A gastrointestinal stromal tumor organoid culture solution and methods of use and applications thereof

Abstract

The application discloses a gastrointestinal stromal tumor organoid culture solution and a use method and application thereof, and belongs to the technical field of gastrointestinal stromal tumor organoid culture. The gastrointestinal stromal tumor organoid obtained by using the culture solution and the culture method provided by the application can retain the histological characteristics and genetic heterogeneity of the original tumor of a patient, can be used as a drug screening model and a disease research model for gastrointestinal stromal tumor treatment, and can promote the progress and development of precise tumor medicine; the gastrointestinal stromal tumor organoid obtained by culture has a high proliferation speed, a high cell survival rate, can be long-term subcultured and amplified, and a gastrointestinal stromal tumor organoid with a diameter of more than 100 microns and good activity can be obtained in 8-10 days; the culture solution is also suitable for culturing wild-type gastrointestinal stromal tumors such as SDH-deficient gastrointestinal stromal tumors; the culture solution does not contain serum and other components with unclear compositions, is not affected by different batches, can maintain a high uniformity in the culture process, can be used for culturing gastrointestinal stromal tumor organoids derived from tumor tissues or malignant cell lines of patients, and is suitable for drug sensitivity detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gastrointestinal stromal tumor organoid culture, in particular to a gastrointestinal stromal tumor organoid culture medium and a use method and application thereof. BACKGROUND

[0002] Gastrointestinal stromal tumor (GIST) is a common soft tissue sarcoma in the gastrointestinal tract, which is believed to originate from interstitial cells of Cajal (ICC) responsible for pacing and mediating nerve transmission in the gastrointestinal tract or multipotent stem cells differentiated from Cajal cells. The tumor tissue is mainly composed of a certain number of spindle cells or epithelioid cells, and immunohistochemistry is commonly positive for CD117, DOG-1 and CD34. Molecular genetic studies of gastrointestinal stromal tumors show that more than 85-90% of patients have acquired type III receptor tyrosine kinase mutations (C-KIT or PDGFRA mutations), and 10-15% of KIT and PDGFRA wild-type patients are mainly RAS-related pathway gene mutations and function loss of succinate dehydrogenase SDH and neurofibromatosis type 1 NF1 gene (Kelly et al., 2021).

[0003] Gastrointestinal stromal tumor can occur in any part of the digestive system such as stomach, intestine, duodenum, esophagus and mesentery, and often metastasizes to adjacent tissues and organs such as abdominal cavity, pelvic cavity and liver. There are no obvious symptoms in the early stage of the disease, and as the disease progresses, symptoms such as abdominal distension, vomiting of blood and melena may occur. According to the statistics worldwide, the incidence of gastrointestinal stromal tumor is 10-15 per 1 million, and for patients who can undergo radical resection, the 5-year survival rate is 54%, and for patients with adjuvant therapy and recurrence and metastasis, the median survival period is only 29 months (Soreide et al., 2016, DeMatteo et al., 2000). Gastrointestinal stromal tumor is not sensitive to conventional chemotherapy and radiotherapy, although the development of drugs such as Gleevec has effectively prolonged the survival period of patients, but the heterogeneity between individuals makes it difficult for current treatment methods to benefit all patients, and it is urgent to find a clinically relevant drug model to guide patients for individualized treatment and improve the prognosis of patients.

[0004] Organoid is a kind of 3D cell complex in vitro driven by stem cells and self-assembled, which can reproduce part of the structure and physiological function of the organ. Patient-derived organoid (PDO) is a kind of mini-tumor formed by three-dimensional culture of tumor tissue obtained from surgery or biopsy in vitro, which has the characteristics of high reduction of original tumor histological heterogeneity and genetic map, long-term expansion and maintenance of genomic stability. As a drug screening model and disease research model of precision medicine, it has been verified in the culture system of multiple cancer subtypes. However, there are few reports on gastrointestinal stromal tumor organoid culture medium and culture method. Therefore, it is necessary to develop an effective method for constructing gastrointestinal stromal tumor organoid in vitro, to make up for the shortcomings of the existing technology, and to meet the needs of gastrointestinal stromal tumor basic research and clinical diagnosis and treatment. SUMMARY

[0005] The purpose of the present application is to provide a gastrointestinal stromal tumor organoid culture solution and its use method and application, in order to make up for the blank of no gastrointestinal stromal tumor organoid culture medium and culture method at present.

[0006] In order to achieve the above-mentioned purpose, the present application provides a gastrointestinal stromal tumor organoid culture solution, which comprises the following components: Advanced DMEM / F12 culture medium, StemPro MSC SFM culture medium, Wnt conditioned medium, HEPES buffer, L-alanyl-L-glutamine, N-acetylcysteine, sodium pyruvate, nicotinamide, forskolin, MEM-NEAA solution, B27 supplement, N2 supplement, epidermal growth factor EGF, fibroblast growth factor FGF, insulin-like growth factor IGF-1, hepatocyte growth factor HGF, stem cell growth factor SCF, platelet-derived growth factor PDGF, R-spondin1, A83-01, Y-27632, ML-098, MHY1485.

[0007] Preferably, it comprises the following components: Advanced DMEM / F12 medium for a total volume of 30-57.5%, StemPro MSC SFM medium for a total volume of 10-25%, Wnt conditioned medium for a total volume of 30-40%, MEM-NEAA solution for a total volume of 1-2%, B27 supplement for a total volume of 1-2%, N2 supplement for a total volume of 0.5-1%, 5-10 mM HEPES buffer, 500-600 mg / L L-alanyl-L-glutamine, 1-1.5 mM N-acetyl cysteine, 1-1.5 mM sodium pyruvate, 1-1.5 mM nicotinamide, 5-10 mM forskolin, 10-25 ng / mL epidermal growth factor EGF, 50-100 ng / mL fibroblast growth factor FGF, 25-50 ng / mL insulin-like growth factor IGF-1, 10-25 ng / mL hepatocyte growth factor HGF, 25-100 ng / mL stem cell factor SCF, 25-50 ng / mL platelet-derived growth factor PDGF, 200-500 ng / mL R-spondin, 50-500 ng / mL A83-01, 5-10 mM Y-27632, 100-200 nM ML-098, 0.5-2 mM MHY1485.

[0008] Preferably, it comprises the following components: Advanced DMEM / F12 medium for a total volume of 30-57.5%, StemPro MSC SFM medium for a total volume of 10-25%, Wnt conditioned medium for a total volume of 30-40%, MEM-NEAA solution for a total volume of 1-2%, B27 supplement for a total volume of 1-2%, N2 supplement for a total volume of 0.5-1%, 5-10 mM HEPES buffer, 500-600 mg / L L-alanyl-L-glutamine, 1-1.5 mM N-acetyl cysteine, 1-1.5 mM sodium pyruvate, 1-1.5 mM nicotinamide, 5-10 mM forskolin, 10-25 ng / mL epidermal growth factor EGF, 50-100 ng / mL fibroblast growth factor FGF, 25-50 ng / mL insulin-like growth factor IGF-1, 10-25 ng / mL hepatocyte growth factor HGF, 25-100 ng / mL stem cell factor SCF, 25-50 ng / mL platelet-derived growth factor PDGF, 200-500 ng / mL R-spondin, 50-500 ng / mL A83-01, 5-10 mM Y-27632, 100-200 nM ML-098, 0.5-2 mM MHY1485.

