Method for constructing brain glioma organoid with reserved immune microenvironment

Abstract

The invention provides a method for constructing a brain glioma organ retaining an immune microenvironment, and the culture medium, the culture system and the method provided by the invention overcome the shortages that a large number of tumor immune cells are lost and the tumor immune microenvironment is greatly damaged along with the prolonging of culture time of a current glioma organ model. The tumor microenvironment of the glioblastoma can be more completely reserved for a longer time, the heterogeneity of organoid tissues is greatly reserved, and the model is suitable for being used as a new generation of glioblastoma organoid models; a more reliable research tool is provided for the tumor microenvironment research, the tumor generation mechanism research, the drug effect screening, the development and application of a disease model and the like of the glioblastoma.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro culture and detection of organoids, and in particular to a method for constructing brain glioma organoids that retain an immune microenvironment. Background Art

[0002] Glioblastoma (GBM) is the most common malignant primary intracranial tumor, accounting for 57% of all gliomas and 48% of primary malignant tumors of the central nervous system. The current multimodal comprehensive treatment for GBM includes surgery, radiotherapy, and systemic therapy (chemotherapy, targeted therapy), which has made great progress in recent years, but the overall survival of GBM is short and long-term survival is rare. Although the treatment of GBM is constantly improving, the current 5-year survival rate is only 5.8%, and advanced treatment strategies are still needed, and new treatment directions still need to be developed, such as immunotherapy and precision targeted therapy. This requires a deeper understanding of the molecular information of GBM and its interaction with the immune system.

[0003] In order to better study the pathogenic mechanism and treatment methods of glioma, scientists have constructed glioma organoids. The interaction between tumor cells and immune cells and stromal cells is crucial to restore the real situation in the body. The glioblastoma organoids established by existing methods cannot retain the original composition of the parental tissue and the tumor microenvironment of the tissue, which limits the application scenarios. There are currently no reports on the construction of methods that can retain the glioblastoma microenvironment. So far, some glioblastoma organoid models have been established. Some researchers have used patient tumor tissue fragments and constructed patient-derived glioblastoma organoids using suspension culture. The results show that with the increase of culture time, tumor immune cells will be lost in large quantities, and the tumor immune microenvironment is greatly damaged. Some researchers use human induced pluripotent stem cells and use CRISPR-Cas9 technology to knock out genes such as PTEN and TP53, thereby generating glioblastoma-like organoids. This organoid does not contain non-tumor components such as immune cells, and cannot retain the glioblastoma immune microenvironment.

[0004] In view of this, the present invention aims to provide a method for constructing glioblastoma organoids that can better retain immune cells and maintain the immune microenvironment, without introducing any exogenous cells and without genetic modification, thereby achieving a longer-term retention of immune cells, especially macrophages in the original tissue. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a method for constructing glioblastoma organoids that retain the immune microenvironment.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: A first aspect of the present invention provides a culture medium for constructing glioblastoma organoids that retain an immune microenvironment, the culture medium comprising a basal culture medium and additives.

[0007] The basic culture medium includes at least one of DMEM / F-12 and Neurobasal.

[0008] The additives include at least one of B-27 additives, N2 additives, non-essential amino acids, L-glutamine, antibiotics, insulin, and B-mercaptoethanol.

[0009] Furthermore, the ratio of DMEM / F-12 to Neurobasal in the basal culture medium is 1:1.

[0010] Furthermore, the non-essential amino acids include at least one of L-alanine, L-glutamic acid, L-aspartic acid, L-asparagine, L-proline, L-serine and glycine.

[0011] Furthermore, the non-essential amino acids are MEM non-essential amino acid solution.

[0012] Furthermore, the L-glutamine may also be a GlutaMAX additive.

[0013] Furthermore, the antibiotic is selected from at least one of penicillin, streptomycin and amphotericin B.

[0014] Furthermore, the antibiotic is penicillin-streptomycin.

[0015] Furthermore, the concentration of the penicillin-streptomycin is 10,000 U / mL.

