stromal tumor cells containing SCF with final concentrations of 20 ng / ml, 50 ng / ml, and 100 ng / ml, respectively. Culture media containing various concentrations of SCF were added to a 24-well plate coated with collagen I in a volume of 500 μl per well, in triplicate wells for each concentration. Primary gastrointestinal stromal tumor cells (GIST-1) isolated from gastrointestinal stromal tumor tissue in section (1) of this Example were cultured at 4 × 10 per well in a 24-well culture plate coated with collagen I. 4 The cells were seeded at a cell density of 100 x 100 and incubated at 37 °C in 5% CO in media formulations containing various concentrations of SCF. 2 The primary GIST cells were cultured at 37 °C and 2% oxygen concentration. Cell counts were performed on days 3, 7, and 12 for primary GIST cells cultured with various media formulations. The results are shown in Figure 2A.

[0047] Similarly, culture media for gastrointestinal stromal tumor cells containing various concentrations of platelet-derived growth factor AA (PDGFAA, R&D) and basic fibroblast growth factor (bFGF, R&D) were prepared using basal medium 2 at final concentrations of 20 ng / ml, 50 ng / ml, and 100 ng / ml, respectively. Primary cell culture and counting were performed as above. The results are shown in Figure 2B and Figure 2C.

[0048] These results demonstrated that the addition of various concentrations of the receptor tyrosine kinase ligands SCF, bFGF, and PDGFAA to basal medium 2 had a time- and dose-dependent proliferative effect on the in vitro growth of primary GIST cells.

[0049] (5) Study on the proliferation effect of various factors added at various concentrations to the culture medium for primary gastrointestinal stromal tumor cells Six different media formulations were also prepared: 1. Basal medium (hereinafter referred to as BM): the basal medium prepared in section (3) of Example 1; 2. Formulation 2: basal medium supplemented with 1:100 diluted N2, 1 nM gastrin, 100 ng / ml PDGFAA, and 20 ng / ml bFGF, respectively; 3. Formulation 3: basal medium supplemented with 1:100 diluted N2, 1 nM gastrin, 100 ng / ml PDGFAA, and 50 ng / ml SCF; 4. Formulation 4: basal medium supplemented with 1:100 diluted N2, 1 nM gastrin, 50 ng / ml SCF, and 20 ng / ml bFGF; 5. Formulation 5: basal medium supplemented with 1:100 diluted N2, 100 ng / ml PDGFAA, 50 ng / ml SCF, and 20 ng / ml bFGF, respectively; 6. Formulation 6: Basal medium supplemented with 1 nM gastrin, 100 ng / ml PDGFAA, 50 ng / ml SCF, and 20 ng / ml bFGF; 7. Complete medium (hereafter referred to as CM): basal medium supplemented with 1:100 diluted N2, 1 nM gastrin, 100 ng / ml PDGFAA, 50 ng / ml SCF, and 20 ng / ml bFGF.

[0050] First, we compared the in vitro proliferation effects of basal and complete media on primary GIST cells. Basal and complete media were added to collagen I-coated 24-well plates in a volume of 500 μl per well, with two wells in each group.

[0051] According to the method of Example 1, section (1), GIST cells (GIST-2) isolated from gastrointestinal stromal tumor tissue were cultured at 4 × 10 cells / well in a 24-well culture plate coated with collagen I. 4 The cells were seeded at a cell density of 100 x 100 cells and incubated at 37 °C in 5% CO with two different media formulations. 2 The GIST cells were cultured at 100-200 ng / ml concentration and 2% oxygen concentration. On the 10th day of culture, cell counting was performed on the GIST cells under different medium formulations. The number of GIST cells cultured using the basal medium was 100%. The results are shown in Figure 3A. These results revealed that the use of complete medium increased the cell number by approximately 70% in just 10 days compared to the use of the basal medium.