[0009] Preferably, the fibroblast growth factor is FGF4 capable of specifically binding FGFR1; the platelet-derived growth factor PDGF is one of PDGF-AA, PDGF-BB and PDGF-AB; and the HEPES buffer has a pH of 7.2-7.4.

[0010] Preferably, the FGF is FGF4 and the platelet-derived growth factor is PDGF-AA.

[0011] Preferably, the Wnt conditioned medium is a medium containing secreted Wnt ligands harvested at appropriate time intervals according to standard methods using L-Wnt 3a cell lines.

[0012] Preferably, the antibacterial component is primary antibiotic Primocin, penicillin-streptomycin double antibiotic and amphotericin b.

[0013] Preferably, the concentration of primary antibiotic Primocin is 100 μg / mL, the concentration of penicillin-streptomycin double antibiotic is 100 μg / mL, and the concentration of amphotericin b solution is 25 μg / mL.

[0014] The method for culturing gastrointestinal stromal tumor organoids using the gastrointestinal stromal tumor organoid culture solution described above comprises the following steps:

[0015] S1, obtaining gastrointestinal stromal tumor tissue to be cultured, rinsing with pre-cooled DPBS buffer, and uniformly cutting into 1-3 mm 3 size tissue pieces;

[0016] S2, adding tissue digestion solution, and digesting the gastrointestinal stromal tumor tissue pieces obtained in step S1 at 37℃ and 100 rpm on a constant temperature shaker; the composition of the tissue digestion solution is collagenase type II 1 mg / mL, hyaluronidase 20 μg / mL, dispase 0.5 mg / mL, and dithiothreitol 0.5 mM;

[0017] S3, monitoring the digestion status in real time, and when a large number of dissociated cell clusters can be observed under a microscope, adding an equal volume of culture solution containing 5% FBS to terminate the digestion, centrifuging to discard the supernatant, and obtaining cell precipitate;

[0018] S4, resuspending the cell precipitate with fresh gastrointestinal stromal tumor culture medium, filtering and collecting the cell suspension using a 100 μm cell sieve, and centrifuging again to remove the supernatant;

[0019] S5, resuspending the precipitate with a small amount of organoid culture solution, counting the cells and centrifuging to concentrate, and ensuring that each 1 μL of the suspension contains 15,000-20,000 cell clusters;

[0020] S6, mix the concentrated cell suspension with Matrigel at a ratio of 1:4 by volume, inoculate in the form of small droplets in a 24-well plate, and place in a 37℃ cell incubator for 0.5h to allow the droplets to solidify;

[0021] S7, after the droplets solidify, carefully add 500μL gastrointestinal stromal tumor organoid culture solution along the wall of the hole, and place it back in a 37℃ cell incubator with a CO2 concentration of 5% for culture, and replace the culture solution every 3-4 days.

[0022] The gastrointestinal stromal tumor organoid culture solution as described above is applied in organoid culture.

[0023] StemPro MSC SFM medium (purchased from Gibco Company) is a serum-free human mesenchymal stem cell culture medium that can promote the growth and proliferation of mesenchymal stem cells and maintain their differentiation potential.

[0024] L-alanyl-L-glutamine, N-acetylcysteine, nicotinamide, B27, N2, MEM-NEAA, and sodium pyruvate are all nutritional additives for the culture solution. L-alanyl-L-glutamine can serve as a nitrogen source to support the synthesis of a large amount of protein and nucleic acid, and maintain cell growth. Nicotinamide is a B3 vitamin that participates in various enzyme-mediated oxidation-reduction reactions, can promote energy metabolism, and accelerate the formation of organoids. N-acetylcysteine, as an antioxidant, can scavenge intracellular ROS and inhibit apoptosis. MEM-NEAA solution contains various non-essential amino acids such as glycine, proline, and L-aspartic acid, which can effectively improve the cell culture medium ratio and reduce the synthetic metabolic burden of mesenchymal cells. B27 and N2 additives contain various nutritional elements such as human transferrin, insulin, progesterone, sodium selenite, and putrescine, which can be used as a substitute for serum for serum-free culture of organoids. Pyruvate is an intermediate metabolite of glycolysis, which can generate acetyl-CoA through the pyruvate dehydrogenase system to enter the tricarboxylic acid cycle, and serve as a precursor for succinic acid synthesis. In SDH-deficient gastrointestinal stromal tumors, the addition of pyruvate helps to increase the accumulation of succinic acid in tumor cells.

[0025] Stem cell factor (SCF) and platelet-derived growth factor (PDGF-AA) both belong to the platelet-derived growth factor family, and as ligands can bind to their respective type III receptor tyrosine kinases C-Kit and PDGFR, inducing autophosphorylation of the transmembrane segment tyrosine residues, and activating downstream signaling pathways such as SRC, Ras-Raf-Erk, and PLC-γ. Activation of SCF / C-Kit and PDGF / PDGFR signals plays an important role in the maintenance of the phenotype, proliferation, and differentiation of gastrointestinal stromal tumor cells and their precursors, interstitial cells of Cajal.

[0026] Fibroblast growth factor 4 (FGF-4) is a heparin-binding growth factor that can bind to the receptor tyrosine kinase FGFR1C as a ligand, initiate downstream signals, and promote the proliferation of mesenchymal stem cells. It has been reported that extensive overexpression and persistent activation of FGF4 signals can be found in SDH-deficient GIST and quadruple wild-type GIST tumor cells (Flavahan et al., 2019, Urbini et al., 2019).

[0027] Insulin-like growth factor 1 (IGF-1), also known as somatomedin C, is a mitogenic polypeptide growth hormone with a tertiary structure similar to insulin. It can bind to the insulin receptor IR and the insulin-like growth factor receptor IGFR, phosphorylate IRS, CRK, SHC, and other proteins to activate downstream pathways such as PI3K / Akt and MAPK, increase the uptake of nutrients by gastrointestinal stromal tumor organoids, promote cell proliferation, accelerate the cell cycle and inhibit apoptosis, and increase the volume and construction survival rate of gastrointestinal stromal tumor organoids.

[0028] Hepatocyte growth factor (HGF) is a ligand for the receptor tyrosine kinase C-MET expressed by interstitial cells. HGF / C-MET signal activation can coordinate cell processes such as cell migration dispersion, proliferation, angiogenesis, and epithelial-mesenchymal transition, and enhance GIST cell viability. Compensatory activation of C-MET signals is common in most imatinib-resistant GIST patients (Cohen et al., 2015, Mahadevan et al., 2015).

