Method for culturing vascularized and immunized glioma organs

Abstract

The invention relates to the technical field of tumor organoid culture, in particular to a method for culturing vascularized and immunized glioma organoid. The invention provides a method for culturing vascularized and immunized glioma organs, which comprises the following steps: cutting glioblastoma tissues into blocks, and splitting red blood cells to obtain treated tissue blocks; carrying out suspension culture on the treated tissue blocks in a gliocyte organoid culture medium; a round spherical organoid with dense cells is formed within 7-14 days after the culture, and if the organoid is spherical, the survival time is longer than or equal to 2 weeks and the diameter is continuously increased, the glioma organoid is successfully established; and detecting and verifying the glioma organoid. The glioma organoid obtained through the culture method is consistent with primary glioblastoma tissue, and the multiplication capacity and the stemness characteristic of glioma are reserved; and the immune infiltration pattern similar to that of the tissue of the patient is shown.

Description

Technical Field

[0001] This invention relates to the field of tumor organoid culture technology, and more particularly to a method for culturing vascularized and immunomodulated glioma organoids. Background Technology

[0002] Gliomas are the most common primary malignant tumors of the central nervous system. They are a large group of tumors originating from glial cells, exhibiting morphological characteristics of astrocytes, oligodendrocytes, and ependymal cells. According to the World Health Organization (WHO) classification, glioblastoma (GBM) is a grade IV glioma, the most common and primary highly aggressive malignant tumor of the brain, and its treatment outcome is the worst among all cancers. Standard treatment for GBM mainly includes surgical intervention, supplemented by temozolomide chemotherapy, radiotherapy (RT), or targeted therapy. However, current treatment outcomes are poor, and there is no standard treatment for recurrence; treatment is mainly supportive and palliative, depending on the patient's specific condition. The median survival time for GBM cases is approximately 12.5–15 months, with 2-year and 5-year survival rates of only 25% and 10%, respectively. Due to its highly dynamic and complex microenvironment and unique intratumoral heterogeneity, GBM urgently requires combination therapies with one or more for precise targeted attack.

[0003] In recent years, the application and development of organoids in tumor therapy and targeted drug screening have been increasingly recognized. Organoids are three-dimensional structures formed through self-organization in an in vitro three-dimensional culture environment from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or patient-derived adult stem cells / tissues. Tumor organoids are derived from tumor tissue or tumor stem cells. The tumor is isolated into single cells and cultured utilizing the unlimited proliferation characteristics of tumor tissue, preserving the histological structure, gene mutations, and heterogeneity of the primary tumor. They serve as in vitro models for studying the malignant biological behavior of tumors. They can be used for tumor disease mechanism research and drug screening, but lack a complete tumor microenvironment (such as blood vessels and immune cells). Patient-Derived Tumor Organoids (PDTOs) are constructed directly from single cells or small tissue blocks isolated from patient tumor biopsy or surgical samples through 3D culture, highly preserving patient-specific genetic variations and drug sensitivity responses. Compared to traditional 2D culture models, these models can more closely represent the cellular composition and physiological behavior of the human body, forming a three-dimensional tumor microenvironment (TME) and preserving the heterogeneity of the parent tumor—key factors for tumor research and treatment. This advanced in vitro model system provides a highly biomimetic experimental platform for organ development research, disease modeling, and drug screening, overcoming the inherent limitations of traditional in vitro cell models and animal experiments while avoiding the ethical controversies associated with human trials. Although animal models can simulate the physiological and pathological processes of multicellular, multi-tissue, and organ interactions, some human disease phenotypes are difficult to simulate in animals due to differences in species and genetic background, presenting significant limitations in simulating the human brain tumor microenvironment. In contrast, brain organoid technology can accurately reproduce the three-dimensional structure and physiological functions of human brain tissue, providing an ideal platform for exploring the biological behavior of tumors in the brain, and has already been widely used in research on various malignant brain tumors. Currently, there is substantial policy support both domestically and internationally.

[0004] Current methods for constructing vascularized organoids primarily rely on endothelial cell co-culture, microfluidic chip perfusion, and induction by exogenous angiogenic factors. These strategies can, to some extent, form capillary-like lumens or endothelial cell arrangements, providing preliminary means for simulating tumor angiogenesis. Some models have already achieved the expression of vascular endothelial markers (such as CD31 and vWF) and morphologically exhibit tubular or reticular structures. However, these methods generally suffer from limitations such as the lack of long-term perfusion capacity of the vascular network, the absence of blood-brain barrier-like features, and the lack of a functional interface with the tumor compartment. Especially in glioma research, the existing abnormal expansion of new blood vessels and the pathological state of partial disruption of the blood-brain barrier make it difficult for current vascularized organoid models to simultaneously reproduce this complex pathological feature of "increased permeability and barrier constraint."