[0052] Next, 20ng / ml, 50ng / ml, and 100ng / ml SCF were added based on formulation 2 to study the proliferation-promoting effect of various concentrations of SCF. The medium of formulation 2 containing various concentrations of SCF was added to a 24-well plate coated with collagen I in duplicate wells at a volume of 500μl per well. Following the same method as in section (1) of this example, GIST-2 was cultured at 4×10 cells / well in a 24-well culture plate coated with collagen I. 4 The cells were seeded at a cell density of 100 x 100 cells and incubated at 37 °C in 5% CO with formulations containing various concentrations of SCF. 2The cells were cultured at 100°C and 2% oxygen concentration. After culturing for 10 days, the gastrointestinal stromal tumor cells were counted under various medium formulations. The number of gastrointestinal stromal tumor cells cultured using the medium of formulation 2 was 100%. The results are shown in Figure 3B.

[0053] These results revealed that the addition of SCF effectively promoted cell proliferation compared to the use of Formulation 2 medium, where at an SCF concentration of 50 ng / ml, the cell number could be increased by about 25% after 10 days of culture compared to the use of Formulation 2 medium.

[0054] Similarly, following the same process as formulation 2 above, based on formulation 3, 20ng / ml, 50ng / ml, and 100ng / ml bFGF were added, respectively, to study the proliferation-promoting effect of various concentrations of bFGF. The results are shown in Figure 3C. These results reveal that the addition of bFGF can effectively promote cell proliferation compared with the use of the medium of formulation 3, where at a bFGF concentration of 20ng / ml, the cell number can be increased by about 17% after 10 days of culture compared with the use of the medium of formulation 3.

[0055] Similarly, following the same process as formulation 2 above, 20ng / ml, 50ng / ml, and 100ng / ml PDGFAA were added based on formulation 4 to study the proliferation-promoting effect of various concentrations of PDGFAA, respectively. The results are shown in FIG. 3D. These results reveal that the addition of PDGFAA can effectively promote cell proliferation compared with the use of formulation 4 medium, where at a PDGFAA concentration of 100ng / ml, the cell number can be increased by about 13% after 10 days of culture compared with the use of formulation 4 medium.

[0056] Similarly, following the same steps as above formulation 2, based on formulation 5, 0.3nM, 1nM and 3nM gastrin I were added to study the proliferation-promoting effect of various concentrations of gastrin I. The results are shown in Figure 3E. These results reveal that compared with the use of the medium of formulation 5, adding gastrin I can effectively promote cell proliferation, where at a concentration of 1nM gastrin I, the cell number can be increased by about 8% after 10 days of culture compared with the use of the medium of formulation 5.

[0057] Similarly, following the same process as formulation 2 above, based on formulation 6, N2 diluted at 1:200, 1:100, and 1:50, respectively, was added to study the proliferation-promoting effect of various concentrations of N2. The results are shown in FIG. 3F. These results revealed that the addition of N2 effectively promoted cell proliferation compared with the use of formulation 6 medium, where the addition of N2 at a dilution rate of 1:100 could increase the cell number by about 8% after 10 days of culture compared with the use of formulation 6 medium.

[0058] [Example 2] Culture of human primary GIST cells and comparison with existing culture methods (1) Culture of primary GIST cells derived from human gastrointestinal stromal tumor tissue Using the same method as in Section (1) of Example 1, primary GIST cells (GIST-1, GIST-2, GIST-3) were isolated from cancer tissues of three patients with gastrointestinal stromal tumors, respectively. Then, the isolated GIST cells were counted using a hemocytometer, and then cultured at 5 × 10 per well in a 12-well plate coated with collagen I (Corning). 4The cells were seeded at a cell density of 1000 cells / well, where the coating method was as follows: Collagen I was prepared into collagen dilution solution by diluting collagen I with ultrapure water at a ratio of 1:50, and 1 ml of collagen dilution solution was added per well to a 12-well culture plate to completely cover the bottom of the well of the plate, and the plate was left in an incubator at 37°C for 1 hour, after which the collagen dilution solution was removed to obtain a collagen-coated culture plate.

[0059] Complete medium (CM) prepared according to section (5) of Example 1 was added to a 12-well plate at 2 mL per well and incubated at 37° C. in 5% CO 2 The medium was replaced every 3 days after the start of the culture.

[0060] FIG. 4 is a 100x phase contrast micrograph of cells cultured up to day 14.

