Culture medium for culturing 3D breast cancer tumor organoid model and model construction method

Abstract

The invention belongs to the technical field of organoid culture, discloses a culture medium for culturing a 3D breast cancer tumor organoid model and a model construction method, and particularly discloses a culture medium. Comprising an L-glutamine additive, an antibiotic, a Wnt signaling pathway activator, a bone morphogenetic protein inhibitor, a growth factor, a regulatory factor, a serum substitute, nicotinamide, acetylcysteine, hydrocortisone, estradiol, a eriochin, a TGF-beta signaling pathway inhibitor, a Rock inhibitor, a buffer solution and a basal culture medium. The invention provides the culture medium for culturing the breast cancer tumor organoid, and the culture medium can effectively simulate a tumor microenvironment, promote formation and growth of the breast cancer tumor organoid and ensure heterogeneity and genome stability of patient-derived tumors by adding specific growth factors, thereby laying a solid foundation for subsequent research and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and specifically relates to a culture medium for culturing a 3D breast cancer tumor organoid model and a model construction method. Background Art

[0002] Breast cancer is the most common malignant tumor among women worldwide, and its age of onset is showing a trend of younger age. Although advances in early screening, targeted therapy and immunotherapy have significantly improved patient survival rates, drug resistance, recurrence and metastasis remain the main causes of treatment failure. Breast cancer is divided into four subtypes based on the expression levels of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki67: Luminal A, Luminal B, HER2 overexpression and triple-negative breast cancer. These subtype classifications provide an important basis for clinical treatment, but due to the high heterogeneity and complexity of breast cancer, the treatment responses of different patients vary significantly and are difficult to predict, which may lead to unnecessary side effects and economic burden. Therefore, personalized treatment has become a key need in the clinical diagnosis and treatment of breast cancer.

[0003] Tumor organoid technology simulates the in vivo tumor microenvironment through three-dimensional culture, preserving the heterogeneity, tissue specificity, and genetic mutation information of tumor cells, providing an important tool for tumor research. Breast cancer organoids can simulate the growth and biological characteristics of tumors in vitro, and have the advantages of simple operation, short cycle time, and high throughput. They are widely used in drug sensitivity screening, personalized medication, and tumor biology research. However, in long-term or multi-generation culture, the structural and functional stability of organoids is difficult to maintain, which may affect the continuity and accuracy of research. Summary of the Invention

[0004] This invention aims to provide a culture medium and method for constructing 3D breast cancer organoid models. This approach addresses the slow growth rate and low yield of existing organoids derived from breast cancer tissue. This approach effectively improves the success rate and efficiency of breast cancer organoid model construction, providing a foundation for drug sensitivity testing and personalized treatment plans, thereby promoting the development of precision medicine for breast cancer.

[0005] The first aspect of the present invention aims to provide a culture medium.

[0006] The second aspect of the present invention aims to provide use of the culture medium of the first aspect of the present invention in preparing organoids.

[0007] The third aspect of the present invention aims to provide a method for preparing tumor organoids.

[0008] The fourth aspect of the present invention aims to provide the use of tumor organoids prepared by the method of the third aspect of the present invention in screening anti-tumor drugs.

[0009] The fifth aspect of the present invention aims to provide a drug screening method.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a culture medium comprising an L-glutamine additive, an antibiotic, a Wnt signaling pathway activator, a bone morphogenetic protein inhibitor, a growth factor, a regulatory factor, a serum replacement, nicotinamide, acetylcysteine, hydrocortisone, estradiol, forskolin, a TGF-β signaling pathway inhibitor, a Rock inhibitor, a buffer, and a basal culture medium.

[0012] In some embodiments of the present invention, the Wnt signaling pathway activator is selected from at least one of Wnt3a, Wnt4, Wnt5a, Rspondin 1, Rspondin 2, Rspondin 3, Rspondin 4, CHIR99021, CHIR98014, SB 216763, SB415286 and TCS2002; preferably Wnt3a and Rspondin 3.

[0013] In some embodiments of the present invention, the antibiotic is selected from at least one of penicillin / streptomycin dual antibiotic, penicillin, streptomycin and amphotericin B; preferably penicillin / streptomycin dual antibiotic.

[0014] In some embodiments of the present invention, the bone morphogenetic protein inhibitor is selected from at least one of Noggin, Gastrin, Connective Tissue Growth Factor, Inhibin, BMP-3 and Dorsomorphin; preferably Noggin.

[0015] In some embodiments of the present invention, the regulatory factor is selected from at least one of NRG1 and KGF; preferably NRG1.

[0016] In some embodiments of the present invention, the serum replacement is selected from at least one of B27, KSR, and KOSR; preferably B27 and KSR.