[0029] Wnt CM is a conditioned medium containing Wnt3a protein activity obtained by fermentation of L-Wnt 3A cell lines, which can activate the Wnt / β-Catenin pathway to maintain the stemness of tumor cells. R-spondin, as a ligand for LGR4-6 receptors, can act as an agonist for Wnt signals to improve β-catenin signaling, allowing long-term stable subculture of organoids.

[0030] Forskolin is a potent adenylyl cyclase activator that can increase the concentration of intracellular second messenger cAMP and enhance cell signaling.

[0031] Y-27632 is a small molecule inhibitor of Rho-associated protein kinase p160ROCK, which can target the catalytic site of ROCK-1 and ROCK-2 to inhibit the anoikis of cells, and can also induce the differentiation of pluripotent stem cells into mesodermal cell lineage through epithelial-mesenchymal transition (EMT).

[0032] ML-098 is a small molecule agonist of Ras and related binding protein Rab, and the addition of ML-098 in the culture solution can strengthen the upstream activated RTK-RAS signal by increasing the affinity of GTPase protein in tumor cells to guanine nucleotide, and promote the growth and self-assembly of gastrointestinal stromal tumor organoids.

[0033] MHY1485 is an mTOR activator with a morpholine triazine structure, which can phosphorylate the ser2448 site of mTOR to activate mTOR protein and its downstream proteins ribosomal S6K1 and cell translation initiation factor 4EP1, thereby enhancing cell protein synthesis translation and glycolysis metabolism, and promoting the further growth of gastrointestinal stromal tumor organoids. Studies have found that mTOR signal activation can be detected in metastatic gastrointestinal stromal tumors and KIT inhibitor-resistant gastrointestinal stromal tumors (Duan et al., 2020, Li et al., 2015).

[0034] Therefore, the gastrointestinal stromal tumor organoid culture solution and the use method and application thereof provided by the present application have the following specific technical effects:

[0035] (1) The gastrointestinal stromal tumor organoids obtained by using the culture solution and the culture method provided by the present application can retain the histological characteristics and genetic heterogeneity of the original tumor of the patient, and can be used as a drug screening model and a disease research model for the treatment of gastrointestinal stromal tumors, thereby promoting the progress and development of precision medicine for tumors;

[0036] (2) The gastrointestinal stromal tumor culture solution provided by the present application contains a plurality of gastrointestinal stromal tumor-specific cytokines, and the synergistic effect of the gastrointestinal stromal tumor-specific cytokines can activate the gastrointestinal stromal tumor cell proliferation and survival related pathways, maintain the self-renewal of tumor mesenchymal stem cells, improve the proliferation rate and cell viability of the gastrointestinal stromal tumor organoids, and realize long-term subculture and expansion. By using the culture solution and the culture method provided by the present application, a gastrointestinal stromal tumor organoid with a diameter of more than 100 μm and good activity can be obtained in 8-10 days;

[0037] (3) The gastrointestinal stromal tumor culture solution provided by the present application is suitable for culturing gastrointestinal stromal tumors with type III receptor tyrosine kinase mutations, and is also suitable for culturing wild-type gastrointestinal stromal tumors such as SDH-deficient gastrointestinal stromal tumors;

[0038] (4) The gastrointestinal stromal tumor culture solution provided by the application does not contain components with unclear components such as serum, is not affected by the quality of different batches of serum, and can maintain a high uniformity during the culture process; the culture solution and the culture method can be used for culturing gastrointestinal stromal tumor organoids derived from tumor tissues or malignant cell lines of patients, and are suitable for drug sensitivity detection.

[0039] The technical solutions of the application will be further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings required to be used in the description of the embodiments of the application will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor under the premise of not paying creative labor.

[0041] Figure 1 FIG. 1 is an inverted microscope photograph of gastrointestinal stromal tumor organoids cultured for 4 days and 8 days in Example 1 of the application, wherein 10x represents 10 times magnification, and 20x represents 20 times magnification;

[0042] Figure 2 FIG. 2 is an inverted microscope photograph of gastrointestinal stromal tumor organoids cultured for 4 days and 8 days in Example 2 of the application, wherein 10x represents 10 times magnification, and 20x represents 20 times magnification;

[0043] Figure 3 FIG. 3 is an inverted microscope photograph of SDH wild-type gastrointestinal stromal tumor organoids cultured for 4 days and 8 days in Example 3 of the application, wherein 10x represents 10 times magnification, and 20x represents 20 times magnification;

[0044] Figure 4 FIG. 4 is an inverted microscope photograph of gastrointestinal stromal tumor organoids after continuous subculture for 4 days in Example 4 of the application, wherein P0 is a non-subcultured gastrointestinal stromal tumor organoid, P1 is a gastrointestinal stromal tumor organoid subcultured once, P2 is a gastrointestinal stromal tumor organoid subcultured twice, 10x represents 10 times magnification, and 20x represents 20 times magnification;

[0045] Figure 5 FIG. 5 is an identification diagram of HE and IHC staining of gastrointestinal stromal tumor organoids cultured in Example 5 of the application, wherein a is an HE staining diagram of gastrointestinal stromal tumor organoids, b is an IHC staining diagram of CD117 marker of gastrointestinal stromal tumor organoids, c is an IHC staining of DOG-1 marker of gastrointestinal stromal tumor organoids, and 20x represents 20 times magnification;

[0046] Figure 6Figure 6 is a photograph of the inverted microscope of the gastrointestinal stromal tumor organoids cultured in Example 6 of the present application, wherein 10x represents 10 times magnification and 20x represents 20 times magnification;

[0047] Figure 7 Figure 4 is a photograph of the inverted microscope of the gastrointestinal stromal tumor organoids cultured in Comparative Example 1 of the present application on the 4th day and the 8th day, wherein 10x represents 10 times magnification and 20x represents 20 times magnification;

[0048] Figure 8 Figure 5 is a photograph of the inverted microscope of the gastrointestinal stromal tumor organoids cultured in Comparative Example 2 of the present application on the 4th day and the 8th day, wherein 10x represents 10 times magnification and 20x represents 20 times magnification;

[0049] Figure 9 Figure 6 is a photograph of the inverted microscope of the gastrointestinal stromal tumor organoids cultured in Comparative Example 3 of the present application on the 4th day and the 8th day, wherein 10x represents 10 times magnification and 20x represents 20 times magnification;

[0050] Figure 10 Figure 7 is a column chart of the cell viability of the gastrointestinal stromal tumor organoids cultured in Example 1 and Comparative Examples 1-3 of the present application;

[0051] Figure 11 Figure 8 is a result of the drug sensitivity test of the gastrointestinal stromal tumor organoids in Example 7 of the present application, wherein A is a photograph of the morphological changes of the gastrointestinal stromal tumor organoids before and after 96 hours of drug addition at different concentrations of regorafenib, and B is a regorafenib drug sensitivity dose-effect curve of the gastrointestinal stromal tumor. DETAILED DESCRIPTION

[0052] The gastrointestinal stromal tumor organoid culture solution provided by the present application comprises Advanced DMEM / F12 culture medium, StemPro MSC SFM culture medium, Wnt conditioned medium, HEPES buffer, L-alanyl-L-glutamine, N-acetyl cysteine, sodium pyruvate, nicotinamide, MEM-NEAA solution, B27 supplement, N2 supplement, primary antibiotic Primocin, penicillin-streptomycin-amphotericin b solution, epidermal growth factor EGF, fibroblast growth factor FGF4, insulin-like growth factor IGF-1, hepatocyte growth factor HGF, stem cell growth factor SCF, platelet-derived growth factor PDGF-AA, forskolin, R-spondin1, A83-01, Y-27632, ML-098, and MHY1485.