[0005] To mimic the tumor immune microenvironment, researchers have attempted to co-culture peripheral blood mononuclear cells (PBMCs), tumor-infiltrating lymphocytes (TILs), or specific immune effector cells with organoids. These methods can observe T cell infiltration, cytotoxic responses, and the secretion of related factors within a short period, thus enhancing the value of organoids in the evaluation of immunotherapeutic drugs. However, these immunomodulatory strategies are mostly limited to short-term co-culture and exogenous supplementation, making it difficult to maintain the long-term activity and spatial distribution of immune cells. Especially in the glioma scenario, how to stably reconstruct the synergistic effects of microglia, macrophages, and T cells, and simulate their infiltration trajectory within the vascular pathway and tumor compartment, remains a bottleneck that existing models struggle to overcome. Summary of the Invention

[0006] The purpose of this invention is to provide a method for culturing vascularized and immunomodulated glioma organoids. The glioma organoids cultured using this method are identical to primary glioblastoma tissue, retaining the proliferative capacity and stemness characteristics of gliomas; moreover, they exhibit an immune infiltration pattern similar to that of patient tissues.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for culturing vascularized and immunomodulated glioma organoids, comprising the following steps: (1) The glioblastoma tissue was cut into pieces to obtain glioblastoma tissue blocks; the glioblastoma tissue blocks were subjected to erythrocyte lysis to obtain processed tissue blocks; (2) The treated tissue blocks are cultured in suspension in glioblastoma organoid culture medium; during the suspension culture, 70-80% of the culture medium is replaced every 46-50 hours. (3) Within 7 to 14 days of culture, round, densely cellular spherical organoids are formed. If the organoids are spherical in shape, survive for ≥2 weeks and their diameter continues to increase, then the glioma organoids are successfully established. (4) Detection and verification of glioma organoids.

[0008] Preferably, in step (1), the glioblastoma tissue needs to be washed 2 to 4 times with calcium-magnesium phosphate-buffered saline before being cut into pieces.

[0009] Preferably, in step (1), the length of the glioblastoma tissue block is 0.5~1mm.

[0010] Preferably, in step (1), necrotic, hemagglutinous, and non-tumor brain tissue needs to be removed before the red blood cells are lysed.

[0011] Preferably, in step (1), the method of red blood cell lysis is as follows: mix the glioblastoma tissue block and the red blood cell lysis solution, incubate on a shaker for 8-12 minutes, discard the red blood cell lysis solution, and then wash to obtain the processed tissue block.

[0012] Preferably, the shaking speed is 8-12 times / minute; the incubation temperature is 20-30°C.

[0013] Preferably, the washing solution is DMEM culture medium; the washing is performed 2 to 4 times.

[0014] Preferably, in step (2), the culture temperature is 35~39℃; the CO2 concentration is 4~6%; and the humidity is ≥90%.

[0015] Preferably, in step (4), the detection and verification method is as follows: prepare frozen or paraffin sections from cultured glioma organoids and use multiplex immunofluorescence and immunohistochemistry for detection.

[0016] Beneficial effects: (1) Preservation of vascularization features: After culturing for 14 days using the culture method of this application, positive expression of vascular markers such as CD31 and vWF can be detected, proving that it has vascular-like structure, and the glioma organoids obtained by culture are consistent with the primary glioblastoma tissue; (2) Reproduction of the immune microenvironment: Microglia, T lymphocytes and other immune components can be detected in glioma organoids, showing an immune infiltration pattern similar to that of patient tissues; (3) Enhanced tumor authenticity: Markers such as GFAP, SOX2, and Ki67 remained positive in glioma organoids, indicating that they retained the proliferative capacity and stemness characteristics of gliomas; (4) Wide range of applications: This glioma organoid model is not only suitable for basic research, but can also serve as a preclinical platform to evaluate the efficacy prediction of anti-angiogenic drugs, immunotherapy and their combination therapies, and provide support for precision medicine. Attached Figure Description

