Culture medium of rhabdomyosarcoma organ, construction method and culture method of rhabdomyosarcoma organ

Abstract

The invention discloses a culture medium for rhabdomyosarcoma organoid and a construction and culture method, and belongs to the technical field of organoid model construction. The culture medium comprises a basic culture medium, a basic component, a specific factor component and an additive component, wherein the specific factor component is prepared from the following components in final concentration: 5 to 20 [mu] g / mL of Heparin, 50 to 200 ng / mL of IGF1, 10 to 100 ng / mL of HGF, 30 to 80 ng / mL of FGF-2, and 83 to 01 of 20 to 90 [mu] M of A; the culture medium has the beneficial effects that the culture medium for culturing the rhabdomyosarcoma organ is provided, the rhabdomyosarcoma tissue cut by an operation can be efficiently converted into the rhabdomyosarcoma organ, the construction success rate can reach 95% or above, and the HE staining result of the obtained rhabdomyosarcoma organ is consistent with that of the original operation tissue of the rhabdomyosarcoma organ. According to the method, the technical problems are solved, a rapid and effective rhabdomyosarcoma construction scheme is established, assistance is provided for personalized medication of patients, a new platform is provided for disease research and drug research and development of the patients, and the method has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid model construction, and specifically relates to a culture medium, construction and culture method of rhabdomyosarcoma organoids. Background Art

[0002] Rhabdomyosarcoma is a mesenchymal cell that originates from or differentiates into skeletal muscle cells. It is the most common soft tissue tumor in childhood and is a highly malignant skeletal muscle myoblast-like tumor. Although skeletal muscle has failed to completely differentiate, it still expresses myogenic markers. It can be divided into embryonic rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma, and grape-shaped rhabdomyosarcoma. It is more common in children and extremely rare in adults. There are fewer male children than female children with the disease. It is more common in the head and neck area. According to the age, tumor size, pathology, and clinical stage of rhabdomyosarcoma patients, they can be divided into three groups: low, medium, and high risk, for stratified and comprehensive treatment.

[0003] Patients often experience pain and bleeding due to tumor compression, and are prone to early metastasis and recurrence, which seriously affects the treatment outcome and long-term prognosis. The main treatment is surgical resection accompanied by chemoradiotherapy. Only 10% of patients' rhabdomyosarcoma can be completely removed, and up to 37.7% of rhabdomyosarcoma may metastasize. Therefore, even for patients with complete resection, chemotherapy and radiotherapy are necessary. Chemotherapy drugs target all cells in the human body, including tumor cells and normal cells, which means that the drugs used may have side effects on normal cells. Most side effects will disappear after stopping the medication. For most patients with rhabdomyosarcoma who are children, some drugs can cause permanent damage to the kidneys and bladder, and some permanently damage cells in the ovaries and testicles, making it difficult or even impossible to have children. At the same time, the 5-year survival rate of 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 are developed from treated tumors or distant metastases. Cell lines from untreated tumors are severely lacking, and it is crucial to seek additional model systems to better study the pathobiology of rhabdomyosarcoma. Therefore, alternative models that can capture the complexity of the TME are needed to advance preclinical research on related therapies. This is of great significance for finding new treatments, identifying individuals who respond to immunotherapy, and stratifying patients.

[0005] Genetic analysis and histopathological analysis have shown that patient-derived tumor organoids (PDTOs) retain the genetic diversity and phenotypic heterogeneity of the original tumor more faithfully than cancer cell line models. In addition, PDTOs have become a more reliable in vitro model for simulating human cancer, predicting in vivo drug sensitivity, and monitoring tumor progression. In addition to its powerful scientific research uses, PDTOs can be used as preclinical personalized drug surrogates to conduct in vitro high-throughput drug screening within the patient's limited time window, and customize the most suitable clinical therapy for patients to prolong their survival cycle and improve their quality of survival. However, there are few reports on the construction and culture protocols of rhabdomyosarcoma organoids or the construction efficiency is low. Summary of the invention

[0006] The technical problem to be solved by the present 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 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, a basal component, a specific factor component, and an additive component;

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

[0010] The specific factor composition includes the following components at 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;

[0011] The additive composition includes 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 proteolytic degradation and inducing oligomerization of FGF molecules, thereby binding to FGF receptors to form dimers and activating them. Adding Heparin to the culture system can inhibit the growth of other cells.

