Synovial sarcoma three-dimensional model and construction method and application thereof

Abstract

The invention relates to a synovial sarcoma three-dimensional model and a construction method and application thereof. The construction method comprises the following steps: extracting synovial sarcoma cells; performing suspension culture on synovial sarcoma organs; performing three-dimensional culture on synovial sarcoma organs; wherein a culture medium used in the three-dimensional culture of the synovial sarcoma organoid is a mixed culture medium and comprises a synovial sarcoma organoid culture medium and an endothelial cell culture medium. Compared with the prior art, the synovial sarcoma organ culture medium and the endothelial cell culture medium are mixed to form the mixed culture medium for three-dimensional culture, when the synovial sarcoma organ is subjected to three-dimensional culture, the cell survival rate is increased, a three-dimensional model with a vascular structure is formed, and the simulation degree is high.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a three-dimensional model of synovial sarcoma and a construction method and application thereof. Background Art

[0002] Synovial sarcoma (SS) is a relatively malignant soft tissue sarcoma that often occurs in adolescents. Due to the extremely low level of T cell infiltration in its tumor microenvironment (TME), it is able to escape the immune system. Currently, the main treatment for SS is surgical resection, supplemented by radiotherapy and chemotherapy. However, this conventional therapy has a very low cure rate, and nearly half of patients develop incurable metastatic disease, with a poor prognosis and high mortality. In the study of synovial sarcoma, TME cells and the factors they secrete play a key role in tumor pathogenesis and may be effective targets for immunotherapy. Therefore, the complex interactions between tumor cells and their mediators are crucial for the rational development of effective anticancer drugs.

[0003] In SS research, the complex tissue origins and variable pathological morphology of synovial sarcoma lead to challenges in developing synovial sarcoma xenograft models, including long production cycles, low tumor formation rates, small tumor size, and difficult tissue sampling. Furthermore, significant genetic differences exist between humans and animal models, which can lead to significant discrepancies between the efficacy of candidate drugs in animal models and later human trials. Furthermore, animal model development is time-consuming and expensive, making it difficult to use for high-throughput drug screening. Compared to two-dimensional (2D) models established with immortalized cell lines, tumor organoids better simulate tissue development and pathological states. However, due to their lack of a complete microenvironment, they struggle to fully characterize the original tissue. Therefore, constructing tumor organoid models with a microenvironment for drug testing more closely resembles the human body's true response to drug action. Three-dimensional (3D) models not only mimic the 3D environment in which cancer cells grow but also simulate the interaction between cancer cells and the TME (including their extracellular matrix), making them ideal tools for drug screening. In particular, the 3D model constructed with cells derived from the original SS tumor tissue retains specific tumor characteristics and has broad application prospects in scientific research and drug development.

[0004] Currently, when culturing synovial sarcoma organoids, they are mostly cultured by adding synovial sarcoma organoid-related culture medium. The synovial sarcoma organoids cultured in this way lack vascular structure and have a low survival rate. Summary of the Invention

[0005] The purpose of the present invention is to provide a three-dimensional model of synovial sarcoma and its construction method and application, so as to form a three-dimensional model with a vascular structure.

[0006] The purpose of the present invention can be achieved by the following technical solution: a method for constructing a three-dimensional model of synovial sarcoma, the method comprising the following steps:

[0007] Synovial sarcoma cell extraction;

[0008] Synovial sarcoma organoid suspension culture;

[0009] Three-dimensional culture of synovial sarcoma organoids;

[0010] Among them, the culture medium used in the three-dimensional culture of synovial sarcoma organoids is a mixed culture medium, including: synovial sarcoma organoid culture medium and endothelial cell culture medium.

[0011] The present invention uses a culture medium formed by mixing synovial sarcoma organoid culture medium and endothelial cell culture medium for three-dimensional culture during the synovial sarcoma three-dimensional model culture stage, thereby achieving co-culture of vascular endothelial cells and synovial sarcoma cells, effectively improving cell survival rate, and forming a highly simulated three-dimensional synovial sarcoma model with a vascular structure.

[0012] Preferably, the synovial sarcoma organoid culture medium comprises a first basal culture medium and a first specific supplemental factor.

[0013] Further preferably, the first basal culture medium is DMEM or Advanced DMEM / F12.

[0014] Further preferably, the first specific additive factor comprises: 1x Glutamax, 1x HEPES, 50-200 U / mL penicillin-streptomycin, 1v / v%-5v / v% B27, 5-20 μM Nicotinamide, 1-5 μM N-Acetylcysteine, 10-100 ng / mL EGF, 10-100 ng / mL FGF, 10-100 ng / mL IGF1, 5v / v%-10v / v% FBS (Fetal Bovine Serum).