[0061] (2) Preparation of existing culture medium for primary gastrointestinal stromal tumor cells As a control, a culture medium for cell conditional reprogramming technology (hereinafter also referred to as "F medium") was prepared. The preparation procedure was as described in (Non-Patent Document 2), and the medium composition is shown in Table 2.

[0062] [Table 2]

[0063] As another control, a culture medium for primary gastrointestinal stromal tumor cells reported in the literature was prepared. For the specific steps of preparation and culture, see Non-Patent Document 4. The composition of the culture medium was as follows: 20% calf serum was added to RPMI 1640 (manufactured by Corning), followed by a commercially available penicillin-streptomycin solution (manufactured by Corning) at a volume ratio of 1%, and finally SCF was added at a final concentration of 10 ng / ml. Hereinafter, it is referred to as "known composition 1".

[0064] (3) Comparison of various culture techniques for gastrointestinal stromal tumor cells In order to investigate the culture effects of the culture medium and culture method of the present invention and the two existing culture media and culture methods, primary GIST cells (GIST-2) were isolated from cancer tissues of patients suffering from gastrointestinal stromal tumors using the same method as in section (1) of Example 1, and GIST-2 were cultured in vitro using the following three culture conditions in parallel:

[0065] A. Technology of the present invention: Primary GIST cells (GIST-2) were cultured at 5×10 per well in 12-well plates with or without collagen I (Corning) coating. 4 The cells were seeded at a seeding density of 100 x 100 cells / well and incubated at 37°C in 5% CO with 2 mL of complete medium CM prepared in section (5) of Example 1 per well. 2 The cells were cultured in vitro at 100 and 2% oxygen.

[0066] B. Conditional cell reprogramming technique: Primary GIST cells (GIST-2) were cultured at 5 × 10 per well in 12-well plates with or without collagen I (Corning) coating. 4 Seed the cells at an inoculation density of 100 x 100 cells per well and incubate at 37 °C with 5% CO in 2 mL of F medium per well. 2 The cells were cultured in vitro at 100 and 2% oxygen.

[0067] C. Another reported technique: Primary GIST cells (GIST-2) were cultured at 5 × 10 per well in 12-well plates with or without collagen I (Corning) coating. 4 Inoculate the wells at a seeding density of 100 x 100 cells and incubate at 37 °C in 5% CO with 2 mL of "known formulation 1" per well. 2 The cells were cultured in vitro at 20% oxygen and 20% concentration.

[0068] FIG. 5 shows cell photographs taken under a phase contrast microscope at 100× magnification of GIST-2 cells cultured under various culture conditions for up to 8 days.

[0069] 5, it can be seen that primary GIST cells derived from gastrointestinal stromal tumor tissue can be efficiently cultured in vitro by using the culture medium and culture method for gastrointestinal stromal tumor cells of the present invention. Furthermore, when using the GIST culture medium of the present invention, the culture plate coated with collagen I can produce better growth efficiency than the culture plate not coated with collagen I.

[0070] Also, as shown in Figure 5, compared with the culture medium and culture method of the present invention, the number of GIST cells cultured using the known formulation 1 is fewer, there are more cell fragments, and the culture effect without collagen I coating is poorer than that with coating treatment. The number of GIST cells cultured using F medium is also fewer than that cultured using the culture method of the present invention, and many fibroblasts with thick shapes and unclear outlines can be seen under the microscope, indicating that the cultured cells contain a large proportion of fibroblasts, and there is no obvious difference in the culture effect of F medium regardless of whether it is coated with collagen I or not.

[0071] [Example 3] Identification of immune markers for gastrointestinal stromal tumor cells (1) The complete medium CM in section (5) of Example 1, the culture medium F for gastrointestinal stromal tumor cells in section (2) of Example 2, and the known formulation 1 were used. (2) Cancer tissues the size of a soybean were collected from clinical surgical resection samples of patients with gastrointestinal stromal tumors. Primary gastrointestinal stromal tumor cells (GIST-1) were isolated using the same method as in Section (1) of Example 1, and the primary gastrointestinal stromal tumor cells (GIST-1) were cultured using Culture Method A, Culture Method B, and Culture Method C in Section (3) of Example 2, respectively. (3) Immunofluorescence was used to detect the expression of important cancer-related biomarkers on human gastrointestinal stromal tumor cells.