[0014] IGF1 is a highly anabolic and anti-catabolic compound. After binding to the receptors in cells, IGF1 can stimulate cell growth and inhibit cell death. At the same time, in the presence of sufficient protein, it can promote the growth of new muscle cells and improve the proliferation capacity of muscle cells.

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

[0016] The combination of HGF and IGF1 can promote the expression of myogenic compounds in MSCs;

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

[0018] A 83-01 is an effective inhibitor that blocks Smad2 phosphorylation and inhibits TGF-β-induced epithelial-mesenchymal transition. It also inhibits Smad2 phosphorylation to maintain 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 cell apoptosis and aging.

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

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

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

[0022] The 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, sample digestion solution, and pretreat rhabdomyosarcoma samples;

[0024] (2) Preparation of rhabdomyosarcoma sample cells: Cut the rhabdomyosarcoma sample into small pieces, place them in sample digestion solution for digestion, then filter them with a filter, centrifuge them, 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 washing and preservation solution is prepared by adding 2× penicillin-streptomycin-glutamine and 10 μM Y-27632 to DMEM culture medium.

[0027] Preferably, 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 to Advanced DMEM / F12.

[0028] Preferably, the rhabdomyosarcoma sample pretreatment specifically comprises: washing the rhabdomyosarcoma sample with a sample washing and preservation solution.

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

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

[0031] The beneficial effects of the present invention are:

[0032] The present invention provides a culture medium, construction and culture method for rhabdomyosarcoma organoids, which can efficiently transform surgically removed rhabdomyosarcoma tissue into rhabdomyosarcoma organoids, with a construction success rate of more than 95%, good morphology, and the obtained rhabdomyosarcoma organoids are consistent with the HE staining results of their original surgical tissues. The invention intends to overcome the above technical difficulties, establish a fast and effective rhabdomyosarcoma construction plan, provide assistance for personalized medication for patients, and provide a new platform for patient disease research and drug development, with good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0034] Figure 2 This is a HE staining comparison of the original tumor tissue of rhabdomyosarcoma and the rhabdomyosarcoma organoid cultured in Example 1 of the present invention;

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

[0036] Figure 4 This is a morphological diagram of rhabdomyosarcoma organoids cultured in Comparative Example 1;

[0037] Figure 5 This is the morphological diagram of the rhabdomyosarcoma organoid cultured in Comparative Example 2;

[0038] Figure 6 This is the morphological diagram of the rhabdomyosarcoma organoids cultured in Comparative Example 3. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0041] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

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

[0043] Embodiment 1:

[0044] A rhabdomyosarcoma organoid culture medium (denoted as culture medium A) that does not contain Y-27632, comprising a basal culture medium, a basal component, a specific factor component and an additive component; the basal culture medium is Advanced DMEM / F12;

[0045] The basic components specifically consisted of the following components at final concentrations: 20 mM GlutaMAX, 50 U / mL penicillin-streptomycin, 10 mM HEPES;

[0046] The specific factor components are specifically 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,

[0047] The additive components specifically include 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, and 20 ng / mL EGF.

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

[0049] A rhabdomyosarcoma organoid culture medium containing Y-27632 (referred to as culture medium B), which differs from the above-mentioned culture medium A in that the additive component also contains 10 μM Y-27632, and the remaining components and preparation method are the same as those of culture medium A.

[0050] A method for culturing rhabdomyosarcoma organoids, using the above-mentioned culture medium A and culture medium B for culturing, specifically comprising the following steps:

[0051] (1) Prepare sample cleaning and storage solution: add 2× penicillin-streptomycin-glutamine and 10 μM Y-27632 to DMEM medium, prepare before use, and store or transport at 0-8°C;

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

[0053] (3) Rhabdomyosarcoma sample pretreatment: After the rhabdomyosarcoma surgical sample is removed from the body, it is washed once with a sample cleaning and preservation solution 5 times the sample volume, and then transferred to a new sample cleaning and preservation solution 5 times the sample volume, and the sample is transported to the laboratory at a 0-8°C cold chain;

