Solid tumor three-dimensional model of bone and soft tissue tumor and construction method, application, detection method and culture reagent combination thereof

Abstract

The invention provides a bone and soft tissue solid tumor three-dimensional model as well as a construction method, application, a detection method and a culture reagent combination thereof, and belongs to the technical field of bioengineering. The construction method comprises the following steps: acquiring solid tumor cells of bone and soft tissue tumors; inoculating the solid tumor cells into a culture container, adding a solid tumor two-dimensional cell culture medium, and performing two-dimensional culture; the method comprises the following steps: mixing solid tumor cells subjected to two-dimensional culture with vascular endothelial cells according to a certain cell proportion, carrying out balling culture to form a globule polymer, adding an extracellular matrix into the globule polymer, gelling, adding into a solid tumor three-dimensional cell culture medium, and culturing to form a bone and soft tissue solid tumor three-dimensional model with a vascular structure. According to the construction method, 2D culture is carried out firstly, then 3D balling culture is carried out, the survival rate is effectively increased, and the formed three-dimensional model has a tumor microenvironment formed by a vascular structure and can be used for drug detection and tumor invasion assessment analysis.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a three-dimensional model of a bone and soft tissue tumor solid tumor, and a construction method, application, detection method, and culture reagent combination thereof. Background Art

[0002] Solid bone and soft tissue tumors are common primary bone cancers with many types, and most of them are radioresistant and highly genetically heterogeneous. Neither tumor resection nor chemotherapy can control the metastasis and spread of the tumor, so the mortality rate of patients with this type of tumor is high. Secondly, due to its high genetic heterogeneity, lack of effective targets, and poor availability of in vitro and in vivo models for studying tumor progression and metastasis cascade mechanisms, the pathophysiological mechanisms of solid bone and soft tissue tumors are still poorly understood, and the clinical transformation of new drug research and development has stagnated.

[0003] The tumor microenvironment (TME) plays an important role in the development of cancer metastasis. It includes a variety of cells. TME cells are involved in every stage of cancer progression, affecting the occurrence, progression, invasion, intravasation, metastatic spread and growth of tumors. TME cells and the molecules they secrete are believed to play a key role in the pathogenesis of cancer. Therefore, models that can closely reflect these interactions are essential for a better understanding of cancer-related events and drug development.

[0004] In the research on bone and soft tissue solid tumors, there are mainly two-dimensional culture and three-dimensional culture methods. Among them, two-dimensional culture is to culture the treated tumor cells on the wall. The method is too simple and cannot reproduce the dynamic environment of cancer cell growth, including the cell-cell and cell-extracellular matrix that affect cancer progression and drug resistance.

[0005] (Extracellular matrix, ECM) interaction. Secondly, three-dimensional culture is to mix the treated tumor cells with matrix gel and add them to the culture container, then culture them in a cell culture incubator, add organoid complete culture medium to form an organoid model. Although this method can simulate the dynamic environment of cancer cell growth and the interaction between cancer cells and TME (including its extracellular matrix), it makes up for the oversimplified 2D experiment and can be used as a tool for drug screening. However, since bone and soft tissue tumors are of mesenchymal origin, when using the organoid three-dimensional culture method to culture them into a three-dimensional organoid model, the survival rate is low and there is a lack of vascular structure. As the volume of the organoid increases, hypoxia and metabolic waste accumulation lead to cell apoptosis, which eventually leads to tissue necrosis and the vascularization of the tissue cannot be achieved. Summary of the invention

[0006] The present disclosure aims to solve at least one of the technical problems existing in the prior art, and provides a three-dimensional model of solid tumors of bone and soft tissue tumors, and a construction method, application, detection method, and culture reagent combination thereof.

[0007] In one aspect of the present disclosure, a method for constructing a three-dimensional model of a bone and soft tissue tumor solid tumor is provided, the construction method comprising:

[0008] Obtain solid tumor cells from bone and soft tissue tumors;

[0009] Inoculating the tumor solid tumor cells into a culture container, adding a tumor solid tumor two-dimensional cell culture medium, and performing two-dimensional culture;

[0010] The solid tumor cells after two-dimensional culture are mixed with vascular endothelial cells at a certain cell ratio for spheroid culture to form spheroid aggregates, and extracellular matrix is ​​added to the spheroid aggregates. After gelation, solid tumor three-dimensional cell culture medium is added to culture to form a three-dimensional model of bone and soft tissue tumor solid tumors with vascular structure.

[0011] Optionally, the solid tumor three-dimensional cell culture medium includes solid tumor two-dimensional cell culture medium and endothelial cell culture medium;

[0012] Wherein, the two-dimensional cell culture medium for solid tumors includes AdvancedDMEM / F12, FBS, PS, and one or more of EGF, basicFGF, IGF, Glutamax, and HEPES; wherein,

[0013] The concentration of the FBS ranges from 5% to 20%;

[0014] The concentration range of the PS is 1-3%;

[0015] The concentration range of EGF is 50ng / mL-200ng / mL;

[0016] The concentration range of basicFGF is 10ng / mL-100ng / mL;

[0017] The concentration range of the IGF is 10 ng / mL-50 ng / mL;

[0018] The concentration range of Glutamax is 0.5%-5%;

[0019] The concentration range of HEPES is 0.5%-5%;

[0020] The endothelial cell culture medium includes ECM, FBS, EGF, basic FGF, and PS; wherein,

[0021] The concentration of the FBS ranges from 5% to 20%;

[0022] The concentration range of EGF is 10ng / mL-100ng / mL;

[0023] The concentration range of basic FGF is 10ng / mL-100ng / mL;

[0024] The concentration of PS is in the range of 1%-3%.

[0025] Optionally, in the tumor solid tumor three-dimensional cell culture medium, the volume ratio of the tumor solid tumor two-dimensional cell culture medium and the endothelial cell culture medium is (1-2):1.

[0026] Optionally, the two-dimensionally cultured solid tumor cells are mixed with vascular endothelial cells and cultured into spheres to form spheroids, comprising:

[0027] Use the passage digestion solution to digest the vascular endothelial cells and the solid tumor cells after two-dimensional culture respectively;

[0028] The digested solid tumor cells and vascular endothelial cells were resuspended respectively using solid tumor three-dimensional cell culture medium;

[0029] Mixing the resuspended solid tumor cell suspension and the vascular endothelial cell suspension to obtain a mixed suspension; wherein, in the mixed suspension, the vascular endothelial cells account for 1%-30% of the total number of the vascular endothelial cells and the solid tumor cells;

[0030] The mixed suspension is added into a low-adsorption U-shaped bottom well plate, or a culture container containing a low-adsorption U-shaped bottom structure, and is cultured to form spherulomers.

[0031] Optionally, the passage digestion solution is at least one of TrypLE, Trypsin, and Accutase.