[0016] Furthermore, the additives include B-27 additive, N2 additive, MEM non-essential amino acid solution, GlutaMAX additive, antibiotics, insulin, and B-mercaptoethanol.

[0017] Furthermore, the additives include B-27 additive 50×, N2 additive 100×, MEM non-essential amino acid solution 100×, GlutaMAX additive 100×, penicillin-streptomycin 100×, insulin 2.5 μg / mL, and B-mercaptoethanol 1000×.

[0018] The second aspect of the present invention provides a matrix gel-culture medium mixture, wherein the matrix gel-culture medium mixture comprises matrix gel and the culture medium described in the first aspect of the present invention.

[0019] Furthermore, the ratio of the matrix gel to the culture medium described in the first aspect of the present invention is 1:1.

[0020] A third aspect of the present invention provides a culture system for constructing glioblastoma organoids that retain an immune microenvironment, wherein the culture system comprises a first culture system and a second culture system.

[0021] The first culture system comprises a chamber, the matrix gel-culture medium mixture described in the second aspect of the present invention, and an organoid completely coated in the matrix gel-culture medium mixture.

[0022] The second culture system comprises the culture medium and the outer culture dish described in the first aspect of the present invention.

[0023] The first culture system and the second culture system are connected via a recombinant basement membrane.

[0024] Furthermore, the complete coating refers to the amount of the matrix gel-culture medium mixture used so that 100% of the volume of the organoid coated with the matrix gel-culture medium mixture is immersed therein.

[0025] Furthermore, the organoids completely coated in the matrix gel-culture medium mixture are derived from the glioblastoma tissue to be cultured.

[0026] Furthermore, the number of the organoids completely coated in the matrix gel-culture medium mixture is 20 to 30.

[0027] Furthermore, the diameter of the chamber is 1.5 cm.

[0028] Furthermore, the diameter of the outer culture dish is 3 to 5 times that of the chamber.

[0029] Furthermore, the diameter of the outer culture dish is 4 times that of the chamber.

[0030] The fourth aspect of the present invention provides a kit for constructing a glioblastoma organoid that retains an immune microenvironment, the kit comprising the culture medium described in the first aspect of the present invention, the matrix gel-culture medium mixture described in the second aspect of the present invention and / or the culture system described in the third aspect of the present invention.

[0031] The fifth aspect of the present invention provides a method for constructing a glioblastoma organoid that retains an immune microenvironment, the method comprising completely coating the glioblastoma tissue to be cultured in the matrix gel-culture medium mixture described in the second aspect of the present invention and placing it in the small chamber described in the third aspect of the present invention, placing the small chamber in the outer culture dish described in the third aspect of the present invention, and adding the culture medium described in the first aspect of the present invention for culturing.

[0032] Furthermore, the glioblastoma tissue to be cultured is a pre-treated tissue.

[0033] Furthermore, the pretreatment step includes: removing vascular tissue and necrotic tissue from the glioblastoma specimen, chopping it up and washing it, removing residual red blood cells, adding the culture medium described in the first aspect of the present invention, removing the supernatant after standing, and obtaining the glioblastoma tissue to be cultured.

[0034] Furthermore, the cutting and shredding refers to cutting the glioblastoma specimen into fragments with a diameter of 0.5-1.0 mm.

[0035] Furthermore, the culturing step includes: static culturing in a cell culture incubator and regularly replacing the culture medium in the outer culture dish.

[0036] Furthermore, the interval of regular replacement is 2 to 3 days.

[0037] Furthermore, replacing the culture medium in the outer culture dish means retaining 1 / 3 of the original culture medium in the outer culture dish.

[0038] Furthermore, the conditions for static culture in the cell culture incubator are 37°C, 5% CO 2 .

[0039] The sixth aspect of the present invention provides an application of the culture medium described in the first aspect of the present invention, the matrix gel-culture medium mixture described in the second aspect of the present invention, the culture system described in the third aspect of the present invention, the kit described in the fourth aspect of the present invention, or the glioblastoma organoid constructed by the method described in the fifth aspect of the present invention, the application comprising: 1) Application in screening therapeutic drugs for glioblastoma, predicting the sensitivity of subjects to glioblastoma therapeutic drugs, or detecting the responsiveness of subjects to glioblastoma therapy.

