Culture medium and method for constructing immune organoid model and application

Abstract

The invention discloses a culture medium and a method for constructing an immune organ-like model and application, belongs to the field of biological medicines, and mainly solves the problem that in-vitro human immune organ model construction and human immunology process simulation are difficult to realize. According to the method, human peripheral blood mononuclear cells (PBMC), thymus cells, tonsil cells, spleen cells or lymph node primary cells are inoculated in a culture plate with a specific structure, and an immune environment with a physiological three-dimensional (3-dimensional) structure is formed by applying a specific culture medium and conditions, so that long-term, efficient and convenient culture of the immune organ is realized; the immune tissue function of a human body is simulated. The technology involved in the invention can be applied to the fields of deep research of accelerated immunology mechanism, drug screening and evaluation, patient selection, disease detection and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a culture medium, method and application for constructing an immune organoid model. Background Art

[0002] Immunotherapy has revolutionized diseases such as tumors and viral infections. However, a limitation of immunotherapy is that only 10% to 30% of patients respond. This phenomenon is closely related to the fact that preclinical evaluation of vaccines and immunotherapeutic drugs relies primarily on animal models. Due to genetic and environmental differences in immune responses between different species, animal models may not effectively assess the true interaction between drugs and humans, and may even lead to unpredictable toxicity or efficacy failure in clinical trials. A key reason for the failure of immunotherapy is that immune responses in the body typically occur within the highly specialized tissue microenvironment of lymphoid follicles (LFs), which are the core structures of the adaptive immune system. The synergistic action of different immune cell populations can promote the recognition of pathogens or cancer cells and precisely regulate the balance between immune activation and immune regulation, thereby shaping the characteristics of the immune response and supporting immune cell function. In recent years, research on the construction of in vitro models of immune organs has developed rapidly. However, explant cultures prepared from small pieces or slices of isolated immune organ tissue samples are not suitable for the study of antibody production due to their short half-life. In vitro lymph node organoid models constructed based on primary cells isolated from human immune organs or human peripheral blood mononuclear cells (PBMCs) have shown promise, but they only contain a few cells among T lymphocytes, B lymphocytes or dendritic cells (DCs), and cannot replicate the complexity of adaptive immunity or capture all the characteristics of adaptive immune responses. Summary of the Invention

[0003] In response to the shortcomings of the existing technology, the present invention provides a culture medium, method and application for constructing an immune organoid model. Immune organoids cultured from the patient's own tissue cells are used to detect the immune response of lymph node organoids induced by different antigens, or to conduct sensitivity tests on possible therapeutic drugs or regimens, to quickly determine the optimal treatment regimen and its optimal dosage, as well as to identify drug targets and screen patients who respond to immunotherapy.

[0004] The technical solution provided by the present invention is as follows:

[0005] The present invention provides a culture medium for constructing an immune organoid model, comprising an upper chamber culture medium and a lower chamber culture medium, wherein the upper chamber culture medium and the lower chamber culture medium both comprise a basal culture medium, wherein the basal culture medium is supplemented with the following components: HEPES, Glutamax, an antibacterial agent, an antioxidant, sodium pyruvate, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, a GSK-3 inhibitor, insulin-transferrin-selenium, B-27, and human AB serum, and the basal culture medium for the lower chamber culture is further supplemented with a B lymphocyte stimulating factor.

[0006] Furthermore, the basal culture medium is RPMI1640 culture medium; the antibacterial agent is penicillin / streptomycin dual antibiotic and gentamicin; the antioxidant is N-acetylcysteine; and the GSK-3 inhibitor is selected from any one or more of AZD2858, AZD1080, B216763, CHIR99021, CHIR98014, LY2090314, NP031112, SB216763, SB415286, and TWS119.