[0053] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0054] In order to make the purpose, technical scheme and advantages of the present application more clear, thorough and complete, the technical scheme of the present application is described clearly and completely below through the drawings and examples. The following detailed description is a description of the examples, which aims to provide further detailed description of the present application. Unless otherwise specified, all technical terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0055] In the description of the present application, it should be noted that, unless otherwise specified, the examples are carried out under conventional conditions or manufacturer's recommended conditions. The reagents or instruments used are not specified by the manufacturer, but are conventional products that can be purchased on the market.

[0056] Example 1

[0057] S1.1, preparation of a gastrointestinal stromal tumor organoid culture solution, comprising the following specific components, concentrations and preparation methods:

[0058] The mother liquor of each medium component is prepared using ultrapure water and DMSO solvent in a strictly sterile environment, and the concentration of each component mother liquor is as follows:

[0059] L-alanyl-L-glutamine 55 g / mL, N-acetyl cysteine 100 mM, nicotinamide 125 mM, sodium pyruvate 1 M, HEPES buffer mother liquor 1 M, forskolin 5 mM, EGF 10 μg / mL, FGF 450 μg / mL, HGF 20 μg / mL, IGF-1 50 μg / mL, SCF 50 μg / mL, PDGF-AA 25 μg / mL, 200 μg / mL R-spondin, 50 μg / mL A83-01, Y-27632 10 mM, MHY1485 1 M, ML-098 100 μM, primary antibiotic Primocin 50 mg / mL, penicillin-streptomycin double antibiotic 100 mg / mL, amphotericin b 25 mg / mL.

[0060] Prepare 500 mL Advanced DMEM / F12 medium, and then add 100 mL StemPro MSC SFM medium, 300 mL Wnt conditioned medium, 20 mL B27 supplement, 10 mL N2 supplement, 10 mL MEM-NEAA solution, 10 mL L-alanyl-L-glutamine stock solution, 10 mL N-acetyl cysteine stock solution, 10 mL nicotinamide stock solution, 5 mL HEPES buffer stock solution, 1 mL sodium pyruvate stock solution, 1 mL forskolin stock solution, 1 mL EGF stock solution, 1 mL FGF4 stock solution, 1 mL HGF stock solution, 1 mL IGF-1 stock solution, 1 mL SCF stock solution, 1 mL PDGF-AA stock solution, 1 mL R-spondin stock solution, 1 mL A-8301 stock solution, 1 mL Y-27632 stock solution, 1 mL MHY1485 stock solution, 1 mL ML-098 stock solution, 2 mL primocin stock solution, 10 mL penicillin-streptomycin stock solution, and 1 mL amphotericin B stock solution into the medium, mix well, and then dilute to 1 L with Advanced DMEM / F12 medium. The medium is ready for use after being filtered through a 0.22-μm filter in a clean bench.

[0061] S1.2. In vitro organoid culture of the patient-derived gastrointestinal stromal tumor tissue using the gastrointestinal stromal tumor organoid culture medium prepared in step S1.1, and the specific steps are as follows:

[0062] (1) Obtain 5-10 mm 3 The patient-derived gastrointestinal stromal tumor tissue is rinsed with 4℃ pre-cooled DPBS buffer, and then cut into 1-3 mm 3 size tissue pieces.

[0063] (2) Add 2.5 mL tissue digestion solution, and digest the tissue into a large number of cell clusters composed of 10-20 cells on a constant-temperature shaker at 37℃ and 100 rpm. Add 5 mL culture medium containing 5% FBS to terminate the digestion, centrifuge at 1500 rpm for 5 min to remove the supernatant, and obtain a cell pellet.

[0064] (3) Resuspend the cell pellet by adding 1 mL of the gastrointestinal stromal tumor culture medium prepared in step S1.1, filter the cell suspension through a 100-μm cell sieve, and collect the cell suspension. Centrifuge at 1500 rpm for 5 min to remove the supernatant.

[0065] (4) Resuspend the pellet by adding 100 μL of organoid culture medium, count the cells using a hemocytometer, and centrifuge to concentrate, to ensure that 1 μL of the suspension contains 15000-20000 cell clusters.

[0066] (5) 10 μL of the gastrointestinal stromal tumor cell suspension obtained in step (4) is mixed with 40 μL of Matrigel liquid drop on ice, and the mixed gel drop is inoculated into a 24-well plate and allowed to stand in a 37°C incubator for 30 min to solidify the gel drop.

[0067] (6) After the gel drop is solidified, 500 μL of the gastrointestinal stromal tumor organoid culture solution prepared in step S1 is carefully added along the wall of the well, and the 24-well plate is placed back into the cell incubator at 37°C and a CO2concentration of 5% for continued culture, with the culture solution being replaced every 4 days.

[0068] On the 4th day and the 8th day of culture, the cultured gastrointestinal stromal tumor organoids are photographed using an inverted microscope, and the photographs are as shown in FIG. 2. Figure 1 As shown in FIG. 2, the diameter of the obtained organoids is large, with a size of about 125-150 μm, the organoid cells have good coagulation, and the organoids are dense solid spheres with obvious dendritic bifurcation on the periphery, clear boundaries, and good growth activity.

[0069] Example 2

[0070] S2.1, a gastrointestinal stromal tumor organoid culture solution is prepared, and the mother solution is prepared in the same manner as in Example 1, and the specific method is as follows:

[0071] Prepare 500 mL of Advanced DMEM / F12 medium, and sequentially add 100 mL of StemPro MSC SFM medium, 300 mL of Wnt conditioned medium, 20 mL of B27 supplement, 10 mL of N2 supplement, 10 mL of MEM-NEAA solution, 10 mL of L-alanyl-L-glutamine mother solution, 10 mL of N-acetylcysteine mother solution, 10 mL of nicotinamide mother solution, 5 mL of HEPES buffer mother solution, 1 mL of sodium pyruvate mother solution, 1 mL of forskolin mother solution, 1 mL of EGF mother solution, 1 mL of FGF4 mother solution, 1 mL of HGF mother solution, 1 mL of IGF-1 mother solution, 1 mL of SCF mother solution, 1 mL of PDGF-AA mother solution, 1 mL of R-spondin mother solution, 1 mL of A-8301 mother solution, 1 mL of Y-27632 mother solution, 1 mL of MHY1485 mother solution, 2 mL of ML-098 mother solution, 2 mL of primocin mother solution, 10 mL of amphotericin B mother solution, and 1 mL of fluconazole mother solution, and then mix well, and then dilute to 1 L with Advanced DMEM / F12 medium, and then pass through a 0.22 μm filter membrane in a clean bench.

