A medium, method of constructing and culturing a rhabdomyosarcoma organoid

Abstract

The application discloses a rhabdomyosarcoma organoid culture medium, a construction method and a culture method, and belongs to the technical field of organoid model construction. The culture medium comprises a basic culture medium, basic components, specific factor components and additive components; the specific factor components comprise the following components with terminal concentrations: 5-20 mu g / mL heparin, 50-200 ng / mL IGF1, 10-100 ng / mL HGF, 30-80 ng / mL FGF-2 and 20-90 mu M A 83-01. The application has the beneficial effects that the application proposes a culture medium for culturing rhabdomyosarcoma organoids, can efficiently transform the rhabdomyosarcoma tissues removed by surgery into rhabdomyosarcoma organoids, the construction success rate can be as high as 95% or more, and the rhabdomyosarcoma organoids obtained are consistent with the HE staining results of the original surgical tissues. The application intends to solve the above technical problems, establish a rapid and effective rhabdomyosarcoma construction scheme, provide help for personalized medication of patients, provide a new platform for disease research and drug research of patients, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of organoid model construction technology, specifically relating to a culture medium, construction and culture method for rhabdomyosarcoma organoids. Background Technology

[0002] Rhabdomyosarcoma originates from skeletal muscle cells or mesenchymal cells differentiating into skeletal muscle cells. It is the most common soft tissue tumor in childhood and is a highly malignant myoblastic tumor of skeletal muscle. Although complete differentiation of skeletal muscle fails, myogenic markers are still expressed. It can be classified into embryonal rhabdomyosarcoma, acinar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma, and botryoid rhabdomyosarcoma. It is more common in children and extremely rare in adults. Males are less likely to be affected than females. It commonly occurs in the head and neck region. Based on the patient's age, tumor size, pathology, and clinical stage, rhabdomyosarcoma can be divided into low-, intermediate-, and high-risk groups for stratified and comprehensive treatment.

[0003] Patients often experience pain and bleeding due to tumor compression, and are prone to early metastasis and recurrence, severely impacting treatment outcomes and long-term prognosis. The primary treatment is surgical resection followed by radiotherapy and chemotherapy. Only 10% of rhabdomyosarcomas can be completely removed, and up to 37.7% may metastasize. Therefore, even for patients with complete resection, chemotherapy and radiotherapy are necessary. Chemotherapy drugs target all cells in the body, including tumor cells and normal cells, meaning that the drugs may cause side effects on normal cells. Most side effects disappear after discontinuation of the medication. For rhabdomyosarcoma patients, most of whom are children, some drugs can cause permanent damage to the kidneys and bladder, and some can permanently damage cells in the ovaries and testes, making fertility difficult or even impossible. Furthermore, the 5-year survival rate for patients with recurrent or metastatic rhabdomyosarcoma is only 20%. Therefore, it is necessary to optimize the rhabdomyosarcoma disease model and innovate treatment strategies.

[0004] Currently, there are 30 commonly used rhabdomyosarcoma cell lines, most of which develop from treated tumors or distant metastases. Cell lines from untreated tumors are severely lacking, making the search for additional model systems crucial for better studying the pathobiology of rhabdomyosarcoma. Therefore, alternative models capable of capturing the complexity of the TME are needed to advance preclinical studies of related therapies. This is significant for finding new treatments, identifying individuals who respond to immunotherapy, and stratifying patients.

[0005] Genetic and histopathological analyses have shown that patient-derived tumor organoids (PDTOs) more faithfully preserve the genetic diversity and phenotypic heterogeneity of the original tumor than cancer cell line models. Furthermore, PDTOs have become a more reliable in vitro model for simulating human cancer, predicting in vivo drug sensitivity, and monitoring tumor progression. Beyond their powerful research applications, PDTOs can serve as personalized preclinical drug screening surrogates for high-throughput in vitro drug screening within a patient's limited time window, allowing for the customization of the most suitable clinical therapy to prolong patient survival and improve their quality of life. However, there are few reports on the construction and culture protocols for rhabdomyosarcoma organoids, or the construction efficiency is low. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to solve the problems of low survival rate and poor morphology in the culture of rhabdomyosarcoma organoids.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] The first aspect of the present invention provides a culture medium for culturing rhabdomyosarcoma organoids, comprising a basal culture medium, basic components, specific factor components, and additive components.