[0072] The primary antibodies used in this experiment were CKIT (CD117) (Cell Signaling Technology) and α-SMA (Cell Signaling Technology). Secondary antibody anti-mouse IgG (H+L), F(ab')2 fragment (Alexa Fluor™ 488 conjugate) (Cell Signaling Technology) was used to target CKIT (CD117), and secondary antibody anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor™ 594 conjugate) (Cell Signaling Technology) was used to target α-SMA. Here, CKIT is an important biomarker for gastrointestinal stromal tumors, and fibroblasts do not express CKIT protein. In clinical practice, CKIT is commonly used in the differential diagnosis of gastrointestinal stromal tumors. α-SMA has been recognized as an important biomarker for fibroblasts.

[0073] In detail, when the GIST cells occupied about 80% of the bottom area of ​​the culture plate, the culture supernatant of the original 12-well plate was discarded, 1 mL of 0.05% trypsin (Thermo Fisher) was added to digest the cells, and after incubation at 37°C for 15 minutes, the digested cells were resuspended in 5 mL of DMEM / F12 medium containing 10% (volume / volume) calf serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, then collected in a centrifuge tube and centrifuged at 300 g / min for 5 minutes. The cell precipitates of each group after centrifugation were resuspended using the complete medium CM of the present invention, F medium, and known formulation 1, respectively, and the cells in the cell suspension were counted using a hemocytometer. 4 × 10 cells per cover glass were cultured on a cover glass coated with collagen I. 4The cells were seeded at a density of 100×100 cells / well, and the coating method was the same as in section (2) of Example 1. Each group of cells was cultured using the complete medium CM of the present invention, F medium, and known formulation 1, respectively, to grow the cells on the cover glass.

[0074] After 14 days of culture, the cells were rinsed twice with PBS buffer, fixed with 4% paraformaldehyde for 15 minutes, and then incubated with TBST (TBS + 0.1% Tween 20) containing 1% BSA (Shanghai Sangon Biotech) and 1% Triton X-100 for 1 hour at room temperature. The cells were rinsed three times with TBST buffer for 3 minutes each time. After removing the TBST buffer, 50 μL of the primary antibody dilution (CKIT antibody diluted 1:2000 times) was dropped onto the slide, the result was incubated at 4 °C for 12 to 16 hours, then rinsed three times with PBS for 3 minutes each time, and the secondary antibody anti-rabbit IgG (H + L), F (ab') 2 fragment (Alexa Fluor ™ 594 conjugate) (8 μg / ml) was dropped onto the slide, the result was incubated at room temperature for 60 minutes, and then rinsed three times with PBS for 3 minutes each time. Secondary antibody anti-rabbit IgG(H+L), F(ab')2 fragment (Alexa Fluor™ 594 conjugate) (8 μg / ml) was then dropped onto the slide and the result was incubated at room temperature for 60 minutes, then rinsed three times with PBS for 3 minutes each time. The cells were incubated with 1 μg / ml DAPI dye (Sigma) for 10 minutes. The cells were rinsed once with PBS. After mounting the cover slip with a drop of mounting medium (Thermo Fisher Scientific), the result was photographed under a fluorescent microscope at 100x.

[0075] As a positive control, a recognized gastrointestinal stromal tumor cell line, GIST-T1 (purchased from ATCC), was used. Primary cells were cultured for 13 days, and then 8 × 10 4GIST-T1 cells were placed on a cover slip and the cells were allowed to grow on the cover slip for 24 hours. These cells were subjected to immunofluorescence staining and photographed in the same manner as the above-mentioned GIST-1 cells cultured by various methods.

[0076] The results are shown in Figure 6A.

