Method for culturing glioma organoids by high-throughput gas-liquid interface method

Abstract

The present invention relates to the technical field of tissue culture, and specifically relates to a method for culturing glioma organoids by a high-throughput air-liquid interface method. The present invention uses a self-developed 96-well high-throughput air-liquid interface organoid culture device and a supporting system to culture and passage glioma organoids. Compared with traditional culture methods, the system of the present invention is small in size and high in throughput, and can directly achieve in-situ inoculation, medium replacement and drug sensitivity testing of organoids, greatly improving the efficiency of experiments and saving economic and labor costs. At the same time, the method of the present invention has a high cell tissue viability rate and a short cycle, retains the heterogeneity of the organoid tissue, is more conducive to predicting the drug reactivity of patients, and is very important for studying the effect of drugs on glioma.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue culture, and particularly to a method for culturing glioma organoids by a high-throughput air-liquid interface method. Background Art

[0002] Tumor organoids (Patient derived organoids, PDOs) are miniature in vitro organ models that are cultured from tumor tissues taken from patients in the laboratory, highly mimic the characteristics of the source tumor tissues, and retain the genetic characteristics and tumor heterogeneity of the primary tissues. Tumor organoids are usually used for disease model construction, mechanism research, and screening of personalized precision medications for patients.

[0003] Glioma is the most common malignant tumor in the central nervous system. Currently, for the treatment of glioma, it is advocated to mainly perform surgical resection, combined with radiotherapy, chemotherapy, electric field therapy, and immunotherapy. Organoids play an important role in personalized precision medicine for glioma patients, especially in the formulation of chemotherapy regimens. With the in-depth understanding of the tumorigenesis mechanism, more and more evidence shows that the occurrence of tumors is inseparable from immune escape. Immunotherapy is undoubtedly the focus of glioma treatment research and an important supplement to future clinical glioma treatment regimens.

[0004] The tumor microenvironment (Tumormicroenvironment, TME) is composed of tumor-associated stromal cells, immune cells, and the secreted products (such as cytokines and chemokines) of the corresponding cells and non-cellular components in the extracellular matrix (ECM). The tumor microenvironment provides a good growth environment and nutrients for tumors, promoting tumor progression and metastasis, which is also the reason for the failure of conventional treatments for many individual patients in clinical practice. Compared with the PDX animal model, the in vitro culture environment of tumor organoids is relatively simple. However, for organoid models lacking the tumor microenvironment, it is difficult to fully simulate the drug reactivity of tumors. In particular, with the culture of tumor organoids, immune cells will gradually decline in function, decrease in number, and even gradually disappear.

[0005] Currently, the culturing method for glioma organoids is the mechanical fragmentation method, using a spring scissors to cut the tumor tissue into 1 mm 3Fragments were added to the culture medium and cultured with shaking on a horizontal shaker. The research results proved that a large number of tumor immune cells were lost during the culture of the organoids, and the tumor immune microenvironment was greatly damaged. It has been reported in the literature that culturing tissue-derived organoids using the air-liquid interface (ALI) method can, to a certain extent, preserve the immune microenvironment in situ. Culturing glioma organoids using the ALI method does not require a shaker, and the tissue processing method is simple. However, the amplification efficiency of glioma organoids is low, the culture time is long, and it is difficult to achieve high-throughput drug sensitivity testing. Therefore, a method that can improve the amplification efficiency of glioma organoids, reduce the culture duration, and can complete high-throughput drug sensitivity testing needs to be developed. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method for culturing glioma organoids by high-throughput air-liquid interface method.

[0007] The present invention provides a method for culturing glioma organoids, characterized in that it is to culture and passage glioma organoids in a culture container adhered with a matrix gel culture medium in the presence of a culture medium;

[0008] The culture container includes a container body and an experimental tank located in the body; the experimental tank includes a groove and a convex structure; the side of the groove is the groove side wall, the convex structure is connected to the bottom surface of the groove, the convex structure is located in the groove, and the convex structure has a horizontal top surface;

[0009] Further, the groove is filled with a culture medium; the horizontal top surface of the convex structure is adhered with a matrix gel culture medium.