[0032] Optionally, obtaining bone and soft tissue tumor solid tumor cells comprises:

[0033] Use buffer or a mixture of buffer and penicillin-streptomycin and gentamicin to wash primary tissues of solid tumors of bone and soft tissue;

[0034] The primary tissue of solid tumor is crushed, resuspended and left to stand;

[0035] Digesting the primary tissue of the solid tumor with a primary tissue digestion solution to obtain a cell suspension; wherein the primary tissue digestion solution comprises: Advanced DMEM / F12 culture medium, and one or more of collagenase I, collagenase II, and collagenase IV;

[0036] Resuspend the digested cell suspension in solid tumor 2D cell culture medium.

[0037] Optionally, the concentration range of collagenase I is 0.5 mg / mL-5 mg / mL;

[0038] The concentration range of the collagenase II is 0.5 mg / mL-5 mg / mL;

[0039] The concentration range of the collagenase IV is 0.5 mg / mL-5 mg / mL;

[0040] The primary tissue digestion solution also includes one or more of hyaluronidase and Y27632; wherein,

[0041] The concentration range of the hyaluronidase is 100 μg / mL-1 mg / mL;

[0042] The concentration range of Y27632 is 1 μM-10 μM.

[0043] In another aspect of the present disclosure, a three-dimensional model of a solid tumor of bone and soft tissue tumor is provided, which is formed by the construction method described above.

[0044] In another aspect of the present disclosure, a three-dimensional model of solid tumors of bone and soft tissue tumors described above is provided for use in compound analysis and / or treatment screening;

[0045] The compound analysis includes at least one of drug screening, drug sensitivity testing, and drug toxicity testing;

[0046] The treatment modality includes at least one of radiation therapy and thermal therapy.

[0047] Another aspect of the present disclosure provides a method for detecting a target object using the above-mentioned three-dimensional model of a solid tumor of a bone and soft tissue tumor, the detection method comprising:

[0048] Introducing a target object to be tested into the three-dimensional model of bone and soft tissue tumor solid tumor;

[0049] Obtain the effect of the target object on the three-dimensional model of the bone and soft tissue tumor solid tumor.

[0050] Optionally, the target comprises a drug and / or a treatment;

[0051] The drug includes at least one of a tumor chemotherapy drug, a targeted drug, and a tumor vascular inhibitor;

[0052] The treatment modalities include radiotherapy and / or thermal therapy.

[0053] In another aspect of the present disclosure, a culture reagent combination is provided, which comprises a two-dimensional cell culture medium for solid tumors and a three-dimensional cell culture medium for solid tumors;

[0054] Wherein, the two-dimensional culture medium for solid tumors includes Advanced DMEM / F12, FBS, PS, and one or more of EGF, basicFGF, IGF, Glutamax, and HEPES; wherein,

[0055] The concentration of the FBS ranges from 5% to 20%;

[0056] The concentration range of the PS is 1-3%;

[0057] The concentration range of EGF is 50ng / mL-200ng / mL;

[0058] The concentration range of basicFGF is 10ng / mL-100ng / mL;

[0059] The concentration range of the IGF is 10 ng / mL-50 ng / mL;

[0060] The concentration range of Glutamax is 0.5%-5%;

[0061] The concentration range of HEPES is 0.5%-5%;

[0062] Wherein, the solid tumor three-dimensional cell culture medium is obtained by mixing the solid tumor two-dimensional cell culture medium and the endothelial cell culture medium in a volume ratio of (1-2):1;

[0063] The endothelial cell culture medium includes ECM, FBS, EGF, basic FGF, and PS; wherein,

[0064] The concentration of the FBS ranges from 5% to 20%;

[0065] The concentration range of EGF is 10ng / mL-100ng / mL;

[0066] The concentration range of basic FGF is 10ng / mL-100ng / mL;

[0067] The concentration range of PS is 1%-3%;

[0068] The two-dimensional cell culture medium for solid tumors and the three-dimensional cell culture medium for solid tumors are used in the aforementioned method for constructing a three-dimensional model of a solid tumor of a bone and soft tissue tumor, or in the construction of the aforementioned three-dimensional model of a solid tumor of a bone and soft tissue tumor.

[0069] The present disclosure provides a three-dimensional model of a solid tumor of bone and soft tissue tumors and a construction method, application, detection method, and culture reagent combination thereof, which has the following beneficial effects compared to the prior art:

[0070] First, the three-dimensional model construction method disclosed in the present invention adopts 2D culture first and then 3D sphere culture, which effectively improves the survival rate, and the 2D culture stage can continue to pass and proliferate, further improving the survival rate;

[0071] Second, the construction method disclosed in the present invention can realize the vascularized culture of solid tumors of bone and soft tissue tumors, and the constructed tumor sphere model has a tumor microenvironment composed of vascular structures, with a high degree of simulation and high throughput, and can be used to evaluate tumor invasion and drug sensitivity detection, especially to evaluate the effectiveness of anti-tumor angiogenesis drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 This is a flowchart of a method for constructing a three-dimensional model of a solid tumor of bone and soft tissue tumors according to an embodiment of the present disclosure;

[0073] Figure 2 A flowchart of a method for detecting a target object using a three-dimensional model of a bone and soft tissue tumor solid tumor according to another embodiment of the present disclosure;

[0074] Figure 3 This is a bright field image and a fluorescent staining image of an osteosarcoma tumor sphere according to Example 1 of the present disclosure; wherein, Figure 3 (A) is a bright field image of osteosarcoma tumor spheres. Figure 3 (B) is a fluorescent color image of Epcam expression (marking tumor cells) in osteosarcoma tumor spheres. Figure 3 (C) is a fluorescent color image of osteosarcoma tumor spheres with DAPI (labeling cell nuclei). Figure 3 (D) is a fluorescent color image of osteosarcoma tumor spheres with Epcam and DAPI superimposed;

[0075] Figure 4 This is an observation diagram of the osteosarcoma tumor sphere biological model in Example 2 of the present disclosure during the drug sensitivity test;

[0076] Figure 5 The cell viability results of the osteosarcoma tumor sphere biological model after drug administration in Example 2 of the present disclosure;

[0077] Figure 6 This is an observation diagram of the osteosarcoma tumor sphere biological model in Example 3 of the present disclosure during the drug sensitivity test;

[0078] Figure 7 The cell viability results of the osteosarcoma tumor sphere biological model after drug administration in Example 3 of the present disclosure. DETAILED DESCRIPTION

[0079] In order to enable those skilled in the art to better understand the technical solution of the present disclosure, the present disclosure is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0080] Unless otherwise specified, the technical terms or scientific terms used in the present disclosure shall have the usual meanings understood by persons with ordinary skills in the field to which the present disclosure belongs. The words "include" or "comprises" used in the present disclosure neither limit the mentioned numbers, steps, actions, operations and / or their groups, nor exclude the presence or addition of one or more other different numbers, steps, actions, operations and / or their groups.