[0040] 2) Application in the preparation of products for screening therapeutic drugs for glioblastoma, predicting the sensitivity of a subject to a glioblastoma therapeutic drug, or detecting the responsiveness of a subject to a glioblastoma therapy.

[0041] Advantages and beneficial effects of the present invention: The organoid culture method provided by the present invention overcomes the shortcomings of the current glioma organoid model, that is, as the culture time is prolonged, a large number of tumor immune cells are lost and the tumor immune microenvironment is greatly damaged. It can preserve the tumor microenvironment of glioma more completely and for a longer period of time, and greatly preserves the heterogeneity of organoid tissue. It is suitable as a new generation of glioma organoid model, and provides a more reliable research tool for tumor microenvironment research, tumor occurrence mechanism research, drug efficacy screening, development and application of disease models, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a microscope view of the organoid cultured by the method described in the embodiment of the present invention; Figure 2 This is a microscope view of the organoid cultured using the method described in CN116064395A; Figure 3 This is a comparison chart of cell activity after culturing organoids for 2 weeks using the method described in CN116064395A and the method described in the embodiment of the present invention. Figure 3 A in FIG. 1 is an activity diagram of organoid cells cultured by the method described in CN116064395A. Figure 3 B is a graph showing the activity of organoid cells cultured by the method described in the embodiment of the present invention; Figure 4 This is a comparison of immunofluorescence between the method described in CN116064395A and the method described in the embodiment of the present invention after culturing organoids for 2 weeks. Figure 4 A in FIG. 1 is an immunofluorescence image of an organoid cultured by the method described in CN116064395A. Figure 4 B is an immunofluorescence image of the organoid cultured in Example; Figure 5 This is an example diagram of the organoid culture system. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0044] In the present invention, glioblastoma (GBM) is the most common malignant tumor of the nervous system and the most common malignant primary tumor in the brain, accounting for 57% of all gliomas and 48% of primary malignant tumors of the central nervous system. The tumor is usually located under the cortex, and most of them grow in various parts of the supratentorial cerebral hemisphere, showing infiltrative growth, often invading several lobes, and invading deep structures, and can also spread to the contralateral cerebral hemisphere through the corpus callosum. The most common site of occurrence is the frontal lobe. Studies have found that the molecular mechanisms of primary glioblastoma and secondary glioblastoma are different. The molecular changes of primary glioblastoma are mainly amplification and overexpression of epidermal growth factor receptor (EGFR), while secondary glioblastoma is mainly mutation of p53. Primary glioblastoma accounts for the vast majority of GBM, nearly 90%, and is common in elderly patients, with rapid clinical progression. In a specific embodiment of the present invention, glioblastoma refers to primary glioblastoma.

[0045] In the present invention, organoids are a model based on a 3D in vitro cell culture system that is highly similar to the source tissue or organ in the body. These 3D in vitro culture systems can replicate the complex spatial morphology of differentiated tissues, and can show the interaction and spatial position morphology between cells and between cells and their surrounding matrix. And it can also have similar physiological responses to differentiated tissues and organs in the body, and has a high similarity with the source tissue. Compared with the traditional 2D cell culture model, the 3D cultured organoids contain a variety of cell types, breaking through the simple physical contact between cells, forming a closer interaction between cells and between cells and matrices, forming a functional "micro-organ", which can be better used to simulate the occurrence process and physiological and pathological state of organ tissues, and therefore has broad application prospects in basic research and clinical diagnosis and treatment. In a specific embodiment of the present invention, a method for constructing glioblastoma organoids is provided, and the glioblastoma organoids cultured by this method have a high survival rate, strong activity, and can retain the tumor immune microenvironment to a great extent, and are ideal glioblastoma research tools.

