Gastrointestinal stromal tumor organ culture solution as well as use method and application thereof

Abstract

The invention discloses a gastrointestinal stromal tumor organ culture solution as well as a use method and application thereof, and belongs to the technical field of gastrointestinal stromal tumor organ culture. The gastrointestinal stromal tumor organs obtained by adopting the culture solution and the culture method provided by the invention can retain histological characteristics and genetic heterogeneity of original tumors of patients, can be used as a medicine sieve model and a disease research model for gastrointestinal stromal tumor treatment, and can promote progressive development of tumor precision medicine; the cultured gastrointestinal stromal tumor organ is high in proliferation speed and cell viability, long-term passage and amplification can be realized, and the gastrointestinal stromal tumor organ with the diameter of more than 100 microns and good activity can be obtained in 8-10 days; the method is also suitable for culturing wild gastrointestinal stromal tumors such as SDH defect type gastrointestinal stromal tumors; the culture medium does not contain components with indefinite components such as serum, is not influenced by different batches, and can maintain relatively high homogenization in the culture process; the cell culture medium can be used for culturing gastrointestinal stromal tumor organs derived from tumor tissues or malignant cell lines of patients, and is suitable for drug sensitivity detection.

Description

Technical Field

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

[0002] Gastrointestinal stromal tumor (GIST) is a common soft tissue sarcoma of the gastrointestinal tract. It is believed to originate from interstitial cells of Cajal (ICC) or multipotent stem cells that differentiate into Cajal cells, which are responsible for pacemaking and mediating neurotransmission in the gastrointestinal tract. Its tumor tissue is mainly composed of varying numbers of spindle cells or epithelial-like cells, and immunohistochemistry often shows positive results for CD117, DOG-1, and CD34. Molecular genetic correlation studies of gastrointestinal stromal tumors have shown 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 mainly have RAS-related pathway gene mutations and succinate dehydrogenase SDH, neurofibromatosis type 1 NF1 gene function loss, etc. (Kelly et al., 2021).

[0003] Gastrointestinal stromal tumors can occur in any part of the digestive system, including the stomach, intestines, duodenum, esophagus, and mesentery, and often metastasize to adjacent tissues and organs such as the abdominal cavity, pelvic cavity, and liver. There are no obvious symptoms in the early stage of the disease. As the disease progresses, symptoms such as abdominal distension, hematemesis, and black stools may occur. According to statistics worldwide, the incidence of gastrointestinal stromal tumors is 10-15 people per 1 million. For patients who can undergo radical resection, the 5-year survival rate after surgery is 54%. For patients with adjuvant therapy and recurrence and metastasis, the median survival is only 29 months (Soreide et al., 2016, DeMatteo et al., 2000). Gastrointestinal stromal tumors are insensitive to conventional chemotherapy and radiotherapy. Although the development of drugs such as Gleevec has effectively extended the survival of patients, the heterogeneity between individuals makes it difficult for current treatments to benefit all patients. It is urgent to find a drug model with high clinical relevance to guide patients in personalized treatment and improve their prognosis.

[0004] Organoids are 3D cell complexes in vitro that are spontaneously assembled by stem cells and can reproduce some of the structures and physiological functions of organs. Patient-derived organoids (PDOs) are mini tumors formed by in vitro three-dimensional culture of tumor tissues obtained from patients through surgery or biopsy. They have the characteristics of highly restoring the histological heterogeneity and genetic map of the original tumor, and can be amplified for a long time and maintain genome stability. They have been verified as drug screening models and disease research models for precision medicine in culture systems of multiple cancer subtypes. However, there are few reports on gastrointestinal stromal tumor organoid culture media and culture methods. In view of this, it is necessary to develop effective methods for constructing gastrointestinal stromal tumor organoids in vitro to make up for the shortcomings of existing technologies and meet the needs of basic research and clinical diagnosis and treatment of gastrointestinal stromal tumors. Summary of the invention

[0005] The purpose of the present invention is to provide a gastrointestinal stromal tumor organoid culture medium and its use method and application, so as to fill the gap of the current lack of gastrointestinal stromal tumor organoid culture medium and culture method.

[0006] To achieve the above-mentioned purpose, the present invention provides a gastrointestinal stromal tumor organoid culture fluid, comprising the following components: Advanced DMEM / F12 culture medium, StemPro MSC SFM culture medium, Wnt conditioned culture medium, HEPES buffer, L-alanyl-L-glutamine, N-acetylcysteine, sodium pyruvate, nicotinamide, forskolin, MEM-NEAA solution, B27 additive, N2 additive, 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, and MHY1485.

[0007] Preferably, the invention comprises the following components: 30-57.5% of Advanced DMEM / F12 medium in total volume, 10-25% of StemPro MSC SFM medium in total volume, 30-40% of Wnt conditioned medium in total volume, 1-2% of MEM-NEAA solution in total volume, 1-2% of B27 additive in total volume, 0.5-1% of N2 additive in total volume, 5-10 mM HEPES buffer, 500-600 mg / L L-alanyl-L-glutamine, 1-1.25 mM N-acetylcysteine, 1-1.5 mM sodium pyruvate, 1-1.5 mM nicotinamide, 5-10 μM forskolin, 10-25 ng / mL epidermal growth factor EGF, 50-10 0ng / mL fibroblast growth factor FGF, 25-50ng / mL insulin-like growth factor IGF-1, 10-25ng / mL hepatocyte growth factor HGF, 25-100ng / mL stem cell growth factor SCF, 25-50ng / mL platelet-derived growth factor PDGF, 200-500ng / mL R-spondin, 50-500ng / mL A83-01, 5-10μM Y-27632, 100-200nM ML-098, 0.5-2μM MHY1485.