[0009] The basic components include the following components at final concentrations: 10-30 mM GlutaMAX, 50-100 U / mL penicillin-streptomycin, and 10-25 mM HEPES;

[0010] The specific factor components include the following components at final concentrations: 5-20 μg / mL Heparin, 50-200 ng / mL LIFG1, 10-100 ng / mL HGF, 30-80 ng / mL FGF-2, and 20-90 μM A 83-01;

[0011] The additive components include the following components at final concentrations: (1-2)×B27 reagent, 100-200 μg / mL Primocin, (1-2)×N2 reagent, 1-2 mM N-Acetylcysteine, 100-200 ng / mL R-spondin 1, and 10-50 ng / mL EGF.

[0012] illustrate:

[0013] Heparin can stabilize the activity of growth factors by preventing protein degradation and inducing FGF molecule oligomerization, thereby binding to FGF receptors to form dimers and activating them. Adding Heparin to the culture system can inhibit the growth of other hybrid cells.

[0014] IGF1 is a highly anabolic and anticatabolic compound. After binding to its receptors in cells, IGF1 stimulates cell growth and inhibits cell death. In the presence of sufficient protein, it can promote the growth of new muscle cells and enhance the proliferative capacity of muscle cells.

[0015] HGF has a significant effect on promoting the occurrence, survival and regeneration of tissue cells and inhibiting apoptosis;

[0016] The combined use of HGF and IGF1 can promote the expression of myoblastic compounds in MSCs;

[0017] FGF-2 plays an important role in regulating cell survival, division, differentiation and migration. It is a key mitogen in tissue homeostasis and cancer and can regulate the self-renewal of various stem cell types.

[0018] A 83-01 is an effective inhibitor of Smad2 phosphorylation and TGF-β-induced epithelial-mesenchymal transition. It also inhibits Smad2 phosphorylation, thereby maintaining the self-renewal and proliferation of tumor stem cells. Therefore, A 83-01 plays an important role in promoting cell proliferation, preventing cell differentiation, maintaining stem cell stemness, and inhibiting apoptosis and senescence.

[0019] Preferably, the basal culture medium is Advanced DMEM / F12.

[0020] Preferably, the additive further includes 5-30 μM Y-27632.

[0021] A second aspect of the present invention provides a method for preparing the above-mentioned culture medium, comprising the following steps: adding basic components, specific factor components and additive components to a basic culture medium and mixing them evenly to obtain the culture medium.

[0022] A third aspect of the present invention provides a method for culturing rhabdomyosarcoma organoids, comprising the following steps:

[0023] (1) Prepare sample cleaning and preservation solution and sample digestion solution, and pretreat rhabdomyosarcoma samples;

[0024] (2) Preparation of rhabdomyosarcoma sample cells: Cut the rhabdomyosarcoma sample into small pieces of minced meat, place them in sample digestion solution for digestion, then filter them with a filter screen, centrifuge, remove the supernatant, and collect the sample cells.

[0025] (3) Add the sample cells to the above culture medium for culture.

[0026] Preferably, the sample cleaning and preservation solution is prepared by adding 2× penicillin-streptomycin-glutamine and 10μM Y-27632 to DMEM culture medium.

[0027] Preferably, the sample digestion solution is prepared by adding collagenase IV 1 mg / mL, elastase 1 U / mL, DNase I 0.1 mg / mL, and Y-27632 10 μM to Advaced DMEM / F12.

[0028] Preferably, the pretreatment of the rhabdomyosarcoma sample specifically involves cleaning the rhabdomyosarcoma sample using a sample cleaning and preservation solution.