[0010] Even further,

[0011] The liquid level of the culture medium in the groove contacts the matrix gel culture medium but does not submerge the matrix gel culture medium; there is a gap between the convex structure and the groove side wall of the groove, and the height of the convex structure is less than the height of the groove side wall;

[0012] Optionally, the groove is a cylindrical groove;

[0013] Optionally, the convex structure is a cylindrical convex, and further, the diameter of the convex is 3 - 3.5 mm.

[0014] Optionally, the culture container body has a plurality of the experimental tanks;

[0015] Optionally, the plurality of experimental tanks are arranged in an array on the container body.

[0016] In the present invention, in the described culture method, the Matrigel culture medium comprises a substrate and primary glioma tissue;

[0017] The substrate comprises a culture solution and Matrigel, and the volume ratio of the culture solution to Matrigel is 1:1; the substrate adheres to the horizontal top surface of the boss;

[0018] The primary glioma tissue adheres to the substrate.

[0019] Furthermore,

[0020] The step of the substrate adhering to the horizontal top surface of the boss is: mixing the culture solution and Matrigel and placing them on the horizontal top surface of the boss structure to solidify, and the solidification conditions are 30°C to 37°C for 5 minutes.

[0021] In the present invention, the solidification time condition of the Matrigel is about 30 minutes to 5 minutes. This condition can achieve semi-solidification of the substrate, enabling a part of the primary glioma tissue to be embedded in the semi-solidified gel, playing a role in fixation.

[0022] In the Matrigel culture medium of the present invention,

[0023] The Matrigel of the substrate comprises 60 wt% laminin, 30 wt% type IV collagen, 8 wt% nestin, and 1.8 wt% to 2 wt% heparan sulfate proteoglycan.

[0024] The present invention provides a drug sensitivity detection method, which includes: culturing glioma organoids by the culture method described in the present invention. Among them, after adding a culture solution to the groove of the culture container and culturing for 4 to 7 days, replacing the culture solution in the groove with a culture solution containing the drug to be tested, and obtaining a drug sensitivity detection result according to the growth state of the glioma organoids.

[0025] Furthermore, the culture solution includes but is not limited to any one of Advanced-DMEM cell culture solution, MEM cell culture solution, DMEM cell culture solution, RPMI1640 cell culture solution, or F-12 cell culture solution; the drug to be tested includes but is not limited to at least one of cytotoxic drugs, targeted drugs, immunological agents, and / or traditional Chinese medicine preparations; the cytotoxic drugs include but are not limited to cisplatin, carboplatin, paclitaxel, fluorouracil, and / or mitomycin; the targeted drugs include but are not limited to icotinib, erlotinib, gefitinib, anlotinib, bevacizumab, cetuximab, trastuzumab, and / or rituximab, etc.; the immunological agents include pembrolizumab and / or nivolumab; the traditional Chinese medicine preparations include but are not limited to Brucea javanica oil emulsion and / or cinobufacini.

[0026] Furthermore, the judgment criteria for the drug sensitivity test can be directly determined based on the size change of the organoids. The drug sensitivity results can be determined by comparing the size and growth of the organoids before and after drug administration. The judgment criteria for the drug sensitivity test can also be determined based on the morphology of the organoids under the microscope. If the organoids shrink and cell disintegration appears at the edge under the microscope, it indicates that the drug is effective. In addition, the judgment criteria for the drug sensitivity test can also be determined by using a kit to detect cell viability and calculating the cell death rate after the experiment. It can also be determined by tissue embedding section staining to detect apoptosis after the experiment.