[0008] Preferably, the following components are included: 50% of the total volume of Advanced DMEM / F12 culture medium, 10% of the total volume of StemPro MSC SFM medium, 30% Wnt conditioned medium in total volume, 1% MEM-NEAA solution in total volume, 2% B27 supplement in total volume, 1% N2 supplement in total volume, 5mM HEPES buffer, 550mg / L L-alanyl-L-glutamine, 1mM N-acetylcysteine, 1mM sodium pyruvate, 1.25mM nicotinamide, 5μM forskolin, 10ng / mL epidermal growth factor EGF, 50ng / mL fibroblast growth factor FGF, 50ng / mL insulin-like growth factor IGF-1, 20ng / mL hepatocyte growth factor HGF, 50ng / mL stem cell growth factor SCF, 25ng / mL platelet-derived growth factor PDGF, 200ng / mL R-spondin, 50ng / mL A83-01, 10μM Y-27632, 100nM ML-098, 1μM MHY1485.

[0009] Preferably, the fibroblast growth factor is FGF4 that can specifically bind to FGFR1; the platelet-derived growth factor PDGF is one of PDGF-AA, PDGF-BB and PDGF-AB; and the pH of the HEPES buffer is 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 using L-Wnt 3a cell line according to standard methods.

[0012] Preferably, the composition further comprises antibacterial components, which are primary antibiotics Primocin, penicillin-streptomycin dual antibiotics and amphotericin B.

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

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

[0015] S1. Obtain the gastrointestinal stromal tumor tissue to be cultured, rinse it with pre-cooled DPBS buffer, and evenly cut it into 1-3 mm 3 Size of tissue blocks;

[0016] S2, adding tissue digestion solution, and digesting the gastrointestinal stromal tumor tissue block obtained in step S1 at 37° C. and 100 rpm in a constant temperature shaker; the tissue digestion solution comprises 1 mg / mL type II collagenase, 20 μg / mL hyaluronidase, 0.5 mg / mL dispase, and 0.5 mM dithiothreitol;

[0017] S3. Real-time monitoring of the digestion status. When a large number of dissociated cell clusters can be observed under a microscope in the digestion supernatant, an equal volume of culture medium containing 5% FBS is added to terminate the digestion, and the supernatant is discarded by centrifugation to obtain a cell pellet.

[0018] S4. Resuspend the cell pellet 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;

[0019] S5. Add a small amount of organoid culture medium to resuspend the pellet, count the cells and concentrate by centrifugation to ensure that each 1 μL suspension contains 15,000 to 20,000 cell clusters;

[0020] S6. The concentrated cell suspension was mixed evenly with matrix gel at a volume ratio of 1:4, inoculated into a 24-well plate in the form of small gel droplets, and placed in a cell culture incubator at 37°C for 0.5 h to allow the gel droplets to solidify;

[0021] S7. After the gel drop solidifies, carefully add 500 μL of gastrointestinal stromal tumor organoid culture medium along the wall of the hole, return to the cell culture incubator at 37°C and 5% CO2 concentration, and replace the culture medium every 3 to 4 days.

[0022] A use of the gastrointestinal stromal tumor organoid culture medium as described above in organoid culture.

[0023] StemPro MSC SFM medium (purchased from Gibco) 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 culture medium. Among them, L-alanyl-L-glutamine can be used as a nitrogen source to support the synthesis of a large amount of proteins and nucleic acids and maintain cell growth. Nicotinamide is a B3 vitamin that participates in a variety of enzymatic redox reactions, promotes energy metabolism, and accelerates the formation of organoids. N-acetylcysteine, as an antioxidant, can remove ROS in cells and inhibit cell apoptosis. MEM-NEAA solution contains a variety of non-essential amino acids such as glycine, proline, and L-aspartic acid. As a nutritional additive, it can effectively improve the ratio of cell culture medium and reduce the anabolism burden of mesenchymal cells. B27 additives and N2 additives contain a variety of nutrients such as human transferrin, insulin, progesterone, sodium selenite, putrescine, etc., which can be used as substitutes for serum for serum-free culture of organoids. Pyruvate is an intermediate metabolite of glycolysis. It can generate acetyl-CoA through the pyruvate dehydrogenase system and enter the tricarboxylic acid cycle. As a precursor for succinate synthesis, in SDH-deficient gastrointestinal stromal tumors, the addition of pyruvate helps to increase the accumulation of succinate in tumor cells.

[0025] Both stem cell factor (SCF) and platelet-derived growth factor (PDGF-AA) belong to the platelet-derived growth factor family. As ligands, they can bind to their respective type III receptor tyrosine kinases C-Kit and PDGFR, induce autophosphorylation of tyrosine residues in the transmembrane segment, and activate downstream signaling pathways such as SRC, Ras-Raf-Erk, and PLC-γ. The activation of SCF / C-Kit and PDGF / PDGFR signals plays an important role in the phenotypic maintenance, 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 sustained activation of FGF4 signals can be found in both 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 insulin receptor IR and insulin-like growth factor receptor IGFR, phosphorylate IRS, CRK, SHC and other proteins to activate PI3K / Akt, MAPK and other downstream pathways, improve the uptake of nutrients by gastrointestinal stromal tumor organoids, promote cell proliferation, accelerate cell cycle and inhibit apoptosis, and increase the volume and survival rate of gastrointestinal stromal tumor organoids.