[0081] like Figure 1 As shown, in one aspect of the present disclosure, a method for constructing a three-dimensional model of a bone and soft tissue tumor solid tumor is provided, which specifically includes the following steps S110 to S130:

[0082] S110. Obtain solid tumor cells of bone and soft tissue tumors.

[0083] Specifically, in step S110, obtaining solid tumor cells includes the following steps S1101 to S1104:

[0084] S1101. Cleaning: Use primary tissue cleaning solution to clean the primary tissue of solid tumor cells and remove the surface mucosa.

[0085] In some preferred embodiments, the primary tissue washing solution is PBS buffer.

[0086] In other preferred embodiments, the primary tissue washing solution is PBS buffer with penicillin-streptomycin and gentamicin, that is, penicillin-streptomycin and gentamicin are added to PBS buffer.

[0087] As a further preferred embodiment, the concentration of penicillin-streptomycin may be preferably 3 to 5%, and the concentration of gentamicin may be preferably 100 to 200 μg / mL.

[0088] S1102, crushing: mince the tissue washed in step S1101 into a meat paste, resuspend it in a buffer solution, let it stand and discard the supernatant.

[0089] S1103. Digestion: Add primary tissue digestion solution of solid tumor cells, mix well by blowing, and then use a metal bath shaker to shake and digest at 37°C for 30-60 minutes to digest into single cells. Finally, add buffer to terminate digestion and obtain a cell suspension.

[0090] It should be understood that, in addition to the metal bath shaker, other similar devices may be used during the shaking digestion, and no specific limitation is made to this.

[0091] It should be noted that the present embodiment does not specifically limit the digestion time, and needs to be determined according to the specific conditions of the digestion. For example, when the digestion time is reached, a small amount of digestion solution is taken and observed under a microscope, and when a large number of single cells are seen, the digestion is terminated. If there are still many cell clusters, the digestion time needs to be extended to continue digestion until a large number of single cells appear.

[0092] In some preferred embodiments, the primary tissue digestion solution of solid tumor cells is Advanced DMEM / F12 culture medium supplemented with collagenase.

[0093] As a further preferred embodiment, collagenase can be one or more of collagenase I, collagenase II, and collagenase IV. Wherein, the concentration range of collagenase I can be preferably 0.5mg / mL-5mg / mL, the concentration range of collagenase II can be preferably 0.5mg / mL-5mg / mL, and the concentration range of collagenase IV can be preferably 0.5mg / mL-5mg / mL. More preferably, the concentration range of collagenase I can be preferably 1mg / mL-2mg / mL, the concentration range of collagenase II can be preferably 1mg / mL-2mg / mL, and the concentration range of collagenase IV can be preferably 1mg / mL-2mg / mL. Using the above-mentioned preferred collagenase concentration, the viability is higher during cell digestion, and the amount of cells obtained is more. Of course, in other preferred embodiments, collagenase I, collagenase II and collagenase IV of other concentration ranges can also be adopted.

[0094] In other preferred embodiments, the primary tissue digestion fluid of solid tumor cells further comprises one or more of hyaluronidase and Y27632.

[0095] As a further preferred embodiment, the concentration range of hyaluronidase may preferably be 100 μg / mL-1 mg / mL, for example, 100 μg / mL, 300 μg / mL, 500 μg / mL, 800 μg / mL, 1 mg / mL, etc., and the concentration range of Y27632 may preferably be 1 μM-10 μM, for example, 1 μM, 3 μM, 5 μM, 7 μM, 10 μM, etc.

[0096] S1104, resuspension: filter the digested cell suspension through a cell mesh, resuspend the cells in a solid tumor two-dimensional cell culture medium, and count them.

[0097] In some preferred embodiments, the two-dimensional cell culture medium for solid tumors includes Advanced DMEM / F12, FBS and PS. Among them, Advanced DMEM / F12 is used as the basic culture medium, fetal bovine serum (FBS) is rich in growth factors, proteins, nutrients and other components to promote cell growth and proliferation, and PS is a double antibody (penicillin plus streptomycin) to reduce bacterial contamination and avoid interference or contamination of target cells.

[0098] As a further preferred embodiment, the concentration range of FBS is preferably 5%-20%, for example, 5%, 10%, 15%, 20%, etc., and the concentration range of PS is preferably 1%-3%, for example, 1%, 2%, 3%, etc.

[0099] In other preferred embodiments, the two-dimensional cell culture medium for solid tumors also includes one or more of EGF, basicFGF, IGF, Glutamax, and HEPES. Among them, EGF and basicFGF, as growth factors, synergize with fetal bovine serum FBS to promote the proliferation, metabolic activity and differentiation of tumor cells and maintain pathological characteristics. IGF is a human recombinant protein that helps to induce cell proliferation, migration and differentiation. Glutamax can improve cell viability and growth, and helps to maintain stability in a wide temperature range. HEPES is used to provide pH stability, anti-oxidation and improve the cell growth environment.

[0100] As a further preferred embodiment, the concentration range of EGF is preferably 50ng / mL-200ng / mL, for example, 50ng / mL, 100ng / mL, 150ng / mL, 200ng / mL, etc. The concentration range of basic FGF is preferably 10ng / mL-100ng / mL, for example, 10ng / mL, 30ng / mL, 50ng / mL, 70ng / mL, 100ng / mL, etc. The concentration range of IGF is preferably 10ng / mL-50ng / mL, for example, 10ng / mL, 20ng / mL, 30ng / mL, 40ng / mL, 50ng / mL, etc. The concentration range of Glutamax is preferably 0.5%-5%, for example, 0.5%, 1%, 2%, 3%, 4%, 5%, etc. The concentration of HEPES (1M) is preferably 0.5%-5%, for example, 0.5%, 1%, 2%, 3%, 4%, 5%, etc.

[0101] It should be noted that in this embodiment, there is no specific limitation on solid tumors of bone and soft tissue tumors. For example, bone tumors include osteosarcoma, Ewing sarcoma, chondrosarcoma, fibrosarcoma, multiple myeloma, chordoma, reticular cell sarcoma, etc., and soft tissue malignant tumors include synovial sarcoma, fibrosarcoma, liposarcoma, rhabdomyosarcoma, etc.

[0102] In some preferred embodiments, the solid tumor cells may preferably be osteosarcoma.

[0103] S120, inoculating solid tumor cells into a culture container, adding a two-dimensional cell culture medium for solid tumor cells, and performing two-dimensional culture.

[0104] Specifically, the solid tumor cells obtained in step S110 are inoculated into a culture container (e.g., a T25 flask, at 6×10 5 / mL density was inoculated into T25 culture flasks), and solid tumor 2D cell culture medium was added into the culture flasks. The culture was cultured in an incubator for three days or the culture medium was replaced when most of the cells adhered to the wall.

[0105] It should be noted that the solid tumor two-dimensional cell culture medium in step S120 is the same as the two-dimensional cell culture medium used in step S110 . Please refer to the above description for specific components.