[0029] Preferably, the filter is a 100μm filter.

[0030] Preferably, the culture temperature in step (3) is 37°C, and the culture medium is changed every 3 days.

[0031] The beneficial effects of this invention are as follows:

[0032] This invention provides a culture medium, construction, and culture method for rhabdomyosarcoma organoids. It can efficiently transform surgically removed rhabdomyosarcoma tissue into rhabdomyosarcoma organoids, achieving a success rate of over 95%. The resulting organoids exhibit good morphology, and their HE staining results are consistent with the original surgical tissue. This invention aims to overcome the aforementioned technical challenges, establish a rapid and effective rhabdomyosarcoma construction protocol, facilitate personalized medicine for patients, and provide a new platform for disease research and drug development, demonstrating promising application prospects. Attached Figure Description

[0033] Figure 1 This is a morphological image of the rhabdomyosarcoma organoid cultured in Example 1 of the present invention;

[0034] Figure 2 HE staining comparison image of rhabdomyosarcoma prototum tissue and rhabdomyosarcoma organoid cultured in Example 1 of this invention;

[0035] Figure 3 This is a morphological image of the rhabdomyosarcoma organoid cultured in Example 2 of the present invention;

[0036] Figure 4 The image shows the morphology of the rhabdomyosarcoma organoid cultured in Comparative Example 1.

[0037] Figure 5 The image shows the morphology of the rhabdomyosarcoma organoid cultured in Comparative Example 2.

[0038] Figure 6 This is a morphological image of the rhabdomyosarcoma organoid cultured in Comparative Example 3. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0041] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0042] All reagents and raw materials used in the examples can be purchased from the market or prepared by known methods.

[0043] Example 1:

[0044] A culture medium for rhabdomyosarcoma organoids that does not contain Y-27632 (denoted as medium A) includes basal medium, basic components, specific factor components, and additive components; the basal medium is Advanced DMEM / F12;

[0045] The basic components are as follows, with the following final concentrations: 20 mM GlutaMAX, 50 U / mL penicillin-streptomycin, and 10 mM HEPES;

[0046] The specific factor components are as follows, with the following final concentrations: 10 μg / mL Heparin, 100 ng / mL IGF1, 60 ng / mL HGF, 50 ng / mL FGF-2, and 50 μM A 83-01.

[0047] The additive components are specifically the following components at the following final concentrations: 1×B27 reagent, 100μg / mL Primocin, 1×N2 reagent, 1.25mM N-Acetylcysteine, 200ng / mL R-spondin 1, and 20ng / mL EGF.

[0048] The preparation method of the above-mentioned culture medium A includes the following steps: adding the basic components, specific factor components, and additive components into Advanced DMEM / F12 and mixing them evenly to obtain the final product.

[0049] A culture medium for rhabdomyosarcoma organoids containing Y-27632 (denoted as culture medium B) differs from culture medium A in that the additives also contain 10 μM Y-27632, while the other components and preparation method are the same as those of culture medium A.

[0050] A method for culturing rhabdomyosarcoma organoids, using the aforementioned culture medium A and culture medium B, specifically includes the following steps:

[0051] (1) Preparation of sample cleaning and preservation solution: Add 2× penicillin-streptomycin-glutamine and 10μM Y-27632 to DMEM medium. Prepare fresh before use and store or transport at 0-8℃.

[0052] (2) Preparation of sample digestion solution: Add collagenase IV 1 mg / mL, elastase 1 U / mL, DNase I 0.1 mg / mL, and Y-27632 10 μM to Advaced DMEM / F12. Prepare fresh before use.

[0053] (3) Pretreatment of rhabdomyosarcoma samples: After the rhabdomyosarcoma surgical samples were removed from the body, they were washed once with 5 times the sample volume of sample cleaning and preservation solution, and then transferred to a new 5 times the sample volume of sample cleaning and preservation solution. The samples were transported to the laboratory via cold chain at 0-8℃.