[0017] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is selected from at least one of A-83-01, LY2157299, LY3200882, SB-431542, LY2109761, SB-525334, SB-505124 and LY364947; preferably A-83-01.

[0018] In some embodiments of the present invention, the Rock inhibitor is selected from at least one of Y-27632, Thiazovivin and Azaindole1, preferably Y-27632.

[0019] In some embodiments of the present invention, the buffer is selected from HEPES and Tirs-HC; preferably HEPES.

[0020] In some embodiments of the present invention, the basal culture medium is selected from at least one of Advanced DMEM / F12, DMEM, and RMPI1640; preferably Advanced DMEM / F12.

[0021] In some embodiments of the present invention, the growth factors include epidermal growth factor EGF, fibroblast growth factor and insulin-like growth factor.

[0022] In some embodiments of the present invention, the fibroblast growth factor is selected from at least one of FGF1, FGF2, FGF4, FGF7, FGF9 and FGF10, preferably FGF10.

[0023] In some embodiments of the present invention, the insulin-like growth factor is selected from at least one of IGF-1 and IGF-2, preferably IGF-1.

[0024] In some embodiments of the present invention, the culture medium is Advanced DMEM / F12 containing L-glutamine supplement, penicillin / streptomycin double antibody, Rspondin 3, Wnt3a, Noggin, EGF, serum substitute B27, serum substitute KSR, nicotinamide, acetylcysteine, hydrocortisone, estradiol, forskolin, NRG1, FGF10, A83-01, IGF-1, Y-27632, and HEPES buffer.

[0025] In some embodiments of the present invention, the culture medium contains 1-2X L-glutamine supplement, 1-2X penicillin / streptomycin double antibody, 50-1000 ng / mL Rspondin 3, 10-100 ng / mL Wnt3a, 10-100 ng / mL Noggin, 5-50 ng / mL EGF, 0.5-1X serum substitute B27, 5%-30% serum substitute KSR, 5-20 mM nicotinamide, 1-10 mM acetylcysteine, 0.1-2 μg / mL hydrocortisone, 50-250 nM estradiol, 5-20 μM forskolin, 10-100 ng / mL NRG1, 10-150 ng / mL FGF10, 200-900 nM A83-01, 10-100 ng / mL IGF-1, 5-10 μM Y-27632, 1% to 2% HEPES buffered Advanced DMEM / F12.

[0026] In some embodiments of the present invention, the culture medium contains 1-2X L-glutamine supplement, 1-2X penicillin / streptomycin double antibody, 100-1000 ng / mL Rspondin 3, 30-100 ng / mL Wnt3a, 30-100 ng / mL Noggin, 5-30 ng / mL EGF, 0.5-1X serum substitute B27, 5%-20% serum substitute KSR, 5-15 mM nicotinamide, 1-5 mM acetylcysteine, 0.1-1 μg / mL hydrocortisone, 50-200 nM estradiol, 5-15 μM forskolin, 30-100 ng / mL NRG1, 10-100 ng / mL FGF10, 200-800 nM A83-01, 20-100 ng / mL IGF-1, 7-10 μM Y-27632, 1% to 2% HEPES buffered Advanced DMEM / F12.

[0027] In some embodiments of the present invention, the culture medium contains 1-2X L-glutamine supplement, 1-2X penicillin / streptomycin double antibody, 200-1000 ng / mL Rspondin 3, 50-100 ng / mL Wnt3a, 50-100 ng / mL Noggin, 5-20 ng / mL EGF, 0.5-1X serum substitute B27, 5%-15% serum substitute KSR, 5-10 mM nicotinamide, 1-2 mM acetylcysteine, 0.5-1 μg / mL hydrocortisone, 100-200 nM estradiol, 5-10 μM forskolin, 50-100 ng / mL NRG1, 20-100 ng / mL FGF10, 300-800 nM A83-01, 30-100 ng / mL IGF-1, 8-10 μM Y-27632, 1% to 2% HEPES buffered Advanced DMEM / F12.

[0028] The second aspect of the present invention provides use of the culture medium of the first aspect of the present invention in preparing organoids.

[0029] In some embodiments of the invention, the organoid comprises a tumor organoid.

[0030] In some embodiments of the invention, the organoid is a breast cancer tumor organoid.

[0031] A third aspect of the present invention provides a method for preparing tumor organoids, wherein tumor cells are cultured in the culture medium of the first aspect of the present invention to obtain tumor organoids, wherein the culture is a three-dimensional culture.