[0028] Hepatocyte growth factor (HGF) is a ligand of the receptor tyrosine kinase C-MET expressed by mesenchymal cells. HGF / C-MET signaling activation can coordinate cell migration, proliferation, angiogenesis, epithelial-mesenchymal transition and other cellular processes, and enhance GIST cell viability. Compensatory activation of C-MET signaling 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 line, which can activate the Wnt / β-Catenin pathway to maintain the stemness of tumor cells. R-spondin, as a ligand of LGR4-6 receptor, can act as an agonist of Wnt signaling to enhance the signal transduction of β-catenin, allowing organoids to be cultured stably for a long time.

[0030] Forskolin is an effective adenylate cyclase activator that can increase the concentration of the second messenger cAMP in cells and enhance the conduction of cell signals.

[0031] Y-27632 is a small molecule inhibitor of the Rho-associated protein kinase p160ROCK. It can target and bind to the catalytic sites of ROCK-1 and ROCK-2, inhibit cell anoikis, and induce pluripotent stem cells to selectively differentiate into mesenchymal cell lineages through epithelial-mesenchymal transition (EMT).

[0032] ML-098 is a small molecule agonist of Ras and related binding protein Rab. The present invention adds ML-098 to the culture medium, which can enhance the upstream activated RTK-RAS signal by increasing the affinity of GTPase protein in tumor cells to guanine nucleotides, thereby promoting the growth and self-assembly of gastrointestinal stromal tumor organoids.

[0033] MHY1485 is an mTOR activator with a morpholinotriazine structure, which can phosphorylate the ser2448 site of mTOR to activate the mTOR protein and its downstream protein ribosomal S6K1 and cellular translation initiation factor 4EP1, thereby enhancing cellular protein synthesis and translation and glycolysis metabolism, and promoting further growth of gastrointestinal stromal tumor organoids. Studies have found that the activation of mTOR signaling 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 medium and its use method and application provided by the present invention have the following specific technical effects:

[0035] (1) Gastrointestinal stromal tumor organoids can be obtained by in vitro culture using the culture medium and culture method provided by the present invention, which 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, thereby promoting the advancement of precision medicine for tumors;

[0036] (2) The gastrointestinal stromal tumor culture fluid of the present invention contains a variety of gastrointestinal stromal tumor-specific cytokines, the synergistic effect of which can activate gastrointestinal stromal tumor cell proliferation and survival-related pathways, maintain the self-renewal of tumor mesenchymal stem cells, increase the proliferation rate and cell viability of gastrointestinal stromal tumor organoids, and achieve long-term passage and expansion. Using the culture fluid and culture method of the present invention, gastrointestinal stromal tumor organoids with a diameter of more than 100 μm and good activity can be obtained in 8 to 10 days;

[0037] (3) The gastrointestinal stromal tumor culture medium provided by the present invention 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 medium provided by the present invention does not contain components with unclear composition such as serum, is not affected by the quality of serum from different batches, and can maintain a high degree of uniformity during the culture process; the culture medium and culture method of the present invention can be used to culture gastrointestinal stromal tumor organoids derived from patient tumor tissues or malignant cell lines, and are suitable for drug sensitivity testing.

[0039] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0041] Figure 1 These are inverted microscope photos of gastrointestinal stromal tumor organoids on the 4th and 8th days of culture in Example 1 of the present invention, where 10× represents 10 times magnification and 20× represents 20 times magnification;

[0042] Figure 2 These are inverted microscope photos of gastrointestinal stromal tumor organoids on the 4th and 8th days of culture in Example 2 of the present invention, where 10× represents 10 times magnification and 20× represents 20 times magnification;

[0043] Figure 3 These are inverted microscope photos of SDH wild-type gastrointestinal stromal tumor organoids on the 4th and 8th days of gastrointestinal stromal tumor organoid culture in Example 3 of the present invention, where 10× represents 10 times magnification and 20× represents 20 times magnification;

[0044] Figure 4 This is an inverted microscope photograph of the gastrointestinal stromal tumor organoid after continuous subculture for 4 days in Example 4 of the present invention, wherein P0 is a gastrointestinal stromal tumor organoid that has not been subcultured, P1 is a gastrointestinal stromal tumor organoid that has been subcultured once, and P2 is a gastrointestinal stromal tumor organoid that has been subcultured twice. 10× indicates a magnification of 10 times, and 20× indicates a magnification of 20 times;

[0045] Figure 5 HE and IHC staining identification images of gastrointestinal stromal tumor organoids cultured in Example 5 of the present invention, wherein a is a HE staining image of gastrointestinal stromal tumor organoids, b is an IHC staining image of the CD117 marker of gastrointestinal stromal tumor organoids, and c is an IHC staining image of the DOG-1 marker of gastrointestinal stromal tumor organoids, and 20× indicates a magnification of 20 times;

[0046] Figure 6This is an immunofluorescence staining identification diagram of the CD117 marker of the gastrointestinal stromal tumor organoid cultured in Example 6 of the present invention, wherein 20× means 20 times magnification;