[0015] Still more preferably, the first specific additive factor comprises: 1x Glutamax, 1x HEPES, 100 U / mL penicillin-streptomycin, 2 v / v% B27, 10 μM Nicotinamide, 1 μM N-Acetylcysteine, 50 ng / mL EGF, 50 ng / mL FGF, 50 ng / mL IGF1, and 10 v / v% FBS (Fetal Bovine Serum).

[0016] Preferably, the endothelial cell culture medium comprises a second basal culture medium and a second specific supplemental factor.

[0017] Further preferably, the second basal culture medium is ECM.

[0018] Further preferably, the second specific additional factor comprises: 5v / v%-20v / v% FBS (Fetal Bovine Serum), 10-100ng / mL EGF, 10-100ng / mL basic FGF, and 50-200U / mL penicillin-streptomycin.

[0019] More preferably, the second specific additional factor comprises: 10 v / v% FBS (Fetal Bovine Serum), 10 ng / mL EGF, 10 ng / mL basic FGF, and 100 U / mL penicillin-streptomycin.

[0020] Preferably, in the mixed culture medium, the volume ratio of synovial sarcoma organoid culture medium to endothelial cell culture medium is (1-2):1.

[0021] Preferably, the three-dimensional culture of synovial sarcoma organoids specifically comprises the following steps:

[0022] digesting the synovial sarcoma organoids obtained by suspension culture using synovial sarcoma passage digestion fluid, collecting the synovial sarcoma cell suspension, and resuspending the synovial sarcoma cell suspension using the mixed culture medium to obtain a synovial sarcoma organoid cell suspension;

[0023] digesting endothelial cells with trypsin, collecting an endothelial cell suspension, and resuspending the endothelial cell suspension with the mixed culture medium to obtain an endothelial cell suspension;

[0024] mixing the synovial sarcoma organoid cell suspension with the endothelial cell suspension to obtain a mixed suspension;

[0025] The mixed suspension is added to a low-adsorption U-shaped bottom well plate or a culture container with a low-adsorption U-shaped bottom structure, placed in an incubator and cultured into spheres to form a synovial sarcoma three-dimensional model.

[0026] Further preferably, in the mixed suspension, the ratio of the number of synovial sarcoma organoid cells to the number of endothelial cells is 1:(0.05-0.25).

[0027] Further preferably, the synovial sarcoma passage digestion fluid is at least one of TrypLE, Trypsin, and Accutase.

[0028] Preferably, the synovial sarcoma cell extraction specifically comprises the following steps:

[0029] The primary synovial sarcoma tissue was cleaned and broken to obtain synovial sarcoma tissue fragments;

[0030] digesting the synovial sarcoma tissue fragments using synovial sarcoma primary tissue digestion fluid to obtain a synovial sarcoma cell suspension;

[0031] The synovial sarcoma cell suspension was resuspended in synovial sarcoma organoid culture medium.

[0032] Further preferably, the synovial sarcoma primary tissue digestion fluid comprises a first basal culture medium and one or more of collagenase I, collagenase II, collagenase III, and collagenase IV, and the first basal culture medium is DMEM or Advanced DMEM / F12.

[0033] More preferably, the final concentration of any collagenase in the synovial sarcoma primary tissue digestion solution is 0.5 mg / mL-2 mg / mL.

[0034] Further preferably, the synovial sarcoma organoid culture medium used in the synovial sarcoma cell extraction is the same as the synovial sarcoma organoid culture medium in the mixed culture medium.

[0035] Preferably, the synovial sarcoma organoid suspension culture specifically comprises the following steps:

[0036] Perform anti-adhesion treatment on the culture well plates;

[0037] The synovial sarcoma cells extracted from the synovial sarcoma cells were inoculated into the culture well plate, and the number of synovial sarcoma cells in each well was (1-2)×10 6 , add 1-3 mL of synovial sarcoma organoid culture medium to culture and obtain synovial sarcoma organoids.

[0038] Preferably, the synovial sarcoma organoid culture medium used in the synovial sarcoma organoid suspension culture is the same as the synovial sarcoma organoid culture medium in the mixed culture medium.

[0039] A three-dimensional model of synovial sarcoma is obtained using the above construction method.

[0040] An application of the above-mentioned synovial sarcoma three-dimensional model is to apply the synovial sarcoma three-dimensional model to compound analysis.

[0041] Preferably, the compound analysis includes any one of drug screening, drug sensitivity testing and drug toxicity testing.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The present invention forms a mixed culture medium for three-dimensional culture by mixing synovial sarcoma organoid culture medium with endothelial cell culture medium. When synovial sarcoma organoids are cultured in three dimensions, a three-dimensional model with a vascular structure, a tumor microenvironment, a high degree of simulation, and a high survival rate can be formed.