[0046] In the present invention, culture medium refers to a nutrient matrix prepared by combining different nutrients for the growth and reproduction of microorganisms, plants or animals (or tissues). Generally, it contains several major types of substances such as carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements), vitamins and water. The culture medium is not only a basic substance for providing cell nutrition and promoting cell proliferation, but also a living environment for cell growth and reproduction. Organoids contain a variety of differentiated cell types, which also exist in the corresponding organs or tissues in the body. The signal pathways that mediate organoid formation are the same as the signal pathways for the development and homeostasis of organs or tumors in the body. During the culture, factors, growth factors and small molecules need to be added to the culture medium to activate or inhibit the specific signal pathways involved in the formation of organoids. Therefore, different combinations of additives are required to prepare different organoids. Even for the small intestine and colon, which are very similar tissues, the combinations of additives required for the preparation of organoids are not the same. Organoid culture medium is mainly composed of basal culture medium, culture medium supplements, amino acids, cytokines, small molecule inhibitors, hormones, buffers, etc., and there are more than 30 different ingredients in common additives other than basal culture medium.

[0047] B-27 Supplement is a serum-free supplement optimized to support low- or high-density growth and short- or long-term survival of embryonic, postnatal, and adult hippocampal and other CNS neurons.

[0048] N2 Supplement is a serum-free supplement based on Bottenstein's N-1 formulation, designed to support the growth and expression of primary neuroblastoma cells and postmitotic neurons derived from the peripheral and central nervous systems.

[0049] Amino acids are the basic units of protein and are also important components of life. According to the ability of cells to synthesize them, amino acids can be divided into three categories: essential amino acids, semi-essential amino acids, and non-essential amino acids. Non-essential amino acids are usually added to the culture medium to reduce the side effects of the cells' own production of non-essential amino acids during cell culture and promote cell proliferation and metabolism.

[0050] L-glutamine is an essential amino acid that serves as an energy source for cultured cells and participates in protein synthesis and nucleic acid metabolism. GlutaMAX is an alternative to L-glutamine with better stability, which can significantly reduce the accumulation of toxic ammonia and improve cell viability and growth.

[0051] Insulin plays an important role in many cell activities and is a commonly used additive in mammalian cell culture media. Insulin can accelerate the utilization of glucose and inhibit the production of glucose, inhibit the decomposition of fat, promote the synthesis and storage of fat, promote protein synthesis, prevent protein decomposition, and promote potassium and magnesium ions to pass through the cell membrane into the cell; promote the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and adenosine triphosphate (ATP).

[0052] Antibiotics can prevent microbial contamination in cell culture, promote better cell growth, proliferation and differentiation, and reduce cytotoxic reactions caused by microbial metabolites.

[0053] B-mercaptoethanol, also known as β-mercaptoethanol, plays an important role in cell growth. It can reduce sulfur-containing compounds in serum to glutathione, induce cell proliferation and delay cell senescence. It is a non-specific activation effect; at the same time, it avoids damage to cells by peroxides.

[0054] In the most preferred embodiment of the present invention, a culture medium is provided for constructing glioblastoma organoids that retain an immune microenvironment, the specific components of which include DMEM / F-12 and Neurobasal basal culture medium, B27 additive 50×, N2 additive 100×, MEM non-essential amino acid solution 100×, GlutaMAX additive 100×, penicillin-streptomycin (10,000U / mL) 100×, insulin 2.5 μg / mL, and B-mercaptoethanol 1000×.

[0055] In the present invention, Matrigel refers to a soluble basement membrane preparation extracted from EHS mouse tumors rich in extracellular matrix proteins, and its main components are composed of laminin, type IV collagen, heparan sulfate proteoglycan (HSPG) and nestin, and also include various growth factors such as TGF-β, EGF, IGF, FGF, etc. Under room temperature conditions, Matrigel polymerizes to form a three-dimensional matrix with biological activity, which can simulate the structure, composition, physical properties and function of the basement membrane of cells in vivo, is conducive to the culture and differentiation of cells in vitro, and is a commonly used tool in organoid construction. The present invention has no special restrictions on the source of Matrigel, that is, it can be a self-prepared Matrigel, or it can be a commercially customized or purchased Matrigel product.