[0106] S130, mixing the two-dimensionally cultured solid tumor cells with vascular endothelial cells for spheroid culture to form spheroid aggregates, adding extracellular matrix to the spheroid aggregates, adding solid tumor three-dimensional cell culture medium to the spheroid aggregates after gelation, and culturing to form a three-dimensional model of bone and soft tissue tumor solid tumors with vascular structures.

[0107] Specifically, in step S130, the following steps S1301 to S1305 are also included:

[0108] S1301. Digestion: Use the passage digestion solution to digest the vascular endothelial cells and the solid tumor cells after two-dimensional culture, respectively, and add DMEM medium containing 10% FBS to terminate the digestion.

[0109] It should be understood that during digestion, the vascular endothelial cells and the solid tumor cells should be located in different culture flasks (eg, T25 culture flasks), and the vascular endothelial cells and the solid tumor cells in different culture flasks should be digested separately using the passage digestion solution to form a cell suspension.

[0110] In some preferred embodiments, the digestion solution for passage is at least one of TrypLE, Trypsin, and Accutase.

[0111] S1302, resuspension: adding a two-dimensional cell culture medium for solid tumors to the digested solid tumor cells, adding a vascular endothelial cell culture medium to the digested vascular endothelial cells, centrifuging, discarding the supernatant, and obtaining a solid tumor cell pellet and a vascular endothelial cell pellet, respectively; then, resuspending the solid tumor cell pellet and the vascular endothelial cell pellet, respectively, using a three-dimensional cell culture medium for solid tumors, and counting and detecting cell viability.

[0112] In some preferred embodiments, the tumor solid tumor three-dimensional cell culture medium includes a tumor solid tumor two-dimensional cell culture medium and an endothelial cell culture medium, wherein the volume ratio of the tumor solid tumor two-dimensional cell culture medium and the endothelial cell culture medium is preferably (1-2):1, for example, 1:1, 1.5:1, 2:1, etc.

[0113] As a further preferred embodiment, the two-dimensional cell culture medium for solid tumors is the same as that described above, that is, it includes Advanced DMEM / F12, FBS, PS, and one or more of EGF, basicFGF, IGF, Glutamax, and HEPES, wherein the concentration range of FBS is 5%-20%; the concentration range of PS is 1-3%; the concentration range of EGF is 50ng / mL-200ng / mL; the concentration range of basicFGF is 10ng / mL-100ng / mL; the concentration range of IGF is 10ng / mL-50ng / mL; the concentration range of Glutamax is 0.5%-5%; and the concentration range of HEPES is 0.5%-5%.

[0114] As a further preferred embodiment, the endothelial cell culture medium comprises: ECM, FBS, EGF, basic FGF, and PS. The concentration range of FBS is 5%-20%, for example, 5%, 10%, 15%, 20%, etc., the concentration range of EGF is 10ng / mL-100ng / mL, for example, 10ng / mL, 20ng / mL, 50ng / mL, 80ng / mL, 100ng / mL, etc., the concentration range of basic FGF is 10ng / mL-100ng / mL, for example, 10ng / mL, 20ng / mL, 50ng / mL, 80ng / mL, 100ng / mL, etc., and the concentration range of PS is 1%-3%, for example, 1%, 2%, 3%, etc.

[0115] S1303, mixing the resuspended solid tumor cell suspension and the vascular endothelial cell suspension in a certain ratio to obtain a mixed suspension.

[0116] In some preferred embodiments, in the mixed suspension, vascular endothelial cells account for 1%-30% of the total number of vascular endothelial cells and tumor solid tumor cells, for example, 1%, 5%, 10%, 15%, 20%, 25%, 30%, etc.

[0117] As a further preferred embodiment, tumor-associated vascular endothelial cells account for 2%-15% of the total cells.

[0118] S1304, spheroidization: adding the above mixed suspension to a low-adsorption U-bottom well plate, or a culture container with a low-adsorption U-bottom structure, centrifuging and culturing in an incubator for two days to form spheroids.

[0119] In this embodiment, the spheroidization stage is equivalent to agglomerating the mixed suspension of the two cells and adding a tumor solid tumor three-dimensional cell culture medium for culture. After culture, the tumor solid tumor cells and vascular endothelial cells in the mixed suspension are agglomerated to obtain a spheroid with clear boundaries and a vascular structure.

[0120] In some preferred embodiments, the centrifugal treatment condition may preferably be centrifugal treatment at a rotation speed of 1000 rpm for 5 minutes.

[0121] S1305, plating: discard excess cell culture medium, add extracellular matrix to a low-adsorption U-bottom well plate, or a culture container with a low-adsorption U-bottom structure, blow to make the matrix gel evenly wrap the spheroids, and transfer the spheroids to the culture well plate or culture chip. After gelation, add tumor solid tumor three-dimensional cell culture medium and culture to form a three-dimensional model of bone and soft tissue tumor solid tumors.

[0122] It should be noted that when the three-dimensional model is used for drug sensitivity testing, the drug solution to be tested is added after the tumor spheres are transferred to the culture well plate or culture chip to form a gel. Therefore, the drug solution to be tested can also be mixed with the three-dimensional cell culture medium and added.

[0123] In some preferred embodiments, when the tumor spheres are transferred to a culture well plate for culture, a 96-well plate may be preferred, and a three-dimensional cell culture medium is added to the culture wells. The well plate can provide multiple independent experimental units to improve experimental efficiency.

[0124] In other preferred embodiments, when transferring the tumor spheres to the culture chip for culture, a drug sensitivity chip may be preferred, and the tumor spheres are transferred to the middle hole of the drug sensitivity chip. After gelation, three-dimensional cell culture medium is added to the middle hole and the left and right side holes of the drug sensitivity chip respectively.

[0125] In other preferred embodiments, the extracellular matrix includes hydrogel materials such as matrix glue, natural biomaterials, synthetic biomaterials and mixed materials thereof, which are not specifically limited and can be specifically selected according to actual needs.

[0126] In other preferred embodiments, the gelling method can be any one of temperature, light, and pH adjustment, or a combination of the above methods, which is not specifically limited and can be specifically selected according to different hydrogel materials.

[0127] In this embodiment, by adding extracellular matrix to the spherulomer, the growth environment of cells in the body can be more accurately simulated, tissue heterogeneity can be reflected, and analysis such as drug screening and toxicity testing can be facilitated.

[0128] In the method for constructing a three-dimensional model of a solid tumor of a bone and soft tissue tumor provided by the present disclosure, the solid tumor cells are first cultured in two dimensions and then cultured in three dimensions to effectively improve the cell survival rate. At the same time, the cells can continue to be passaged and proliferated in the two-dimensional culture stage to further improve the survival rate. Secondly, in the three-dimensional sphere culture stage, vascular endothelial cells are co-cultured with solid tumor cells to form a tumor sphere model with a high degree of simulation, high throughput and a tumor microenvironment.