[0044] 2. The 3D synovial sarcoma model of the present invention includes synovial sarcoma cells and vascular endothelial cells, has a complete tumor microenvironment, can represent the original tissue, is closer to the human body's true response to drug effects, and can be used for drug testing;

[0045] 3. The present invention uses suspension culture followed by three-dimensional culture to improve the success rate of culturing synovial sarcoma organoids;

[0046] 4. The present invention can promote the proliferation of synovial sarcoma organoids by specifically adding factors EGF, basic FGF and IGF1 to the synovial sarcoma organoid culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a flowchart of the method for constructing a three-dimensional model of synovial sarcoma according to the present invention;

[0048] Figure 2 This is a schematic diagram of the first example of synovial sarcoma organoids growing in clusters in Example 2 of the present invention;

[0049] Figure 3 This is a schematic diagram of a three-dimensional model of the first example of synovial sarcoma organoid formation in Example 2 of the present invention;

[0050] Figure 4 This is the immunofluorescence image of the first synovial sarcoma organoid in Example 2 of the present invention;

[0051] Figure 5 This is a schematic diagram of a three-dimensional model of the second example of synovial sarcoma organoid formation in Example 3 of the present invention;

[0052] Figure 6 This is a schematic diagram of a three-dimensional model of the third example of synovial sarcoma organoid formation in Example 4 of the present invention;

[0053] Figure 7 This is a comparative diagram of drug screening in Example 5 of the present invention;

[0054] Figure 8 This is a bright field comparison of the synovial sarcoma three-dimensional model cultured using the culture medium of Comparative Example 1 and Example 1 for 5 days. DETAILED DESCRIPTION

[0055] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0056] like Figure 1 As shown, the present invention proposes a method S100 for constructing a synovial sarcoma three-dimensional model, comprising the following specific steps S110 to S130:

[0057] S110. Synovial sarcoma cell extraction, comprising the following specific steps:

[0058] (1) Washing: Use primary tissue washing solution to wash the primary synovial sarcoma tissue and remove the surface mucosa. The primary tissue washing solution is a buffer solution, preferably a buffer solution with penicillin and streptomycin or gentamicin added.

[0059] (2) Crushing: mince the primary synovial sarcoma tissue into a minced meat, resuspend it in buffer, let it stand, and discard the supernatant.

[0060] (3) Digestion: Add synovial sarcoma primary tissue digestion solution, blow and mix, and then use a metal bath shaker at 37°C (other similar equipment can also be used here) to shake and digest for 15 minutes to 2 hours (preferably 30-60 minutes, more preferably 30 minutes, 45 minutes, 60 minutes) until digested into single cells, and finally add buffer to terminate digestion.

[0061] In some preferred embodiments, the synovial sarcoma primary tissue digestion solution includes collagenase and a first basal culture medium, wherein the collagenase includes one or more of collagenase I, collagenase II, collagenase III, and collagenase IV.

[0062] In other preferred embodiments, the final concentration of any one of collagenase I, collagenase II, collagenase III, and collagenase IV in the primary synovial sarcoma digestion fluid is 0.5 mg / mL-2 mg / mL (e.g., 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, etc.), and the first basal culture medium is any one of DMEM and Advanced DMEM / F12.

[0063] (4) Resuspension: The digested cell suspension was filtered through a cell sieve, and the cells were resuspended in synovial sarcoma cell culture medium and counted.

[0064] In some preferred embodiments, the synovial sarcoma organoid culture medium used for resuspending cells includes a first basal culture medium and a first specific added factor, that is, Advanced DMEM / F12 supplemented with 0.5%-5% Glutamax, 0.5%-5% HEPES (1M), 0.5%-5% double antibody, 1%-10% B27 additive, 5-20 μM Nicotinamide, 1-5 μM N-Acetylcysteine, 10-100 ng / mL EGF, 10-100 ng / mL FGF, 10-100 ng / mL IGF1, and 5-10% FBS.

[0065] S120, Synovial sarcoma organoid suspension culture, comprising the following specific steps:

[0066] (1) Anti-adhesion treatment of 12-well plates: Add 0.5-1 mL of anti-adhesion reagent to each well of a 12-well plate, let it stand at room temperature for 10-30 min, and then rinse twice with 1-2 mL of PBS for later use.