[0007] Furthermore, the concentrations of the components in the upper chamber culture medium and the lower chamber culture medium are as follows: HEPES: 1X, Glutamax: 1X, N-acetylcysteine: 1~1.5mM, penicillin / streptomycin dual antibody: 1X, gentamicin: 50μg / mL, sodium pyruvate: 1~1.2mM, glycine: 0.5~1.5mM, L-alanine: 0.5~1.5mM, L-asparagine: 0.5~1.5mM, L-aspartic acid: 0.5~1.5mM, L-glutamate: 0.5~1.5mM, L-proline: 0.5~1.5mM, GSK-3 inhibitor: 6~10nM, insulin-transferrin-selenium: 1X, B-27: 1X, human AB serum concentration is 10%, B lymphocyte stimulating factor: 0.5~1μg / mL; the aforementioned percentage concentrations are volume concentrations.

[0008] The present invention also provides a use of the above-mentioned culture medium in constructing an immune organoid model.

[0009] The present invention also provides a method for constructing an immune organoid model based on the above-mentioned culture medium, characterized in that it comprises the following steps: S1. Isolate PBMCs from blood or directly resuscitate frozen PBMCs, and obtain a primary PBMC cell pellet after centrifugation; or mechanically process the obtained thymus, tonsil, spleen, or lymph node tissue, and obtain a primary thymus, tonsil, spleen, or lymph node cell pellet after centrifugation; S2. Resuspend the initial cell pellet with upper chamber culture medium, adjust the cell density after cell counting, centrifuge, discard the supernatant, and resuspend the cell pellet (after counting and adjusting the cell density) with upper chamber culture medium containing type I collagen to obtain a gel suspension; S3, placing a Transwell chamber on a culture plate, and coating the polycarbonate membrane of the Transwell chamber with type I collagen to obtain a Matrigel layer; S4, inoculating the gel suspension onto the matrigel layer to form a cell gel layer after solidification; S5. Add the culture medium of the upper chamber containing the antigen into the Transwell chamber, and add the culture medium of the lower chamber containing the antigen into the culture plate below the Transwell chamber. Culture for 6 to 7 days to obtain immune organoids.

[0010] Furthermore, the coating method includes: Dilute type I collagen to 100-150 μg / mL, take 100 μL and add it to the Transwell chamber. Incubate in a 37°C, 5% CO2 incubator for 1-1.5 hours to form a matrix gel layer.

[0011] Furthermore, each area is 0.33 cm 2 The cell density in the Transwell chamber was 1.75 × 10 7 The matrix gel layer was washed twice with PBS before inoculation.

[0012] Furthermore, when resuspended with upper chamber culture medium containing type I collagen, the volume ratio of the upper chamber culture medium to type I collagen was 1:1.

[0013] The present invention also provides an immune organoid model, which is constructed using the above-described method.

[0014] The present invention also provides the use of the immune organoid model and tumor organoid model in constructing a co-culture model.

[0015] Beneficial effects

[0016] The Transwell cell culture plate used in the present invention can be used for various cell cultures and for research on co-culture, cell chemotaxis, cell migration, cell invasion, and other aspects. The Transwell is placed in a culture plate, with the inner chamber being called the upper chamber and the inner chamber being called the lower chamber. The upper chamber contains the upper chamber culture medium, while the lower chamber contains the lower chamber culture medium. The upper and lower chamber culture mediums are separated by a permeable polycarbonate membrane. Cells are seeded in the upper chamber, and the components of the lower chamber culture medium can interact with the cells in the upper chamber, thereby enabling the study of the effects of the components of the lower chamber culture medium on cell growth, motility, etc., as well as the chemotaxis and migration functions of the cells in the upper chamber.