[0056] In the present invention, the basement membrane is a continuous membrane layer formed by specialization of the extracellular matrix, which can form an interface between endothelial cells, epithelial cells, muscle or neuronal cells and their adjacent matrix. The recombinant basement membrane is an artificially constructed soluble basement membrane purified from a tumor, and the extract is gelled at 37°C to form a recombinant basement membrane. The main components include laminin, type IV collagen, entactin and heparin sulfate proteoglycan.

[0057] In the present invention, screening for therapeutic drugs for glioblastoma refers to treating the glioblastoma organoids provided by the present invention with the substance to be screened. If the activity of the glioblastoma organoids is inhibited or apoptotic, the substance to be screened may be a therapeutic drug for glioblastoma; if the activity of the glioblastoma organoids is not inhibited, the substance to be screened is not a therapeutic drug for glioblastoma. The substance to be screened refers to a substance that may be used as a drug, including chemical drugs, biological drugs, and traditional Chinese medicine.

[0058] In the present invention, predicting the sensitivity of the subject to the glioblastoma therapeutic drug refers to treating the glioblastoma organoids derived from the subject with the glioblastoma therapeutic drug. If the activity of the glioblastoma organoids is inhibited or apoptotic, the subject is sensitive to the therapeutic drug; if the activity of the glioblastoma organoids is not inhibited, the subject is insensitive to the therapeutic drug.

[0059] In the present invention, detecting the responsiveness of the subject to the glioblastoma therapy means treating the glioblastoma organoids derived from the subject with the therapy. If the activity of the glioblastoma organoids is inhibited or apoptotic, the subject has a good responsiveness to the therapy; if the activity of the glioblastoma organoids is not inhibited, the subject has a poor responsiveness to the therapy. The therapy includes radiotherapy, chemotherapy, etc.

[0060] In the present invention, immunofluorescence staining, also known as fluorescent antibody technology, refers to a technology that combines antibody molecules with some fluorescent tracer substances and uses antigen-antibody reaction to locate antigen substances in tissues or cells. The method of tracing or examining the corresponding antigen with fluorescent antibodies is called fluorescent antibody method; the method of tracing or examining the corresponding antibody with known fluorescent antigen markers is called fluorescent antigen method. Fluorescent tracer substances include fluorescein isothiocyanate, tetraethyl rhodamine, tetramethyl rhodamine isothiocyanate and other fluorescent substances such as 4-methylumbelliferyl phosphate, a substrate of alkaline phosphatase, and p-hydroxyphenylacetic acid, a substrate of horseradish peroxidase.

[0061] Glioma-associated microglia / macrophages are the main immune cell types in the tumor microenvironment (TME), and they increase significantly with tumor grade, accounting for up to 30% to 50% of tumor cells. The immune microenvironment-retaining glioblastoma organoids constructed by the method provided by the present invention use two fluorescent probes, IBA1 and CD11b, to locate the microglia / macrophages therein. IBA1, also known as AIF-1, is an evolutionarily conserved calcium-binding protein. As an F-actin binding protein, IBA1 may reshape the actin cytoskeleton of microglia. IBA1 is uniquely expressed in cells of the monocytic lineage, and therefore, antibodies targeting this protein are widely used to mark microglia / macrophages in the brain and other tissues. CD11b refers to the transmembrane protein, cluster of differentiation molecules 11b, also known as integrin αM (ITGAM), which is composed of α and β heterodimers expressed in cells of the innate immune system. Antibodies that detect CD11b are often used to identify cells of the myeloid lineage, including neutrophils, monocytes, macrophages, and microglia.

[0062] To make the technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The experimental methods in the following examples are conventional methods unless otherwise specified, and the materials and reagents used in the following examples are obtained from commercial sources unless otherwise specified. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Simple improvements to the present invention made according to the essence of the present invention all belong to the scope of protection claimed in the present invention.