[0054] The sample was removed and stained in sterile 0.04% trypan blue dye for 1 min, and then the stained part was removed using a sterile scalpel;

[0055] The unstained part was transferred to a sample washing and preservation solution with a volume 10 times that of the sample, and incubated at 180 rpm, 25°C on a shaker for 30 min, with the sample washing and preservation solution replaced every 5 min;

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

[0057] The digested cell suspension was collected and filtered through a 100 μm filter;

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

[0059] (5) Adding culture medium for culture: Add the sample cells to the above culture medium B to form a cell suspension, mix it thoroughly with Matrigel, and evenly drip it into the well plate at 35 μL / drop;

[0060] After incubation at 37°C for 30 min to allow the matrix gel to fully solidify, the plate was removed and medium B was added, and the plate was placed in the incubator for continuous culture. The medium was replaced with medium A the next day.

[0061] The culture was continued in a 37°C incubator for 10 to 20 days, and the medium was changed every 3 days.

[0062] The morphology of the organoids in this example after 10 days of culture is shown in Figure 1 As shown in the figure, it can be seen that 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 larger size.

[0063] The rhabdomyosarcoma surgical specimens and organoids were stained with HE. The staining results are shown in Figure 2 As shown in the figure, it can be seen that the pathological characteristics of the rhabdomyosarcoma organoids cultured by the culture medium and culture method of the present invention are consistent with those of the original rhabdomyosarcoma tissue.

[0064] Embodiment 2:

[0065] A rhabdomyosarcoma organoid culture medium (denoted as culture medium A) that does not contain Y-27632, comprising a basal culture medium, a basal component, a specific factor component and an additive component; the basal culture medium is Advanced DMEM / F12;

[0066] The basic components specifically consisted of the following components at final concentrations: 10 mM GlutaMAX, 80 U / mL penicillin-streptomycin, 25 mM HEPES;

[0067] The specific factor components are specifically the following components at the final concentrations: 20 μg / mL Heparin, 200 ng / mL IGF1, 10 ng / mL HGF, 80 ng / mL FGF-2, 20 μM A 83-01,

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

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

[0070] A rhabdomyosarcoma organoid culture medium containing Y-27632 (referred to as culture medium B), which differs from culture medium A of this embodiment in that the additive component also contains 30 μM Y-27632, and the remaining components and preparation method are the same as culture medium A of this embodiment.

[0071] A method for culturing rhabdomyosarcoma organoids, using culture medium A and culture medium B of this embodiment for culturing, specifically comprising the following steps:

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

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

[0074] (3) Rhabdomyosarcoma sample pretreatment: same as Example 1.

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

[0076] (5) Adding culture medium for culturing: Add culture medium B of this example, form a cell suspension, mix thoroughly with Matrigel, and drip evenly into the well plate at 35 μL / drop;

[0077] After incubation at 37°C for 30 minutes to allow the matrix gel to fully solidify, the plate was taken out and medium B of this example was added, and the plate was placed in an incubator for continuous culture. The medium A of this example was replaced the next day.

[0078] The culture was continued in a 37°C incubator for 10 to 20 days, and the medium was changed every 3 days.

[0079] The morphology of the organoids in this example after 10 days of culture is shown in Figure 3 As shown in the figure, it can be seen that 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 larger size.

[0080] Comparative Example 1:

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

[0082] A rhabdomyosarcoma organoid culture medium containing Y-27632 (referred to as culture medium D), which differs from culture medium B in Example 1 in that it lacks specific factor components, and the remaining components and preparation method 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 of this comparative example for culturing, specifically comprising the following steps:

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

[0085] (2) Prepare sample digestion solution: add 10 μM Y-27632 to 1×TrypLE enzyme and prepare it immediately before use. The difference from Example 1 is that 1×TrypLE enzyme is used to replace collagenase, elastase, and DNaseⅠ;

[0086] (3) Rhabdomyosarcoma sample pretreatment: same as Example 1.

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

[0088] (5) Adding culture medium for cultivation: adding culture medium D of this comparative example, mixing with Matrigel to form a cell suspension, and evenly dropping into the well plate at 35 μL / drop;

[0089] After incubation at 37°C for 30 minutes to allow the matrix gel to fully solidify, the plate was taken out and the medium D of the comparative example was added, and the plate was placed in the incubator for continuous culture. The medium C of the comparative example was replaced the next day.