[0067] (2) Inoculation and culture: Synovial sarcoma organoids were inoculated into 12-well plates, with the number of synovial sarcoma organoids inoculated per well being (1-2) × 10 6 (For example, 1×10 6 ), add 1-3 mL (for example, preferably 1 mL) of synovial sarcoma organoid culture medium, and culture for three days or passage when the diameter of the cell cluster is greater than 300 μm under microscopy.

[0068] It should be noted that, during the suspension culture of synovial sarcoma organoids, the synovial sarcoma organoid culture medium used is the same as that in step S110, including a first basal culture medium and a first specific additive factor.

[0069] S130, 3D culture of synovial sarcoma organoids, including the following specific steps:

[0070] (1) Organoid collection: Collect synovial sarcoma organoids from the culture dish, centrifuge at 400-600g for 4-6 minutes at room temperature, discard the supernatant, wash the cell pellet with buffer, and centrifuge at 400-600g for 4-6 minutes at room temperature.

[0071] (2) Digestion of synovial sarcoma organoids: Discard the supernatant and digest the synovial sarcoma organoids in a 12-well plate with synovial sarcoma passage digestion solution. Observe the cell clumps under a microscope every 3 minutes until they become single cells. Terminate the digestion with 2-3 volumes of serum-containing synovial sarcoma organoid culture medium. Collect the cell suspension, centrifuge at 400-600g for 4-6 minutes at room temperature, discard the supernatant, and resuspend in mixed culture medium to obtain a synovial sarcoma organoid cell suspension. Count the cells and calculate the cell concentration of the synovial sarcoma organoid suspension.

[0072] In some preferred embodiments, the digestion solution for passage is at least one of TrypLE, Trypsin, and Accutase.

[0073] In other preferred embodiments, the culture medium used for resuspending synovial sarcoma organoids includes synovial sarcoma organoid culture medium and endothelial cell culture medium, that is, a mixed culture medium formed by mixing the two culture media, and the mixed volume ratio of the two culture media is preferably (1-2:1), for example, 1:1, 1.5:1, 2:1.

[0074] Among them, the synovial sarcoma organoid culture medium includes a first basal culture medium and a first specific additive factor, the first basal culture medium is DMEM or Advanced DMEM / F12; the first specific additive factor includes: 1xGlutamax, 1x HEPES (1M), 50-200U / mL penicillin-streptomycin, 1v / v%-5v / v% B27 additive, 5-20μM Nicotinamide, 1-5μM N-Acetylcysteine, 10-100ng / mL EGF, 10-100ng / mL FGF, 10-100ng / mL IGF1, 5v / v%-10v / v% FBS (Fetal Bovine Serum).

[0075] Secondly, the endothelial cell culture medium includes: a second basal culture medium and a second specific additive factor, the second basal culture medium is ECM; the second specific additive factor includes: 5v / v%-20v / v% FBS, 10-100ng / mL EGF, 10-100ng / mL basic FGF, 50-200U / mL penicillin-streptomycin, and the percentage concentrations given above represent mass concentrations.

[0076] In the culture medium, the basal medium can provide basic support for cell growth and metabolism, promote the adhesion, growth, and differentiation of stem cells, and thus support the formation and growth of organoids and endothelial cells. Specific added factors contain growth factors, antioxidants, and specific fatty acids required by various cells, which can improve cell viability, cell growth, and the cell growth environment. During the cell culture process, they can scavenge free radicals and other oxidizing substances in the culture medium, help protect cells, prevent bacterial contamination of cell culture, and promote cell growth and differentiation.

[0077] In this embodiment, by mixing the synovial sarcoma organoid culture medium with the endothelial cell culture medium to form a three-dimensional culture mixed culture medium, it is beneficial to improve the cell survival rate when the synovial sarcoma organoid is cultured three-dimensionally, and a three-dimensional synovial sarcoma model with a vascular structure can be formed, which has a tumor microenvironment and a high degree of simulation.

[0078] In some preferred embodiments, the volume ratio of synovial sarcoma organoid culture medium to endothelial cell culture medium is (1-2):1, preferably 1:1, 1.5:1, 2:1, etc. This volume ratio is beneficial to improving cell survival rate and constructing a three-dimensional synovial sarcoma organoid model with a vascular structure.

[0079] In other preferred embodiments, the first basal culture medium is preferably Advanced DMEM / F12, which helps to reduce the amount of serum added and can maintain the normal growth rate and morphology of cells.