[0017] Figure 1 Morphology of glioblastoma tissue cultured for 0, 1, 3 and 6 days under a 4× microscope; Figure 2 Morphology of glioblastoma tissue cultured for 1 day and 6 days under macroscopic visualization; Figure 3 The morphology of glioblastoma tissue cultured for 7 days, 10 days, and 14 days under 4× and 10× microscopes; Figure 4 HE staining results for cultured at 0d; Figure 5Immunofluorescence-vascular (Ki67, Vimentin, GFAP, SOX2, CD31, vWF) results after 14 days of culture; Figure 6 Results of immunofluorescence-microglia (CD11b, IBA1, TMEM119) cultured for 14 days; Figure 7 Results of immunofluorescence-immunotherapy T cells (CD3, CD4, CD8) cultured for 14 days; Figure 8 Results of immunofluorescence of immune cells, blood vessels and tumors (CD45, CD31, GFAP) after 14 days of culture; Figure 9 Results of immunofluorescence-macrophages (CD68, CD86, CD206) cultured for 14 days; Figure 10 Immunohistochemical (vWF and CD31) results after 14 days of culture; Figure 11 HE staining results for cultured cells at 0d and 20d; Figure 12 Immunofluorescence-angiography results after 20 days of culture; Figure 13 Immunofluorescence-tumor results after 20 days of culture; Figure 14 Results of immunofluorescence-immunomicroenvironment cultured for 20 days; Figure 15 Results of immunofluorescence-immunomicroenvironment cultured for 20 days; Figure 16 Results of immunofluorescence-immunomicroenvironment cultured for 20 days. Detailed Implementation

[0018] This invention provides a method for culturing vascularized and immunomodulated glioma organoids, comprising the following steps: (1) The glioblastoma tissue was cut into pieces to obtain glioblastoma tissue blocks; the glioblastoma tissue blocks were subjected to erythrocyte lysis to obtain processed tissue blocks; (2) The treated tissue blocks are cultured in suspension in glioblastoma organoid culture medium; during the suspension culture, 70-80% of the culture medium is replaced every 46-50 hours. (3) Within 7 to 14 days of culture, round, densely cellular spherical organoids are formed. If the organoids are spherical in shape, survive for ≥2 weeks and their diameter continues to increase, then the glioma organoids are successfully established. (4) Detection and verification of glioma organoids.

[0019] In this invention, in step (1), the glioblastoma tissue obtained by surgical resection needs to be immediately transferred to a 15mL centrifuge tube containing 5mL of ice-cold glioblastoma organoid culture medium for short-term storage during transportation; before culturing glioblastoma organoids, the tissue needs to be cleaned: carefully aspirate the culture medium above the glioblastoma tissue in a laminar flow hood; wash the glioblastoma tissue three times with 10mL of 4℃ PBS, allowing the glioblastoma tissue to sink to the bottom of the centrifuge tube, and discard the supernatant.

[0020] Before the glioblastoma tissue is cut into blocks, it needs to be washed 2 to 4 times with calcium and magnesium phosphate buffered saline, preferably 3 times.

[0021] In this invention, in step (1), the length of the glioblastoma tissue block is 0.5~1mm, preferably 0.7~0.8mm, and more preferably 0.75mm; The cut pieces need to be cut into pieces in a 10cm culture dish containing glioblastoma organoid culture medium; The method of cutting the tissue is as follows: first, cut the entire tissue into large pieces, and then cut each large piece into smaller pieces. This will quickly expose the tissue to the nutrients in the culture medium. Tear-and-shred the tissue, and only cut it in a straight line to reduce mechanical damage.

[0022] In this invention, in step (1), necrotic, blood clots and non-tumor brain tissue need to be removed before the red blood cells are lysed in order to improve the quality of subsequent clumping and growth; necrotic masses are mostly dark and loose, blood clots are deep red, and brain tissue cells have low density and are pinkish-yellow; brain tissue may be difficult to distinguish from tumor tissue at first, and usually does not aggregate or grow for 1 to 2 weeks.

[0023] In this invention, in step (1), the method of red blood cell lysis is as follows: glioblastoma tissue block and red blood cell lysis solution are mixed, incubated on a shaker for 8-12 minutes, the red blood cell lysis solution is discarded, and the tissue block is washed to obtain the processed tissue block; The incubation time is preferably 9 to 11 minutes, and more preferably 10 minutes.

[0024] In this invention, the speed of the shaker is 8-12 times / min, preferably 9-11 times / min, and more preferably 10 times / min; the incubation temperature is 20-30℃, preferably 23-27℃, and more preferably 25 min.

[0025] In this invention, the washing solution is DMEM culture medium; the number of washing cycles is 2 to 4, preferably 3.