[0072] Compared with Example 1, the amount of ML-098 mother solution added in Example 2 is 2 mL, and the concentration is increased to 200 nM.

[0073] S2.2, using the gastrointestinal stromal tumor organoid culture solution prepared in step S2.1, the patient-derived gastrointestinal stromal tumor tissue obtained is cultured in vitro in the form of an organoid, and the specific steps are completely the same as those in Example 1. On the 4th day and the 8th day of culture, the gastrointestinal stromal tumor organoids are photographed using an inverted microscope, and the photographs are as shown in FIG. 2. Figure 2 As shown in FIG. 2, using the culture solution and the culture method of the present embodiment, the diameter of the cultured organoids is not significantly different from that of Example 1, and the size is about 125-150 μm. The organoid cell aggregation is good, and the organoids are dense solid spheres. The outer periphery can be seen to have obvious branching dendrites, the boundary is clear, and the growth activity is good.

[0074] Example 3

[0075] A gastrointestinal stromal tumor organoid culture solution is prepared for culturing SDH-deficient wild-type gastrointestinal stromal tumors. The preparation method of the mother liquor is the same as that of the culture solution in Example 1. The addition amount of the FGF4 mother liquor is increased to 2 mL, and the concentration is 100 ng / mL. The addition amount of the SCF mother liquor is adjusted to 0.5 mL, and the concentration is 25 ng / mL.

[0076] The culture method of the gastrointestinal stromal tumor organoids is completely the same as that in Example 1.

[0077] On the 4th day and the 8th day of culture, the gastrointestinal stromal tumor organoids are photographed using an inverted microscope, and the photographs are as shown in FIG. 3. Figure 3 As shown in FIG. 3, using the culture solution and the culture method of the present embodiment, the diameter of the cultured SDH-deficient gastrointestinal stromal tumor organoids is about 100-150 μm. The gastrointestinal stromal tumor organoid cell aggregation is good, and the organoids are mostly dense elliptical spheres. The outer periphery can be seen to have thick branching dendrites.

[0078] Example 4

[0079] The primary gastrointestinal stromal tumor organoids cultured in Example 1 are subcultured, and the specific steps are as follows:

[0080] (1) Select the culture wells with good growth, carefully aspirate the organoid culture solution in the culture wells with a pipette, rinse with 1 mL of DPBS buffer solution for 3 times, and then discard.

[0081] (2) Add 500 μL of organoid recovery solution (Corning Cat#354253) to each well, and gently blow and suck up and down for 10-20 times to disperse the matrix glue. Collect the suspension into a 1.5 mL centrifuge tube, and place it on ice for 30-60 min to dissolve the matrix glue.

[0082] (3) After the matrix glue is completely dissolved, remove the supernatant after centrifugation at 4°C and 250g for 5 min.

[0083] (4) Add TrypLE digestion enzyme to digest for 5 min at room temperature, gently blow to mix, add equal volume of organoid culture medium containing 5% FBS to terminate digestion, centrifuge at 250 g for 5 min at 4°C to remove supernatant.

[0084] (5) Resuspend the organoids in liquid Matrigel at a 1:2 to 1:3 passage ratio, and reseed in a 24-well cell culture plate at a volume of 50 μL per well.

[0085] (6) After the Matrigel droplets solidify, add 500 μL of the gastrointestinal stromal tumor organoid culture medium prepared in Example 1 to each well, and return to a cell culture incubator at 37°C with a CO2concentration of 5% for continued culture to complete the passage.

[0086] The light microscope photograph of the gastrointestinal stromal tumor organoids after 4 days of subculture is shown in FIG. 5. The gastrointestinal stromal tumor organoids maintained good cell aggregation after 2 passages using the culture medium and subculture method of this example, and were mostly dense spheres or ellipsoidal spheres with clear boundaries. After 4 days of subculture, the diameter was 125-150 μm. Figure 4 Example 5

[0087]

[0088] Immunohistochemical identification of the gastrointestinal stromal tumor organoids obtained in Example 1 was performed by embedding the organoids obtained in Example 1, and performing H&E staining and IHC staining of CD117 and DOG-1, according to the following steps:

[0089] (1) Collection and fixation of organoids: The organoids were recovered from Matrigel using the method of steps (1)-(4) of Example 4, and fixed with 4% paraformaldehyde at room temperature for 1 h. The fixed organoids were centrifuged at 250 g for 5 min at 4°C to remove the fixing solution. The organoid pellet was rinsed with 1 mL of PBS buffer for 3 times. The centrifugation step was repeated, and the supernatant was removed, and the organoid pellet was retained.

[0090] (2) Agarose embedding of organoids: The 3% agarose gel was heated in a microwave oven to completely melt, and the agarose solution was cooled to 55-60°C. 200 μL of the agarose solution was added to resuspend the organoid pellet, and transferred to a 200 μL mold, and placed on ice for 30 min until it solidified.

[0091] (3) Dehydration and permeation: The agarose block containing the organoid pellet that had solidified was removed and placed in a dehydration box, and then placed in a dehydration machine for gradient alcohol dehydration. The dehydrated agarose block was transferred to a tissue embedding box, and treated with anhydrous ethanol and xylene mixture (1:1, by volume) for 10 min, and then treated with xylene for 2 times, 10 min each time. ​

[0092] (4) Paraffin embedding: immediately transfer the agar block after the completion of the permeabilization treatment into the melted wax jar, immerse in wax for 3 times at 60℃, each time for 1 h. Wrap the organoid with embedding mold, prepare and trim the wax block.

[0093] (5) Section deparaffinization: fix the embedded wax block on the microtome, cut into 5-7 μm thick slices, and place on the glass slide after water bath. Place the section into xylene for 12 min-xylene for 12 min-absolute ethanol for 6 min-95% ethanol for 6 min-85% ethanol for 6 min, and complete deparaffinization by rinsing with distilled water.

[0094] (6) H&E staining: stain with hematoxylin for 6 min, rinse with distilled water for 1 min, then place in 1% hydrochloric acid alcohol for 2 s, then rinse with distilled water for 30 min, then immerse in 1% eosin for 2 min, and dehydrate and mount.

[0095] (7) Antigen retrieval: place the tissue section in a retrieval box filled with TRIS-EDTA retrieval solution or citrate buffer (pH 6.0), and cook in a microwave oven for antigen retrieval.

[0096] (8) Block endogenous peroxidase: add 3% hydrogen peroxide to the section, incubate at room temperature for 25 min, and shake the glass slide in PBS buffer on a decolorizing shaker for 3 times, each time for 5 min.

[0097] (9) Serum blocking: wipe the glass slide, add PBS buffer containing 5% serum (the serum needs to be consistent with the species source of the secondary antibody) to evenly cover the tissue, and block at room temperature for 1 h.