[0080] In other preferred embodiments, in the first specific additive factor, the concentration of the dual antibody (penicillin-streptomycin dual antibody) is preferably 100 U / mL, 150 U / mL, or 200 U / mL; the volume percentage of the B27 additive is preferably 1 v / v%, or 2 v / v%; the concentration of nicotinamide is preferably 5 μM, 10 μM, 15 μM, or 20 μM; the concentration of N-acetylcysteine ​​is preferably 1 μM, 2 μM, or 5 μM; the concentration of EGF is preferably 10 ng / mL, 20 ng / mL, 50 ng / mL, or 100 ng / mL; the concentration of FGF is preferably 10 ng / mL, 20 ng / mL, 50 ng / mL, or 100 ng / mL; the concentration of IGF1 is preferably 10 ng / mL, 20 ng / mL, 50 ng / mL, or 100 ng / mL; and the volume percentage of FBS is preferably 5 v / v%, or 10 v / v%. Of course, in other preferred embodiments, other concentrations of the first specific additive factor can also be selected. The specific added factors formed by mixing the above components help promote the proliferation, differentiation, and maturation of synovial sarcoma organoid cells, inhibit cell apoptosis, and improve cell survival rate.

[0081] As a further preferred embodiment, the first specific additive factor includes: 1x Glutamax, 1x HEPES (1M), 100U / mL penicillin-streptomycin, 2v / v% B27 additive, 10μM Nicotinamide, 1μM N-Acetylcysteine, 50ng / mL EGF, 50ng / mL FGF, 50ng / mL IGF1, and 10v / v% FBS.

[0082] In other preferred embodiments, the volume percentage of Fetal Bovine Serum in the second specific additive factor is preferably 5 v / v%, 10 v / v%, 15 v / v%, or 20 v / v%, the concentration of EGF is preferably 10 ng / mL, 20 ng / mL, 50 ng / mL, or 100 ng / mL, the concentration of basic FGF is preferably 10 ng / mL, 20 ng / mL, 50 ng / mL, or 100 ng / mL, and the concentration of penicillin-streptomycin is preferably 100 U / mL, 150 U / mL, or 200 U / mL. Similarly, in other preferred embodiments, other concentrations of the second specific additive factor may also be selected. The specific additive factor formed by mixing the above components helps promote the growth and proliferation of endothelial cells while maintaining the function and morphology of endothelial cells.

[0083] As a further preferred embodiment, the second specific additional factor includes: 10 v / v% FBS, 10 ng / mL EGF, 10 ng / mL basic FGF, and 100 U / mL penicillin-streptomycin.

[0084] (3) Digestion of endothelial cells: Use trypsin to digest the endothelial cells in the culture flask and add endothelial cell culture medium to terminate the digestion. Collect the cell suspension, centrifuge at 400-600g for 4-6 minutes at room temperature, discard the supernatant, add mixed culture medium (the mixed culture medium here also includes synovial sarcoma organoid culture medium and endothelial cell culture medium, please refer to the previous description for specific components and concentrations) and resuspend to obtain endothelial cell suspension. Count the cells and calculate the cell concentration of the endothelial cell suspension.

[0085] (4) Mixing: The above-mentioned synovial sarcoma organoids and endothelial cells are mixed in a certain ratio to obtain a mixed suspension, wherein the number of tumor organoids and endothelial cells in the mixed suspension is 1: (0.05-0.25).

[0086] (5) Spheroidization: Add the above mixed suspension to a low-adsorption U-bottom well plate or a culture container with a low-adsorption U-bottom structure, centrifuge (600-800g, 4-6min), and then culture in an incubator to form a three-dimensional model of synovial sarcoma.

[0087] The following will further illustrate the method for constructing a synovial sarcoma three-dimensional model and the culture medium used in conjunction with specific examples:

[0088] Unless otherwise specified, the reagents, methods, instruments and equipment used in the present invention are conventional reagents, methods, instruments and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0089] The main reagents and product numbers used in the following examples are shown in Table 1. Reagents not included in Table 1 are also conventional products that can be purchased commercially.

[0090] Table 1 Main reagent manufacturers and product numbers

[0091]

[0092]

[0093] Example 1

[0094] This embodiment provides a culture medium for constructing a synovial sarcoma three-dimensional model, wherein the culture medium includes a synovial sarcoma organoid culture medium and an endothelial cell culture medium.

[0095] The synovial sarcoma organoid culture medium comprises: a first basal culture medium and a first specific additive factor, wherein the first basal culture medium is Advanced DMEM / F12, and the first specific additive factor comprises the following components at final concentrations: Fetal Bovine Serum, 10 v / v%; Penicillin-Streptomycin, 100 U / mL; EGF, 50 ng / mL; Basic FGF, 50 ng / mL; IGF1, 50 ng / mL; Glutamax, 1x; HEPES (1 M), 1x; B27 additive, 2 v / v%; Nicotinamide, 10 μM; N-Acetylcysteine, 1 μM;

[0096] The endothelial cell culture medium includes a second basal culture medium and a second specific additive factor, wherein the second basal culture medium is ECM; the second specific additive factor includes the following components at final concentrations: Fetal Bovine Serum, 10%; EGF, 10 ng / mL; basic FGF, 10 ng / mL; and penicillin-streptomycin, 100 U / mL.