[0032] In some embodiments of the present invention, the three-dimensional culture comprises resuspending the tumor cells in the culture medium of the first aspect of the present invention, mixing with the matrix gel, placing on a plate and letting it stand, adding the culture medium of the first aspect of the present invention after solidification, and placing in a cell culture incubator for culture.

[0033] In some embodiments of the present invention, the culture conditions are culturing at 37° C. in a 5% CO 2 incubator, and replacing the culture medium every 3 to 4 days until 3D cancer tumor organoids are generated.

[0034] In some embodiments of the invention, the tumor organoid comprises a breast cancer tumor organoid.

[0035] In some embodiments of the present invention, the tumor cells are obtained by the following steps: dissociating tumor-related tissue, adding DMEM medium to terminate the tumor tissue dissociation reaction, collecting the cells, and washing them, thereby isolating the tumor cells. The tumor cells may also be commercially available tumor cells or tumor cells isolated from tumor tissue using other methods.

[0036] The fourth aspect of the present invention provides the use of tumor organoids prepared by the method of the third aspect of the present invention in screening anti-tumor drugs, personalized drug sensitivity testing, tumor molecular phenotype analysis, and gene-drug association studies.

[0037] A fifth aspect of the present invention provides a drug screening method, comprising adding an anti-tumor drug to the tumor organoid prepared by the method of the fourth aspect of the present invention to evaluate the efficacy of the anti-tumor drug.

[0038] In some embodiments of the present invention, during the drug screening process, a positive drug control group and a negative control group are set up simultaneously.

[0039] The beneficial effects of the present invention are:

[0040] The present invention provides a culture medium for culturing breast cancer organoids. By adding specific growth factors, this culture medium can effectively simulate the tumor microenvironment, promote the formation and growth of breast cancer organoids, and ensure the heterogeneity and genomic stability of patient-derived tumors, laying a solid foundation for subsequent research and application. Furthermore, KSR is used instead of FBS, which has a clear chemical composition, high batch consistency, and is free of animal-derived ingredients. This avoids contamination by viruses, mycoplasmas, and other bacteria that may be carried by FBS, reduces the risk of interference from unknown components in serum FBS, and improves the repeatability and stability of experiments.

[0041] This invention provides a method for preparing breast cancer tumor organoids. This method can efficiently and stably cultivate breast cancer tumor organoids with high physiological relevance. This model can be used for a variety of important research applications, including personalized drug sensitivity testing, tumor molecular phenotyping, gene-drug association studies, and immunotherapy screening. It also facilitates high-throughput drug screening studies. The resulting breast cancer tumor organoids provide an important technical platform for personalized breast cancer treatment and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the process for constructing breast cancer tumor organoids from breast cancer tumor tissue.

[0043] Figure 2 This is a morphological image of the breast cancer tumor organoid constructed from breast cancer tumor tissue in Example 5 of the present invention. The scale is 100 μm.

[0044] Figure 3 This is a morphological image of the breast cancer tumor organoid constructed from breast cancer tumor tissue in Example 6 of the present invention. The scale is 100 μm.

[0045] Figure 4This is a morphological image of the breast cancer tumor organoid constructed from breast cancer tumor tissue in Example 3 of the present invention. The scale is 100 μm.

[0046] Figure 5 This is a morphological image of the breast cancer tumor organoid constructed from breast cancer tumor tissue in Example 8 of the present invention. The scale is 100 μm.

[0047] Figure 6 This is a morphological image of the breast cancer tumor organoid constructed from the breast cancer tumor tissue in Comparative Example 5 of the present invention, with a scale of 200 μm.

[0048] Figure 7 This is a morphological image of the breast cancer tumor organoid constructed from breast cancer tumor tissue in Comparative Example 6 of the present invention, with a scale of 100 μm.

[0049] Figure 8 This is a morphological image of the breast cancer tumor organoid constructed from the breast cancer tumor tissue in Comparative Example 7 of the present invention. The scale is 100 μm.

[0050] Figure 9 This is a morphological image of the breast cancer tumor organoid constructed from the breast cancer tumor tissue in Comparative Example 8 of the present invention, with a scale of 100 μm.

[0051] Figure 10 Graphs showing hematoxylin-eosin (HE) and immunohistochemistry (IHC) staining results of patient-derived breast cancer tumor tissue and breast cancer tumor organoids in Example 7 of the present invention, with a scale of 100 μm.

[0052] Figure 11 The figure shows a comparison of the number of organoid cells obtained when breast cancer tumor organoids were constructed using the culture medium of Example 3 and Comparative Examples 1-4 (i.e., using the construction methods of Example 7 and Comparative Examples 5-8). DETAILED DESCRIPTION

[0053] The present invention is further described in detail below through specific examples.