[0090] The culture was continued in a 37°C incubator for 10 to 20 days, and the medium was changed every 3 days.

[0091] The morphology of the organoids in this comparative example after 10 days of culture is shown in Figure 4 As shown, it can be seen that after the culture medium is subtracted from specific factors, the cultured cells are hollow in morphology, extremely small in number, and small in size, and are almost unable to form rhabdomyosarcoma organoids.

[0092] Comparative Example 2:

[0093] A rhabdomyosarcoma organoid culture medium (denoted as culture medium C) that does not contain Y-27632 is different from culture medium A in Example 2 in that it lacks specific factor components, and the remaining components and preparation method are the same as culture medium A in Example 2.

[0094] A rhabdomyosarcoma organoid culture medium containing Y-27632 (referred to as culture medium D), which differs from culture medium B in Example 2 in that it lacks specific factor components, and the remaining components and preparation method are the same as culture medium B in Example 2.

[0095] A method for culturing rhabdomyosarcoma organoids, using the culture medium C and the culture medium D of this comparative example for culturing, specifically comprising the steps of:

[0096] (1) to (4) are the same as in Example 1.

[0097] (5) Adding culture medium for cultivation: adding sample cells to the culture medium D of this comparative example, forming a cell suspension, and then fully mixing with Matrigel, and dripping evenly into the well plate at 35 μL / drop;

[0098] After incubation at 37°C for 30 min to allow the matrix gel to fully solidify, the plate was removed and medium D was added, and the plate was placed in the incubator for continuous culture. The next day, medium C was replaced.

[0099] The culture was continued in a 37°C incubator for 10 to 20 days, and the medium was changed every 3 days.

[0100] The morphology of the organoids in this comparative example after 10 days of culture is shown in Figure 5 As shown in the figure, it can be seen that after subtracting specific factors from the culture medium, the cultured tissues obtained are hollow in morphology, extremely small in number, and small in size, and are almost unable to form rhabdomyosarcoma organoids.

[0101] Comparative Example 3:

[0102] A rhabdomyosarcoma organoid culture medium not containing Y-27632 (referred to as culture medium E), which differs from the culture medium of Example 1 in that the specific factor "Heparin is increased to 50 μg / mL, IGF1 is reduced to 10 ng / mL, and HGF is missing", and the remaining components and preparation method are the same as those of culture medium A of Example 1.

[0103] A rhabdomyosarcoma organoid culture medium containing Y-27632 (referred to as culture medium F) is different from the culture medium E of this comparative example in that the additive component also contains 10 μM Y-27632, and the remaining components and preparation method are the same as those of the culture medium E of this comparative example.

[0104] A method for culturing rhabdomyosarcoma organoids, using the above-mentioned culture medium E and culture medium F for culturing, specifically comprising the following steps:

[0105] (1) to (4) are the same as in Example 1.

[0106] (5) Adding culture medium for cultivation: adding sample cells to the culture medium F of this comparative example, forming a cell suspension, and then fully mixing with Matrigel, and dripping evenly into the well plate at 35 μL / drop;

[0107] After incubation at 37°C for 30 min to allow the matrix gel to fully solidify, the plate was removed and medium F was added, and the plate was placed in the incubator for continuous culture. The next day, medium E was replaced;

[0108] The culture was continued in a 37°C incubator for 10 to 20 days, and the medium was changed every 3 days.

[0109] The morphology of the organoids in this comparative example after 10 days of culture is shown in Figure 6 As shown in the figure, it can be seen that the cultured tissues are hollow in morphology, extremely small in number, small in size, and can hardly form rhabdomyosarcoma organoids.

[0110] Application examples:

[0111] During the two years, a total of 20 clinical rhabdomyosarcoma samples that met ethical requirements were collected. The culture media and culture methods of Examples 1 to 2 and Comparative Examples 1 to 3 were used to construct and culture rhabdomyosarcoma organoid models, and the organoid formation was recorded and counted using a microscope.

[0112] Standard: The successful modeling of organoids was determined by the formation of 5,000 organoids with an average diameter greater than 80 μm within 45 days. The statistical results are shown in Table 1 below.