[0047] Figure 7 These are inverted microscope photos of gastrointestinal stromal tumor organoids cultured on the 4th and 8th days in Comparative Example 1 of the present invention, where 10× means 10 times magnification and 20× means 20 times magnification;

[0048] Figure 8 These are inverted microscope photos of gastrointestinal stromal tumor organoids cultured on the 4th and 8th days in Comparative Example 2 of the present invention, where 10× means 10 times magnification and 20× means 20 times magnification;

[0049] Fig. 9 These are inverted microscope photos of gastrointestinal stromal tumor organoids cultured on the 4th and 8th days in Comparative Example 3 of the present invention, where 10× means 10 times magnification and 20× means 20 times magnification;

[0050] Fig.10 This is a bar graph of cell viability of gastrointestinal stromal tumor organoids cultured in Example 1 and Comparative Examples 1 to 3 of the present invention;

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

[0052] The gastrointestinal stromal tumor organoid culture fluid provided by the present invention comprises Advanced DMEM / F12 culture medium, StemPro MSC SFM culture medium, Wnt conditioned culture medium, HEPES buffer, L-alanyl-L-glutamine, N-acetylcysteine, sodium pyruvate, nicotinamide, MEM-NEAA solution, B27 additive, N2 additive, 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 solution of the present invention is further described below through the accompanying drawings and embodiments.

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

[0055] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0056] Example 1

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

[0058] Use ultrapure water and DMSO solvent to prepare the stock solutions of each culture medium component in a strictly sterile environment. The concentrations of the stock solutions of each component are as follows:

[0059] L-Alanyl-L-glutamine 55g / mL, N-acetylcysteine ​​100mM, Nicotinamide 125mM, Sodium pyruvate 1M, HEPES buffer stock solution 1M, Forskolin 5mM, EGF 10μg / mL, FGF450μg / mL, HGF 20μg / mL, IGF-150μg / mL, SCF 50μg / mL, PDGF-AA25μg / mL, 200μg / mL R-spondin, 50μg / mL A83-01, Y-2763210mM, MHY14851M, ML-098100μM, primary antibiotic Primocin 50mg / mL, Penicillin-Streptomycin dual antibody 100mg / mL, Amphotericin B 25mg / mL.

[0060] Prepare 500 mL Advanced DMEM / F12 medium, and 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-acetylcysteine ​​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 L IGF-1 stock solution, 1 mL SCF stock solution, 1 mL PDGF-AA stock solution, and 1 mL R-spondin stock solution, 1mL A-8301 stock solution, 1mL Y-27632 stock solution, 1mL MHY1485 stock solution, 1mL ML-098 stock solution, 2mL primary antibiotic primocin stock solution, 10mL penicillin-streptomycin dual antibody stock solution, 1mL amphotericin B stock solution, stir and mix well, then use Advanced DMEM / F12 culture medium to make up to 1L, and then filter through a 0.22μm filter membrane in an ultra-clean workbench.

[0061] S1.2. Using the gastrointestinal stromal tumor organoid culture medium prepared in step S1.1, perform in vitro organoid culture on the gastrointestinal stromal tumor tissue obtained from the patient, and the specific steps are as follows:

[0062] (1) Obtain 5-10 mm 3 Gastrointestinal stromal tumor tissue from patients was rinsed with 4°C precooled DPBS buffer and evenly cut into 1-3 mm 3 Size of tissue blocks.

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

[0064] (3) Add 1 mL of the gastrointestinal stromal tumor culture medium prepared in step S1.1 to resuspend the cell pellet, then filter and collect the cell suspension using a 100 μm cell sieve, and centrifuge again at 1500 rpm for 5 min to remove the supernatant.

[0065] (4) Add 100 μL of organoid culture medium to resuspend the pellet, count the cells using a hemocytometer, and concentrate by centrifugation to ensure that each 1 μL of suspension contains 15,000 to 20,000 cell clusters.

[0066] (5) 10 μL of the gastrointestinal stromal tumor cell suspension obtained in step (4) was taken and mixed evenly with 40 μL of matrix gel droplets on ice. The mixed gel droplets were inoculated into a 24-well plate and placed in a 37°C incubator for 30 min to allow the gel droplets to solidify.

[0067] (6) After the gel drop solidifies, carefully add 500 μL of the gastrointestinal stromal tumor organoid culture medium prepared in step S1 and preheated at 37°C along the well wall, and place the 24-well plate back into a cell culture incubator at 37°C and a CO2 concentration of 5% for continued culture, with the culture medium replaced every 4 days.

[0068] On the 4th and 8th day of culture, the cultured gastrointestinal stromal tumor organoids were photographed using an inverted microscope. Figure 1 As shown, using the culture medium and culture method provided by the present invention, the obtained organoids have a larger diameter, with a size of about 125 to 150 μm, and the organoid cells have good cohesion, appearing as dense solid spheres with obvious dendritic bifurcations on the periphery, clear boundaries, and good growth activity.