[0027] The present invention uses a self-developed 96-well high-throughput air-liquid interface organoid culture device and a supporting system for the culture and passage of glioma organoids. Compared with the traditional culture method, the system of the present invention is small in size and high in throughput, and can directly achieve in-situ inoculation, medium replacement, and drug sensitivity testing of organoids, greatly improving the experimental efficiency and saving economic and labor costs. At the same time, the method of the present invention has a high cell tissue viability rate and a short cycle, retains the heterogeneity of the organoid tissue, is more conducive to predicting the drug reactivity of patients, and is very important for studying the effect of antibody drugs on glioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Showing the front-sectional structure diagram of the 96-well sterile air-liquid interface organoid culture device;

[0029] Figure 2 Showing the Matrigel-coated surface at the top of the culture column;

[0030] Figure 3 Showing the inoculation and culture of organoids;

[0031] Figure 4 Showing the histological features and immune cells of glioma organoids, where A, B: glioma organoids; C: GFAP is glial fibrillary acidic protein, mainly distributed in astrocytes of the central nervous system and is a major marker of glioma; D, E: CD3 and CD11b are major immune cell markers in glioma tumors;

[0032] Figure 5 Showing the high-throughput in-situ drug sensitivity test of glioma organoids;

[0033] Figure 6 Showing glioma organoids cultured at the air-liquid interface. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] The present invention provides a method for culturing glioma organoids by the high-throughput air-liquid interface method. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0035] Currently, the multi-well containers applied to organoid culture do not support the air-liquid interface method. If one wants to use the air-liquid interface method, it needs to be cultured in a petri dish, and the process is relatively cumbersome. Compared with multi-well containers, the efficiency and output of organoid culture in a petri dish are insufficient, and the output is also the throughput. A petri dish usually requires more culture medium than a multi-well container, resulting in higher costs. It is difficult to adapt to the actual application situation by culturing in a petri dish. When performing drug sensitivity tests on organoids using the air-liquid interface method, the existing multi-well containers for organoid culture do not support the air-liquid interface method, and the efficiency and output are insufficient when using a petri dish for culture.

[0036] In view of this, the present invention uses a self-developed culture container to culture organoids. Specifically, the culture container can culture organoids by the air-liquid interface method. The culture container has an experimental well, and there is a boss structure in the experimental well. A covering layer for tissue culture is provided on the horizontal top surface of the boss structure. When the situation of performing drug sensitivity tests on organoids using the air-liquid interface method occurs, the organoids can be lifted and suspended for culture through the boss structure, and a certain amount of culture medium is injected to perform drug sensitivity tests on the organoids by the air-liquid interface method. The culture container can have multiple experimental wells and can simultaneously perform sensitivity tests on organoids with multiple drugs, improving the efficiency and output of organoid culture compared with using existing containers.

[0037] In order to enable those in the technical field to better understand this culture method, the technical solutions will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0038] Figure 1 It is a schematic diagram of the culture container.

[0039] The schematic diagram of the container includes the container body 100 and the experimental well 101 shown by a solid line in the body. Figure 1The schematic diagram of the container shown includes numbers and letters located on the container body, and the numbers and letters are used to label the experimental wells.

[0040] Among them, the experimental well includes a groove 201 and a boss structure 202. The boss structure is located inside the groove, and there is a gap between the boss structure and the side wall of the groove. Here, the groove is a cylindrical groove, and the boss structure is a cylindrical boss.

[0041] Among them, the boss structure 301 is connected to the bottom 303 of the groove. The boss structure 301 has a horizontal top surface. There is a certain gap between the boss structure 301 and the side wall 302 of the groove. The boss structure has a horizontal top surface, and the height of the boss structure is less than the height of the side wall of the groove.

[0042] In Figure 1 In the described culture container, there are multiple experimental wells. Here, a container with 96 experimental wells is used as an example. Of course, it can also have other numbers of experimental wells. The center spacing between two experimental wells of a common 96-well cell culture plate is 9 mm. Considering the distribution of experimental wells on a common 96-well cell culture plate, the experimental wells in the culture container can be distributed in the same way as those on a common 96-well cell culture plate. Of course, it can also be distributed in the same way as those in other common multi-well cell culture plates, or the experimental wells can be arranged according to experimental requirements. The size of the experimental wells can also be designed according to a common 96-well cell culture plate, or can be designed separately.