[0054] Remove the sample, immerse it in sterile 0.04% trypan blue staining solution for 1 minute, and then use a sterile scalpel to remove the stained part;

[0055] Transfer the unstained portion to 10 times the sample volume of sample cleaning and preservation solution, and incubate at 180 rpm and 25°C on a shaker for 30 min, changing the sample cleaning and preservation solution every 5 min.

[0056] (4) Preparation of rhabdomyosarcoma sample cells: Collect the cleaned sample, cut the sample into small pieces with a side length of less than 1 mm using a sterile scalpel and surgical scissors, transfer the sample to 5 times the sample volume of sample digestion solution, digest at 200 rpm and 37°C for 3-5 min on a constant temperature shaker, and terminate digestion with 10 times the sample digestion solution volume of Advaced DMEM / F12.

[0057] Collect the digested cell suspension and filter it through a 100μm filter screen;

[0058] After filtration, centrifuge at 500×g, 4℃ for 8 min, discard the supernatant, and collect the sample cell pellet.

[0059] (5) Culture medium addition: Add the sample cells to the above culture medium B, and after forming a cell suspension, mix them thoroughly with Matrigel and add them evenly to the well plate at a rate of 35 μL / drop.

[0060] After incubating at 37℃ for 30 minutes to allow the matrix gel to fully solidify, remove the well plate, add culture medium B, and place it in the incubator for continuous incubation. Replace with culture medium A the next day.

[0061] Incubate at 37℃ for 10–20 days, changing the medium every 3 days.

[0062] The morphology of the organoids in this embodiment after 10 days of culture is shown in the figure. Figure 1 As shown in the figure, the organoids obtained by using the rhabdomyosarcoma organoid culture medium and culture method provided by the present invention have full morphology, considerable quantity, and can be cultured to a large size.

[0063] Hematoxylin and eosin (HE) staining was performed on surgical samples of rhabdomyosarcoma and organoids. The staining results are shown in the figure. Figure 2 As shown in the figure, the rhabdomyosarcoma organoids obtained by the culture medium and culture method of the present invention have the same pathological characteristics as the original rhabdomyosarcoma tissue.

[0064] Example 2:

[0065] A culture medium for rhabdomyosarcoma organoids that does not contain Y-27632 (denoted as medium A) includes basal medium, basic components, specific factor components, and additive components; the basal medium is Advanced DMEM / F12;

[0066] The basic components are as follows, with the following final concentrations: 10 mM GlutaMAX, 80 U / mL penicillin-streptomycin, and 25 mM HEPES;

[0067] The specific factor components are as follows, with final concentrations of: 20 μg / mL Heparin, 200 ng / mL IGF1, 10 ng / mL HGF, 80 ng / mL FGF-2, and 20 μM A 83-01.

[0068] The additive components are specifically the following components at the following final concentrations: 1.5×B27 reagent, 200μg / mL Primocin, 2×N2 reagent, 2mM N-Acetylcysteine, 100ng / mL R-spondin 1, and 10ng / mL EGF.

[0069] The preparation method of culture medium A in this embodiment is the same as that in Example 1.

[0070] A culture medium for rhabdomyosarcoma organoids containing Y-27632 (denoted as culture medium B) differs from culture medium A in that the additives also contain 30 μM Y-27632, while the other components and preparation method are the same as those of culture medium A in this embodiment.

[0071] A method for culturing rhabdomyosarcoma organoids, using culture medium A and culture medium B as described in this embodiment, specifically includes the following steps:

[0072] (1) Preparation of sample cleaning and preservation solution: Same as in Example 1.

[0073] (2) Preparation of sample digestion solution: Same as in Example 1.

[0074] (3) Pretreatment of rhabdomyosarcoma samples: Same as in Example 1.

[0075] (4) Preparation of rhabdomyosarcoma sample cells: Same as in Example 1.

[0076] (5) Culture medium addition: Add culture medium B of this example, and after forming a cell suspension, mix it thoroughly with Matrigel and drip it evenly into the well plate at a rate of 35 μL / drop.