[0097] Furthermore, the method for preparing the culture medium includes: mixing a synovial sarcoma organoid culture medium and an endothelial cell culture medium in a volume ratio of 1:1 to obtain a culture medium for constructing a synovial sarcoma three-dimensional model.

[0098] Example 2

[0099] This example provides a culture method for constructing a three-dimensional synovial sarcoma model based on the culture medium of Example 1, which specifically includes the following steps:

[0100] Step 1: Extraction of the first synovial sarcoma cell:

[0101] (1) Cleaning: Weigh the primary synovial sarcoma tissue sample, take three sterile 60 mm culture dishes, add 5-6 mL of 4°C PBS (10% PS + 2% gentamicin sulfate) to each dish, use forceps to wash the tissue three times in each dish, remove the surface mucosa and fat, and transfer to a new dish.

[0102] (2) Mincing: Place the culture dish containing the tissue on an ice box and mince it with a scalpel until it becomes minced meat. Add 3-10 mL of PBS to the culture dish and transfer the minced tissue to a 15 mL centrifuge tube. Let it stand and remove the supernatant. Then wash it twice with PBS.

[0103] (3) Discard PBS, add 0.1 mL of digestion solution to 1 mg of synovial sarcoma tissue sample, and place it in a 37°C metal bath for digestion. The dissociation time is about 45 minutes. Take 10 μL every 15 minutes and observe the digestion process under a microscope until a large number of single cells are observed. The digestion solution is 1 mg / mL collagenase I and 1 mg / mL collagenase II, prepared using Ad-DMEM / F12.

[0104] (4) Termination of digestion: Add 2-3 times the volume of serum-containing synovial sarcoma organoid culture medium to terminate digestion, collect the cell suspension, filter the filtrate through a 100 μm molecular sieve, centrifuge at 1000 r, 4°C for 10 min, and discard the supernatant.

[0105] (5) Removal of red blood cells: If the cell pellet contains a large number of red blood cells, add 500 μL of red blood cell lysis buffer according to the amount of red blood cells, react for 2 minutes, add 5 times the volume of serum-containing synovial sarcoma organoid culture medium to terminate the reaction, and centrifuge to remove the supernatant.

[0106] (6) Resuspend the cells in synovial sarcoma organoid culture medium and count them.

[0107] In step (1), the synovial sarcoma sample was obtained from a surgical resection specimen in a hospital. The surgical resection specimen was immersed in tissue preservation fluid and transferred from the hospital to the laboratory. Before obtaining the specimen, informed consent was obtained from the patient.

[0108] Step 2: Synovial sarcoma organoid suspension culture:

[0109] (1) Anti-adhesion treatment: Add 500 μL of anti-adhesion reagent to each well of a 12-well plate and gently shake to evenly cover the bottom of the plate. After standing at room temperature for 20 min, aspirate the anti-adhesion reagent and add 1 mL of PBS to rinse for 5 min. Repeat this cycle twice. The anti-adhesion reagent used is Anti-Adherence Rinsing Solution (Stemcell, catalog number #07010).

[0110] (2) Plating: 1×106 Cells were cultured in a 5% CO2 incubator with 1 mL of synovial sarcoma organoid culture medium. Suspension culture was performed for 5 days.

[0111] Step 3: Synovial sarcoma 3D model cultivation:

[0112] (1) Collect synovial sarcoma organoids in the culture dish, centrifuge 500g of synovial sarcoma organoids at room temperature for 5 minutes, discard the supernatant, wash the cell clumps with sterile PBS solution, centrifuge at 500g at room temperature for 5 minutes, and discard the supernatant. Digest the cells with TryplE digestion solution at 37°C, and observe the digestion of the cell clumps under a microscope every 3 minutes until they become single cells. Terminate the digestion with 2-3 times the volume of serum-containing culture medium (10% FBS+Ad-DMEM / F12), collect the cell suspension and centrifuge at 500g, centrifuge at room temperature for 5 minutes, discard the supernatant, add mixed culture medium (synovial sarcoma organoid culture medium and endothelial cell culture medium in a volume ratio of 1:1) to resuspend, count the cells, and obtain a synovial sarcoma organoid cell suspension;

[0113] (2) HUVEC cells were obtained, digested into single cells using TrypE, resuspended in mixed culture medium, and counted to obtain a vascular endothelial cell suspension;

[0114] (3) The synovial sarcoma organoid cell suspension was mixed with the endothelial cell suspension. In the mixed suspension, the number of tumor organoid cells and the number of endothelial cells was 5:1; the total number of cells was 10,000, of which 8,333 were synovial sarcoma organoids and 1,667 were endothelial cells.