[0069] Example 2

[0070] S2.1. Prepare a gastrointestinal stromal tumor organoid culture medium. The mother liquid preparation method is the same as that in Example 1. The specific method is as follows:

[0071] Prepare 500 mL Advanced DMEM / F12 medium, and 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-acetylcysteine ​​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 L IGF-1 stock solution, 1 mL SCF stock solution, 1 mL PDGF-AA stock solution, and 1 mL R-spondin stock solution, 1mL A-8301 stock solution, 1mL Y-27632 stock solution, 1mL MHY1485 stock solution, 2mL ML-098 stock solution, 2mL primary antibiotic primocin stock solution, 10mL penicillin-streptomycin dual antibody stock solution, 1mL amphotericin B stock solution, stir and mix well, then make up to 1L with Advanced DMEM / F12 culture medium, and then filter through a 0.22μm 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 medium prepared in step S2.1, the obtained patient-derived gastrointestinal stromal tumor tissue is cultured in vitro, and the specific steps are exactly the same as those in Example 1. On the 4th day and the 8th day of culture, the cultured gastrointestinal stromal tumor organoids are photographed using an inverted microscope, as shown in the following figure: Figure 2 As shown, using the culture medium and culture method of this example, the diameter of the cultured organoids is no different from that of Example 1, and the size is about 125-150 μm. The organoid cells have good cohesion and are dense solid spheres. Obvious dendritic bifurcation can be seen on the periphery, with clear boundaries and good growth activity.

[0074] Example 3

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

[0076] The method for culturing gastrointestinal stromal tumor organoids is exactly the same as in Example 1.

[0077] On the 4th and 8th day of culture, the cultured gastrointestinal stromal tumor organoids were photographed using an inverted microscope. Figure 3 As shown, the SDH-deficient gastrointestinal stromal tumor cultured using the culture medium and culture method of this example has a gastrointestinal stromal tumor organoid diameter of about 100-150 μm, and the gastrointestinal stromal tumor organoid cells have good cohesion, mostly appearing as dense elliptical spheres, with thick branch-like branches visible on the periphery.

[0078] Example 4

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

[0080] (1) Select a well with good growth, carefully remove the organoid culture medium in the well with a pipette, add 1 mL of DPBS buffer to rinse three times, and then discard it again.

[0081] (2) Add 500 μL of organoid recovery solution (Corning Cat#354253) to each well and gently pipette up and down 10 to 20 times to break up the matrix gel. Collect the suspension into a 1.5 mL centrifuge tube and place on ice for 30 to 60 min to allow the matrix gel to decompose.

[0082] (3) After the matrix gel was completely dissolved, centrifuge at 4°C and 250 g for 5 min and remove the supernatant.

[0083] (4) Add TrypLE digestion enzyme and digest at room temperature for 5 min. Gently pipette to mix evenly. Add an equal volume of organoid culture medium containing 5% FBS to terminate digestion. Centrifuge again at 4°C and 250 g for 5 min and remove the supernatant.

[0084] (5) Resuspend the organoid pellet in liquid Matrigel at a subculture ratio of 1:2 to 1:3 and re-seed in a 24-well cell culture plate at a volume of 50 μL per well.

[0085] (6) After the matrix gel droplets solidified, 500 μL of the gastrointestinal stromal tumor organoid culture medium prepared in Example 1 was added to each well, and the cells were returned to the cell culture incubator at 37° C. and a CO 2 concentration of 5% for continued culture to complete the subculture.

[0086] Light microscopic photograph of gastrointestinal stromal tumor organoids cultured for 4 days Figure 4 As shown, using the culture medium and subculture method of this example, after two subcultures, the gastrointestinal stromal tumor organoids still maintained good cell cohesion, mostly dense spheres or ellipsoids with clear boundaries. After 4 days of culture after subculture, the diameter can reach 125-150 μm.

[0087] Example 5

[0088] The gastrointestinal stromal tumor organoids obtained in Example 1 were subjected to immunohistochemical identification. After the organoids obtained in Example 1 were embedded and sliced, H&E staining and IHC staining of CD117 and DOG-1 were performed. The specific steps are as follows:

[0089] (1) Collection and fixation of organoids: Organoids were recovered from the matrix gel using the method of steps (1) to (4) of Example 4 using organoid recovery solution (Corning Cat#354253), fixed with 4% paraformaldehyde at room temperature for 1 h, and centrifuged at 4°C and 250 g for 5 min to remove the fixative. 1 mL of PBS buffer was added to rinse the organoid pellet three times. The centrifugation step was repeated, the supernatant was removed, and the organoid pellet was retained.

[0090] (2) Agarose embedding of organoids: Heat 3% agarose gel in a microwave oven until it is completely melted. When the agarose solution cools to 55-60°C, add 200 μL of agarose solution to resuspend the organoid pellet, transfer it to a 200 μL mold, and place it on ice for 30 min to solidify.

[0091] (3) Dehydration and permeabilization: Take out the solidified agarose block containing the organoid precipitate and place it in a dehydration box. Then place the dehydration box in a dehydrator and perform gradient alcohol dehydration in sequence. After dehydration, transfer the agarose block to a tissue embedding box and treat it with anhydrous ethanol and xylene mixture (anhydrous ethanol: xylene volume ratio is 1:1) for 10 minutes, then move it to xylene for treatment twice, each time for 10 minutes.

[0092] (4) Paraffin embedding: After permeabilization, transfer the agar block immediately into a melted wax tank and immerse it in wax at 60°C for 3 times, each time for 1 hour. Wrap the organoids with an embedding mold and prepare and trim the wax block.

[0093] (5) Dewaxing of sections: Fix the embedded wax block on the microtome, cut into thin slices of 5-7 μm thickness, flatten in a water bath and place on a glass slide. Place the sections in xylene for 12 min, xylene for 12 min, anhydrous ethanol for 6 min, 95% ethanol for 6 min, and 85% ethanol for 6 min, and rinse with distilled water to complete dewaxing.