[0054] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0055] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0056] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0057] Example 1

[0058] A culture medium for culturing breast cancer tumor organoids, the culture medium comprising the following components at final concentrations: Advanced DMEM / F12 (purchased from Gibco), 500 mL; HEPES buffer (purchased from Gibco), 1%; L-glutamine supplement (purchased from Gibco), 1X; Penicillin Streptomycin antibiotics (purchased from Gibco), 1X; Rspondin 3 (purchased from Novoprotein), 200 ng / mL; Wnt3a (purchased from Novoprotein), 50 ng / mL; bone morphogenetic protein inhibitor Noggin (purchased from Novoprotein), 100 ng / mL; epidermal growth factor EGF (purchased from Novoprotein), 20 ng / mL; serum substitute B27 (purchased from Gibco), 1X; serum substitute KSR (purchased from Gibco), 5 v / v%; nicotinamide (purchased from Sigma), 10 mM; acetylcysteine ​​(purchased from MacLean), 2 mM; hydrogenated Pyrrolidone (purchased from MCE), 0.5μg / mL; estradiol (purchased from Sigma), 200nM; forskolin (purchased from Peprotech), 5μM; NRG1 (purchased from Novoprotein), 50ng / mL; fibroblast growth factor FGF10 (purchased from Novoprotein), 100ng / mL; TGF-β growth inhibitor A83-01 (purchased from MCE), 800nM; insulin-like growth factor IGF-1 (purchased from Novoprotein), 30ng / mL; Rock inhibitor Y-27632 (purchased from MCE), 10μM.

[0059] Example 2

[0060] A culture medium for culturing breast cancer tumor organoids, the culture medium comprising the following components at final concentrations: Advanced DMEM / F12, 500 mL; HEPES buffer, 1%; L-glutamine additive, 1X; Penicillin Streptomycin antibiotic, 1X; Rspondin 3, 300ng / mL; Wnt3a, 100ng / mL; bone morphogenetic protein inhibitor Noggin, 50ng / mL; epidermal growth factor EGF, 10ng / mL; serum substitute B27, 0.5X; serum substitute KSR, 10%; nicotinamide, 5mM; acetylcysteine, 1mM; hydrocortisone, 1μg / mL; estradiol, 100nM; forskolin, 8μM; NRG1, 100ng / mL; fibroblast growth factor FGF10, 50ng / mL; TGF-β growth inhibitor A83-01, 300nM; insulin-like growth factor IGF-1, 30ng / mL; Rock inhibitor Y-27632, 10μM.

[0061] Example 3

[0062] A culture medium for culturing breast cancer tumor organoids, the culture medium comprising the following components at final concentrations: Advanced DMEM / F12, 500 mL; HEPES buffer, 1%; L-glutamine additive, 1X; Penicillin Streptomycin antibiotic, 1X; Rspondin 3, 500ng / mL; Wnt3a, 50ng / mL; bone morphogenetic protein inhibitor Noggin, 50ng / mL; epidermal growth factor EGF, 5ng / mL; serum substitute B27, 1X; serum substitute KSR, 10%; nicotinamide, 5mM; acetylcysteine, 1mM; hydrocortisone, 0.5μg / ml; estradiol, 100nM; forskolin, 10μM; NRG1, 50ng / mL; fibroblast growth factor FGF10, 20ng / mL; TGF-β growth inhibitor A83-01, 400nM; insulin-like growth factor IGF-1, 100ng / mL; Rock inhibitor Y-27632, 10μM.

[0063] Example 4

[0064] A culture medium for culturing breast cancer tumor organoids, the culture medium comprising the following components at final concentrations: Advanced DMEM / F12, 500 mL; HEPES buffer, 1%; L-glutamine additive, 1X; Penicillin Streptomycin antibiotic, 1X; Rspondin 3, 1000ng / mL; Wnt3a, 50ng / mL; bone morphogenetic protein inhibitor Noggin, 50ng / mL; epidermal growth factor EGF, 5ng / mL; serum substitute B27, 1X; serum substitute KSR, 15%; nicotinamide, 5mM; acetylcysteine, 1mM; hydrocortisone, 0.5μg / ml; estradiol, 100nM; forskolin, 10μM; NRG1, 50ng / mL; fibroblast growth factor FGF10, 20ng / mL; TGF-β growth inhibitor A83-01, 400nM; insulin-like growth factor IGF-1, 100ng / mL; Rock inhibitor Y-27632, 10μM.