[0115] (4) The mixed suspension was added to a low-adsorption U-bottom plate and centrifuged at 700 g for 5 min;

[0116] After centrifugation, the cells were placed in an incubator at 37°C and 5% CO2 for 3 days to form a 3D model of synovial sarcoma.

[0117] like Figure 2 As shown, for the first cultured synovial sarcoma organoid, the bright field image shows that the synovial sarcoma organoids grow in clusters.

[0118] like Figure 3 As shown in Figure 1, this is the first three-dimensional model of synovial sarcoma organoid formation, forming a dense spheroid. Figure 4 As shown, immunofluorescence staining reveals that green marks the synovial sarcoma organoid, red marks the endothelial cells, and blue marks the cell nuclei. Microvessels can be seen within the 3D synovial sarcoma spheroid model.

[0119] Example 3

[0120] This example uses the same construction method and culture medium as Example 2 to form a three-dimensional model of the second synovial sarcoma organoid. Figure 5 Shown is a second 3D model of synovial sarcoma organoid formation, forming dense spheroids.

[0121] Example 4

[0122] This example uses the same construction method and culture medium as Example 2 to form a three-dimensional model of the third synovial sarcoma organoid. Figure 6 As shown, the third 3D model of synovial sarcoma organoid formation also formed dense spheroids.

[0123] In summary, combined with Examples 2-4, it can be seen that different examples of synovial sarcoma organoids all formed dense spheroids under the same culture conditions, and a synovial sarcoma three-dimensional model was successfully constructed using the construction method of the present invention.

[0124] Example 5

[0125] This example uses the synovial sarcoma three-dimensional model constructed in Example 2 for drug screening. The specific steps are as follows:

[0126] (1) Day 1: Observe and record the growth status of the 3D synovial sarcoma model. Prepare the drug-containing culture medium: 2 μM doxorubicin + 100 μM ifosfamide, 1 μM anlotinib. Aspirate the culture medium from the well plate and add 100 μL of drug-containing culture medium to each well. Place in a 37°C incubator for culture.

[0127] (2) Day 4: 3 days after drug treatment, activity was detected using a CTG detection kit.

[0128] (3) Discard all the culture medium in the well plate, add 100 μL of CTG diluent (1:1 dilution of CTG reagent stock solution and PBS) to each well, and mix thoroughly by pipetting.

[0129] (4) Use an oscillator to shake for 60 minutes.

[0130] (5) Use a chemiluminescence microplate reader to detect and read the values ​​on the well plate.

[0131] like Figure 7 As shown, both doxorubicin + ifosfamide (a combination drug) and anlotinib have a certain inhibitory effect on the synovial sarcoma model constructed by the present invention, that is, both doxorubicin + ifosfamide and anlotinib can be used for screening synovial sarcoma drugs, and the combination drug of doxorubicin + ifosfamide is more sensitive to synovial sarcoma.

[0132] Comparative Example 1

[0133] This comparative example differs from Example 1 in that the synovial sarcoma organoid culture medium used in this comparative example includes: a basal medium Ad-DMEM / F12; a first specific additive factor comprising the following components at the following final concentrations: Fetal Bovine Serum, 10 v / v%; Penicillin-Streptomycin, 100 U / mL; and EGF, 20 ng / mL. The remaining culture medium remained the same as in Example 1.

[0134] The bright field image of the synovial sarcoma three-dimensional model cultured for 5 days in comparative example 1 is shown in FIG. Figure 8 -(a).

[0135] The bright field image of the synovial sarcoma three-dimensional model cultured for 5 days in Example 1 is shown in FIG. Figure 8 -(b).

[0136] pass Figure 8 It can be seen that using the culture medium of the present invention, synovial sarcoma spheroids have a better growth state and proliferate faster.

[0137] The present invention proposes a three-dimensional model of synovial sarcoma and its construction method and application, which have the following beneficial effects compared with the existing technology: in the three-dimensional culture stage of synovial sarcoma, the culture medium used in the present invention is a mixed culture medium, which forms a three-dimensional model with a vascular structure after cultivation, and can simulate the tumor microenvironment. By optimizing the components and concentration of the culture medium, it helps to improve the cell survival rate; in addition, the constructed three-dimensional model of synovial sarcoma can not only simulate the three-dimensional environment of cancer cell growth, but also simulate the interaction between cancer cells and TME, which is closer to the human body's real response to drug effects and can realize drug screening.