[0026] In this invention, in step (2), the culture temperature is 35~39℃, preferably 36~38℃, and more preferably 37℃; the CO2 concentration of the culture is 4~6%, preferably 5%; and the humidity of the culture is ≥90%, preferably 92~98%, and more preferably 95%. The culture medium is preferably replaced at a time of 47-49 hours, more preferably at 48 hours. The amount of culture medium replaced is preferably 73-77%, more preferably 75%.

[0027] In this invention, the detection and verification method in step (4) is as follows: prepare frozen or paraffin sections from cultured glioma organoids and use multiplex immunofluorescence and immunohistochemistry for detection; Multiplex immunofluorescence and immunohistochemistry assays include the following specific detection indicators: Tumor cell characteristics: GFAP, SOX2, Ki67, Vimentin; Vascularization markers: CD31, vWF; Immune microenvironment markers: CD11b, IBA1, TMEM119 (microglia), CD68, CD86, CD206 (macrophages), CD3, CD4, CD8 (T lymphocytes), CD45 (total immune cells).

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] In an embodiment of the present invention, the glioblastoma organoid culture medium was purchased from Accuroid Glioma Organoid Culture Medium Co., Ltd. (Guangzhou), and the erythrocyte lysis buffer was purchased from Beijing Solarbio Technology Co., Ltd.

[0030] Ki67 (rabbit anti-human), GFAP (rabbit anti-human), SOX2 (rabbit anti-human), Abcam (rabbit anti-human), Vimentin (rabbit anti-human), CD31 (rabbit anti-human), and vWF (mouse anti-human) were purchased from Abcam; CD11b (rabbit anti-human), IBA1 (rabbit anti-human), CD3 (rabbit anti-human), CD4 (rabbit anti-human), CD45 (rabbit anti-human), CD68 (rabbit anti-human), CD86 (rabbit anti-human), and CD206 (rabbit anti-human) were purchased from Huilan Biotechnology; TMEM119 (rabbit anti-human) and CD8 (rabbit anti-human) were purchased from Huaan Biotechnology; Neun (rabbit anti-human) was purchased from Beijing Solarbio Technology Co., Ltd.; anti-quenching mounting medium was purchased from Thermo Fisher Scientific; TSA kit (containing goat anti-mouse / rabbit universal HRP enzyme-labeled secondary antibody) was purchased from Hunan Aifang Biotechnology Co., Ltd.; and blocking solution (bovine serum albumin BSA-V) was purchased from Solarbio.

[0031] Example 1: Culture of an vascularized, immunomodulated glioma organoid

[0032] (1) The glioblastoma tissue obtained after surgical resection needs to be immediately transferred to a 15mL centrifuge tube containing 5mL of ice-cold glioblastoma organoid culture medium for short-term storage during transportation; then, carefully aspirate the culture medium above the glioblastoma tissue in a laminar flow hood; wash the glioblastoma tissue three times with 10mL of 4℃ PBS, allowing the glioblastoma tissue to settle to the bottom of the centrifuge tube, and discard the supernatant; transfer the glioblastoma tissue to a 10cm culture dish containing glioblastoma organoid culture medium (with a suitable dissecting microscope), and use sharp dissecting scissors and forceps to remove the glioblastoma tissue. The tumor tissue was cut into large pieces, and then each large piece was cut into regular 1mm pieces. This quickly exposed the tissue to the nutrients in the culture medium. Tear-like dissection was prohibited; only straight-line shearing was used to reduce mechanical damage. The qualified tumor pieces were collected into 50mL centrifuge tubes using a large-diameter pipette tip. The supernatant was discarded to obtain glioblastoma tissue blocks. The glioblastoma tissue blocks were washed three times with 10mL of calcium-magnesium phosphate-buffered saline at room temperature. Then, 10mL of erythrocyte lysis buffer was added, and the mixture was incubated on a shaker at 25℃ and 10 shakes / min for 10min. When there was a lot of blood, the solution turned red. The lysis buffer was then discarded, and the tissue blocks were washed three times with 10mL of DMEM culture medium to obtain the processed tissue blocks. (2) The treated tissue blocks were placed in glioblastoma organoid culture medium and cultured in suspension at 37°C, 5% CO2 and 95% humidity; during the suspension culture, 75% of the culture medium was replaced every 48 hours. (3) The culture medium was divided into three groups (7-day group, 10-day group, and 14-day group, i.e., cultured for only 7 days, 10 days, and 14 days respectively), and the changes in organoid culture were observed with the naked eye and under a microscope. Figures 1-5 By day 6 of culture, glioblastomas visibly enlarge, and within 7-14 days of culture, they form round, densely cellular spherical organoids. If the organoids are spherical, survive for ≥2 weeks, and their diameter continues to increase, then the glioblastoma organoids have been successfully established. (4) Detection and verification of glioma organoids.