[0129] Another aspect of the present disclosure provides a three-dimensional model of a solid tumor of bone and soft tissue tumors. The three-dimensional model is formed by the construction method described above. Please refer to the above description for the specific process, which will not be repeated here.

[0130] The three-dimensional model of bone and soft tissue tumor solid tumor obtained in this embodiment includes solid tumor cells and vascular endothelial cells.

[0131] In some preferred embodiments, the three-dimensional model of a solid tumor of bone and soft tissue tumor is preferably a three-dimensional model of an osteosarcoma tumor sphere.

[0132] Another aspect of the present disclosure provides an application of a three-dimensional model of a bone and soft tissue tumor solid tumor in compound analysis and / or treatment screening. For the specific application process, please refer to the target detection method below.

[0133] In some preferred embodiments, compound analysis includes at least one of drug screening, drug sensitivity testing, and drug toxicity testing.

[0134] In other preferred embodiments, the treatment modalities include radiotherapy, thermal therapy, and other treatment modalities.

[0135] The three-dimensional spheroid model disclosed in the present invention is a tumor spheroid model with a tumor microenvironment, which has a high degree of simulation and can evaluate tumor invasion and cell activity. It is applied to drug sensitivity detection, and in particular, can effectively evaluate the effectiveness of anti-tumor angiogenesis drugs.

[0136] like Figure 2 As shown, another aspect of the present disclosure provides a method S200 for detecting a target object using the above-mentioned three-dimensional model of bone and soft tissue tumor solid tumors, comprising the following specific steps S210 to S220:

[0137] S210, introducing the target object to be measured into the three-dimensional model of bone and soft tissue tumor solid tumor.

[0138] Specifically, the tumor spheres transferred to the 96-well plate or drug sensitivity chip in step S130 described above are cultured in a carbon dioxide incubator for 1-2 days, taken out and observed under a microscope, and the target to be tested can be added when buds appear around the sphere aggregates.

[0139] It should be understood that the target to be tested here should be understood in a broad sense, and can be a drug to be tested, for example, a tumor chemotherapy drug, a targeted drug, a tumor vascular inhibitor, and other drugs. The target to be tested can also be other treatment methods such as radiotherapy and hyperthermia. Of course, the target to be tested can also be a drug and a treatment method. In other words, the target is a possible factor that affects the change of the three-dimensional model index.

[0140] S220, obtaining the effect of the target object on the three-dimensional model of bone and soft tissue tumor solid tumor.

[0141] It should be noted that the present embodiment evaluates the effect of the target object to be measured on the three-dimensional model by changing the indicators of the three-dimensional model of the solid tumor of bone and soft tissue tumors, wherein the indicator changes include at least one of the shape changes, size changes, activity changes, vascular morphology changes, protein molecule expression changes or distribution changes of the three-dimensional model of the solid tumor of bone and soft tissue tumors.

[0142] Specifically, taking the use of drugs as the target to be tested as an example, the day of drug addition is recorded as day 0, that is, the three-dimensional model is first photographed on day 0 to record the growth status of the solid tumor tumor spheres of bone and soft tissue tumors when no drugs are added. The culture medium in the culture chamber is aspirated, and the three-dimensional cell culture medium containing the drug solution prepared in advance is added, and the cells are placed in an incubator at 37°C and 5% CO2 for culture. Afterwards, on the third day of culture, take another photo to record the growth status of the solid tumor tumor spheres of bone and soft tissue tumors on the third day after drug addition. The culture medium in the culture chamber is aspirated, and the three-dimensional cell culture medium containing the drug solution is added, and the cells are placed in an incubator at 37°C and 5% CO2 for culture. Take another photo to observe and record the growth status of the solid tumor tumor spheres of bone and soft tissue tumors on the fifth day after drug addition. At the same time, the CTG detection kit can be used for activity detection.

[0143] It should be understood that the detection of solid tumor spheres of bone and soft tissue tumors is not limited to image analysis and activity detection, but can also be subjected to a variety of detection methods such as pathological detection, marker detection, and genetic detection.

[0144] It should be further explained that, in other embodiments, radiotherapy or thermotherapy can also be used as the target to be tested to determine the appropriate treatment method. Of course, radiotherapy or thermotherapy can also be used together with drugs as the target to be tested to screen out the appropriate treatment plan, which is suitable for precision medicine, drug development and other fields.

[0145] In another aspect of the present disclosure, a culture reagent combination is provided, which comprises a two-dimensional cell culture medium for solid tumors and a three-dimensional cell culture medium for solid tumors;

[0146] Wherein, the two-dimensional culture medium for solid tumors includes Advanced DMEM / F12, FBS, PS, and one or more of EGF, basicFGF, IGF, Glutamax, and HEPES; wherein,

[0147] The concentration of the FBS ranges from 5% to 20%;

[0148] The concentration range of the PS is 1-3%;

[0149] The concentration range of EGF is 50ng / mL-200ng / mL;

[0150] The concentration range of basicFGF is 10ng / mL-100ng / mL;

[0151] The concentration range of the IGF is 10 ng / mL-50 ng / mL;

[0152] The concentration range of Glutamax is 0.5%-5%;

[0153] The concentration range of HEPES is 0.5%-5%;

[0154] Wherein, the solid tumor three-dimensional cell culture medium is obtained by mixing the solid tumor two-dimensional cell culture medium and the endothelial cell culture medium in a volume ratio of (1-2):1;

[0155] The endothelial cell culture medium includes ECM, FBS, EGF, basic FGF, and PS; wherein,

[0156] The concentration of the FBS ranges from 5% to 20%;

[0157] The concentration range of EGF is 10ng / mL-100ng / mL;

[0158] The concentration range of basic FGF is 10ng / mL-100ng / mL;

[0159] The concentration range of the PS is 1%-3%;

[0160] The two-dimensional cell culture medium for solid tumors and the three-dimensional cell culture medium for solid tumors are used in the aforementioned method for constructing a three-dimensional model of a solid tumor of a bone and soft tissue tumor, or in the construction of the aforementioned three-dimensional model of a solid tumor of a bone and soft tissue tumor.

[0161] The following will further illustrate the construction method and application of the three-dimensional model of bone and soft tissue tumor solid tumors in combination with several specific examples:

[0162] Example 1

[0163] This example takes the construction of a 3D model of osteosarcoma tumor spheres on a well plate as an example. The construction method includes the following steps:

[0164] Step 1: Extraction of osteosarcoma cells

[0165] S1. Two osteosarcoma cell culture medium additives were taken out from -20°C, restored to room temperature, mixed with the basal culture medium to obtain osteosarcoma two-dimensional cell culture medium, preheated to room temperature and set aside; wherein, the osteosarcoma two-dimensional cell culture medium includes the following components with final concentrations: the basal culture medium is Advanced DMEM / F12, and Fetal Bovine Serum, 10%; Penicillin-Streptomycin, 1%; EGF, 50ng / mL; basic FGF, 50ng / mL; Glutamax, 1%; HEPES (1M), 1%; the aforementioned percentage concentrations represent mass concentrations.