[0065] Example 5

[0066] A method for constructing breast cancer tumor organoids (schematic diagram of the construction method is shown in FIG. Figure 1 ), comprising the following steps:

[0067] (1) Breast cancer tumor tissue collected from surgery / puncture biopsy was resuspended and washed three times with DPBS and minced into 0.5-1 mm 3 The size was determined by adding 10 mL of DMEM + collagenase II (final concentration 1 mg / mL) + DNase I (final concentration 0.1 mg / mL) into the digestion solution and further dissociating the tumor tissue in a 37°C constant temperature shaker. The dissociation time was controlled between 20 and 50 min.

[0068] (2) After tumor tissue dissociation was completed, two volumes of DMEM medium were added to terminate the breast cancer tumor tissue dissociation reaction;

[0069] (3) Take a 100 μm cell strainer and hang it on the mouth of a 50 mL centrifuge tube, and filter the suspension obtained in step (2) to collect tumor epithelial cells;

[0070] (4) Transfer the filtered cell suspension to a new 15 mL centrifuge tube, centrifuge at 300 x g for 5 min, discard the supernatant, and resuspend and wash twice with DPBS;

[0071] (5) Resuspend the cells using the culture medium used to culture breast cancer tumor organoids in Example 1 and count the cells. Mix Matrigel and cell suspension at a ratio of 5:2 and inoculate into 12-well plates, with 5 Matrigel domes inoculated per well. Each Matrigel dome has a volume of 25 μL, and each μL of Matrigel and cell suspension mixture contains approximately 500 cells.

[0072] (6) After waiting for 30 minutes for the Matrigel to solidify, 1 mL of the culture medium used to culture breast cancer tumor organoids in Example 1 was added to completely cover the Matrigel dome. The cells were placed in a 37°C, 5% CO2 incubator and cultured for 10 to 15 days. The culture medium was replaced every 3 to 4 days until 3D breast cancer tumor organoids were generated.

[0073] Example 6

[0074] A method for constructing breast cancer tumor organoids, comprising the following steps:

[0075] (1) Breast cancer tumor tissue collected from surgery / puncture biopsy was resuspended and washed three times with DPBS and minced into 0.5-1 mm 3 size, tissue digestion solution was added and the tumor tissue was further dissociated in a 37°C constant temperature shaker;

[0076] (2) After tumor tissue dissociation was completed, two volumes of DMEM medium were added to terminate the breast cancer tumor tissue dissociation reaction;

[0077] (3) Take a 100 μm cell strainer and hang it on the mouth of a 50 mL centrifuge tube, and filter the suspension obtained in step (2) to collect tumor epithelial cells;

[0078] (4) Transfer the filtered cell suspension to a new 15 mL centrifuge tube, centrifuge at 300 x g for 5 min, discard the supernatant, and resuspend and wash twice with DPBS;

[0079] (5) Resuspend the cells using the culture medium used to culture breast cancer tumor organoids in Example 2 and count the cells. Mix Matrigel and cell suspension at a ratio of 5:2 and inoculate into 12-well plates, with 5 Matrigel domes inoculated per well. Each Matrigel dome has a volume of 25 μL, and each μL of Matrigel and cell suspension mixture contains approximately 500 cells.

[0080] (6) After waiting for 30 minutes for the Matrigel to solidify, 1 mL of the culture medium used to culture breast cancer tumor organoids in Example 2 was added to completely cover the Matrigel dome. The cells were placed in a 37°C, 5% CO2 incubator and cultured for 10 to 15 days. The culture medium was replaced every 3 to 4 days until 3D breast cancer tumor organoids were generated.

[0081] Example 7

[0082] A method for constructing breast cancer tumor organoids, comprising the following steps:

[0083] (1) Breast cancer tumor tissue collected from surgery / puncture biopsy was resuspended and washed three times with DPBS and minced into 0.5-1 mm 3 size, tissue digestion solution was added and the tumor tissue was further dissociated in a 37°C constant temperature shaker;

[0084] (2) After tumor tissue dissociation was completed, two volumes of DMEM medium were added to terminate the breast cancer tumor tissue dissociation reaction;

[0085] (3) Take a 100 μm cell strainer and hang it on the mouth of a 50 mL centrifuge tube, and filter the suspension obtained in step (2) to collect tumor epithelial cells;

[0086] (4) Transfer the filtered cell suspension to a new 15 mL centrifuge tube, centrifuge at 300 x g for 5 min, discard the supernatant, and resuspend and wash twice with DPBS;

[0087] (5) Resuspend the cells using the culture medium used to culture breast cancer tumor organoids in Example 3 and count the cells. Mix Matrigel and cell suspension at a ratio of 5:2 and inoculate into 12-well plates, with 5 Matrigel domes inoculated per well. Each Matrigel dome has a volume of 25 μL, and each μL of Matrigel and cell suspension mixture contains approximately 500 cells.