[0077] After incubating at 37°C for 30 minutes to allow the matrix gel to fully solidify, remove the well plate, add culture medium B of this embodiment, and place it in the incubator for continuous incubation. Replace with culture medium A of this embodiment the next day.

[0078] Incubate at 37℃ for 10–20 days, changing the medium every 3 days.

[0079] The morphology of the organoids in this embodiment after 10 days of culture is shown in the figure. Figure 3 As shown in the figure, the organoids obtained by using the rhabdomyosarcoma organoid culture medium and culture method provided by the present invention have full morphology, considerable quantity, and can be cultured to a large size.

[0080] Comparative Example 1:

[0081] A rhabdomyosarcoma organoid culture medium (denoted as medium C) that does not contain Y-27632 differs from medium A in Example 1 in that it lacks specific factor components, while the other components and preparation methods are the same as those of medium A in Example 1.

[0082] A culture medium for rhabdomyosarcoma organoids containing Y-27632 (denoted as culture medium D) differs from culture medium B in Example 1 in that it lacks specific factor components, while the other components and preparation methods are the same as those of culture medium B in Example 1.

[0083] A method for culturing rhabdomyosarcoma organoids, using culture medium C and culture medium D as described in this comparative example, specifically includes the following steps:

[0084] (1) Preparation of sample cleaning and preservation solution: Same as in Example 1.

[0085] (2) Preparation of sample digestion solution: Add 10 μM Y-27632 to 1×TrypL E enzyme. Prepare fresh before use. The difference from Example 1 is that 1×TrypL E enzyme is used instead of collagenase, elastase and DNase I.

[0086] (3) Pretreatment of rhabdomyosarcoma samples: Same as in Example 1.

[0087] (4) Preparation of rhabdomyosarcoma sample cells: Same as in Example 1.

[0088] (5) Culture medium addition: Add culture medium D of this comparative example, and after forming a cell suspension, mix it thoroughly with Matrigel and add it evenly to the well plate at a rate of 35 μL / drop.

[0089] After incubating at 37℃ for 30 minutes to allow the matrix gel to fully solidify, remove the well plate, add the comparative culture medium D, and place it in the incubator for continuous incubation. The next day, replace it with the comparative culture medium C.

[0090] Incubate at 37℃ for 10–20 days, changing the medium every 3 days.

[0091] The morphology of the organoids in this comparative example after 10 days of culture is shown in [image]. Figure 4 As shown, after removing the specific factor from the culture medium, the resulting cultures are shriveled, few in number, and small in size, making it almost impossible for them to form rhabdomyosarcoma organoids.

[0092] Comparative Example 2:

[0093] A culture medium for rhabdomyosarcoma organoids that does not contain Y-27632 (denoted as culture medium C) differs from culture medium A in Example 2 in that it lacks specific factor components, while the other components and preparation methods are the same as those of culture medium A in Example 2.

[0094] A culture medium for rhabdomyosarcoma organoids containing Y-27632 (denoted as culture medium D) differs from culture medium B in Example 2 in that it lacks specific factor components, while the other components and preparation methods are the same as those of culture medium B in Example 2.

[0095] A method for culturing rhabdomyosarcoma organoids, using culture medium C and culture medium D as described in this comparative example, specifically includes the following steps:

[0096] (1)~(4) Same as Example 1.

[0097] (5) Culture medium addition: Add the sample cells to the culture medium D of this comparative example, and after forming a cell suspension, mix it thoroughly with Matrigel and add it evenly to the well plate at a rate of 35 μL / drop.

[0098] After incubating in a 37℃ incubator for 30 minutes to allow the matrix gel to fully solidify, remove the well plate, add culture medium D, and place it in the incubator for continuous incubation. Replace with culture medium C the next day.

[0099] Incubate at 37℃ for 10–20 days, changing the medium every 3 days.

[0100] The morphology of the organoids in this comparative example after 10 days of culture is shown in [image]. Figure 5 As shown in the figure, after removing the specific factor from the culture medium, the resulting cultures are shriveled, few in number, and small in size, making it almost impossible to form rhabdomyosarcoma organoids.