[0077] According to Fig. 6A, the gastrointestinal stromal tumor cell line GIST-T1 highly expresses CKIT protein, which is a biomarker specific to GIST cells, but does not express α-SMA protein, and the cells cultured using the culture medium and culture method of the present invention also highly express CKIT protein, which is a biomarker specific to GIST cells, but do not express α-SMA protein.On the other hand, the primary GIST cells derived from the same sample and cultured using F medium or known formulation 1 hardly express CKIT protein, and most of them only express α-SMA protein, which is a biomarker specific to fibroblasts, and these cells have a sheet-like and muscle fiber-like morphology, which indicates that these two culture techniques cannot efficiently culture GIST cells, and most of the cultured cells are fibroblasts, which indicates that the culture effect of the culture medium of the present invention cannot be realized.

[0078] Figure 6B shows the data analysis results by counting CKIT-positive and CKIT-negative labeled cells under a microscope after staining the cells obtained by the three different culture methods mentioned above. The results are counting statistics of three random fields under observation of microscopic photographs.

[0079] From Figures 6A and 6B, it can be confirmed that, compared with the two reported culture techniques, the technique of the present invention can achieve efficient proliferation of patient-derived primary GIST cells in vitro, and the cultured cells are not affected by the interference of fibroblasts.

[0080] [Example 4] Continuous in vitro culture of primary GIST cells Following the same method as in Section (1) of Example 2, GIST-3 cells were continuously cultured using the complete medium CM in Section (5) of Example 1.

[0081] For cells cultured under the conditions of the present invention, the culture medium was changed every 3 days.

[0082] When the GIST cells grew to cover about 80% of the bottom area in the culture plate, the medium supernatant in the original 12-well plate was discarded. 1 mL of 0.05% trypsin (Thermo Fisher: 25300062) was added to digest the cells, and the cells were incubated at 37°C for 15 minutes. After the cells were completely digested, the digested cells were resuspended in 5 mL of DMEM / F12 medium containing 10% (vol / vol) calf serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, then collected in a centrifuge tube and centrifuged at 350 g / min for 5 minutes. The cell pellet after centrifugation was resuspended using complete medium CM, and the cells in the cell suspension were counted using a hemocytometer. 5 × 10 per well were transferred to another 12-well culture plate coated with collagen I. 4 The cells were seeded at a cell density of 1000 cells and further cultured.

[0083] When the growth of the subcultured cells again reached approximately 80% of the bottom area of ​​the culture plate, the cultured cells were digested again according to the procedure described above, harvested, and counted. Again, 5 × 10 cells were cultured per well. 4 The cells were seeded at a density of 100x100 cells and further cultured.

[0084] Below is the formula to calculate the cell population doublings of primary gastrointestinal stromal tumor epithelial cells under various culture conditions: Population doubling number = [log(N / X 0 )] / log2 (where N is the number of cells to be passaged, and X 0 is the number of cells at the time of initial inoculation) (see Greenwood et al., Environ Mol Mutagen 2004, 43(1): 36-44).

[0085] FIG. 7 is a growth curve of an example of gastrointestinal stromal tumor cells under the culture conditions of the present invention, plotted by Graphpad Prism 7.0 software, in which the horizontal axis is the number of days of culture and the vertical axis is the doubling number of cell population. From FIG. 7, it can be confirmed that GIST cells cultured by the culture medium for gastrointestinal stromal tumor cells of the present invention can grow continuously for at least 30 days. In addition, when primary GIST cells are cultured using the existing F medium and known formulation 1, according to the results of Example 3, purified GIST cells cannot be cultured, and the cultured cells are interfered with by a large number of fibroblasts. Therefore, the sustained growth effect of the existing F medium and known formulation 1 on primary GIST cells in vitro cannot be investigated.

[0086] [Example 5] Analysis of gene mutation concordance in primary GIST cells According to the same method as in section (1) of Example 2, primary GIST cells, GIST-1, GIST-2, and GIST-3, were cultured using the complete medium CM in section (5) of Example 1, respectively.

[0087] On day 20 of culture, gastrointestinal stromal tumor cells were collected, and the genomic DNA of the cells was extracted using the DNeasy blood & tissue kit (QIAGEN). In addition, gastrointestinal stromal tumor tissue from the patient corresponding to the source of the cells was collected, and the genomic DNA of the cells was extracted using the DNeasy blood & tissue kit (QIAGEN). Two milliliters of peripheral blood was collected from the patient who provided the cells, and blood genomic DNA was extracted using the DNeasy blood & tissue kit (QIAGEN) as a background control.