[0088] (6) After waiting for 30 minutes for the Matrigel to solidify, 1 mL of the culture medium used to culture breast cancer tumor organoids in Example 3 was added to completely cover the Matrigel dome. The cells were placed in a 37°C, 5% CO2 incubator and cultured for 10 to 15 days. The culture medium was replaced every 3 to 4 days until 3D breast cancer tumor organoids were generated.

[0089] Example 8

[0090] A method for constructing breast cancer tumor organoids, comprising the following steps:

[0091] (1) Breast cancer tumor tissue collected from surgery / puncture biopsy was resuspended and washed three times with DPBS and minced into 0.5-1 mm 3 size, tissue digestion solution was added and the tumor tissue was further dissociated in a 37°C constant temperature shaker;

[0092] (2) After tumor tissue dissociation was completed, two volumes of DMEM medium were added to terminate the breast cancer tumor tissue dissociation reaction;

[0093] (3) Take a 100 μm cell strainer and hang it on the mouth of a 50 mL centrifuge tube, and filter the suspension obtained in step (2) to collect tumor epithelial cells;

[0094] (4) Transfer the filtered cell suspension to a new 15 mL centrifuge tube, centrifuge at 300 x g for 5 min, discard the supernatant, and resuspend and wash twice with DPBS;

[0095] (5) Resuspend the cells using the culture medium used to culture breast cancer tumor organoids in Example 4 and count the cells. Mix Matrigel and cell suspension at a ratio of 5:2 and inoculate into 12-well plates, with 5 Matrigel domes inoculated per well. Each Matrigel dome has a volume of 25 μL, and each μL of Matrigel and cell suspension mixture contains approximately 500 cells.

[0096] (6) After waiting for 30 minutes for the Matrigel to solidify, 1 mL of the culture medium used to culture breast cancer tumor organoids in Example 4 was added to completely cover the Matrigel dome. The cells were placed in a 37°C, 5% CO2 incubator and cultured for 10 to 15 days. The culture medium was replaced every 3 to 4 days until 3D breast cancer tumor organoids were generated.

[0097] Comparative Example 1

[0098] A culture medium for culturing breast cancer tumor organoids, compared with Example 3, differs in that the additives Rspondin 3, Wnt3a, epidermal growth factor EGF, serum substitute KSR, NRG1, fibroblast growth factor FGF10, and insulin-like growth factor IGF-1 are not added, and the composition and proportions of other components are the same.

[0099] Comparative Example 2

[0100] A culture medium for culturing breast cancer tumor organoids, compared with Example 3, except that Wnt3a is not added, and the compositions and proportions of other components are the same.

[0101] Comparative Example 3

[0102] A culture medium for culturing breast cancer tumor organoids, which differs from Example 3 in that the serum substitute KSR is not added, and the compositions and proportions of the other components are the same.

[0103] Comparative Example 4

[0104] A culture medium for culturing breast cancer tumor organoids, compared with Example 3, differs in that the serum substitute KSR is not added, and FBS is added with a final concentration of 10%, while the composition and proportions of other components are the same.

[0105] Comparative Example 5

[0106] A method for constructing breast cancer tumor organoids, compared with Example 7, except that the culture medium is the culture medium for culturing breast cancer tumor organoids in Comparative Example 1.

[0107] Comparative Example 6

[0108] A method for constructing breast cancer tumor organoids, compared with Example 7, except that the culture medium is the culture medium for culturing breast cancer tumor organoids in Comparative Example 2.

[0109] Comparative Example 7

[0110] A method for constructing breast cancer tumor organoids, compared with Example 7, except that the culture medium is the culture medium for culturing breast cancer tumor organoids in Comparative Example 3.

[0111] Comparative Example 8

[0112] A method for constructing breast cancer tumor organoids, compared with Example 7, except that the culture medium is the culture medium for culturing breast cancer tumor organoids in Comparative Example 4.

[0113] Effect embodiment

[0114] 1. Morphology of breast cancer tumor organoids

[0115] The breast cancer tumor organoids constructed in Examples 5 to 8 and Comparative Examples 5 to 8 were examined microscopically using a Nikon inverted microscope to observe the morphology of the breast cancer tumor organoids constructed by each method.