[0166] S2. Add 3-4% penicillin-streptomycin and 100-200 μg / mL gentamicin to the buffer to obtain a primary tissue washing solution for later use;

[0167] S3, the primary tissue digestion solution additive is taken out from -20°C, restored to room temperature, added to the primary tissue digestion solution in a certain proportion and mixed to obtain the osteosarcoma primary tissue digestion solution, heated to 37°C in a water bath, and set aside;

[0168] S4. Add 5-6 mL of primary tissue washing solution to three sterile 60 mm culture dishes, wash the osteosarcoma primary tissue three times in each culture dish to remove the surface mucosa;

[0169] S5. Use a set of sterile surgical knife handles, surgical blades, and disposable forceps to mince the tissue in a 60 mm culture dish until it is minced. Then collect the tissue into a 15 mL centrifuge tube, add 3-5 mL of buffer to resuspend, centrifuge at 1000 rpm, 4°C for 5 min, and remove the supernatant.

[0170] S6. Add 5 mL of primary osteosarcoma tissue digestion solution (per 1 cm 3 Tissue) to a 15 mL centrifuge tube and mix by pipetting;

[0171] S7. Digest at 37°C using a metal bath shaker for 30-60 min. During the shaking process, invert and mix twice every 5 min.

[0172] S8. When the digestion time is reached, take a small amount of digestion solution and observe it under the microscope. When a large number of single cells are seen, add 2 to 3 times the volume of buffer into a 15 mL centrifuge tube to stop the digestion. If there are still many cell clusters, extend the digestion time and continue digestion.

[0173] S9. The digested cell suspension was filtered through a 100 μm cell sieve, and 2 to 3 mL of buffer was used to wash the centrifuge tube and the filter. The filtered filtrate was centrifuged at 4°C and 1000 rpm for 5 min, and the supernatant was removed.

[0174] S10. Resuspend the cells in 1 mL of osteosarcoma 2D cell culture medium, observe the cell status under a microscope, and count the cells.

[0175] Step 2: Two-dimensional culture of osteosarcoma cells

[0176] S11, 6x10 5 The cells were seeded into a T25 culture flask at a density of / mL, 5mL of osteosarcoma 2D cell culture medium was added, and cultured in an incubator at 37°C and 5% CO2;

[0177] S12. After culturing for 3-4 days (or until most cells adhere to the wall), discard the osteosarcoma 2D cell culture medium in the T25 flask and add 5 mL of osteosarcoma 2D cell culture medium again.

[0178] Step 3: Osteosarcoma 3D tumor sphere culture

[0179] S13. Mix the osteosarcoma two-dimensional cell culture medium and the endothelial cell culture medium in a volume ratio of 2:1 to form an osteosarcoma three-dimensional cell culture medium, and restore the three-dimensional cell culture medium from -20°C to room temperature for use; wherein the two-dimensional cell culture medium included in the three-dimensional cell culture medium is the same as that in step S1, and the endothelial cell culture medium includes the following components at final concentrations: basal culture medium ECM; Fetal Bovine Serum, 10%; EGF, 10 ng / mL; basic FGF, 10 ng / mL; penicillin-streptomycin, 1%.

[0180] Take out S14, buffer and subculture digestion solution from the refrigerator at 4°C, return to room temperature and set aside;

[0181] S15. Thaw the matrix gel in advance and place it in a refrigerator at 4°C for later use;

[0182] S16. Use one T25 bottle of osteosarcoma cells and one T25 bottle of endothelial cells with a cell confluence of more than 85%, discard the culture medium, add 2 mL of subculture digestion solution to the T25 bottle of osteosarcoma cells, and add 2 mL of subculture digestion solution to the T25 bottle of endothelial cells, infiltrate the bottom surface, and incubate in a 37°C, 5% CO2 incubator for 1 to 2 minutes;

[0183] S17, add 6 mL of osteosarcoma two-dimensional cell culture medium into the T25 flask of osteosarcoma cells to obtain osteosarcoma cell suspension, add 6 mL of endothelial cell culture medium into the T25 flask of endothelial cells to obtain endothelial cell suspension, then aspirate the suspension into 15 mL centrifuge tubes, centrifuge at 1000 rpm for 5 min, discard the supernatant, and obtain osteosarcoma cell precipitates and endothelial cell precipitates, respectively;

[0184] S18. Resuspend the osteosarcoma cell suspension and endothelial cell suspension in 1 mL of osteosarcoma 3D cell culture medium, and count and detect cell viability;

[0185] S19. Mix the two cell suspensions in proportion, add osteosarcoma three-dimensional cell culture medium to form a mixed suspension, and fill up to the total volume required for plating. The mixing ratio is 200 μL of cell suspension per well, and the total number of cells is 15,000 / well, of which the number of endothelial cells is 1,500 / well and the number of osteosarcoma cells is 13,500 / well. According to the number of plating wells, calculate the total volume required for plating: number of wells × 200 μL × 1.2, calculate the required volume of endothelial cell suspension: 1500 × number of wells × 1.2 ÷ endothelial cell density, calculate the required volume of osteosarcoma cell suspension: 13,500 × number of wells × 1.2 ÷ osteosarcoma cell density;

[0186] S20, adding the mixed suspension to a low-adsorption U-bottom plate, or a culture container with a low-adsorption U-bottom structure, and centrifuging at 1000 rpm for 5 minutes;

[0187] S21, centrifuge the 96-well plate (1000 rpm, 5 min), and then place it in an incubator (37°C, 5% CO2) for two days;

[0188] S22. After two days of culture, remove the excess culture medium in the culture chamber, add 5 μL of matrix gel to each well, gently blow to make the matrix gel evenly wrap the globulomer, transfer to a black flat-bottom 96-well plate, and place in an incubator (37°C, 5% CO2);

[0189] S23. After culturing for 30 minutes, 100 μL of osteosarcoma three-dimensional cell culture medium was added to each well of the 96-well plate after gelation, and the plate was placed in an incubator at 37° C. and 5% CO 2 for culture to form an osteosarcoma tumor sphere biological model.

[0190] Figure 3 (A) is a bright field image of the osteosarcoma tumor sphere. It can be seen that the osteosarcoma tumor sphere obtained in Example 1 has a clear boundary and grows a large area of ​​microvessels with a complex network structure, which indicates that the osteosarcoma tumor sphere constructed in this example has a vascular structure. Figure 3(B) is an immunofluorescence image of Epcam expression (marking tumor cells) in osteosarcoma tumor spheres. Figure 3 (C) is a DAPI (labeling cell nuclei) fluorescence image of osteosarcoma tumor spheres. Figure 3 (D) in the figure is a fluorescence image of Epcam expression and DAPI superimposed on the osteosarcoma tumor sphere. In summary, the osteosarcoma tumor sphere constructed in Example 1 has better simulation performance.