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

[0095] (7) Antigen retrieval: Place the tissue sections 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) Blocking endogenous peroxidase: Add 3% hydrogen peroxide to the sections and incubate at room temperature for 25 min. Place the slides in PBS buffer and wash them three times on a decolorizing shaker for 5 min each time.

[0097] (9) Serum blocking: Wipe the slide clean, add PBS buffer containing 5% serum (the serum must be of the same species as the secondary antibody) to evenly cover the tissue, and block at room temperature for 1 h.

[0098] (10) Antibody incubation: Remove the blocking solution with absorbent paper, add enough primary antibody to cover the tissue, and incubate it flat in a humidified chamber at 4°C overnight. After incubation, place the slide in PBS buffer and wash it on a decolorizing shaker three times, 5 minutes each time. After removing the blocking solution with absorbent paper, add the corresponding secondary antibody to cover the tissue and incubate it at room temperature for 50 minutes.

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

[0100] (12) Counterstaining of cell nuclei: stain with hematoxylin for 3 min, rinse with pure water, differentiate with differentiation solution for 2 s, rinse with tap water, blue with bluing solution for 30 s, rinse with running water, dehydrate and seal the slide.

[0101] The primary antibodies and dilution factors used in this example 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) is ready to use.

[0104] The secondary antibodies and dilution factors 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 slices Figure 5 As shown, the cell morphology of gastrointestinal stromal tumor organoids is mostly spindle-shaped or epithelial-like, and both 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 gastrointestinal stromal tumor organoids obtained in Example 5 were subjected to immunofluorescence identification, and the specific steps were as follows:

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

[0110] (2) Secondary antibody incubation: Place the incubated slides in PBS buffer and wash on a decolorizing shaker for 3 times, 5 minutes each time. After blotting with absorbent paper, add HRP-labeled fluorescent secondary antibody and incubate at room temperature for 50 minutes.

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

[0112] (4) DAPI counterstaining: Wash and clean the slides, add 2 μg / mL DAPI staining solution to counterstain the cell nuclei, and incubate at room temperature for 10 min in the dark.

[0113] (5) Sealing and photographing: Wash the slides three times on a decolorizing shaker. After the slides are dried, seal them with anti-fluorescence quenching sealing agent. Observe and collect images using an inverted fluorescence microscope. The red fluorescent area indicates CD117 positive expression.

[0114] The results are as follows Figure 6 As shown, the CD117 immunofluorescence marker of gastrointestinal stromal tumor organoids was positively expressed, indicating that the cultured gastrointestinal stromal tumor organoids had the histochemical characteristics of the original tumor organoids.

[0115] Example 7

[0116] The gastrointestinal stromal tumor organoids subcultured in Example 4 were tested for regorafenib drug sensitivity, and the specific steps were as follows:

[0117] (1) Select several wells of organoids that have grown well after subculturing (organoids with complete morphology and structure, total number ≥ 500), add organoid recovery solution, and centrifuge to remove the matrix gel.

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

[0119] (3) Calculate the total volume of culture medium required for organoid drug sensitivity plating (set up 3 parallel wells for each concentration, and 90 μL of organoid suspension in each well), add sufficient volume of gastric cancer organoid culture medium containing 10% (vol / vol) matrix gel to resuspend and thoroughly mix the organoids.

[0120] (4) According to the settings of the drug sensitivity test, the organoid suspension was inoculated into a 384-well ultra-low adsorption cell culture plate and cultured in a cell culture incubator at 37°C and 5% CO2.

[0121] (5) Monitor the growth of organoids in real time. If the organoids have good morphology and an average diameter greater than 50 μM 3 days after plating, add the prepared 10× regorafenib drug stock solution at a ratio of 1:9, so that the concentration of regorafenib in each concentration gradient culture 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 a CO2 incubator for continued culture.

[0122] (6) 96 h after drug addition, the 384-well plate was removed and the cell viability of organoids at each drug sensitivity concentration was measured using a multifunctional microplate reader according to the instructions of the CellTiter-Glo 3D Cell Viability Assay Kit.

[0123] (7) Use Graphpad 8.0 software to draw the dose-effect curve and calculate the IC 50 .

[0124] Fig.11 A shows the morphological changes of the constructed gastrointestinal stromal tumor organoids under the treatment of different concentrations of regorafenib. Fig.11 A shows that at the regorafenib treatment concentration of 0.123-1.11 μM, the gastrointestinal stromal tumor organoids showed no obvious morphological changes; at the regorafenib treatment concentrations of 3.33 μM and 10 μM, the gastrointestinal stromal tumor organoids showed only slight shrinkage or dispersion, indicating that the patient's organoid samples 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 the patient's gastrointestinal stromal tumor organoids to regorafenib was 3.758 μM.

[0125] Comparative Example 1

[0126] Gastrointestinal stromal tumor organoids were cultured using typical gastric cancer organoid culture medium. The specific steps are as follows:

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

[0128] To Advanced DMEM / F12 medium, 50% Wnt conditioned medium, 10% R-spondin conditioned medium, 10% noggin conditioned medium, HEPES buffer 10mM, GlutaMax 1X, N-acetylcysteine ​​1mM, penicillin-streptomycin solution volume concentration 1%, B27 supplement volume concentration 2%, EGF 50ng / mL, FGF 10200ng / mL, Gastrin 1nM, A-830 12μM, primocin 1μg / μL, and Y-2763210μM were added in sequence.