[0116] The results showed that the breast cancer tumor organoids derived from breast cancer tumor tissue were cultured using the culture medium of Example 1. Figure 2 It can be observed that due to the low concentration of each component in the additive, the cultured organoids grew well, but the proliferation rate was slow; the breast cancer tumor organoids derived from breast cancer tumor tissue were cultured using the culture medium in Example 2, and the morphological results were shown in FIG. Figure 3, it can be observed that the number of organoids grown in culture is large and the activity is good; the breast cancer tumor organoids derived from breast cancer tumor tissue were cultured using the culture medium in Example 3, and the morphological results are shown in FIG. Figure 4 It can be observed that the number of organoids grown in culture is large, the size is relatively uniform, the diameter is large, the growth rate is fast, and the activity is good; the breast cancer tumor organoids derived from breast cancer tumor tissue were cultured using the culture medium in Example 4, and the morphological results are shown in FIG. Figure 5 It can be observed that although the concentrations of Rspondin 3 and KSR in the additive components are increased relative to those in Example 3, the morphology and activity of the cultured organoids are no different from those in Example 7 (i.e., using the culture medium of Example 3).

[0117] The morphological results of breast cancer tumor organoids cultured using the construction method of Comparative Example 5 are shown in Figure 6 It can be observed that the growth of breast cancer tumor organoids is seriously affected by the lack of supporting organoid growth and proliferation components. The cultured breast cancer tumor organoids are morphologically disintegrated, the number is small, and the vitality is poor. The morphological results of the breast cancer tumor organoids cultured using the construction method of Comparative Example 6 are shown in FIG. Figure 7 It can be observed that when Wnt3a is lacking, the organoid proliferation promoting effect decreases, the number of organoids formed decreases, and the diameter becomes smaller; the morphological results of the ascites-derived lung cancer organoids cultured using the construction method of Comparative Example 3 are shown in Figure 8 It can be observed that when KSR is lacking, the growth rate of organoids slows down and the diameter becomes smaller; the morphological results of breast cancer tumor organoids cultured using the construction method of Comparative Example 8 are shown in Figure 9 It can be observed that the effect of 10% FBS on the proliferation of organoids is basically consistent with that of KSR, but mesenchymal cells can be observed adhering to the bottom of the culture plate, competing with the growth of organoids for nutrients, thereby interfering with the proliferation of breast cancer tumor organoids.

[0118] 2. Hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) identification

[0119] The breast cancer organoids (100-200 μm in diameter) constructed in Example 7 were subjected to hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) identification. The specific process is as follows:

[0120] Hematoxylin and eosin (HE) staining: Once breast cancer organoids reach a diameter of 100-200 μm, they are released from the Matrigel and fixed with 4% PFA. They are then embedded in paraffin and sectioned. Finally, they are stained with hematoxylin (nuclear stain) and eosin (cytoplasmic stain) for histomorphological analysis. These steps are performed simultaneously with the source tissue.

[0121] Immunohistochemistry (IHC) staining: Once breast cancer organoids reach a diameter of 100–200 μm, they are released from Matrigel and fixed with 4% PFA. Embedded in paraffin, sectioned at 4 μm thickness, and then microwave-assisted antigen retrieval with sodium citrate buffer (pH 6.0) to expose target protein epitopes. Endogenous peroxidase activity is blocked with 3% H₂O₂, and nonspecific binding sites are blocked with 5% BSA. The target protein is then incubated with a specific primary antibody (overnight at 4°C) and an HRP-conjugated secondary antibody (1–2 hours at room temperature). DAB colorimetric solution is used to control the development time and label the target protein (brown). Nuclei are counterstained with hematoxylin for analysis of target protein localization and expression levels. These steps are performed simultaneously on the tissue from which the organoids were derived.

[0122] The results of hematoxylin-eosin staining and immunohistochemistry were as follows Figure 10 HE staining showed that the morphological characteristics of breast cancer organoids (such as cell arrangement, nuclear-cytoplasmic ratio, etc.) were highly consistent with the clinical tissue sections of the patient's primary tumor, and the pathological diagnosis was invasive cancer. IHC analysis showed that estrogen receptor (ER, nuclear localization) and human epidermal growth factor receptor 2 (HER2, plasma membrane localization) were positively expressed in both tumor tissues and organoids, and the proliferation marker Ki67 (nuclear localization) was simultaneously highly expressed in both; while progesterone receptor (PR, nuclear localization) was not detected in either tissues or organoids, further verifying the consistency of the molecular phenotypes of organoids and primary tumors.

[0123] 3. Cell survival and cell number of breast cancer organoids

[0124] 30 μL of each Matrigel dome from the breast cancer organoid culture process in Example 7 and Comparative Examples 5-8 was initially seeded with 3000 cells. After 14 days of culture, the breast cancer organoids were digested into single cells, and the cell number of all organoids within each Matrigel dome was counted.