[0017] The immune organoid model constructed by the present invention has the following advantages: (1) High simulation: the lymph node organoids and their 3D microenvironment are closer to natural lymph nodes, which can reproduce the key aspects of innate immunity and adaptive immunity, while reducing dependence on animal experiments; (2) High throughput: based on Transwell, immune organoids can be quickly and successfully constructed with high throughput and low cost, and effective drug combinations and their optimal doses can be quickly and large-scale screened; (3) Personalization: by simulating the patient's own tissue cells, the patient's sensitivity to therapeutic drugs or regimens can be more accurately assessed; (4) Wide application: It has high application value in the fields of antiviral infection treatment, immune system-related disease treatment, tumor treatment, vaccine and adjuvant research and development, and drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the immune organoid model constructed in this invention. A and B represent the isolation of primary immune cells from thymus, spleen, lymph node, and tonsil tissues, respectively, and monocytes from peripheral blood. The gray device represents the Transwell chamber structure. The right-hand extension shows the upper chamber containing culture medium and cells in a 3D Matrigel-based environment, while the lower chamber contains lower chamber culture medium components without cells. The rightmost extension shows the structure of the constructed immune organoid. The bottom section shows the advantages of this model.

[0019] Figure 2 This is a bright field photo of the immune organoids cultured using the method of the present invention.

[0020] Figure 3 The growth activity of immune organoids from different sample sources cultured using the method of the present invention at different time points.

[0021] Figure 4 In the germinal centers (GCs) of the immune organoids cultured from different sample sources during the adaptive immune response, B cells are concentrated in the GC area, while T cells are mainly distributed in the peripheral area.

[0022] Figure 5 The expression patterns of CD38 and CD27 on B cells cultured by RSV mRNA-LNP vaccine showed obvious evolution, with early differentiation into Pre-GC phenotype (CD27 - CD38 + ), and subsequently develop into a GC phenotype (CD27 + CD38 +) population advantage, and then progress to the memory B cell phenotype (CD27 + CD38 - ) and plasmablast differentiation (CD27 + CD38 ++ ).

[0023] Figure 6 This study examined the antibody secretion stimulated by the RSV mRNA-LNP vaccine in immune organoids derived from different sample sources cultured using the present method. This study evaluated whether organoids cultured using the present technology resulted in detectable antibody production and effective immune responses. The bar graph shows quantitative data from fluorescent staining of IgG antibodies at day 14 of culture.

[0024] Figure 7 Figure 3. RSV mRNA-LNP vaccine-stimulated T cell activation in immune organoids cultured using the present method. Activated T cells secrete cytokines. The left graph shows the level of interleukin-6 in the supernatant at day 14 of culture; the right graph shows the level of IFN-γ in the supernatant at day 14 of culture.

[0025] Figure 8 The activation of NLRP3 inflammasome in immune organoids cultured using the method of the present invention was stimulated by LPS / ATP.

[0026] Figure 9 Schematic diagram of the co-culture model of immune organoids and tumor organoids.

[0027] Figure 10 In order to co-culture the immune organoids cultured using the method of the present invention with lung cancer organoids, the lung cancer organoid gel was inoculated in the lower chamber. On the 14th day, the tumor killing rate of the immune organoid and lung cancer organoid co-culture group was significantly higher than that of the group inoculated with only lung cancer organoid cells.

[0028] Figure 11 On the 14th day of co-culture of immune organoids and lung cancer organoids cultured using the method of the present invention, cells were recovered from the upper and lower chambers and quantified. Compared with the immune organoids cultured alone, the average number of lymphocytes found in the lower chamber of the immune organoids and lung cancer organoids co-culture group increased, and the overall distribution of the major lymphocyte subsets in the upper chamber did not change; the CD4 + T and CD8 +The proportion of T cells was significantly higher than that in the group inoculated with only lung cancer organoid cells; the proportion of B cells migrating from the immune organoid layer in the upper chamber to the lower chamber was 3.98% in the group inoculated with only lung cancer organoid cells (left figure), and 6.70% in the group co-cultured with immune organoids and lung cancer organoids (right figure). DETAILED DESCRIPTION

[0029] Unless otherwise specified, percentages in the present invention represent volume percentage concentrations.

[0030] In the description of the present invention, it should be noted that if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention.