[0191] Example 2

[0192] This example shows how to build a 3D model of osteosarcoma tumor spheres on a well plate and perform drug sensitivity testing, including the following steps:

[0193] The preparation method for the biological model on the well plate is as follows:

[0194] Osteosarcoma tumor spheres were constructed in the same manner as in Example 1, and the cultured osteosarcoma tumor spheres were transferred to a 96-well plate and cultured for 1-2 days before being used for drug testing.

[0195] The specific detection method for the drug sensitivity test of the osteosarcoma tumor sphere biological model on the well plate is as follows:

[0196] S1. Prepare appropriate concentrations of drug-containing three-dimensional cell culture medium containing doxorubicin (1 μM), cisplatin (4.8 μM), and doxorubicin + cisplatin (doxorubicin 1 μM + cisplatin 4.8 μM) to form three chemotherapy drug test groups; of course, it should be understood that in order to illustrate the effects of chemotherapy drugs on the tumor sphere biological model, in this embodiment, a blank control group is also set up, such as Figure 4 shown.

[0197] S2. Observe and record the growth status of osteosarcoma tumor spheres, remove the culture medium in the 96-well plate, add 100 μL of three-dimensional cell culture medium containing drug solution to each culture well of the 96-well plate, and place in a 37°C, 5% CO2 incubator for culture (the day of drug addition is recorded as day 0);

[0198] S3. Observe and record the growth status of osteosarcoma tumor spheres on day 1;

[0199] S4. On the third day, the growth status of osteosarcoma tumor spheres was observed and recorded;

[0200] S5. On the 5th day, the growth status of osteosarcoma tumor spheres was observed and recorded, and the activity was detected using the CellTiter-Glo luminescent cell viability detection kit (CTG) detection kit. 100 μL CTG detection reagent was added to each well, shaken for 5 minutes using a shaker, and incubated at room temperature for 25 minutes;

[0201] S6. After the incubation, use a chemiluminescence microplate reader to detect and calculate the cell viability.

[0202] like Figure 4 As shown, the osteosarcoma tumor sphere biological model is given in the drug sensitivity test process. By observing the growth status of the osteosarcoma tumor spheres recorded on day 0 (without adding drugs), day 1, day 3 and day 5, it can be seen that the osteosarcoma tumor sphere biological model is sensitive to multiple compounds. Doxorubicin, cisplatin and the combination of the two drugs will inhibit the tumor, and the inhibition rate is different.

[0203] like Figure 5 As shown, the cell viability quantitative analysis after 5 days of drug treatment is given. The results show that after cisplatin treatment, the viability of osteosarcoma tumor sphere cells is about 60%; after doxorubicin treatment, the viability of osteosarcoma tumor sphere cells is about 4%; after doxorubicin and cisplatin combined treatment, the viability of osteosarcoma tumor sphere cells is about 1%.

[0204] Example 3

[0205] This example shows how to build a 3D model of osteosarcoma tumor spheres on a drug sensitivity chip and perform drug sensitivity testing, which specifically includes the following steps:

[0206] The specific method for constructing the osteosarcoma tumor sphere model on the drug sensitivity chip is as follows:

[0207] S1. After culturing the osteosarcoma tumor spheres formed in Example 1 in a low-adsorption U-bottom plate or a culture container with a low-adsorption U-bottom structure for two days, remove the excess culture medium in the culture chamber, add 5 μL of matrix gel to each well, gently blow to make the matrix gel evenly wrap the spheromer, and transfer the spheromer to the middle well of the drug sensitivity chip, and place it in an incubator (37° C., 5% CO2);

[0208] S2. After 30 minutes of culture, after gelation, add 50 μL of three-dimensional cell culture medium to the middle hole and the left and right holes of the drug sensitivity chip, and place it in an incubator at 37°C and 5% CO2 for culture to form an osteosarcoma tumor sphere chip model.

[0209] The specific method for drug sensitivity test of osteosarcoma tumor sphere model on drug sensitivity chip is as follows:

[0210] S1. Prepare a three-dimensional cell culture medium containing drugs of appropriate concentrations: sorafenib + doxorubicin + cisplatin, doxorubicin + cisplatin (sorafenib 7 μM, doxorubicin 1 μM, cisplatin 4.8 μM), to form two drug test groups, wherein the first group includes chemotherapy drugs doxorubicin + cisplatin, and anti-vascular drug sorafenib, and the second group is a chemotherapy drug test group; of course, it should be understood that in order to illustrate the effect of drugs on the tumor sphere biological model, in this embodiment, a blank control group is also set up, such as Figure 5 shown.

[0211] S2. Observe and record the growth status of osteosarcoma tumor spheres. Remove the culture medium in the drug sensitivity chip and add a three-dimensional cell culture medium containing a drug solution. For example, add 30 μL of a three-dimensional cell culture medium containing a drug solution to the middle well and the left and right wells of the drug sensitivity chip (the day of drug addition is recorded as day 0);

[0212] S3. After adding the drug, put the drug sensitivity chip into the rocking perfusion instrument, 7° / 15min, and put the rocking perfusion instrument into the incubator for flow culture;

[0213] S4. On the third day, the growth status of the osteosarcoma tumor spheres was observed and recorded, and 30 μL of drug-containing three-dimensional cell culture medium was added to the middle well and the left and right wells;

[0214] S5. On the 5th day, the growth status of osteosarcoma tumor spheres was observed and recorded, and the activity was tested using the CTG detection kit;

[0215] S6. Discard all the culture medium from the left and right wells;

[0216] S7: Do not discard the culture medium in the middle well, directly add 50 μL of CTG stock solution, and mix by pipetting;

[0217] S8. Use a shaker to shake for 5 minutes and incubate at room temperature for 25 minutes;

[0218] S9. Use a chemiluminescence microplate reader to detect and calculate the cell viability.

[0219] like Figure 6 As shown in the figure, the osteosarcoma tumor sphere biological model is given in the drug sensitivity test process. By observing the growth status of the osteosarcoma tumor sphere recorded on day 0 (without drug), day 1, day 3, and day 5, it can be seen that the combination of tumor angiogenesis inhibitors and chemotherapy drugs also has drug sensitivity to the osteosarcoma tumor sphere biological model.

[0220] like Figure 7 As shown, compared with the use of chemotherapy drugs alone, the combination of tumor angiogenesis inhibitors and chemotherapy drugs has a stronger inhibitory effect on the osteosarcoma tumor sphere model and a lower cell viability.

[0221] It should be noted that the above embodiment only describes the construction of a three-dimensional model of osteosarcoma tumor spheres and drug testing thereof as an example. The above method can also be used to form three-dimensional models of other types of bone and soft tissue tumor solid tumors, which are not listed one by one here.