[0063] Example 1 Construction of brain glioma organoids that retain the immune microenvironment 1. Culture medium configuration 1. Basic culture medium: DMEM / F-12:Neurobasal = 1:1; 2. Additives: B27 additive 50×, N2 additive 100×, MEM non-essential amino acid solution 100×, GlutaMAX additive 100×, penicillin-streptomycin (10,000 U / mL) 100×, insulin 2.5 μg / mL, B-mercaptoethanol 1000×.

[0064] 2. Cultivation Methods 1. Fresh ex vivo brain glioma specimens obtained through craniotomy (intraoperative frozen pathology suggests a high possibility of glioma, and postoperative pathology confirms glioblastoma) are placed in pre-cooled glioblastoma preservation solution and transported to the laboratory in an ice bath and incubator. Informed consent has been obtained from relevant personnel for specimen acquisition.

[0065] 2. Pour the glioblastoma specimen into a large dish in a clean bench under sterile operation.

[0066] 3. Use surgical blades, high-pressure steam sterilized microscissors and microtweezers to remove the red vascular tissue of the sample and the black necrotic tissue produced by surgical electrocoagulation. Carefully cut the specimen into fragments with a diameter of about 0.5-1.0 mm. Transfer the fragments to a 15 mL centrifuge tube, add 10 mL DPBS to mix thoroughly, let it stand for 1 minute, remove the less active tissue and impurities in the supernatant, and repeat the washing 3 times.

[0067] 4. After removing the supernatant, add 5 mL of 1× RBC lysis buffer to remove the remaining red blood cells. During the removal process, place the centrifuge tube at 37°C and 5% CO 2 On a shaker in the incubator, set the shaker speed to 120 rpm / min for 10 minutes.

[0068] 5. After the cleaning is completed, let the centrifuge tube stand for about 1 minute. The chopped tissue fragments will settle at the bottom. Aspirate the supernatant, add 10 mL of DPBS to mix thoroughly, let it stand to remove the supernatant, repeat the washing twice, add 10 mL of glioblastoma culture medium, let it stand for 1 minute, and remove the supernatant.

[0069] 6. Prepare a mixed solution of matrix gel and organoid culture medium in a ratio of 1:1 (place on ice to prevent the matrix gel from solidifying). Place an appropriate amount of the above-treated glioblastoma tissue to be cultured in 1 mL of the matrix gel-organoid mixed solution, mix well, and then evenly spread it in a small chamber with a diameter of 1.5 cm.

[0070] 7. Place the chamber in a medium dish and add 3 mL of organoid culture medium to the medium dish; place the culture plate with culture medium in a 37°C cell culture incubator for static culture. Figure 5As shown. Change the culture medium in the dish every 2 to 3 days according to the color change of the culture medium. Replace 2 / 3 of the culture medium each time. Be careful not to drip the liquid into the chamber when changing the culture medium. Observe the growth status of the organoids every day, take photos to record the proliferation rate and morphological changes.

[0071] 8. Cell activity detection: Cover the matrix gel with an appropriate amount of cell recovery solution matrix gel digestion solution, place the culture plate at 2-8℃ or on ice, incubate for 30-90 minutes, and shake gently. Add PBS to wash 2-3 times, place it in a 15 ml tube and let it stand for 1-2 minutes. After the organoids have settled, add an appropriate amount of trypsin to the 15 ml tube, digest at 37℃ for 10 minutes, and after the organoids are digested into single cells, use a 70 μm sieve to remove the incompletely digested organoids, leaving the cell suspension, centrifuge at 1000 r for 5 min, remove the supernatant; add 2 ml PBS to resuspend, take 20 μl of the cell suspension, add an equal amount of AOPI dye solution and mix well, count and analyze the cell activity with a cell counter.

[0072] 9. Immunofluorescence staining: Cover the matrix gel with an appropriate amount of cell recovery solution matrix gel digestion solution, place the culture plate at 2-8℃ or on ice, incubate for 30-90 minutes, and shake gently. Add PBS to wash 2-3 times, place in a 15 ml tube and let stand for 1-2 minutes. After the organoids have settled, embed them in OCT and freeze-section them with a thickness of 4 mm. Slide the slices and store them at -80℃.