[0032] Example 1 Preparation of culture medium

[0033] The present invention provides a culture medium for constructing an immune organoid model, specifically comprising:

[0034] (1) Preparation of upper chamber culture medium: Add the following components to RPMI1640 culture medium to make the final concentrations as follows: HEPES: 1X, Glutamax: 1X, N-acetylcysteine: 1mM, Penicillin / Streptomycin: 1X, Gentamycin: 50μg / mL, Sodium Pyruvate: 1mM, Glycine: 1mM, L-Alanine: 1mM, L-Asparagine: 1mM, L-Aspartic Acid: 1mM, L-Glutamic Acid: 1mM, L-Proline: 1mM, CHIR99021: 10nM, Insulin-Transferrin-Selenium: 1X, B-27: 1X, and human AB serum concentration is 10%.

[0035] (2) Preparation of the lower chamber: Add the following components to RPMI1640 medium to make the final concentrations as follows: HEPES: 1X, Glutamax: 1X, N-acetylcysteine: 1mM, Penicillin / Streptomycin Antibody: 1X, Gentamycin: 50μg / mL, Sodium Pyruvate: 1mM, Glycine: 1mM, L-Alanine: 1mM, L-Asparagine: 1mM, L-Aspartic Acid: 1mM, L-Glutamic Acid: 1mM, L-Proline: 1mM, CHIR99021: 10nM, Insulin-Transferrin-Selenium: 1X, B-27: 1X, Human AB Serum Concentration of 10%, B Lymphocyte Stimulating Factor: 1μg / mL.

[0036] Example 2

[0037] The present invention provides a method for constructing an immune organoid model, and uses RSV mRNA-LNP vaccine as an antigen to stimulate immune organoids to observe the physiological and pathological processes and functions of adaptive immunity such as humoral immunity and cellular immunity. Figure 1 As shown, the actual picture of the culture is as follows Figure 2 As shown, immune organoid activity is Figure 3 As shown, the humoral and cellular immune responses Figures 4-7 The whole process specifically includes the following steps:

[0038] 1. Cell Acquisition

[0039] 1.1 Isolation of fresh PBMC cells to obtain PBMC cell pellet:

[0040] 1) Collect 5 mL of blood in an anticoagulant tube (containing 0.2 mL of anticoagulant solution), shake well, and dilute with an equal volume of PBS (containing 2% fetal bovine serum (FBS)) (1:1).

[0041] 2) Take 2 mL of the lymphocyte stratification fluid according to the instructions and place it into a 15 mL centrifuge tube.

[0042] 3) Tilt the centrifuge tube at a 45° angle, draw up the diluted blood with a pipette, and slowly add it along the wall of the tube 1 cm above the stratified liquid surface so that the diluted blood is superimposed on the stratified liquid, keeping a clear interface between the two.

[0043] 4) Centrifuge at 2000 r / min for 20 min. After centrifugation, the contents are separated into four layers: the upper layer is plasma (containing platelets), the middle layer is the layering fluid, and the bottom layer is red blood cells and granulocytes. A milky white turbid mononuclear cell layer (thin) can be seen at the interface between the upper and middle liquid layers.

[0044] 5) Gently insert a capillary tube (or a 1 mL syringe) into the buffy coat layer, aspirate the mononuclear cells in the interface layer along the periphery of the tube wall, and transfer them into another centrifuge tube.

[0045] 6) Wash twice with 5 mL of RPMI1640 medium, mix well, and centrifuge at 1500 rpm and 1000 rpm for 10 min, respectively. Discard the supernatant to obtain the initial PBMC cell pellet.

[0046] 7) Resuspend the initial PBMC pellet in upper chamber culture medium, count the cells, adjust the cell density to obtain a PBMC suspension, and centrifuge to obtain a freshly isolated PBMC pellet.

[0047] 1.2 Thaw cryopreserved PBMCs to obtain PBMC cell pellets:

[0048] 1) Use tweezers to remove the PBMC cryovial from the liquid nitrogen tank. Place the PBMC cryovial in a 37°C water bath and gently stir until the ice crystals in the cryovial are completely melted.