[0043] The culture container can be compatible with conventional equipment such as enzyme-linked immunosorbent assay (ELISA) readers, live cell workstations, and laser confocal microscopes through such an arrangement method. Of course, it can also support other standard equipment, and of course, it can also achieve the same functions as a common 96-well cell culture plate. The ELISA reader, that is, the enzyme-linked immunosorbent assay detector, is a special instrument for enzyme-linked immunosorbent assay, also known as a microplate detector. The live cell workstation is an instrument used for live cell imaging, cell migration assay, optimized cell analysis, cell culture quality control, and cell proliferation analysis. The laser confocal microscope is an instrument that uses a computer for image processing to obtain fluorescence images of the fine internal structure of cells or tissues.

[0044] The culture container is particularly suitable for organoids with a volume greater than 500 μm and simultaneously requiring culturing using the air-liquid interface method, specifically including but not limited to gliomas, brain organoids, etc.

[0045] The cylindrical groove and the cylindrical boss structure are selected as examples for the experimental wells in the culture container. Of course, the groove can also be a groove of other shapes, and the boss structure can also be a boss of other shapes.

[0046] Among them, in order to meet the experimental requirements, the groove can be filled with a culture medium, which can be the tumor microenvironment required for tumor organoids. The tumor microenvironment is composed of tumor cells, tumor-associated stromal cells, immune cells, and the secreted products of the corresponding cells and non-cellular components in the extracellular matrix. The tumor microenvironment provides a good growth environment and nutrients for tumors, promoting tumor progression and metastasis.

[0047] In the cylindrical groove and cylindrical boss structure of the experimental tank in the culture container, the diameter of the horizontal top surface of the boss structure can be less than one-half of the diameter of the groove. In actual application, the groove is filled with a culture medium, and the boss structure occupies a part of the space of the groove. Through the design that the diameter of the horizontal top surface of the boss structure is less than one-half of the diameter of the groove, the groove has sufficient space to place the culture medium. Of course, the diameter of the horizontal top surface of the boss structure can also be not less than one-half of the diameter of the groove.

[0048] The horizontal top surface of the boss structure can have a coating layer that facilitates the attachment of Matrigel. Generally, tumor organoid culture requires placing tumor organoids on a Matrigel substrate. The coating layer on the horizontal top surface of the boss structure can provide space for the Matrigel substrate for tumor organoid culture. During the actual experiment, considering that tumor organoid culture needs to be carried out on the horizontal top surface of the boss structure, the horizontal top surface of the boss structure is made of Tissue treated material, and the horizontal top surface made of Tissue treated material is the coating layer, which facilitates the attachment of the Matrigel substrate. Of course, it can also be a coating layer generated by other materials, and there can also be no coating layer.

[0049] The Matrigel substrate is generally dropped at the position where tumor organoid culture needs to be carried out. In some existing devices, the diameter of the plane for organoid inoculation inside is 30 mm. If organoid drug sensitivity testing is carried out, only 1 plate can be used at a time, and different drugs cannot be compared and screened. In some experimental scenarios of the present invention, it is best that the width of the horizontal top surface of the boss structure of the culture container is not less than the diameter of the smallest droplet of the Matrigel substrate. Preferably, the width is between 3 and 3.5 mm, which can significantly improve the detection throughput and can achieve the simultaneous determination of two patients, 5 drugs, 3 parallel samples, and 3 drug concentrations. At the same time, the design with a diameter of 3 - 3.5 mm can prevent the Matrigel substrate from dripping into the groove, enabling the Matrigel substrate to better adhere to the horizontal top surface and having stronger usability. Considering that the Matrigel substrate generally adheres to the horizontal plane with one side and has an ellipsoidal surface on the other side, considering the shape of the gel droplet, the boss structure here adopts a cylindrical boss, and the use of a cylindrical boss can better adapt to the experimental scenario.