[0098] (10) Antibody incubation: absorb the blocking solution with a water-absorbing paper, add sufficient primary antibody to cover the tissue, and incubate overnight at 4℃ in a wet box. After incubation, place the glass slide in PBS buffer and shake on a decolorizing shaker for 3 times, each time for 5 min. After absorption with a water-absorbing paper, add the corresponding secondary antibody to cover the tissue, and incubate at room temperature for 50 min.

[0099] (11) DAB color development: place the glass slide in PBS buffer and shake on a decolorizing shaker for 3 times, each time for 5 min. After absorption with a water-absorbing paper, add freshly prepared DAB color developing solution, incubate at room temperature in a dark environment for 15-20 min, and monitor and control the color development time in real time under a microscope. When a brownish yellow positive reaction is observed, rinse the section with pure water to stop the color development.

[0100] (12) Counterstain the nucleus: stain with hematoxylin for 3 min, rinse with pure water, differentiate with differentiation solution for 2 s, rinse with tap water, return to blue with return blue solution for 30 s, and dehydrate and mount after rinsing with running water.

[0101] The primary antibody used in this example and the dilution factor information are as follows:

[0102] Rabbit anti-human CD117 antibody (MXB Cat#kit-0029) ready-to-use;

[0103] Rabbit anti-human DOG-1 antibody (Prosci Cat#PSI-33-698) ready-to-use.

[0104] The secondary antibody and dilution factor information used in this example are as follows:

[0105] Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Thermofisher, Cat#21234), dilution factor 1:500.

[0106] Microscope photos of the sections are shown in Figure 5 The cell morphology of the gastrointestinal stromal tumor organoids is mostly spindle-shaped or epithelioid, and the CD117 and DOG-1 markers are positively expressed. CD117 and DOG-1 are often used in the differential diagnosis of gastrointestinal stromal tumors in clinical practice. The gastrointestinal stromal tumor organoids cultured in this example meet the histochemical characteristics of gastrointestinal stromal tumors.

[0107] Example 6

[0108] The embedded sections of the gastrointestinal stromal tumor organoids obtained in Example 5 were identified by immunofluorescence, and the specific steps are as follows:

[0109] (1) Incubate the embedded sections of the gastrointestinal stromal tumor organoids with the primary antibody, and the specific steps are the same as in Example 5.

[0110] (2) Secondary antibody incubation: Place the incubated slides in PBS buffer and shake on a decolorizing shaker for 3 times, 5 min each time. After absorbing the water with a paper towel, add the fluorescent secondary antibody with HRP label, and incubate at room temperature for 50 min.

[0111] (3) Signal amplification: Wash and wipe the slides, and according to the instructions attached to the fluorescent signal amplification kit, add the corresponding color fluorescent reagent and TSA fluorescent signal enhancer, and incubate at room temperature for 5 min.

[0112] (4) DAPI restaining: Wash and wipe the slides, add 2 μg / mL of DAPI staining solution to restain the cell nucleus, and incubate at room temperature for 10 min in the dark.

[0113] (5) Mounting and photographing: Shake the washed slide on the decolorizing shaker for 3 times. After the slide is dried, use anti-fluorescence quenching mounting medium to mount the slide. Observe and collect images under an inverted fluorescence microscope. The red fluorescence area is CD117 positive expression.

[0114] The results are shown in Figure 6 FIG. 6. The CD117 immunofluorescence marker of the gastrointestinal stromal tumor organoid is positively expressed, indicating that the cultured gastrointestinal stromal tumor organoid has histochemical properties similar to the original tumor organoid.

[0115] Example 7

[0116] The regorafenib drug sensitivity test was performed on the gastrointestinal stromal tumor organoid cultured by passage in Example 4, and the specific steps are as follows:

[0117] (1) Select several wells of well-grown organoids after passage (the organoids have complete morphological structure, and the total number is ≥ 500), and add organoid recovery solution to centrifuge and remove Matrigel.

[0118] (2) Add 400 μL of TrypLE enzyme solution to the centrifuge tube, mechanically blow the suspension for 20-30 times with a wide-bore pipette tip, incubate at 37°C for 5 min, and ensure that the organoids are re-digested into numerous uniformly dispersed organoid clusters. Add an equal volume of 5% FBS-containing organoid culture medium to terminate digestion, centrifuge and remove the supernatant.

[0119] (3) Calculate the total volume of culture solution required for organoid drug sensitivity plating (3 parallel wells for each concentration, 90 μL of organoid suspension per well), and add sufficient volume of 10% (vol / vol) Matrigel-containing gastric cancer organoid culture solution to resuspend and mix well the organoids.

[0120] (4) According to the drug sensitivity experiment setting, inoculate the organoid suspension into a 384-well ultra-low attachment cell culture plate and place it in a cell culture incubator at 37°C, 5% CO2.

[0121] (5) Monitor the growth status of the organoids in real time. After 3 days of plating, if the organoids have good morphology and an average diameter greater than 50 μM, add 1:9 of the prepared 10x regorafenib drug stock solution, so that the concentration of regorafenib drug in each concentration gradient well is 0.123 μM, 0.37 μM, 1.11 μM, 3.33 μM, and 10 μM. After adding the drug, place the 384-well plate in the CO2 incubator for continuous culture.

[0122] (6) After 96 hours of drug addition, remove the 384-well plate, and according to the CellTiter-Glo 3D Cell Viability Assay Kit instructions, use a multifunctional enzyme labeler to determine the cell viability of the organoids at each drug sensitivity concentration.

[0123] (7) The dose-effect curve was drawn using Graphpad 8.0 software, and the IC50 was calculated 50 .

[0124] Figure 11 A Morphological changes of the constructed gastrointestinal stromal tumor organoids under different concentrations of regorafenib treatment. The gastrointestinal stromal tumor organoids were treated with different concentrations of regorafenib for 7 days, and the morphological changes of the gastrointestinal stromal tumor organoids were observed under an inverted microscope. Figure 11 As can be seen from A, under the treatment of regorafenib at a concentration of 0.123-1.11 μM, the gastrointestinal stromal tumor organoids showed no obvious morphological changes; under the treatment of regorafenib at a concentration of 3.33 μM and 10 μM, the gastrointestinal stromal tumor organoids only showed slight shrinkage or dispersion, indicating that the organoid sample of this patient had a certain degree of resistance to regorafenib, which was consistent with the trend of the dose-effect curve of regorafenib. The half-inhibitory concentration of regorafenib for the gastrointestinal stromal tumor organoids of this patient was 3.758 μM.

[0125] Comparative Example 1

[0126] The gastrointestinal stromal tumor organoids were cultured using a typical gastric cancer organoid culture medium, and the specific steps were as follows:

[0127] (1) The medium formula provided in Yan et al 2018 is a commonly used gastric cancer organoid culture medium. The medium was prepared as follows:

[0128] In the Advanced DMEM / F12 culture medium, 50% Wnt conditioned medium, 10% R-spondin conditioned medium, 10% noggin conditioned medium, HEPES buffer 10 mM, GlutaMax 1X, N-acetyl cysteine 1 mM, penicillin-streptomycin solution 1%, B27 supplement 2%, EGF 50 ng / mL, FGF10 200 ng / mL, Gastrin 1 nM, A-8301 2 μM, primocin 1 μg / μL, Y-27632 10 μM were added in sequence.