[0073] 1) Remove tissue slides from refrigerator and incubate on hot plate set to 55°C for 30 minutes to dry. Use a hydrophobic isolation pen to outline the tissue section and allow to dry for 5 minutes.

[0074] 2) Prepare the immunohistology moisturizing chamber by adding a thin layer of ultrapure water to the bottom of the open slide staining chamber. Place the tissue slide horizontally onto the mounting groove at the bottom of the immunohistology moisturizing chamber. Wash the tissue sections 3 times with TBST for 5 minutes each, gently pipetting approximately 200 μl of TBST onto each tissue section and carefully aspirating the wash buffer between washes.

[0075] 3) Incubate each tissue section with approximately 100 μl of blocking and permeabilization buffer at room temperature for 1 hour. Aspirate the blocking and permeabilization buffer and incubate each tissue section with approximately 100 μl of primary antibody diluted in antibody dilution buffer overnight at 4°C. Aspirate the primary antibody solution and quickly wash the tissue sections 3 times with TBST for 5 minutes each, carefully aspirating the wash buffer between washes. Incubate each tissue section with 100 μl of secondary antibody and DAPI diluted in antibody dilution buffer at room temperature for 1.5 hours. Aspirate the secondary antibody solution and quickly wash the tissue sections 3 times with TBST for 5 minutes each, carefully aspirating the wash buffer between washes.

[0076] 3. Cultivation Results 1. The organoids were cultured for 2 weeks according to the comparative example and the culture method provided by the present invention. The comparative example culture method was carried out with reference to the invention patent "Method for constructing brain glioma organoids" (publication number CN116064395A).

[0077] 2. Growth status comparison results are as follows Figure 1 , Figure 2 As shown, it can be seen that the organoids cultured by the method described in the embodiment of the present invention are round, and the surrounding proliferating organoid tissue tentacles are more and denser, indicating that they are in good growth state.

[0078] 3. Cell activity comparison results are as follows Figure 3 , as shown in Table 1, Figure 3 It can be seen that the organoids cultured by the method described in the embodiment of the present invention have good activity and uniform distribution. From Table 1, it can be seen that the cell viability of the organoids cultured by the method described in the embodiment of the present invention is much higher than that of the organoids cultured by the method described in the comparative example, and the average diameter, roundness and agglomeration rate are also higher than those of the comparative example.

[0079] Table 1. Cell activity comparison table

[0080] 4. Fluorescence staining comparison results Figure 4 As shown, the green fluorescence is IBA1, and the red fluorescence is CD11b. It can be seen that the organoids cultured by the method described in the comparative example are basically non-specifically stained, and the fluorescent part does not overlap with the cell nucleus, while the organoids cultured by the method described in the embodiment of the present invention have more specific staining, and the fluorescent part basically overlaps with the cell nucleus, indicating that the organoids cultured by the method described in the embodiment of the present invention contain microglia / macrophages, retain immune cells, and maintain the tumor immune microenvironment.

[0081] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A culture medium for constructing glioblastoma organoids that retain an immune microenvironment, characterized in that: The culture medium comprises a basic culture medium and additives; The basic culture medium includes at least one of DMEM / F-12 and Neurobasal; The additives include at least one of B-27 additives, N2 additives, non-essential amino acids, L-glutamine, antibiotics, insulin, and B-mercaptoethanol.

2. The culture medium according to claim 1, characterized in that The ratio of DMEM / F-12 and Neurobasal in the basic culture medium is 1:1; Preferably, the non-essential amino acids include at least one of L-alanine, L-glutamic acid, L-aspartic acid, L-asparagine, L-proline, L-serine and glycine; Preferably, the non-essential amino acids are MEM non-essential amino acid solution; Preferably, the L-glutamine may also be a GlutaMAX additive; Preferably, the antibiotic is selected from at least one of penicillin, streptomycin and amphotericin B; Preferably, the antibiotic is penicillin-streptomycin; Preferably, the concentration of penicillin-streptomycin is 10,000 U / mL; Preferably, the additives include B-27 additive, N2 additive, MEM non-essential amino acid solution, GlutaMAX additive, antibiotics, insulin, and B-mercaptoethanol; Preferably, the additives include B-27 additive 50×, N2 additive 100×, MEM non-essential amino acid solution 100×, GlutaMAX additive 100×, penicillin-streptomycin 100×, insulin 2.5 μg / mL, and B-mercaptoethanol 1000×.