[0129] (2) Gastrointestinal stromal tumor organoids were cultured using the culture method of step S1.2 of Example 1. The cultured gastrointestinal stromal tumor organoids were photographed using an inverted microscope on the 4th and 8th days of culture. The photographs are as follows: Figure 7 As shown, the gastrointestinal stromal tumor organoids cultured with the culture medium provided in this comparative example have poor growth status and poor activity, and the size of the gastrointestinal stromal tumor organoids is significantly smaller than that of the gastrointestinal stromal tumor organoids cultured in Example 1, with a diameter of only 30 to 40 μm.

[0130] Comparative Example 2

[0131] The gastrointestinal stromal tumor organoid culture medium is prepared and the gastrointestinal stromal tumor organoid is cultured using the culture medium. The specific steps are as follows:

[0132] Prepare 500 mL Advanced DMEM / F12 medium, 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-acetylcysteine ​​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 L IGF-1 stock solution, 1 mL R-spondin stock solution, 1 mL A-8301 stock solution, 1 mL L-27632 stock solution, 2 mL primary antibiotic primocin stock solution, 10 mL penicillin-streptomycin dual antibody stock solution, 1 mL amphotericin B stock solution, mix well and use Advanced DMEM / F12 medium was diluted to 1L, and then filtered through a 0.22μm filter membrane in a clean bench.

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

[0134] (2) The gastrointestinal stromal tumor organoids were cultured using the culture method described in step S1.2 of Example 1. The cultured gastrointestinal stromal tumor organoids were photographed using an inverted microscope on the 4th and 8th days of culture. The photographs are as follows: Figure 8 As shown, the organoids cultured in this comparative example grew slowly and had poor activity. The size of the organoids was significantly smaller than that of Example 1, with a diameter of 50 to 75 μm.

[0135] Comparative Example 3

[0136] The gastrointestinal stromal tumor organoid culture medium is prepared and the gastrointestinal stromal tumor organoid is cultured using the culture medium. The specific steps are as follows:

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

[0138] Prepare 500 mL Advanced DMEM / F12 medium, 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-acetylcysteine ​​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 L IGF-1 stock solution, 1 mL SCF stock solution, 1 mL PDGF-AA stock solution, 1 mL R-spondin stock solution, 1mL A-8301 stock solution, 1mL Y-27632 stock solution, 2mL primary antibiotic primocin stock solution, 10mL penicillin-streptomycin dual antibody stock solution, 1mL amphotericin B stock solution, stir and mix well, then dilute to 1L with Advanced DMEM / F12 culture medium, and then filter through a 0.22μm filter membrane in a clean bench.

[0139] The concentration of the component stock solutions used in the gastrointestinal stromal tumor organoid culture medium of this comparative example is the same as that in Example 1. Compared with the culture medium of Example 1, ML-098 and MHY1485 are not added.

[0140] (2) The gastrointestinal stromal tumor organoids were cultured using the culture method described in step S1.2 of Example 1. The cultured gastrointestinal stromal tumor organoids were photographed using an inverted microscope on the 4th and 8th days of culture. The photographs are as follows: Fig. 9 As shown, the gastrointestinal stromal tumor organoids cultured in this comparative example grew slowly, and the size of the organoids was smaller than that of Example 1, with a diameter of about 75 to 100 μm, but the cells had good cohesion and clear boundaries.

[0141] Example 8

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

[0143] For the gastrointestinal stromal tumor organoids cultured in Example 1 and Comparative Examples 1 to 3, respectively, on the 4th day of incubation in a cell culture incubator, a portion was taken and the cell viability of the organoids was measured using the Cell-tiler glo kit according to the attached instructions.

[0144] The results are as follows Fig.10As shown, the gastrointestinal stromal tumor organoids cultured in Example 1 had the highest cell viability, 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 gastrointestinal stromal tumor organoids cultured in Comparative Example 1 had a cell viability reading of 95±8.49 and a relative cell viability of 10.63±1.46%, and the gastrointestinal stromal tumor organoids cultured in Comparative Example 2 had a cell viability reading of 411±2.83 and a cell viability of 45.95±3.00%.

[0145] The following conclusions can be drawn from the inverted microscope photos of Example 1 and Comparative Examples 1 to 3:

[0146] (1) The cell viability and organoid size of gastrointestinal stromal tumor organoids cultured in Comparative Example 1 were significantly lower than those in 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 of non-epithelial origin.

[0147] (2) In Comparative Example 2, HGF, SCF, PDGF-AA, ML-098, and MHY1485 were not added to the culture medium. Compared with Comparative Example 1, the viability of cultured gastrointestinal stromal tumor organoid cells was improved, but still significantly lower than that of Comparative Example 3 and Example 1, indicating that the three cytokines HGF, SCF, and PDGF-AA and the 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 morphological boundaries and good cell cohesion. The viability of the organoids was significantly improved compared with that in Comparative Example 2, which again demonstrated the growth-promoting effects of HGF, SCF, and PDGF-AA on gastrointestinal stromal tumors. However, since ML-098 and MHY1485 were not added, the cell viability was only 69.78% of that in Example 1, indicating that the synergistic effect of ML-098 and MHY1485 can further promote cell proliferation and growth.