[0088] Subsequently, whole-exome sequencing was performed on the genomic DNA of the cell and tissue samples (for detailed procedures, see Hans Clevers et al., Cell, 11; 172(1-2): 373-386, 2018) and the sequencing results were analyzed for tumor hypermutations using the MuSiC software. MuSiC performs statistical tests for each mutation type on genes, interpreting the gene mutations in the peripheral blood of the corresponding patients as background, to detect genes with a significantly higher mutation rate than background. The analysis results were generated using the software available at https: / / bioinfogp.cnb.csic.es / tools / venny / index.html. The analysis results are shown in Figure 8.

[0089] The tumor hypermutation analysis results in Figure 8 show that the hypermutated genes between the GIST cells cultured using the culture medium for gastrointestinal stromal tumor cells of the present invention and the original tissue of gastrointestinal stromal tumor are basically the same. That is, the GIST cells cultured using the culture medium for gastrointestinal stromal tumor cells of the present invention can maintain the original gene mutation characteristics of the patient's cancer tissue.

[0090] [Example 6] Drug sensitivity testing of GIST cells Taking a surgical resection sample from a patient suffering from gastrointestinal stromal tumor as an example, it is demonstrated that GIST cells cultured from a gastrointestinal stromal tumor tissue sample from the patient can be used to test the sensitivity of the patient's tumor cells to various drugs.

[0091] 1. Seeding of primary GIST cells: GIST cells (GIST-1 and GIST-2) cultured by the method described in section (1) of Example 2 using the complete medium CM in section (5) of Example 1 were seeded into 384-well plates at a density of 3,000 to 5,000 cells per well, and the cells were allowed to attach overnight.

[0092] 2. Drug gradient experiments: (1) A drug storage plate was prepared by gradient dilution: 10 μL of each of the tested drug stock solutions (the concentration of the drug stock solution was determined based on twice the maximum blood concentration Cmax of the drug in the human body) was taken and added to a 0.5 mL EP tube containing 20 μL of DMSO, and 10 μL of the solution from the above EP tube was pipetted into a second 0.5 mL EP tube containing 20 μL of DMSO. In other words, the drug was diluted in a ratio of 1:3. The above method was repeated to perform stepwise dilution to obtain six concentrations required for dosing. Different concentrations of the drug were added to a 384-well drug storage plate. 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 were imatinib (manufactured by MCE), sunitinib (manufactured by MCE), and regorafenib (manufactured by MCE), which are clinically approved as therapeutic agents for gastrointestinal stromal tumors.

[0093] (2) Using a high-throughput automated workstation (purchased from Perkin Elmer), various concentrations of drugs and solvent controls in a 384-well drug storage plate were added to a 384-well cell culture plate seeded with gastrointestinal stromal tumor cells. The drug groups and solvent control groups were each arranged in three replicate wells. The volume of drug added to each well was 100 nL.

[0094] (3) Cell viability test: 72 hours after administration, the chemiluminescence value of cultured cells after drug administration was detected using a Cell Titer-Glo assay kit (Promega). The magnitude of the chemiluminescence value reflects the cell viability and the effect of the drug on the cell viability. The prepared Cell Titer-Glo detection solution was added to each well, and the chemiluminescence value was detected after mixing using a microplate reader.

[0095] Graphs were generated using Graphpad Prism 7.0 software, and the half inhibitory concentration IC 50 was calculated.

[0096] (4) The results of the drug susceptibility test are shown in Figures 9A and 9B.

[0097] Figures 9A and 9B show the drug sensitivity of GIST cells cultured from surgically resected cancer tissue samples from two different GIST patients to three targeted drugs, imatinib, sunitinib, and regorafenib, respectively. These results indicate that cells from the same patient have different sensitivities to different drugs, and cells from different patients also have different sensitivities to the same drug.