[0033] Example 2: Preparation of slices

[0034] Paraffin sections: (1) Fixation: Primary tumor (glioblastoma tissue) and organoids obtained after 14 days of culture (glioblastoma organoids) were fixed with 4% paraformaldehyde for 3 days respectively. (2) Dehydration: Gradual dehydration with 70%, 80%, 90%, and 100% alcohol is carried out to gradually remove water from the tissue; for medium-sized tissues, each step takes 1 hour, while for small tissues, it can take 40 minutes. (3) Transparency: First, mix xylene and anhydrous ethanol in a 1:1 ratio, and soak the tissue in xylene twice, 7 minutes each time; the tissue gradually becomes transparent and becomes a semi-transparent gel (judged by visual observation), which facilitates the subsequent penetration of paraffin; if the transparency time is too long, the tissue will harden and the tissue will break during sectioning; (4) Wax impregnation: Transfer the transparent tissue into molten paraffin (65°C); impregnate three times in sequence, one hour each time; (5) Embedding: Using a suitable container, embed the tissue in clean paraffin; mark it with a pencil and let it stand overnight; (6) Sectioning: Before slicing, place the wax block in the refrigerator for 1 hour. Frozen wax blocks are harder and easier to cut. Replace with a new blade, first trim the surface of the wax block to make it flat, and then slice it at a thickness of 4μm until the surface is smooth and there is complete tissue. It is best to slice continuously. (7) Slide preparation: Gently hold the continuous slide with tweezers, slowly move it into the preheated water bath, and quickly spread the tissue slide on the water; (8) Slide preparation: After fully unfolding, attach the slide to the poly-lysine-coated slide, making the slide at a 45° angle to the water surface. When it touches the tissue, pull the slide upwards. It is best to keep the tissue shape on different slides the same. Frozen slices: (1) Fixation: The washed primary tumor (glioblastoma tissue) and organoids obtained after 14 days of culture were placed in a 15 mL centrifuge tube, and 10 times the volume of 4% PFA was added. The tube was then placed at 4°C for 12 h for mild fixation (low temperature can reduce excessive cross-linking and preserve antigenicity). (2) Dehydration: Soak in 10%→20%→30% sucrose (prepared with PBS) in sequence, place at 4℃, 12h for each stage, until the organoids completely sink (sucrose is a osmotic cryoprotectant that can replace water in the tissue and lower the freezing point, prevent freezing and the formation of large ice crystals, and maintain the integrity of tissue cell morphology). (3) Embedding: Use clean disposable filter paper to absorb the residual liquid on the tissue surface, lay a thin layer of pre-cooled OCT in the mold, and place the cut surface of the tissue to be sectioned downwards to ensure that the tissue boundary is parallel to the edge of the mold and avoid tilting; add OCT until it completely covers the tissue without air bubbles; if there are air bubbles, pick them out with pre-cooled forceps. (4) Quick-freeze: Store at -80℃; (5) Sectioning: Take out the OCT frozen block from the -80℃ freezer and put it into the -20℃ cryostat for 30 minutes to equalize the temperature (the temperature of the cryostat is -20℃, and equalizing the temperature of the tissue can prevent cracks or curling due to temperature difference during slicing); use a small amount of OCT glue to stick the frozen block on the sample holder and grind the bottom of the frozen block flat to ensure that the tissue surface is parallel to the cutter. Further trim until a uniform tissue surface appears, and start the formal 10μm thickness one-time slicing. (6) Covering: Gently cover the newly cut slice with a glass slide coated with poly-L-lysine, wait for 2 seconds and then lift it; Frozen sections and paraffin sections were sealed and stored at -80°C for subsequent HE, IHC, mIHC and other experiments within 2 weeks.