[0049] 2) Pre-add 5 mL of upper chamber culture medium to a 15 mL centrifuge tube. Transfer the PBMC sample from the thawed cryotube to the 15 mL centrifuge tube and centrifuge at 400 x g for 10 min to obtain the initial PBMC cell pellet.

[0050] 3) Resuspend the initial PBMC pellet in upper chamber culture medium, count the cells, adjust the cell density to obtain a PBMC suspension, and centrifuge to obtain a PBMC pellet.

[0051] 1.3 Obtaining primary cell pellets from thymus, tonsils, spleen or lymph nodes:

[0052] 1) Soak the tissue sample in tissue protection solution, transport it to the laboratory at 4°C, and rinse thoroughly with saline.

[0053] 2) Wash the tissue sample in cleaning solution and homogenize it into 3 mm × 3 mm × 3 mm tissue fragments. Filter it through a 70 μm pore size cell sieve to obtain a cell suspension. Perform density gradient centrifugation to obtain the initial cell pellet of the primary immune cells.

[0054] 3) Resuspend the initial cell pellet of immune primary cells in upper chamber culture medium, count the cells, adjust the cell density, and obtain a suspension of primary cells from the thymus, tonsil, spleen, or lymph node. Centrifuge to obtain a pellet of primary cells from the thymus, tonsil, spleen, or lymph node.

[0055] It should be noted that the cells can be PBMCs obtained through legal channels, or thymus, tonsil, spleen, or lymph node tissue samples obtained through legal channels; the thymus, tonsil, spleen, or lymph node tissue samples can be of either human or animal origin. When the sample is human tissue, it is obtained from tissue that requires resection or biopsy in collaboration with a hospital and has passed ethical approval; when the sample is animal tissue, it is purchased through legal channels.

[0056] 2. Preparation of gel suspension

[0057] After the upper chamber culture medium and type I collagen were thoroughly mixed at a volume ratio of 1:1, the primary cell pellets of PBMC, thymus, tonsil, spleen, and lymph node whose cell density was adjusted by counting were resuspended to obtain a gel suspension.

[0058] 3. Transwell chamber coating

[0059] PBS was used to prepare a 100 μg / mL type I collagen dilution solution. 100 μL of the type I collagen dilution solution was added to the polycarbonate membrane coated in the Transwell chamber. The culture plate was then placed in a 37°C, 5% CO2 incubator for 1-1.5 h to obtain a matrix gel layer. The culture plate was a 24-well plate, the diameter of the Transwell chamber was 6.5 mm, and the area of ​​the Transwell chamber was 0.33 cm. 2 , the pore size of the polycarbonate membrane is 5~8μm.

[0060] 4. Cell Seeding

[0061] 1) Before inoculating the gel suspension, wash the Matrigel layer twice with PBS.

[0062] 2) Inoculate 1.75 x 10 7 30 μL of the cell suspension was added to the Matrigel layer.

[0063] 3) Place the culture plate in a 37°C, 5% CO2 incubator for 30 minutes to obtain a cell gel layer.

[0064] 5. Cultivation

[0065] The concentration of RSV mRNA-LNP vaccine was adjusted to 0.8~2μg / mL using upper chamber culture medium, and the concentration of RSV mRNA-LNP vaccine was adjusted to 0.8~2μg / mL using lower chamber culture medium. 200μL of upper chamber culture medium was added to the upper chamber in the Transwell chamber, and 500μL of lower chamber culture medium was added to the culture plate located below the Transwell chamber, namely the lower chamber. The cells were cultured in a 37°C, 5% CO2 incubator for 21 days. The culture medium was replaced every 2.5 days to maintain the culture. The immune organoids were obtained after 6~7 days of culture.

[0066] like Figure 2 As shown in the figure, immune organoids were successfully constructed. Figure 3 As shown in Figure 2, immune organoids from different sample sources cultured using the method of the present invention showed good growth activity. Figure 4 As shown, in the germinal centers formed during the adaptive immune response of the immune organoids cultured using the method of the present invention, B cells mainly accumulate in the GC (germinal center) area, while T cells are mainly distributed in the peripheral area.