[0129] (2) The gastrointestinal stromal tumor organoids were cultured using the culture method of step S1.2 of Example 1, and the gastrointestinal stromal tumor organoids were photographed using an inverted microscope on the 4th day and the 8th day of culture, respectively. The photographs are shown in Figure 7 The gastrointestinal stromal tumor organoids cultured using the medium provided in this comparative example grew poorly and had poor activity, and the size of the gastrointestinal stromal tumor organoids was significantly smaller than that of the gastrointestinal stromal tumor organoids cultured in Example 1, with a diameter of only 30-40 μm.

[0130] Comparative Example 2

[0131] The gastrointestinal stromal tumor organoid culture solution was prepared and the gastrointestinal stromal tumor organoids were cultured using the culture solution, and the specific steps were as follows:

[0132] Prepare 500 mL of Advanced DMEM / F12 medium, and then add 100 mL of StemPro MSC SFM medium, 300 mL of Wnt conditioned medium, 20 mL of B27 supplement, 10 mL of N2 supplement, 10 mL of MEM-NEAA solution, 10 mL of L-alanyl-L-glutamine stock solution, 10 mL of N-acetyl cysteine stock solution, 10 mL of nicotinamide stock solution, 5 mL of HEPES buffer stock solution, 1 mL of sodium pyruvate stock solution, 1 mL of forskolin stock solution, 1 mL of EGF stock solution, 1 mL of FGF4 stock solution, 1 mL of IGF-1 stock solution, 1 mL of R-spondin stock solution, 1 mL of A-8301 stock solution, 1 mL of Y-27632 stock solution, 2 mL of primocin stock solution, 10 mL of penicillin-streptomycin stock solution, 1 mL of amphotericin b stock solution, mix well, and then add Advanced DMEM / F12 medium to 1 L, and then pass through a 0.22 μm filter membrane in a clean bench.

[0133] The component stock solution concentration used in the gastrointestinal stromal tumor organoid culture solution of the present comparative example is the same as that of Example 1, and compared with the culture solution of Example 1, HGF, SCF, PDGF-AA, ML-098, and MHY1485 are removed.

[0134] (2) The gastrointestinal stromal tumor organoids were cultured by the culture method described in step S1.2 of Example 1, and the cultured gastrointestinal stromal tumor organoids were photographed by an inverted microscope on the 4th day and the 8th day of culture, respectively. The photographs are shown in FIG. 2, and the organoids cultured in the present comparative example grew slowly and had poor activity, and the size of the organoids was obviously smaller than that of Example 1, with a diameter of 50-75 μm. Figure 8

[0135] Comparative Example 3

[0136] The gastrointestinal stromal tumor organoid culture solution was prepared and the gastrointestinal stromal tumor organoids were cultured using the culture solution, and the specific steps are as follows:

[0137] (1) Preparation of gastrointestinal stromal tumor organoid culture solution

[0138] ​Prepare 500 mL Advanced DMEM / F12 medium, and then add 100 mL StemPro MSC SFM medium, 300 mL Wnt conditioned medium, 20 mL B27 supplement, 10 mL N2 supplement, 10 mL MEM-NEAA solution, 10 mL L-alanyl-L-glutamine stock solution, 10 mL N-acetyl cysteine stock solution, 10 mL nicotinamide stock solution, 5 mL HEPES buffer stock solution, 1 mL sodium pyruvate stock solution, 1 mL forskolin stock solution, 1 mL EGF stock solution, 1 mL FGF4 stock solution, 1 mL HGF stock solution, 1 mL IGF-1 stock solution, 1 mL SCF stock solution, 1 mL PDGF-AA stock solution, 1 mL R-spondin stock solution, 1 mL A-8301 stock solution, 1 mL Y-27632 stock solution, 2 mL primocin stock solution, 10 mL penicillin-streptomycin stock solution, 1 mL amphotericin B stock solution, mix well, and then add Advanced DMEM / F12 medium to 1 L, and then pass through a 0.22 μm filter membrane in a clean bench.

[0139] The component stock solution concentration used in the gastrointestinal stromal tumor organoid culture solution of the present comparative example is the same as that of Example 1, and no ML-098 and MHY1485 are added compared with the culture solution of Example 1.

[0140] (2) The gastrointestinal stromal tumor organoids were cultured by the culture method described in step S1.2 of Example 1, and the cultured gastrointestinal stromal tumor organoids were photographed by an inverted microscope on the 4th day and the 8th day of culture, respectively. The photographs are shown in Figure 9 The gastrointestinal stromal tumor organoids cultured in the present comparative example grow slowly, and the size of the organoids is smaller than that of Example 1, with a diameter of about 75-100 μm, but the cell aggregation is good, and the boundary is clear.

[0141] Example 8

[0142] The activities of the gastrointestinal stromal tumor organoids cultured in Example 1 and Comparative Examples 1-3 were investigated, respectively, and the specific method was as follows:

[0143] The gastrointestinal stromal tumor organoids cultured in Example 1 and Comparative Examples 1-3 were incubated in a cell incubator for 4 days, and then part of them was used to determine the cell viability of the organoids using a Cell-tiler glo kit according to the attached instructions.

[0144] The results are shown in Figure 10As shown, the cell viability of the gastrointestinal stromal tumor organoids cultured in Example 1 was the highest, with a cell viability reading of 963±62 and a relative cell viability of 100±6.44%, followed by the gastrointestinal stromal tumor organoids cultured in Comparative Example 3, with a cell viability reading of 641±41 and a relative cell viability of 69.78±6.34%, the cell viability of the gastrointestinal stromal tumor organoids cultured in Comparative Example 1 was 95±8.49, and the relative cell viability was 10.63±1.46%, and the cell viability of the gastrointestinal stromal tumor organoids cultured in Comparative Example 2 was 411±2.83, and the cell viability was 45.95±3.00%.

[0145] Based on the inverted microscope photos of Example 1 and Comparative Examples 1-3, the following conclusions can be drawn:

[0146] (1) The cell viability and organoid size of the gastrointestinal stromal tumor organoids cultured in Comparative Example 1 were significantly lower than those of the other groups, indicating that the culture medium for culturing gastric cancer organoids derived from gastric epithelium is not suitable for culturing gastrointestinal stromal tumors derived from non-epithelial sources.

[0147] (2) The cell viability of the gastrointestinal stromal tumor organoids cultured in Comparative Example 2, in which HGF, SCF, PDGF-AA, ML-098, and MHY1485 were not added, was higher than that of Comparative Example 1, but still significantly lower than that of Comparative Example 3 and Example 1, indicating that HGF, SCF, PDGF-AA, and two agonists play an important role in the proliferation and growth of gastrointestinal stromal tumor organoids.