[0050] For another aspect of organoid culture, the diameter of the horizontal top surface of the boss is required to be between 3 and 3.5 mm. Firstly, because the size of glioma organoids after primary isolation is about 1 to 1.5 mm, and the volume will increase to about 2 mm after 20 days of culture. Sufficient space needs to be left for the growth of organoids to ensure that the tissue does not fall off during the culture process. In addition, space for adding organoid culture medium also needs to be left. If it is too little, the frequency of changing and replenishing the liquid increases. Moreover, if too little culture medium is added, it will affect tissue nourishment. Therefore, the diameter of the horizontal top surface of the culture in the present invention is obtained through reasonable consideration and experiments, and the volume of the culture medium is also adapted to the culture system, so as to be more conducive to the culture of tissue organs. At the same time, in the centralized culture of some existing devices, 50 to 200 glioma organoids can be cultured simultaneously in one well. There is intense nutritional competition among the samples, resulting in faster growth of the peripheral organoids, poorer vitality and slower growth of the central organoids, and uneven growth conditions. Using the culture container of this solution, the growth conditions of each organoid are the same, the size is uniform, and the cycle is synchronous. The state consistency of organoids is also very important for the accuracy of drug sensitivity detection.

[0051] In a scenario where tumor organoids need to be cultured by the air-liquid interface method, culture medium is injected into the groove, the matrix gel substrate is dropped on the horizontal top surface of the boss structure, and the tumor organoids are placed on the matrix gel substrate. The solidification time of the substrate is controlled so that part of the tissue is embedded in the substrate for fixation. Considering the requirements of the air-liquid interface method, the tumor organoids to be cultured need to be in contact with both the culture medium and air at the same time. Here, the height of the boss structure needs to be less than the height of the groove side wall, and such a design meets the needs of the air-liquid interface method.

[0052] In a scenario where drug sensitivity testing of tumor organoids needs to be carried out using the air-liquid interface method, the drug sensitivity of tumor organoids to one drug can be tested in one experimental well. Since the culture container adopts a multi-experimental well design, the drug sensitivity of tumor organoids to multiple drugs can be tested simultaneously. According to the actual test requirements, the types of drugs to be tested only need to be less than the number of experimental wells of the container. Compared with using a culture dish for drug sensitivity testing, drug sensitivity testing using the culture container improves the test efficiency and output.

[0053] The culture container has multiple experimental wells, and one organoid is cultured in each experimental well. When drug sensitivity testing needs to be carried out on the cultured organoids, the drug sensitivity testing can be directly carried out in the container provided in this application. In contrast, the organoids cultured through existing containers need to be transferred from the culture container to the test container, saving a large amount of time compared with the existing process.

[0054] Compared with organoids cultured in existing containers, organoids cultured in the culture container of the present invention can retain more immune cells in the organoids, and well maintain the heterogeneity of the organoid tissue. Maintaining the heterogeneity of the organoid tissue is very important for studying the effects of antibody drugs on gliomas. Drug screening for tumor antibody drugs in organoids cultured in the culture container of the present invention is compared with drug screening for tumor antibody drugs in organoids cultured in existing containers, and the results obtained are more accurate.

[0055] The organoids cultured in the culture container of the present invention culture the primary tissues on the top surfaces of different boss levels when the organoids are inoculated, reducing the step of independently peeling off the concentrated cultured organoids before the drug sensitivity test. The existing centralized culture method undergoes the matrix gel dissolution and organoid peeling process, and the viability of the organoids drops to 45%, affecting the accuracy of drug sensitivity; while the organoids cultured in the culture container of the present invention ensure that the viability of the organoids reaches more than 75% before the drug sensitivity test. The use of independent culture and high-throughput drug sensitivity test methods can shorten the time from tissue separation to obtaining drug sensitivity test results to 10 days (the original method requires 20 days). At the same time, the drug sensitivity test does not require a transfer plate, and the organoid culture and drug sensitivity test are achieved at one time, further simplifying the operating steps of the drug sensitivity test.