[0138] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for constructing a three-dimensional model of synovial sarcoma, characterized in that: The construction method comprises the following steps: Synovial sarcoma cell extraction; Synovial sarcoma organoid suspension culture; Three-dimensional culture of synovial sarcoma organoids; Among them, the culture medium used in the three-dimensional culture of synovial sarcoma organoids is a mixed culture medium, including: synovial sarcoma organoid culture medium and endothelial cell culture medium.

2. The method for constructing a three-dimensional model of synovial sarcoma according to claim 1, characterized in that: The synovial sarcoma organoid culture medium comprises a first basal culture medium and a first specific additive factor; The first basal culture medium is DMEM or Advanced DMEM / F12, and the first specific additive factor includes: 1xGlutamax, 1x HEPES, 50-200U / mL of penicillin-streptomycin, 1v / v%-5v / v% B27, 5-20μM Nicotinamide, 1-5μM N-Acetylcysteine, 10-100ng / mL of EGF, 10-100ng / mL of FGF, 10-100ng / mL of IGF1, and 5v / v%-10v / v% of FBS.

3. The method for constructing a three-dimensional model of synovial sarcoma according to claim 1, characterized in that: The endothelial cell culture medium includes a second basal culture medium and a second specific added factor; The second basic culture medium is ECM, and the second specific added factors include: 5v / v%-20v / v% FBS, 10-100ng / mL EGF, 10-100ng / mL basic FGF, and 50-200U / mL penicillin-streptomycin.

4. The method for constructing a three-dimensional model of synovial sarcoma according to claim 1, characterized in that: In the mixed culture medium, the volume ratio of synovial sarcoma organoid culture medium to endothelial cell culture medium is (1-2):

1.

5. The method for constructing a three-dimensional model of synovial sarcoma according to claim 1, characterized in that: The three-dimensional culture of synovial sarcoma organoids specifically includes the following steps: digesting the synovial sarcoma organoids obtained by suspension culture using synovial sarcoma passage digestion fluid, collecting the synovial sarcoma cell suspension, and resuspending the synovial sarcoma cell suspension using the mixed culture medium to obtain a synovial sarcoma organoid cell suspension; digesting endothelial cells with trypsin, collecting an endothelial cell suspension, and resuspending the endothelial cell suspension with the mixed culture medium to obtain an endothelial cell suspension; mixing the synovial sarcoma organoid cell suspension with the endothelial cell suspension to obtain a mixed suspension; The mixed suspension is placed in an incubator for culture to form a three-dimensional synovial sarcoma model.

6. The method for constructing a three-dimensional model of synovial sarcoma according to claim 5, characterized in that: In the mixed suspension, the ratio of the number of synovial sarcoma organoid cells to the number of endothelial cells is 1:(0.05-0.25); The synovial sarcoma passage digestion fluid is at least one of TrypLE, Trypsin, and Accutase.

7. The method for constructing a three-dimensional model of synovial sarcoma according to claim 1, characterized in that: The synovial sarcoma cell extraction specifically comprises the following steps: The primary synovial sarcoma tissue was cleaned and broken to obtain synovial sarcoma tissue fragments; digesting the synovial sarcoma tissue fragments using synovial sarcoma primary tissue digestion fluid to obtain a synovial sarcoma cell suspension; resuspending the synovial sarcoma cell suspension using synovial sarcoma organoid culture medium; The synovial sarcoma primary tissue digestion solution comprises a first basal culture medium and one or more of collagenase I, collagenase II, collagenase III, and collagenase IV, wherein the final concentration of any one of the collagenases in the synovial sarcoma primary tissue digestion solution is 0.5 mg / mL-2 mg / mL; The first basal culture medium is DMEM or Advanced DMEM / F12.

8. The method for constructing a three-dimensional model of synovial sarcoma according to claim 1, characterized in that: The synovial sarcoma organoid suspension culture specifically comprises the following steps: Perform anti-adhesion treatment on the culture well plates; The synovial sarcoma cells extracted from the synovial sarcoma cells were inoculated into the culture well plate, and the number of synovial sarcoma cells in each well was (1-2)×10 6 , add 1-3 mL of synovial sarcoma organoid culture medium for culture.

9. A three-dimensional model of synovial sarcoma, characterized in that: The method is obtained by using the construction method according to any one of claims 1 to 8.

10. A use of the synovial sarcoma three-dimensional model according to claim 9, characterized in that: applying the synovial sarcoma three-dimensional model to compound analysis; The compound analysis includes any one of drug screening, drug sensitivity testing and drug toxicity testing.

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