[0148] (3) The organoids in Comparative Example 3 had clear boundaries and good cell aggregation, and the cell viability was significantly improved compared to Comparative Example 2, again indicating that HGF, SCF, and PDGF-AA promote the growth of gastrointestinal stromal tumors. However, due to the absence of ML-098 and MHY1485, the cell viability was only 69.78% of that of Example 1, indicating that the synergistic effect of ML-098 and MHY1485 can further promote the proliferation and growth of cells.

[0149] Therefore, the gastrointestinal stromal tumor organoids obtained by in vitro culture using the culture medium and culture method provided by the present application can retain the histological characteristics and genetic heterogeneity of the patient's original tumor, and can be used as a drug screening model and disease research model for the treatment of gastrointestinal stromal tumors, promoting the progress and development of precision medicine for tumors. The gastrointestinal stromal tumor organoids cultured have a fast proliferation rate, high cell viability, can be passaged and expanded for a long time, and can obtain gastrointestinal stromal tumor organoids with a diameter of more than 100 μm and good activity in 8-10 days. It is also suitable for culturing wild-type gastrointestinal stromal tumors such as SDH-deficient gastrointestinal stromal tumors. It does not contain serum or other components with unclear composition, is not affected by different batches, and can maintain a high degree of uniformity during culture. It can be used to culture gastrointestinal stromal tumor organoids derived from patient tumor tissues or malignant cell lines, and is suitable for drug sensitivity testing.

[0150] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit it, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can still be modified or replaced equivalently, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A gastrointestinal stromal tumor organoid culture medium, characterized by, The components include: Advanced DMEM / F12 medium, StemPro MSC SFM medium, Wnt conditioned medium, HEPES buffer, L-alanyl-L-glutamine, N-acetyl cysteine, sodium pyruvate, nicotinamide, forskolin, MEM-NEAA solution, B27 supplement, N2 supplement, epidermal growth factor EGF, fibroblast growth factor FGF, insulin-like growth factor IGF-1, hepatocyte growth factor HGF, stem cell growth factor SCF, platelet-derived growth factor PDGF, R-spondin1, A83-01, Y-27632, ML-098, MHY1485; The fibroblast growth factor is FGF capable of specifically binding FGFR1; the FGF is FGF4; the platelet-derived growth factor PDGF is PDGF-AA; the HEPES buffer has a pH of 7.2-7.4; The Wnt conditioned medium is a medium containing secreted Wnt ligands harvested according to a standard method using an L-Wnt 3a cell line; The antibacterial component is a primary antibiotic Primocin, penicillin-streptomycin double antibiotic, and amphotericin b; The concentration of the primary antibiotic Primocin is 100 μg / mL, the concentration of the penicillin-streptomycin double antibiotic is 100 μg / mL, and the concentration of the amphotericin b solution is 25 μg / mL.

2. The gastrointestinal stromal tumor organoid culture of claim 1, wherein, The components include: Advanced DMEM / F12 medium accounting for 30-57.5% of the total volume, StemPro MSC SFM medium accounting for 10-25% of the total volume, Wnt conditioned medium accounting for 30-40% of the total volume, MEM-NEAA solution accounting for 1-2% of the total volume, B27 supplement accounting for 1-2% of the total volume, N2 supplement accounting for 0.5-1% of the total volume, 5-10 mM HEPES buffer, 500-600 mg / L L-alanyl-L-glutamine, 1-1.25 mM N-acetyl cysteine, 1-1.5 mM sodium pyruvate, 1-1.5 mM nicotinamide, 5-10 μM forskolin, 10-25 ng / mL epidermal growth factor EGF, 50-100 ng / mL fibroblast growth factor FGF4, 25-50 ng / mL insulin-like growth factor IGF-1, 10-25 ng / mL hepatocyte growth factor HGF, 25-100 ng / mL stem cell growth factor SCF, 25-50 ng / mL platelet-derived growth factor PDGF, 200-500 ng / mL R-spondin1, 50-500 ng / mL A83-01, 5-10 μM Y-27632, 100-200 nM ML-098, 0.5-2 μM MHY1485.

3. The gastrointestinal stromal tumor organoid culture of claim 1, wherein, Comprise the following components: total volume 50% of Advanced DMEM / F12 medium, total volume 10% of StemPro MSC SFM medium, total volume 30% of Wnt conditioned medium, total volume 1% of MEM-NEAA solution, total volume 2% of B27 additive, total volume 1% of N2 additive, 550 mg / L of L-alanyl-L-glutamine, 5 mM of HEPES buffer, 1 mM of N-acetyl cysteine, 1 mM of sodium pyruvate, 1.25 mM of nicotinamide, 5 μM of forskolin, 10 ng / mL of epidermal growth factor EGF, 50 ng / mL of fibroblast growth factor FGF4, 50 ng / mL of insulin-like growth factor IGF-1, 20 ng / mL of hepatocyte growth factor HGF, 50 ng / mL of stem cell growth factor SCF, 25 ng / mL of platelet-derived growth factor PDGF, 200 ng / mL of R-spondin1, 50 ng / mL of A83-01, 10 μM of Y-27632, 100 nM of ML-098, 1 μM of MHY1485.

4. A method of culturing a gastrointestinal stromal tumor organoid using the gastrointestinal stromal tumor organoid culture solution according to any one of claims 1 to 3, characterized in that, The steps are as follows: S1, obtaining the gastrointestinal stromal tumor tissue to be cultured, rinsing with pre-cooled DPBS buffer, and uniformly cutting into 1-3 mm sized tissue blocks; 3 tissue blocks; S2, adding tissue digestion solution, the gastrointestinal stromal tumor tissue block obtained in step S1 is digested at 37°C and 100 rpm on a constant temperature shaker; the composition of the tissue digestion solution is collagenase type II 1 mg / mL, hyaluronidase 20 μg / mL, dispase 0.5 mg / mL, dithiothreitol 0.5 mM; S3, the digestion condition is monitored in real time, when a large number of dissociated cell clusters can be observed under a microscope in the digestion supernatant, the digestion is terminated by adding an equal volume of culture solution containing 5% FBS, centrifuging and discarding the supernatant to obtain cell precipitate; S4, resuspend the cell precipitate with fresh gastrointestinal stromal tumor culture medium, filter and collect the cell suspension using a 100 μm cell sieve, and centrifuge again to remove the supernatant; S5, resuspend the precipitate with a small amount of organoid culture solution, count the cells and centrifuge to concentrate, ensuring that each 1 μL of suspension contains 15,000-20,000 cell clusters; S6, mix the concentrated cell suspension with Matrigel in a volume ratio of 1:4, inoculate in the form of small gel droplets in a 24-well plate, and place in a 37°C cell incubator for 0.5 h to allow the gel droplets to solidify; S7, after the gel droplets solidify, carefully add 500 μL of gastrointestinal stromal tumor organoid culture solution along the well wall, and place it back in a 37°C cell incubator with a CO2 concentration of 5% for culture, and replace the culture solution every 3-4 days.

5. The gastrointestinal stromal tumor organoid culture solution according to any one of claims 1-3 for use in gastrointestinal stromal tumor organoid culture.

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