[0149] Therefore, the gastrointestinal stromal tumor organoids obtained by in vitro culture using the culture medium and culture method provided by the present invention 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, thereby promoting the progress and development of tumor precision medicine; the cultured gastrointestinal stromal tumor organoids have a fast proliferation rate and a high cell viability, and can achieve long-term passage and expansion. Gastrointestinal stromal tumor organoids with a diameter of more than 100 μm and good activity can be obtained in 8 to 10 days; they are also suitable for culturing wild-type gastrointestinal stromal tumors such as SDH-deficient ones; they do not contain components with unclear components such as serum, are not affected by different batches, and can maintain a high degree of uniformity during the culture process; they can be used to culture gastrointestinal stromal tumor organoids derived from patient tumor tissues or malignant cell lines, and are suitable for drug sensitivity detection.

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A gastrointestinal stromal tumor organoid culture medium, characterized in that: Includes the following components: Advanced DMEM / F12 medium, StemPro MSC SFM 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.

2. The gastrointestinal stromal tumor organoid culture medium according to claim 1, characterized in that: The invention comprises the following components: Advanced DMEM / F12 culture medium with a total volume of 30-57.5%, StemPro MSC SFM culture medium with a total volume of 10-25%, Wnt conditioned culture medium with a total volume of 30-40%, MEM-NEAA solution with a total volume of 1-2%, B27 additive with a total volume of 1-2%, N2 additive with a total volume of 0.5-1%, HEPES buffer with a total volume of 5-10 mM, L-alanyl-L-glutamine with a total volume of 500-600 mg / L, N-acetylcysteine ​​with a total volume of 1-1.25 mM, sodium pyruvate with a total volume of 1-1.5 mM, nicotinamide with a total volume of 1-1.5 mM, forskolin with a total volume of 5-10 μM, epidermal growth factor EGF with a total volume of 10-25 ng / mL, and 50-100 ng / mL. / mL of fibroblast growth factor FGF, 25-50ng / mL of insulin-like growth factor IGF-1, 10-25ng / mL of hepatocyte growth factor HGF, 25-100ng / mL of stem cell growth factor SCF, 25-50ng / mL of platelet-derived growth factor PDGF, 200-500ng / mL of R-spondin, 50-500ng / mL of A83-01, 5-10μM Y-27632, 100-200nM ML-098, and 0.5-2μM MHY1485.

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

4. The gastrointestinal stromal tumor organoid culture medium according to any one of claims 1 to 3, characterized in that: The fibroblast growth factor is FGF that can specifically bind to FGFR1; the platelet-derived growth factor PDGF is one of PDGF-AA, PDGF-BB and PDGF-AB; and the pH of the HEPES buffer is 7.2-7.

4.

5. The gastrointestinal stromal tumor organoid culture medium according to claim 4, characterized in that: The FGF is FGF4; the platelet-derived growth factor is PDGF-AA.

6. The gastrointestinal stromal tumor organoid culture medium according to any one of claims 1 to 3, characterized in that: The Wnt conditioned medium is a medium containing secreted Wnt ligands harvested by fermentation using L-Wnt 3a cell line according to standard methods.

7. The gastrointestinal stromal tumor organoid culture medium according to claim 1, characterized in that: It also includes antibacterial components, which are primary antibiotics Primocin, penicillin-streptomycin dual antibiotics and amphotericin B.

8. The gastrointestinal stromal tumor organoid culture medium according to claim 7, characterized in that: The concentration of the primary antibiotic Primocin is 100 μg / mL, the concentration of the penicillin-streptomycin dual antibiotic is 100 μg / mL, and the concentration of the amphotericin B solution is 25 μg / mL.

9. A method for culturing gastrointestinal stromal tumor organoids using the gastrointestinal stromal tumor organoid culture medium according to any one of claims 1 to 8, characterized in that: Here are the steps: S1. Obtain the gastrointestinal stromal tumor tissue to be cultured, rinse it with pre-cooled DPBS buffer, and evenly cut it into 1-3 mm 3 Size of tissue blocks; S2, adding tissue digestion solution, and digesting the gastrointestinal stromal tumor tissue block obtained in step S1 at 37° C. and 100 rpm in a constant temperature shaker; the tissue digestion solution comprises 1 mg / mL type II collagenase, 20 μg / mL hyaluronidase, 0.5 mg / mL dispase, and 0.5 mM dithiothreitol; S3. Real-time monitoring of the digestion status. When a large number of dissociated cell clusters can be observed under a microscope in the digestion supernatant, an equal volume of culture medium containing 5% FBS is added to terminate the digestion, and the supernatant is discarded by centrifugation to obtain a cell pellet. S4. Resuspend the cell pellet 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. Add a small amount of organoid culture medium to resuspend the pellet, count the cells and concentrate by centrifugation to ensure that each 1 μL suspension contains 15,000 to 20,000 cell clusters; S6. The concentrated cell suspension was mixed evenly with matrix gel at a volume ratio of 1:4, inoculated into a 24-well plate in the form of small gel droplets, and placed in a cell culture incubator at 37°C for 0.5 h to allow the gel droplets to solidify; S7. After the gel drop solidifies, carefully add 500 μL of gastrointestinal stromal tumor organoid culture medium along the wall of the hole, return to the cell culture incubator at 37°C and 5% CO2 concentration, and replace the culture medium every 3 to 4 days.

10. Use of the gastrointestinal stromal tumor organoid culture medium according to any one of claims 1 to 8 in organoid culture.

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