[0222] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present disclosure, but the present disclosure is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and substance of the present disclosure, and these modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. A method for constructing a three-dimensional model of a bone and soft tissue tumor solid tumor, characterized in that: The construction method comprises: Obtain solid tumor cells from bone and soft tissue tumors; Inoculating the tumor solid tumor cells into a culture container, adding a tumor solid tumor two-dimensional cell culture medium, and performing two-dimensional culture; The solid tumor cells after two-dimensional culture are mixed with vascular endothelial cells at a certain cell ratio for spheroid culture to form spheroid aggregates, and extracellular matrix is ​​added to the spheroid aggregates. After gelation, solid tumor three-dimensional cell culture medium is added to culture to form a three-dimensional model of bone and soft tissue tumor solid tumors with vascular structure.

2. The construction method according to claim 1, characterized in that: The solid tumor three-dimensional cell culture medium comprises a solid tumor two-dimensional cell culture medium and an endothelial cell culture medium; Wherein, the two-dimensional cell culture medium for solid tumors includes Advanced DMEM / F12, FBS, PS, and one or more of EGF, basicFGF, IGF, Glutamax, and HEPES; wherein, The concentration of the FBS ranges from 5% to 20%; The concentration range of the PS is 1-3%; The concentration range of EGF is 50ng / mL-200ng / mL; The concentration range of basicFGF is 10ng / mL-100ng / mL; The concentration range of the IGF is 10 ng / mL-50 ng / mL; The concentration range of Glutamax is 0.5%-5%; The concentration range of HEPES is 0.5%-5%; The endothelial cell culture medium includes ECM, FBS, EGF, basic FGF, and PS; wherein, The concentration of the FBS ranges from 5% to 20%; The concentration range of EGF is 10ng / mL-100ng / mL; The concentration range of basic FGF is 10ng / mL-100ng / mL; The concentration of PS is in the range of 1%-3%.

3. The construction method according to claim 1, characterized in that: In the tumor solid tumor three-dimensional cell culture medium, the volume ratio of the tumor solid tumor two-dimensional cell culture medium and the endothelial cell culture medium is (1-2):

1.

4. The construction method according to claim 1, characterized in that: The method of mixing the two-dimensionally cultured solid tumor cells with vascular endothelial cells for spheroid culture to form spheroid aggregates comprises: Use the passage digestion solution to digest the vascular endothelial cells and the solid tumor cells after two-dimensional culture respectively; The digested solid tumor cells and vascular endothelial cells were resuspended respectively using solid tumor three-dimensional cell culture medium; Mixing the resuspended solid tumor cell suspension and the vascular endothelial cell suspension to obtain a mixed suspension; wherein, in the mixed suspension, the vascular endothelial cells account for 1%-30% of the total number of the vascular endothelial cells and the solid tumor cells; The mixed suspension is added into a low-adsorption U-shaped bottom well plate, or a culture container containing a low-adsorption U-shaped bottom structure, and is cultured to form spherulomers.

5. The construction method according to claim 4, characterized in that: The passage digestion solution is at least one of TrypLE, Trypsin, and Accutase.

6. The construction method according to claim 1, characterized in that: The method of obtaining bone and soft tissue tumor solid tumor cells comprises: Use buffer or a mixture of buffer and penicillin-streptomycin and gentamicin to wash primary tissues of solid tumors of bone and soft tissue; The primary tissue of solid tumor is crushed, resuspended and left to stand; The primary tissue of the solid tumor is digested with a primary tissue digestion solution to obtain a cell suspension; wherein the primary tissue digestion solution comprises: Advanced DMEM / F12, and one or more of collagenase I, collagenase II, and collagenase IV; Resuspend the digested cell suspension in solid tumor 2D cell culture medium.

7. The construction method according to claim 6, characterized in that: The concentration range of the collagenase I is 0.5 mg / mL-5 mg / mL; The concentration range of the collagenase II is 0.5 mg / mL-5 mg / mL; The concentration range of the collagenase IV is 0.5 mg / mL-5 mg / mL; The primary tissue digestion solution also includes one or more of hyaluronidase and Y27632; wherein, The concentration range of the hyaluronidase is 100 μg / mL-1 mg / mL; The concentration range of Y27632 is 1 μM-10 μM.

8. A three-dimensional model of solid tumors of bone and soft tissue tumors, characterized in that: It is formed by the construction method described in any one of claims 1 to 7.

9. An application of a three-dimensional model of a bone and soft tissue tumor solid tumor, characterized in that: The three-dimensional model of solid tumors of bone and soft tissue tumors according to claim 8 is applied to compound analysis and / or treatment screening; wherein, The compound analysis includes at least one of drug screening, drug sensitivity testing, and drug toxicity testing; The treatment modality includes at least one of radiation therapy and thermal therapy.

10. A method for detecting a target object using the three-dimensional model of bone and soft tissue tumor solid tumors according to claim 9, characterized in that: The detection method comprises: Introducing a target object to be tested into the three-dimensional model of bone and soft tissue tumor solid tumor; Obtain the effect of the target object on the three-dimensional model of the bone and soft tissue tumor solid tumor.

11. The detection method according to claim 10, characterized in that: The target includes drugs and / or treatment methods; The drug includes at least one of a tumor chemotherapy drug, a targeted drug, and a tumor vascular inhibitor; The treatment modalities include radiotherapy and / or thermal therapy.

12. A culture reagent combination, characterized in that: The culture reagent combination comprises a two-dimensional cell culture medium for solid tumors and a three-dimensional cell culture medium for solid tumors; Wherein, the two-dimensional culture medium for solid tumors includes Advanced DMEM / F12, FBS, PS, and one or more of EGF, basicFGF, IGF, Glutamax, and HEPES; wherein, The concentration of the FBS ranges from 5% to 20%; The concentration range of the PS is 1-3%; The concentration range of EGF is 50ng / mL-200ng / mL; The concentration range of basicFGF is 10ng / mL-100ng / mL; The concentration range of the IGF is 10 ng / mL-50 ng / mL; The concentration range of Glutamax is 0.5%-5%; The concentration range of HEPES is 0.5%-5%; Wherein, the solid tumor three-dimensional cell culture medium is obtained by mixing the solid tumor two-dimensional cell culture medium and the endothelial cell culture medium in a volume ratio of (1-2):1; The endothelial cell culture medium includes ECM, FBS, EGF, basic FGF, and PS; wherein, The concentration of the FBS ranges from 5% to 20%; The concentration range of EGF is 10ng / mL-100ng / mL; The concentration range of basic FGF is 10ng / mL-100ng / mL; The concentration range of the PS is 1%-3%; The two-dimensional cell culture medium for solid tumors and the three-dimensional cell culture medium for solid tumors are used in the construction method according to any one of claims 1 to 7, or in the construction of the model according to claim 8.

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