[0035] Example 3: Detection and Validation of Glioma Organoids Cultured for 14 Days

[0036] HE staining: (1) Principle: Hematoxylin (basic dye) carries a positive charge in staining solution with pH≈6. It binds to the phosphate group (negative charge) of DNA double strand through ionic bonds, making basophilic substances such as chromatin in the cell nucleus and nucleic acid in the cytoplasm turn purple-blue; Eosin (acid dye) dissociates into negative ions in solution, which bind to the amino group (positive charge) of protein, staining eosinophilic substances such as cytoplasm, collagen fibers, and hemoglobin red; (2) Dewaxing: Xylene I → Xylene II → Xylene III → Xylene IV, 5 minutes each time, to remove wax; (3) Hydration: Anhydrous ethanol → 95% → 80% → 70%, 5 min each time, to wash away xylene; (4) Rinsing: Soak in tap water for 5 minutes to remove ethanol, then rinse with pure water. (5) Hematoxylin: Soak for 15 minutes for staining; (6) Wash off excess color with water: rinse directly with water for 10 seconds to remove residual hematoxylin; (7) Differentiation: Soak in 1% hydrochloric acid alcohol for 1 second, then immediately take it out and put it in water to wash away the hematoxylin that has not been fully combined; (8) Blueing: Soak in tap water for 15 minutes (weakly alkaline liquid, for blueing). (9) Eosin: Soak for 10 minutes; (10) Washing off the floating color: Rinse directly with water for 10 seconds to remove residual eosin; (11) Gradient alcohols: 70% (10s), 80% (30s), 95% (2min), anhydrous ethanol (5min); (12) Mounting: Add neutral resin to the slide, cover it with a coverslip starting from the edge of the tissue, let it stand until the coverslip completely covers the tissue, and allow it to air dry at room temperature to complete the mounting; observe the slide using a microscope. Figure 6 ); The results showed that glioblastoma cells were densely packed and structurally disordered; nuclear atypia (large, deeply stained nuclei with prominent nucleoli); and microvascular proliferation (multiple irregular blood vessels).

[0037] Multiplex immunofluorescence mIHC: (1) Principle: The primary antibody specifically recognizes the target antigen epitope, and the secondary antibody (labeled enzyme) binds to the primary antibody, amplifying the signal. The enzyme needs to be added with a substrate to develop color and form a visible localization marker. Significance: ① Clarify the distribution of the target in organoids (such as cell membrane, cytoplasm, and nucleus); ② Targets: CD31, vWF, CD11b, IBA1, TMEM119, CD3, CD4, CD8, CD45, GFAP, SOX2, Ki67, Vimentin.

[0038] (2) Baking the slides (to evaporate the moisture from the wax slides and melt the paraffin, allowing the slides to adhere better to the glass slides and to remove impurities such as xylene): Paraffin tissue sections stored at 4°C were allowed to warm naturally at room temperature and then baked at 65°C for 2 hours (or 70°C for 1 hour). (3) Dewaxing and hydration: 1) Dewaxing: Xylene I → Xylene II → Xylene III → Xylene IV, 5 minutes each time, to remove wax; 2) Hydration: Anhydrous ethanol → 95% → 80% → 70%, 5 minutes each time, to wash away xylene; 3) Rinsing: Soak in tap water (pure water is better) for 5 minutes to remove ethanol; (4) Antigen retrieval: Add an appropriate amount of water and antigen retrieval solution (pH 6.0 sodium citrate / pH 9.0 EDTA) to a pressure cooker, bring to a boil over high heat at 2200°C, then simmer over low heat at 500°C (121°C for 20 minutes). After the cooker has cooled naturally, open the lid and cover it with transparent gloves to allow it to cool naturally. (5) Elimination of endogenous sources (this step is not necessary for directly labeled fluorescence, but is required for HRP-labeled samples): 1) Objective: To eliminate endogenous peroxidase and reduce false positives; 2) Add 3% hydrogen peroxide and incubate at room temperature for 15 minutes; wash 3 times with TBST; Because some cells contain endogenous peroxidases (such as myeloperoxidase), the subsequent addition of hydrogen peroxide will cause the hydrogen peroxide to decompose, resulting in the deposition of non-specific TSA signals, causing false positives, and the background area will also be colored. (6) Site closure: 1) Objective: To block non-specific targets and reduce non-specific antibody adsorption; 2) Use 3% BSA as a sealing agent and seal at room temperature for 30 minutes; (7) Incubation of primary antibody: 1) Objective: To specifically recognize the target antigen; 2) Dilute the primary antibody and add it to the tissue to cover it. Incubate at 37°C for 2 hours or at 4°C overnight. Then wash three times with TBST. (8) Incubation of secondary antibodies: 1) Purpose: To connect the primary antibody and label it with fluorescence (or HRP); 2) Add a fluorescent secondary antibody of the same species as the primary antibody, cover the tissue, incubate in the dark for 30 min, and wash 3 times with TBST; (9) TSA reagent: HRP is applied to the TSA substrate for color development; each session lasts 8 minutes. (10) Antibody elution: 1) Wash away the previously bound antibodies to facilitate antibody binding in the second round; 2) Place paraffin sections in antigen retrieval solution in a 100°C water bath for 40 minutes; for frozen sections, which are easy to detach, it is recommended to cover the sample with an appropriate amount of mIHC antibody elution buffer preheated to 37°C until completely dissolved, incubate at 37°C for 20 minutes, discard the elution buffer, add an appropriate amount of antibody elution buffer again to cover the sample, incubate at 37°C for 20 minutes, discard the elution buffer, and wash three times with TBST for 5 minutes each time. (11) Repeat steps (5) to (10) (using a different fluorescent dye) --- second round of labeling (12) Repeat steps (5) to (9) (using a different fluorescent dye) --- third round of marking (13) Covering: 1) Purpose: To protect the stability of the sample structure and prevent fluorescence quenching; 2) Mount the slide using the built-in nuclear dye (DAPI) and curing mounting medium. Place one drop of mounting medium on the tissue using a pipette, gently place the coverslip onto the slide, and leave it in the dark for 24 hours to cure. The results were observed under a microscope. Figures 7-9 As shown; The results showed that Ki67, Vimentin, GFAP, and SOX2 were expressed in both 0-day glioblastoma and 14-day glioma organoids.