[0101] Comparative Example 3:

[0102] A rhabdomyosarcoma organoid culture medium (denoted as medium E) that does not contain Y-27632 differs from the medium in Example 1 in that the specific factor "Heparin" is increased to 50 μg / mL, IGF1 is reduced to 10 ng / mL, and HGF is absent. The other components and preparation methods are the same as those of medium A in Example 1.

[0103] A rhabdomyosarcoma organoid culture medium containing Y-27632 (denoted as culture medium F) differs from the comparative culture medium E in that the additives also contain 10 μM Y-27632, while the other components and preparation methods are the same as those of the comparative culture medium E.

[0104] A method for culturing rhabdomyosarcoma organoids, using the aforementioned culture medium E and culture medium F, specifically includes the following steps:

[0105] (1)~(4) Same as Example 1.

[0106] (5) Culture medium addition: Add the sample cells to the culture medium F of this comparative example, and after forming a cell suspension, mix it thoroughly with Matrigel and add it evenly to the well plate at a rate of 35 μL / drop.

[0107] After incubating at 37℃ for 30 minutes to allow the matrix gel to fully solidify, remove the well plate, add culture medium F, and place it in the incubator for continuous incubation. Replace with culture medium E the next day.

[0108] Incubate at 37℃ for 10–20 days, changing the medium every 3 days.

[0109] The morphology of organoids in this comparative example after 10 days of culture is shown in [image / description]. Figure 6 As shown in the figure, the cultured organisms are shriveled, few in number, and small in size, making it almost impossible for them to form rhabdomyosarcoma organoids.

[0110] Application example:

[0111] Over two years, a total of 20 clinical rhabdomyosarcoma samples that met ethical requirements were collected. Rhabdomyosarcoma organoid models were constructed and cultured using the culture media and methods of Examples 1-2 and Comparative Examples 1-3, respectively. The organoid formation was recorded and statistically analyzed using a microscope.

[0112] The standard for successful organoid modeling is the formation of 5,000 organoids with an average diameter greater than 80 μm within 45 days. The results are shown in Table 1 below.

[0113] Table 1. Statistical Table of Organoid Modeling for Rhabdomyosarcoma

[0114]

[0115] Table 1 shows that the success rates of rhabdomyosarcoma organoids obtained from different culture media are as follows: Example 2 > Example 1 > Comparative Example 3 > Comparative Example 2 > Comparative Example 1. Furthermore, the success rate of organoids is positively correlated with the construction rate (i.e., a shorter time for the average diameter of the organoids to first reach 80 μm, indicating a faster construction rate). This demonstrates that the rhabdomyosarcoma organoid culture medium and method provided by this invention not only have good reproducibility and high success rate, but also a short cycle time.

[0116] Example 3:

[0117] A culture medium for rhabdomyosarcoma organoids that does not contain Y-27632 (denoted as medium A) includes basal medium, basic components, specific factor components, and additive components; the basal medium is Advanced DMEM / F12;

[0118] The basic components are as follows, with the following final concentrations: 30 mM GlutaMAX, 100 U / mL penicillin-streptomycin, and 18 mM HEPES.

[0119] The specific factor components are as follows, with the following final concentrations: 5 μg / mL Heparin, 50 ng / mL IGF1, 100 ng / mL HGF, 30 ng / mL FGF-2, 90 μM A 83-01.

[0120] The additive components are specifically the following components at the following final concentrations: 2×B27 reagent, 150μg / mL Primocin, 1.5×N2 reagent, 1mM N-Acetylcysteine, 150ng / mL R-spondin 1, and 50ng / mL EGF.

[0121] The preparation method of the above-mentioned culture medium A includes the following steps: adding the basic components, specific factor components, and additive components into Advanced DMEM / F12 and mixing them evenly to obtain the final product.