[0056] In the scenario where tumor organoids are cultured by the gas-liquid interface method, tumor organoids, culture fluid with a tumor microenvironment, and matrix gel are prepared, the culture fluid is placed in a culture fluid injection gun, and the substrate containing matrix gel is placed in a matrix gel gun. The substrate is dripped onto the boss structure of the culture container of the present invention using a matrix gel gun, and a certain treatment is performed to allow the matrix gel to adhere to the horizontal top surface of the boss structure. With the assistance of an observation instrument such as a microscope, the tumor organoid is placed on the substrate containing matrix gel. A certain amount of culture fluid is injected into the groove using a culture fluid injection gun so that the liquid surface of the culture fluid contacts the matrix gel substrate but does not submerge the matrix gel substrate. The tumor organoids are cultured, tested, or observed.

[0057] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0058] The present invention will be further described below in conjunction with embodiments:

[0059] Example 1 High-throughput gas-liquid interface method for constructing glioma organoids

[0060] (1) Preparation of reagents and consumables

[0061] Sterile surgical instruments, 15ml centrifuge tubes, 10cm sterile cell culture dishes, and 96-well sterile air-liquid interface organoid culture devices.

[0062] Primary tissue preservation solution (containing penicillin / streptomycin / amphotericin B), DPBS, red blood cell lysis buffer, DMEM / F12 culture medium, Matrigel, glioma organoid culture medium (Cellada - GMO - 001). Take out Matrigel from -20°C one day in advance, place it at 4°C to melt, and take it out and place it on ice before use.

[0063] Other consumables that directly contact Matrigel are pre - placed at -20°C for freezing.

[0064] (2) Isolation of fresh glioma tumor tissue

[0065] 1) Specimen collection

[0066] As soon as the specimen is removed from the body, collect the specimen as quickly as possible. Using sterile instruments to ensure a sterile environment, put the tumor tissue into a 15 - ml centrifuge tube containing 5 ml of primary tissue preservation solution (containing triple antibiotics), and transfer it at 4°C.

[0067] 2) Washing

[0068] Take out the sample tube in the biosafety cabinet, wipe the outside of the tube with 75% alcohol. After opening the cap, carefully aspirate the tissue preservation solution, add cold DPBS++ containing triple antibiotics, wash repeatedly, and then remove the DPBS++. Repeat the washing 2 to 3 times.

[0069] 3) Tissue fragmentation

[0070] Transfer the washed tissue pieces to a 10 - cm sterile cell culture dish containing 2 ml of cold DPBS++. Under the observation of a stereomicroscope, use a spring scissors to fragment the tumor tissue into tissue fragments with a diameter of about 0.5 mm - 1 mm.

[0071] 4) Lysis of red blood cells

[0072] Transfer the fragmented tissue to a 15 - ml centrifuge tube containing 5 ml of cold DPBS++ and wash 2 times. Add 10 ml of red blood cell lysis buffer, shake and lyse at 60 rpm on a shaker at room temperature for 10 minutes, discard the supernatant, and wash the tissue mass 2 times with normal - temperature DMEM / F12 culture medium.

[0073] (3) Coating with bottom - layer Matrigel

[0074] Dilute and mix Matrigel with glioma organoid culture medium in a volume ratio of 1:1 (Matrigel A, also known as the base), and store it on ice.

[0075] In Figure 1On the upper surface of the culture columns in the 96-well plate shown, add 50 μL of Matrigel. Add it according to the experimental grouping and the expected number of plates to be seeded. Cover the culture plate with Matrigel added, place it in a 37 °C cell incubator, and solidify for 5 minutes. Take it out after the Matrigel is semi-solidified to form a Matrigel A-coated surface as shown in Figure 2 .