[0113] Table 1 Statistics of rhabdomyosarcoma organoid modeling

[0114]

[0115] It can be concluded from Table 1 that the success rates of rhabdomyosarcoma organoids obtained by culturing with different culture media are as follows: Example 2> Example 1> Comparative Example 3> Comparative Example 2> Comparative Example 1. At the same time, the success rate of the organoids is positively correlated with the construction rate (i.e., the time when the average diameter of the organoid reaches 80 μm for the first time is shorter, that is, the construction rate is faster), indicating that the rhabdomyosarcoma organoids obtained using the rhabdomyosarcoma organoid culture medium and culture method provided by the method of the present invention not only have good repeatability, high success rate, but also short cycle.

[0116] Embodiment 3:

[0117] A rhabdomyosarcoma organoid culture medium (denoted as culture medium A) that does not contain Y-27632, comprising a basal culture medium, a basal component, a specific factor component and an additive component; the basal culture medium is Advanced DMEM / F12;

[0118] The basic components specifically consisted of the following final concentrations: 30 mM GlutaMAX, 100 U / mL penicillin-streptomycin, 18 mM HEPES;

[0119] The specific factor components are specifically the following components at the 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 specifically include the following components at the final concentrations: 2×B27 reagent, 150 μg / mL Primocin, 1.5×N2 reagent, 1 mM N-Acetylcysteine, 150 ng / mL R-spondin 1, and 50 ng / mL EGF.

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

[0122] A rhabdomyosarcoma organoid culture medium containing Y-27632 (referred to as culture medium B), which differs from culture medium A of this embodiment in that the additive component also contains 5 μM Y-27632, and the remaining components and preparation method are the same as culture medium A.

[0123] A method for culturing rhabdomyosarcoma organoids, using culture medium A and culture medium B of this example for culturing, and the specific steps are the same as those of Example 1.

[0124] The morphology of the organoids in this example 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, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions 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 in that: Including basic culture medium, basic components, specific factor components and additive components; The base composition includes the following components at final concentrations: 10-30 mM GlutaMAX, 50-100 U / mL penicillin-streptomycin, 10-25 mM HEPES; The specific factor composition includes the following components at 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 composition includes 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.

2. The culture medium for culturing rhabdomyosarcoma organoids according to claim 1, characterized in that: The basic culture medium is Advanced DMEM / F12.

3. The culture medium for culturing rhabdomyosarcoma organoids according to claim 1, characterized in that: The additive also includes 5-30 μM Y-27632.

4. The method for preparing the culture medium according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: adding basic components, specific factor components and additive components into the basic culture medium and mixing them evenly to obtain the product.

5. A method for culturing rhabdomyosarcoma organoids, characterized in that: The following steps are involved: (1) Prepare sample cleaning and preservation solution, sample digestion solution, and pretreat rhabdomyosarcoma samples; (2) Preparation of rhabdomyosarcoma sample cells: Cut the rhabdomyosarcoma sample into small pieces, place them in sample digestion solution for digestion, then filter them with a filter, centrifuge them, remove the supernatant, and collect the sample cells; (3) Adding the sample cells to the culture medium according to any one of claims 1 to 3 for culturing.

6. The method for culturing rhabdomyosarcoma organoids according to claim 5, characterized in that: The sample cleaning and preservation solution is prepared by adding 2× penicillin-streptomycin-glutamine and 10 μM Y-27632 to the DMEM culture medium.

7. The method for culturing rhabdomyosarcoma organoids according to claim 5, characterized in that: The sample digestion solution was prepared by adding 1 mg / mL collagenase IV, 1 U / mL elastase, 0.1 mg / mL DNase I, and 10 μM Y-27632 to Advaced DMEM / F12.

8. The method for culturing rhabdomyosarcoma organoids according to claim 5, characterized in that: The rhabdomyosarcoma sample pretreatment specifically includes: washing the rhabdomyosarcoma sample with a sample washing and preservation solution.

9. The method for culturing rhabdomyosarcoma organoids according to claim 5, characterized in that: The filter is a 100 μm filter.

10. The method for culturing rhabdomyosarcoma organoids according to claim 5, characterized in that: The culture temperature in step (3) is 37°C, and the culture medium is replaced every 3 days.

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