[0067] like Figure 5As shown in the figure, the expression patterns of CD38 and CD27 in B cells cultured after stimulation with the RSV mRNA-LNP vaccine exhibited significant dynamic changes. Specifically, B cells first differentiated into the Pre-GC (pre-germinal center) phenotype (CD27-CD38+), then gradually developed into the GC phenotype (CD27+CD38+) and became dominant, before further differentiating into the memory B cell phenotype (CD27+CD38-) and undergoing plasmablast differentiation (CD27+CD38++).

[0068] like Figure 6 It shows that the organoids from different sources cultured by the technology of the present invention can not only induce the production of detectable levels of antibodies, but also trigger the cellular immune response process. Figure 7 As shown, when the RSV mRNA-LNP vaccine was used to stimulate the immune organoids cultured using the technology of the present invention, a significant increase in the levels of interleukin-6 and gamma interferon factor was detected on the 14th day of culture.

[0069] Example 3

[0070] The present invention provides a method for constructing an immune organoid model, and uses LPS / ATP as an antigen to stimulate the immune organoid to observe the physiological and pathological processes and functions of innate immunity.

[0071] Cell acquisition

[0072] 1.1 Isolation of fresh PBMC cells to obtain PBMC cell pellet (same as Example 2)

[0073] 1.2 Thawing of frozen PBMC to obtain PBMC cell pellet (same as Example 2)

[0074] 1.3 Obtaining primary cell pellets from thymus, tonsils, spleen or lymph nodes (same as Example 2)

[0075] 2. Preparation of gel suspension (same as Example 2)

[0076] 3. Transwell chamber coating (same as Example 2)

[0077] 4. Cell inoculation (same as Example 2)

[0078] 5. Cultivation

[0079] The LPS concentration was adjusted to 500 ng / mL using the upper chamber culture medium and the lower chamber culture medium. 200 μL of upper chamber culture medium was added to the upper chamber and 500 μL of lower chamber culture medium was added to the lower chamber. The cells were cultured in a 37°C, 5% CO2 incubator for 6 h.

[0080] The ATP concentration was adjusted to 5mM using the upper chamber culture medium and 5mM using the lower chamber culture medium. 200μL of upper chamber culture medium was added to the upper chamber, and 500μL of lower chamber culture medium was added to the lower chamber. The cells were cultured in a 37°C, 5% CO2 incubator for 21 days, with the culture medium replaced every 2.5 days. On day 14, the response of the innate immune "signaling hub" - the inflammasome - was detected, such as Figure 8 As shown, the immune organoid model can reproduce the significant expression of NLRP3 in the innate immune response under inflammatory stimulation.

[0081] Example 4

[0082] This example provides a method for constructing an immune organoid and tumor tissue interaction model. In this example, a lung cancer model is selected as a demonstration tumor type for co-culture with immune organoids. Immune organoids are co-cultured with lung cancer organoids to detect the migration of immune cells into tumors and the anti-tumor immune response in the immune organoids. Figure 9 As shown in Figure 2, immune cell killing responses to lung cancer organoids are shown in Figure 2. Figure 10 As shown in Figure 2, the migration of immune cells is as follows: Figure 11 The whole process specifically includes the following steps:

[0083] 1. Cell acquisition.

[0084] 1.1 Isolation of fresh PBMC cells to obtain PBMC cell pellet (same as Example 2)

[0085] 1.2 Thawing of frozen PBMC to obtain PBMC cell pellet (same as Example 2)

[0086] 1.3 Obtaining primary cell pellets from thymus, tonsils, spleen or lymph nodes (same as Example 2)

[0087] 1.4 Isolation of Lung Cancer Organoid Starting Cells:

[0088] 1) Soak lung cancer tissue samples in tissue protection solution, transport them to the laboratory at 4°C, and rinse thoroughly with saline.