3. A matrix gel-culture medium mixture, characterized in that: The matrix gel-culture medium mixture comprises matrix gel and the culture medium according to any one of claims 1 to 2; Preferably, the ratio of the matrix gel to the culture medium according to any one of claims 1 to 2 is 1:

1.

4. A culture system for constructing glioblastoma organoids that retains an immune microenvironment, characterized in that: The culture system includes a first culture system and a second culture system; The first culture system comprises a chamber, the matrix gel-medium mixture of claim 3, and an organoid completely coated in the matrix gel-medium mixture; The second culture system comprises the culture medium and the outer culture dish according to any one of claims 1 to 2; The first culture system and the second culture system are connected via a recombinant basement membrane.

5. The culture system according to claim 4, characterized in that: The complete coating means that the amount of the matrix gel-medium mixture is such that 100% of the volume of the organoid coated with the matrix gel-medium mixture is immersed therein; Preferably, the organoids completely coated in the matrix gel-culture medium mixture are derived from the glioblastoma tissue to be cultured; Preferably, the number of organoids completely coated in the matrix gel-culture medium mixture is 20 to 30; Preferably, the diameter of the chamber is 1.5 cm; Preferably, the diameter of the outer culture dish is 3 to 5 times that of the chamber; Preferably, the diameter of the outer culture dish is 4 times that of the chamber.

6. A kit for constructing glioblastoma organoids retaining an immune microenvironment, characterized in that: The kit comprises the culture medium according to any one of claims 1 to 2, the matrix gel-culture medium mixture according to claim 3, and / or the culture system according to any one of claims 4 to 5.

7. A method for constructing a glioblastoma organoid that retains an immune microenvironment, characterized in that: The method comprises completely coating the glioblastoma tissue to be cultured in the matrix gel-culture medium mixture of claim 3 and placing it in the small chamber of any one of claims 4-5, placing the small chamber in the outer culture dish of claim 4, and adding the culture medium of any one of claims 1-2 for culture.

8. The method according to claim 7, characterized in that The glioblastoma tissue to be cultured is a pre-treated tissue; Preferably, the pretreatment step comprises: removing vascular tissue and necrotic tissue from the glioblastoma specimen, chopping and washing it, removing residual red blood cells, adding the culture medium according to any one of claims 1 to 2, removing the supernatant after standing, and obtaining the glioblastoma tissue to be cultured; Preferably, the cutting and mincing refers to cutting the glioblastoma specimen into fragments with a diameter of 0.5-1.0 mm.

9. The method according to claim 7, characterized in that: The step of culturing comprises: static culturing in a cell culture incubator, and regularly replacing the culture medium in the outer culture dish; Preferably, the interval of regular replacement is 2 to 3 days; Preferably, replacing the culture medium in the outer culture dish means retaining 1 / 3 of the original culture medium in the outer culture dish; Preferably, the conditions for static culture in the cell culture incubator are 37° C., 5% CO 2 .

10. Use of the culture medium according to any one of claims 1-2, the matrix gel-culture medium mixture according to claim 3, the culture system according to any one of claims 4-5, the kit according to claim 6 or the glioblastoma organoid constructed by the method according to any one of claims 7-9, characterized in that: The applications include: 1) Application in screening therapeutic drugs for glioblastoma, predicting the sensitivity of the subject to glioblastoma therapeutic drugs, or detecting the responsiveness of the subject to glioblastoma therapy; 2) Application in the preparation of products for screening therapeutic drugs for glioblastoma, predicting the sensitivity of a subject to a glioblastoma therapeutic drug, or detecting the responsiveness of a subject to a glioblastoma therapy.

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