[0039] Immunohistochemistry (IHC)

[0040] (1) Baked slices: 1) Purpose: To evaporate the moisture in the wax slide and melt the paraffin, allowing the slide to adhere better to the glass slide and to remove impurities such as xylene; 2) Place the paraffin tissue sections stored at 4℃ to room temperature for natural rewarming, and then bake at 65℃ for 2 hours; (2) Dewaxing and hydration: 1) Dewaxing: Xylene I → Xylene II → Xylene III → Xylene IV, 5 minutes each time, to remove wax; 2) Hydration: Anhydrous ethanol → 95% → 80% → 70%, 5 minutes each time, to wash away xylene; 3) Rinse with water: Use tap water / pure water to soak for 5 minutes to remove ethanol; (3) Antigen retrieval: Add an appropriate amount of water and antigen retrieval solution (pH 6.0 sodium citrate / pH 9.0 EDTA) to a pressure cooker, bring to a boil over high heat at 2200°C, then simmer over low heat at 500°C (121°C for 20 minutes). After the cooker has cooled naturally, open the lid and cover it with transparent gloves to allow it to cool naturally. (4) Washing in circles: Place the slide in clean water, take it out and let it dry until it is semi-dry. Use an immunohistochemistry pen to draw the boundary around the tissue, without touching the tissue or interrupting the flow. Use TBST buffer to gently wash 3 times (5 min each time) to remove the antigen retrieval solution. (5) Eliminate endogenous sources: 1) Purpose: To remove peroxidase that is already present in the tissue, so as to avoid it from affecting the color development of subsequent HRP; 2) Cover the tissue with peroxidase inhibitor, incubate at room temperature in the dark for 20 min, and wash three times with TBST (5 min each time). (6) Closed: 1) Objective: To block protein sites and reduce nonspecific results; 2) Cover the tissue with a blocking solution containing 3% BSA (bovine serum albumin) and incubate at room temperature for 30 minutes; (7) Primary and secondary antibodies: 1) Primary antibody: Dilute with blocking agent or primary antibody diluent according to a certain ratio and add dropwise to cover tissue; incubate overnight at 4°C; gently wash 3 times (5 min / time) with TBST buffer to thoroughly wash away the primary antibody; 2) Secondary antibody: Add the secondary antibody labeled with horseradish peroxidase (HRP) to the covered tissue and incubate at room temperature for 30 min; gently wash 3 times (5 min / time) with TBST buffer to thoroughly remove the secondary antibody; (8) Color development: 1) Purpose: To use DAB to develop color in HRP; 2) DAB staining: Prepare DAB working solution (in a certain proportion) and add it dropwise until the slide completely covers the tissue. The staining time needs to be explored through preliminary experiments. The time is between 0.5 and 5 minutes. After observing that the tissue has slightly stained, immediately shake off the DAB solution, put the slide into water and immediately dilute the residual DAB. Gently wash 3 times (5 minutes each time).