[0076] (4) Organoid seeding

[0077] Using a 1 ml pipette tip, gently transfer the glioma primary tissues that are basically the same size after being broken, lysed for red blood cells, and washed one by one to the upper surface of the not yet fully solidified Matrigel A in a biosafety cabinet, as shown in Figure 3 . 1 - 3 glioma organoids can be seeded on each culture column. The transfer process should be as fast as possible and carried out in a low-temperature environment. Place the transferred culture plate in a cell incubator and solidify for 30 minutes. Take out the culture plate, and along the edge of the well, add 100 μL of pre-warmed glioma organoid culture medium to each cell culture well until the top of the culture medium reaches the upper surface of Matrigel A.

[0078] (5) Organoid culture

[0079] Place the culture plate with the added culture medium in a 37 °C cell incubator for static culture. According to the evaporation rate of the culture medium, change the culture medium every 4 - 7 days. When changing the culture medium, pay attention not to directly drop the liquid on the surface of the organoids to prevent flushing the organoids off the culture surface. Observe the growth status of the organoids every day, take pictures to record the proliferation rate, morphological changes, and contamination conditions. The spectral optical photography is as shown in Figure 6 . The immune cells and histological features are as shown in Figure 4 . The formed organoids well retain the immune cells and histological features in the tumor tissue. Maintaining the heterogeneity of the organoid tissue is very important for studying the effect of antibody drugs on glioma and can well predict the drug reactivity of patients.

[0080] (6) In-situ drug sensitivity test of organoids

[0081] For the organoids that need to be tested for drug sensitivity, directly group the well plates (drug types and concentrations), replace and add the culture medium with the corresponding drugs to different culture wells for high-throughput drug testing at the organoid level ( Figure 5 ). There is no need to transfer the organoids a second time, reducing the damage to the organoids.

[0082] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Culture method of glioma organoids, Characterized in that, it is to culture and passage glioma organoids in a culture container adhered with a Matrigel culture medium under the condition of the presence of a culture solution; the culture container includes a container main body and an experimental tank located in the main body; the experimental tank includes a groove and a boss structure; the side of the groove is the groove side wall, the boss structure is connected to the bottom surface of the groove, the boss structure is located in the groove, the boss structure has a horizontal top surface, there is a gap between the boss structure and the groove side wall of the groove, and the height of the boss structure is less than the height of the groove side wall; the groove is filled with a culture solution, and the liquid level of the culture solution in the groove contacts the Matrigel culture medium but does not submerge the Matrigel culture medium; the horizontal top surface of the boss structure is adhered with a Matrigel culture medium; the Matrigel culture medium includes a substrate and glioma primary tissue; the substrate includes a culture solution and Matrigel, the substrate is adhered to the horizontal top surface of the boss; the glioma primary tissue is adhered to the substrate.

2. The culture method according to claim 1, Characterized in that, the groove is a cylindrical groove; the boss structure is a cylindrical boss.

3. The culture method according to claim 2, Characterized in that, the diameter of the boss is 3 - 3.5 mm.

4. The culture method according to claim 1, Characterized in that, the container main body has a plurality of the experimental tanks; the plurality of experimental tanks are arranged in an array on the container main body.

5. The culture method according to any one of claims 1 - 4, Characterized in that, the volume ratio of the culture solution to Matrigel is 1:1; the culture solution includes DMEM cell culture solution or RPMI1640 cell culture solution.

6. The culture method according to claim 5, Characterized in that, the step of adhering the substrate to the horizontal top surface of the boss is: mixing the culture solution and Matrigel and placing them on the horizontal top surface of the boss structure to solidify, and the solidification conditions are 30°C - 37°C, 5 min.

7. The culture method according to claim 6, Characterized in that, the Matrigel of the substrate includes 60 wt% laminin, 30 wt% type IV collagen, 8 wt% nestin, and 1.8 wt% - 2 wt% heparan sulfate proteoglycan.

8. Drug sensitivity detection method, Characterized in that, including: culturing glioma organoids by the culture method according to any one of claims 1 - 7, wherein, after culturing in the culture solution added to the groove of the culture container for 4 - 7 days, replacing the culture solution in the groove with a culture solution containing the drug to be tested, and obtaining the drug sensitivity detection result according to the growth state of the glioma organoids.

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