[0089] 2) Mince the tissue thoroughly and add 3 times the volume of tumor tissue digestion solution. Digest in a 37°C water bath for 30-60 min until the cells are dissociated from the lung cancer tissue. Add 5 times the volume of PBS to terminate the digestion.

[0090] 3) Filter through a cell sieve with a pore size of 70 μm to obtain a cell suspension, and centrifuge to obtain an initial cell pellet of primary lung cancer cells.

[0091] 4) Resuspend the initial lung cancer primary cell pellet in lung cancer organoid culture medium, count the cells, adjust the cell density to obtain a lung cancer primary cell suspension, and centrifuge to obtain a lung cancer primary cell pellet.

[0092] 2. Transwell chamber coating (same as Example 2)

[0093] 3. Immunoprimary cell inoculation

[0094] 1) Before inoculating the primary cell suspension of PBMC, thymus, tonsil, spleen, or lymph node, wash the Matrigel layer twice with PBS.

[0095] 2) Inoculate 1.75 x 10 7 30 μL of the cell suspension was added to the Matrigel layer.

[0096] 3) Place the culture plate in a 37°C, 5% CO2 incubator for 30 minutes to obtain a cell gel layer.

[0097] 4. Lung Cancer Organoid Cell Inoculation

[0098] 1) Resuspend the lung cancer primary cell pellet (cell density adjusted by counting) in Matrigel to obtain a lung cancer organoid gel suspension.

[0099] 2) 30 μL of lung cancer organoid gel suspension (1000 cells) was added to the lower chamber of a Transwell chamber and dispensed into a droplet shape. The culture plate was then placed in a 37°C, 5% CO2 incubator for 30 minutes to obtain an immune organoid and lung cancer organoid co-culture model with a gel layer of primary PBMC, thymus, tonsil, spleen, or lymph node cells in the upper chamber and a gel layer of lung cancer organoid cells in the lower chamber.

[0100] 3) Mix the lower chamber culture medium and tumor organoid culture medium in a 1:1 volume ratio to prepare the culture medium for the immune organoid and tumor organoid co-culture model. Add 200 μL of upper chamber culture medium to the upper chamber and 500 μL of culture medium for the immune organoid and lung cancer organoid co-culture model to the lower chamber. Culture in a 37°C, 5% CO2 incubator for 21 days, replacing the culture medium every 2.5 days to maintain the culture.

[0101] like Figure 10 As shown, on the 14th day, cell activity was detected in the lung cancer organoid single culture group and the immune organoid and lung cancer organoid co-culture group. The live-dead staining results of the lung cancer organoids showed that the tumor cell killing rate in the immune organoid and lung cancer organoid co-culture group was significantly higher than that in the lung cancer organoid single culture group.

[0102] like Figure 11As shown, on the 14th day, the cells in the upper and lower chambers were recovered and quantitatively analyzed. The results showed that compared with the immune organoids cultured alone, the average number of lymphocytes detected in the lower chamber of the immune organoids and lung cancer organoids co-cultured increased, and the overall distribution of lymphocyte subsets in the upper chamber did not change; in addition, in the immune organoids and lung cancer organoids co-cultured group, the CD4 + T and CD8 + The proportion of T cells was significantly higher than that of the culture group inoculated with only lung cancer organoid cells. The proportion of B cells migrating from the upper chamber to the lower chamber was 3.98% in the culture group inoculated with only lung cancer organoid cells, while it reached 6.70% in the immune organoids and lung cancer organoids. These results indicate that immune organoids cultured using the technology of this invention can simulate the physiological process of plasmablasts before returning to the bone marrow or peripheral support niches, that is, first exiting the lymphoid tissue and entering the lymphatic and blood circulation. At the same time, it also demonstrates the physiological function of activated memory B cells retained in the germinal center and T cells chemotaxis and migration to tumor tissue.