[0041] (9) Re-staining & Differentiation Blue Reversal: 1) Purpose: To stain cell nuclei to facilitate the localization of subsequent cell positions; 2) Hematoxylin counterstaining: stain with hematoxylin for 4 minutes, then soak in tap water 3 times, 1 minute each time, to thoroughly wash away the hematoxylin. 3) Differentiation and blueing: Quickly immerse in the differentiation solution for 2 seconds, then rinse gently with running water for 15 minutes; (10) Dehydration & mounting: 1) Dehydration: Gradual dehydration with 75%, 85%, 95%, and 100% alcohol in sequence, allowing the slides to dry at room temperature for more than 2 hours; 2) Mounting: Add mounting medium (neutral resin) to completely cover the tissue, gently press the four sides of the slide, and do not move the coverslip after it is placed on. The experimental procedure was summarized, a negative control was set up to observe the background and eliminate specific interference; isotype IgG antibody was used instead of primary antibody, and subsequent steps were kept consistent. The slide was observed under a microscope, and the results were as follows: Figure 10 As shown; The results showed that both immunohistochemistry and immunofluorescence could stain for vWF and CD31.

[0042] Example 4: Detection and Validation of Glioma Organoids Cultured for 20 Days

[0043] The glioma organoids were cultured for 20 days according to the method described in Example 1; The method described in Example 2 was used to prepare primary tumor (glioblastoma tissue) and sections of glioma organoids obtained after 20 days of culture; The primary tumor sections and glioma organoid sections obtained after 20 days of culture were stained according to the method described in Example 3. The 20-day glioma organoids were then examined. The staining markers included: vWF, CD31, Ki67, Vimentin, Sox2, CD8, CD4, CD45, CD68, CD86, CD206, CD11b, Neun, and GFAP. The HE staining results are shown below. Figure 11 As shown, the indicator detection results are as follows: Figures 12-16 As shown.

[0044] HE staining results showed that glioblastoma cells were densely packed and structurally disordered; nuclear atypia (large, deeply stained nuclei with prominent nucleoli); and microvascular proliferation (multiple irregular blood vessels). Indicator detection results indicated that this culture method can reconstruct complex tissue structures and functions in vitro, successfully reconstructing a three-dimensional model that more closely resembles the original tumor microenvironment, and can more realistically simulate tumor growth, angiogenesis, and immune responses. This culture method will bring new opportunities for the development and optimization of anti-glioblastoma therapies.

[0045] As can be seen from the above embodiments, the present invention provides a method for culturing vascularized and immunomodulated glioma organoids. The glioma organoids obtained by this method are identical to primary glioblastoma tissue, retain the proliferative capacity and stemness characteristics of gliomas, and exhibit an immune infiltration pattern similar to that of patient tissues.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for culturing vascularized and immunomodulated glioma organoids, characterized in that, Includes the following steps: (1) The glioblastoma tissue was cut into pieces to obtain glioblastoma tissue blocks; the glioblastoma tissue blocks were subjected to erythrocyte lysis to obtain processed tissue blocks; (2) The treated tissue blocks were cultured in suspension in glioblastoma organoid culture medium; During the suspension culture process, 70-80% of the culture medium is replaced every 46-50 hours; (3) Within 7 to 14 days of culture, round, densely cellular spherical organoids are formed. If the organoids are spherical in shape, survive for ≥2 weeks and their diameter continues to increase, then the glioma organoids are successfully established. (4) Detection and verification of glioma organoids.

2. The cultivation method according to claim 1, characterized in that, In step (1), the glioblastoma tissue needs to be washed 2 to 4 times with calcium and magnesium phosphate buffered saline before it is cut into pieces.

3. The cultivation method according to claim 1, characterized in that, In step (1), the length of the glioblastoma tissue block is 0.5~1mm.

4. The cultivation method according to claim 1, characterized in that, In step (1), necrotic, hemagglutinous, and non-tumor brain tissue needs to be removed before the red blood cells are lysed.

5. The cultivation method according to claim 1, characterized in that, In step (1), the method of red blood cell lysis is as follows: mix the glioblastoma tissue block and red blood cell lysis solution, incubate on a shaker for 8-12 minutes, discard the red blood cell lysis solution, and wash to obtain the processed tissue block.

6. The cultivation method according to claim 5, characterized in that, The speed of the shaker is 8-12 times / minute; the incubation temperature is 20-30℃.

7. The cultivation method according to claim 6, characterized in that, The washing solution is DMEM culture medium; the washing is performed 2 to 4 times.

8. The cultivation method according to claim 1, characterized in that, In step (2), the culture temperature is 35~39℃; the CO2 concentration is 4~6%; and the humidity is ≥90%.

9. The cultivation method according to claim 1, characterized in that, In step (4), the detection and verification method is as follows: prepare frozen or paraffin sections from cultured glioma organoids and use multiplex immunofluorescence and immunohistochemistry for detection.

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