[0098] In detail, GIST cells derived from a patient with CKIT wild-type GIST (GIST-1) had lower sensitivity to the targeted drug imatinib, which targets CKIT-mutated GIST patients, with a half-inhibitory concentration of 8.42 μM, but were relatively sensitive to the multi-target inhibitor sunitinib, with a half-inhibitory concentration of 2.65 μM. The test results of GIST cells derived from a patient with GIST with CKIT mutation (GIST-2) revealed that they had good sensitivity to the targeted drug imatinib, with a half-inhibitory concentration of 2.59 μM, and better sensitivity to regorafenib, with a half-inhibitory concentration of 0.63 μM.

[0099] According to the results of this example, it can be confirmed that the results of the sensitivity test of GIST cells cultured using the culture medium for gastrointestinal stromal tumor cells of the present invention to targeted drugs are consistent with the clinical pathological typing of patients.Therefore, the culture medium for gastrointestinal stromal tumor cells of the present invention has the potential to be used in predicting the clinical efficacy of GIST patients.

[0100] Although the present invention has been described in detail above through general description and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention should be within the protection scope of the present invention. [Industrial Applicability]

[0101] The present invention provides a cell culture medium for culturing primary GIST cells, and a culture method for culturing primary GIST cells using the culture medium. The cell culture medium and the culture method of the present invention can be used to culture GIST cells with a high success rate and simple operation without interference from non-GIST cells such as fibroblasts, and can be used to construct a primary GIST cell model and obtain GIST cells with the biological characteristics of GIST patients themselves, which can be used in drug efficacy evaluation or screening.

Claims

1. A cell culture medium for culturing primary gastrointestinal stromal tumor cells, comprising gastrin, N2, insulin, platelet-derived growth factor AA, stem cell factor, basic fibroblast growth factor, and Y27632.

2. 2. The cell culture medium according to claim 1, wherein the gastrin content is 0.3 nM to 10 nM.

3. The cell culture medium described in claim 1, characterized in that the gastrin content is 0.3 nM to 3 nM.

4. The volume concentration of N2 in the cell culture medium ranges from 1:25 to 1:200; The insulin content is 2 μg / ml to 20 μg / ml. The contents of platelet-derived growth factor AA, stem cell factor, and basic fibroblast growth factor are 5 ng / ml to 500 ng / ml, respectively.

2. The cell culture medium according to claim 1, wherein the content of Y27632 is 2 μM to 50 μM.

5. The volume concentration of N2 in the cell culture medium is in the range of 1:50 to 1:100; The insulin content is 5 μg / ml to 10 μg / ml. The contents of platelet-derived growth factor AA, stem cell factor, and basic fibroblast growth factor are each 20 ng / ml to 100 ng / ml.

2. The cell culture medium according to claim 1, wherein the content of Y27632 is 5 μM to 10 μM.

6. The cell culture medium of claim 1 , wherein the cell culture medium is serum-free.

7. A method for culturing primary gastrointestinal stromal tumor cells, comprising the steps of: (1) preparing a cell culture medium according to any one of claims 1 to 6; (2) coating the culture vessel with collagen; (3) inoculating primary gastrointestinal stromal tumor cells in the coated culture vessel and culturing the cells under an oxygen concentration of 0.1% to 25% by using the cell culture medium; A culture method comprising the steps of:

8. The collagen is rat tail type I collagen, The collagen is diluted with ultrapure water at a dilution ratio of 1:5 to 1:100; The culture method according to claim 7, wherein the coating method comprises adding the diluted collagen to the culture vessel to completely cover the bottom of the vessel and leaving the diluted collagen for 30 minutes or more.

9. The culture method described in claim 8, wherein the collagen is diluted with ultrapure water at a dilution ratio of 1:10 to 1:

50.

10. The culture method according to claim 7, wherein in the step (3), the culture is carried out under an oxygen concentration of 0.1% to 4%.

11. A method for evaluating the efficacy of a gastrointestinal stromal tumor drug or a method for screening a gastrointestinal stromal tumor drug, comprising the steps of: (1) culturing gastrointestinal stromal tumor cells by using the culture method according to any one of claims 7 to 10; (2) preparing the drug to be tested in the required concentration gradient; (3) adding the various concentrations of the drug prepared in step (2) to the gastrointestinal stromal tumor cells cultured in step (1); (4) detecting cell viability; and A method comprising:

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