[0122] A rhabdomyosarcoma organoid culture medium containing Y-27632 (denoted as culture medium B) differs from culture medium A in that the additives also contain 5 μM Y-27632, while the other components and preparation method are the same as those of culture medium A.

[0123] A method for culturing rhabdomyosarcoma organoids, using culture medium A and culture medium B in this embodiment, with specific steps the same as in Embodiment 1.

[0124] The morphology of the organoids in this embodiment after 10 days of culture is similar to that in Example 1.

[0125] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for culturing rhabdomyosarcoma organoids, characterized by, consisting of basal medium, basal components, specific factor components and additive components; the basal medium is Advanced DMEM / F12; the basal components consist of the following components at the final concentrations: 10-30 mM GlutaMAX, 50-100 U / mL penicillin-streptomycin, 10-25 mM HEPES; the specific factor components consist of the following components at the final concentrations: 5-20 μg / mL Heparin, 50-200 ng / mL IGF1, 10-100 ng / mL HGF, 30-80 ng / mL FGF-2, 20-90 μM A 83-01; the additive components consist of the following components at the final concentrations: 1×-2×B27 reagent, 100-200 μg / mL Primocin, 1×-2×N2 reagent, 1-2 mM N-Acetylcysteine, 100-200 ng / mL R-spondin 1, 10-50 ng / mL EGF; or 1×-2×B27 reagent, 100-200 μg / mL Primocin, 1×-2×N2 reagent, 1-2 mM N-Acetylcysteine, 100-200 ng / mL R-spondin 1, 10-50 ng / mL EGF, 5-30 µM Y-27632.

2. The medium for culturing rhabdomyosarcoma organoids according to claim 1, characterized in that, the specific factor components consist of the following components at the final concentrations: 10 μg / mL Heparin, 100 ng / mL IGF1, 60 ng / mL HGF, 50 ng / mL FGF-2, 50 μM A 83-01.

3. The medium for culturing rhabdomyosarcoma organoids according to claim 1, characterized in that, the additive components consist of the following components at the final concentrations: 1×B27 reagent, 100 μg / mL Primocin, 1×N2 reagent, 1.25 mM N-Acetylcysteine, 200 ng / mL R-spondin 1, 20 ng / mL EGF.

4. Process for the preparation of the medium according to any one of claims 1 to 3, characterized in that, comprising the steps of adding the basal components, the specific factor components and the additive components into the basal medium and mixing uniformly, thus obtaining.

5. A method of culturing a rhabdomyosarcoma organoid, characterized by, comprising the steps of: (1) preparing a sample washing and preserving solution and a sample digestion solution, and pretreating the rhabdomyosarcoma sample; (2) preparing rhabdomyosarcoma sample cells: cutting the rhabdomyosarcoma sample into small pieces of minced meat, digesting in the sample digestion solution, then filtering with a filter screen, centrifuging, removing the supernatant and collecting the sample cells; (3) adding the sample cells into the medium of any one of claims 1-3 for culture, thus obtaining.

6. The method of culturing a rhabdomyosarcoma organoid according to claim 5, wherein, the preparation method of the sample washing and preserving solution is adding 2×penicillin-streptomycin-glutamine and 10 µM Y-27632 into DMEM medium.

7. The method of culturing a rhabdomyosarcoma organoid according to claim 5, wherein, The sample digestion solution is prepared by adding 1 mg / mL collagenase IV, 1 U / mL elastase, 0.1 mg / mL DNase I and 10 µM Y-27632 in Advaced DMEM / F12.

8. The method of culturing a rhabdomyosarcoma organoid according to claim 5, wherein, The rhabdomyosarcoma sample is pretreated by using a sample cleaning and preserving solution to clean the rhabdomyosarcoma sample.

9. The method of culturing a rhabdomyosarcoma organoid according to claim 5, wherein, The filter screen is a 100 μm filter screen.

10. The method of culturing a rhabdomyosarcoma organoid according to claim 5, wherein, The temperature in step (3) is 37℃, and the culture medium is replaced every 3 days.

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