[0125] The comparison results of the number of harvested breast cancer organoid cells are as follows Figure 11 The cell viability and harvested cell number of breast cancer organoids are shown in Table 1. The results show that the cell viability and number of breast cancer organoids constructed using the culture medium of Example 3 are high, indicating that the culture medium can effectively simulate the tumor microenvironment and promote the formation and growth of breast cancer tumor organoids.

[0126] Table 1 Results of organoid viability and cell number harvested from Example 7 and Comparative Examples 5-8

[0127]

[0128] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A culture medium comprising an L-glutamine additive, an antibiotic, a Wnt signaling pathway activator, a bone morphogenetic protein inhibitor, a growth factor, a regulatory factor, a serum replacement, nicotinamide, acetylcysteine, hydrocortisone, estradiol, forskolin, a TGF-β signaling pathway inhibitor, a Rock inhibitor, a buffer, and a basal culture medium.

2. The culture medium according to claim 1, characterized in that The Wnt signaling pathway activator is selected from at least one of Wnt3a, Wnt4, Wnt5a, Rspondin 1, Rspondin 2, Rspondin 3, Rspondin 4, CHIR99021, CHIR98014, SB 216763, SB 415286 and TCS2002; Much better, the antibiotic is selected from at least one of penicillin / streptomycin double antibiotic, penicillin, streptomycin and amphotericin B; Preferably, the bone morphogenetic protein inhibitor is selected from at least one of Noggin, Gastrin, Connective Tissue Growth Factor, Inhibin, BMP-3 and Dorsomorphin; Preferably, the regulatory factor is selected from at least one of NRG1 and KGF; Preferably, the serum replacement is selected from at least one of B27, KSR, and KOSR; Preferably, the TGF-β signaling pathway inhibitor is selected from at least one of A-83-01, LY2157299, LY3200882, SB-431542, LY2109761, SB-525334, SB-505124, and LY364947; Preferably, the Rock inhibitor is selected from at least one of Y-27632, Thiazovivin and Azaindole 1; Preferably, the buffer is selected from HEPES, Tirs-HCl; Preferably, the basal culture medium is selected from at least one of Advanced DMEM / F12, DMEM, and RMPI 1640.

3. The culture medium according to claim 1, characterized in that The growth factors include epidermal growth factor EGF, fibroblast growth factor and insulin-like growth factor; Preferably, the fibroblast growth factor is selected from at least one of FGF1, FGF2, FGF4, FGF7, FGF9 and FGF10; Preferably, the insulin-like growth factor is at least one selected from IGF-1 and IGF-2.

4. The culture medium according to any one of claims 1 to 3, characterized in that The culture medium is Advanced DMEM / F12 containing L-glutamine supplement, penicillin / streptomycin double antibody, Rspondin 3, Wnt3a, Noggin, EGF, serum substitute B27, serum substitute KSR, nicotinamide, acetylcysteine, hydrocortisone, estradiol, forskolin, NRG1, FGF10, A83-01, IGF-1, Y-27632, and HEPES buffer; Preferably, the culture medium contains 1-2X L-glutamine additive, 1-2X penicillin / streptomycin double antibody, 50-1000ng / mL Rspondin 3, 10-100ng / mL Wnt3a, 10-100ng / mL Noggin, 5-50ng / mL EGF, 0.5-1X serum substitute B27, 5%-30% serum substitute KSR, 5-20mM nicotinamide, 1-10mM acetylcysteine, 0.1-2μg / mL hydrocortisone, 50-250nM estradiol, 5-20μM forskolin, 10-100ng / mL NRG1, 10-150ng / mL FGF10, 200-900nM A83-01, 10-100ng / mL IGF-1, 5-10μM Y-27632, 1% to 2% HEPES buffered Advanced DMEM / F12.

5. Use of the culture medium according to any one of claims 1 to 4 in preparing organoids.

6. The use according to claim 5, characterized in that The organoid comprises a tumor organoid, preferably a breast cancer tumor organoid.

7. A method for preparing tumor organoids, comprising culturing tumor cells in the culture medium according to claims 1 to 4 to obtain tumor organoids, wherein the culture is a three-dimensional culture.

8. The method according to claim 7, characterized in that The three-dimensional culture comprises resuspending the tumor cells in the culture medium of any one of claims 1 to 4, mixing with the matrix gel, placing on a plate and letting it stand, adding the culture medium of any one of claims 1 to 4 after solidification, and culturing in a cell culture incubator.

9. Application of the tumor organoids prepared by the method according to claim 7 or 8 in screening anti-tumor drugs, individualized drug sensitivity testing, tumor molecular phenotyping, and gene-drug association studies.

10. A drug screening method, comprising taking the tumor organoid prepared by the method of claim 7 or 8, adding an anti-tumor drug, and evaluating the efficacy of the anti-tumor drug.

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