[0103] The above are merely preferred embodiments of the present invention and are not intended to limit the invention in any other manner. Any person skilled in the art may utilize the technical content disclosed above to modify or modify the above embodiments into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A culture medium for constructing an immune organoid model, characterized in that: The invention comprises an upper chamber culture medium and a lower chamber culture medium, wherein the upper chamber culture medium and the lower chamber culture medium both comprise a basal culture medium, wherein the basal culture medium is supplemented with the following components: HEPES, Glutamax, an antibacterial agent, an antioxidant, sodium pyruvate, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, a GSK-3 inhibitor, insulin-transferrin-selenium, B-27 and human AB serum, and the basal culture medium of the lower chamber culture is further supplemented with a B lymphocyte stimulating factor.

2. The culture medium for constructing an immune organoid model according to claim 1, characterized in that The basal culture medium is RPMI1640 culture medium; the antibacterial agent is penicillin / streptomycin dual antibiotic and gentamicin; the antioxidant is N-acetylcysteine; and the GSK-3 inhibitor is selected from any one or more of AZD2858, AZD1080, B216763, CHIR99021, CHIR98014, LY2090314, NP031112, SB216763, SB415286, and TWS119.

3. The culture medium for constructing an immune organoid model according to claim 2, characterized in that In the upper chamber culture medium and the lower chamber culture medium, the concentrations of the components are as follows: HEPES: 1X, Glutamax: 1X, N-acetylcysteine: 1~1.5mM, penicillin / streptomycin dual antibody: 1X, gentamicin: 50μg / mL, sodium pyruvate: 1~1.2mM, glycine: 0.5~1.5mM, L-alanine: 0.5~1.5mM, L-asparagine: 0.5~1.5mM, L-aspartic acid: 0.5~1.5mM, L-glutamate: 0.5~1.5mM, L-proline: 0.5~1.5mM, GSK-3 inhibitor: 6~10nM, insulin-transferrin-selenium: 1X, B-27: 1X, human AB serum concentration is 10%, B lymphocyte stimulating factor: 0.5~1μg / mL; the aforementioned percentage concentrations are volume concentrations.

4. Use of the culture medium according to any one of claims 1 to 3 in constructing an immune organoid model.

5. A method for constructing an immune organoid model according to the culture medium according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Isolate PBMCs from blood or directly resuscitate frozen PBMCs, and obtain an initial PBMC cell pellet after centrifugation; or mechanically process the obtained thymus, tonsil, spleen, or lymph node tissue, and obtain an initial thymus, tonsil, spleen, or lymph node primary cell pellet after centrifugation; S2. Resuspend the initial cell pellet with upper chamber culture medium, adjust the cell density after cell counting, centrifuge, discard the supernatant, and resuspend the cell pellet (after counting and adjusting the cell density) with upper chamber culture medium containing type I collagen to obtain a gel suspension; S3, placing a Transwell chamber on a culture plate, and coating the polycarbonate membrane of the Transwell chamber with type I collagen to obtain a Matrigel layer; S4, inoculating the gel suspension onto the matrigel layer to form a cell gel layer after solidification; S5. Add the culture medium of the upper chamber containing the antigen into the Transwell chamber, and add the culture medium of the lower chamber containing the antigen into the culture plate below the Transwell chamber. Culture for 6 to 7 days to obtain immune organoids.

6. The method for constructing an immune organoid model according to claim 5, characterized in that: The coating method includes: Dilute type I collagen to 100-150 μg / mL, take 100 μL and add it to the Transwell chamber. Incubate in a 37°C, 5% CO2 incubator for 1-1.5 hours to form a matrix gel layer.

7. The method for constructing an immune organoid model according to claim 5, characterized in that: Each area is 0.33 cm 2 The cell density in the Transwell chamber was 1.75 × 10 7 The matrix gel layer was washed twice with PBS before inoculation.

8. The method for constructing an immune organoid model according to claim 6, wherein: When resuspending with upper chamber medium containing type I collagen, the volume ratio of upper chamber medium to type I collagen is 1:

1.

9. An immune organoid model, characterized in that The method according to any one of claims 5 to 8 is used to construct the present invention.

10. Use of the immune organoid model and tumor organoid model according to claim 9 in constructing a co-culture model.

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