Medium for co-incubation of tumor organoids and immune cells and drug efficacy evaluation method

Abstract

Provided are a serum-free medium for co-incubation of tumor organoids and immune cells, a tumor organoid immune microenvironment system and a construction method therefor, and a method for evaluating the efficacy of drugs based on tumor organoid immune co-culturing. The used medium for co-incubation of tumor organoids and immune cells can well maintain the growth of tumor organoids and immune cells, and can provide nutritional support for these two different types of cells. In addition, the established method for evaluating the efficacy of drugs in a tumor organoid immune co-culture system can be used for evaluating the efficacy and toxic side effects of an immune drug against a tumor, helping to select potentially effective drugs and optimize a treatment scheme. By means of the organoid immune co-culture system, the response of different patients to drugs can be better understood, providing a basis for personalized medicine and precision treatment.

Description

A culture medium for co-incubating tumor organoids and immune cells and a method for evaluating drug efficacy.

[0001] Priority information

[0002] none. Technical Field

[0003] This invention belongs to the field of cell engineering technology. Specifically, this invention relates to a serum-free culture medium for co-incubating tumor organoids and immune cells, a tumor organoid immune microenvironment system and its construction method, and a method for evaluating the efficacy of tumor organoid immune co-culture. Background Technology

[0004] Tumor organoids are three-dimensional tissue structures formed by culturing tumor cells in vitro. They are structures resembling primary tumor tissue, formed in vitro from tumor cells obtained from patient tumor samples under specific culture conditions. Tumor organoids can mimic the characteristics and behavior of real tumors, providing excellent models for studying tumor biology and drug responses.

[0005] The applications of tumor organoids are very broad. First, they can be used to study the biological characteristics and pathological mechanisms of tumors, including tumor origin, development, metastasis, and drug resistance mechanisms. Second, tumor organoids can be used to screen and evaluate personalized treatment strategies for individual patients. By culturing a patient's tumor cells into organoids in vitro, we can use this model to test the efficacy of different drugs, helping doctors choose the most suitable treatment plan, thereby improving the precision and effectiveness of treatment.

[0006] Furthermore, tumor organoids can be used to develop novel anticancer drugs and research therapeutic strategies such as tumor immunotherapy. By mimicking the interaction between tumors and immune cells in organoids, we can better understand the mechanisms of tumor immune escape and develop immunotherapies targeting tumors.

[0007] Tumor organoid immunotherapy co-culture refers to a research method that simulates the structure of tumor tissue in vitro and introduces immune cells, such as T cells and macrophages, for co-culture. Typically, organoids and immune cells are first cultured in their own unique culture media containing specific growth factors to meet their growth needs. Tumor organoids from different cell and tissue types require different nutrients; therefore, there is currently no universal culture medium specifically for tumor organoids and immune cells.

[0008] Therefore, there is an urgent need to develop a culture medium for co-incubating tumor organoids and immune cells, as well as a method for quantitatively analyzing the efficacy evaluation of tumor organoid immune co-culture.

[0009] Summary of the Invention

[0010] The purpose of this invention is to provide a serum-free culture medium for co-incubating tumor organoids and immune cells, a tumor organoid immune microenvironment system and its construction method, and a method for evaluating the efficacy of tumor organoid immune co-culture. The method for evaluating the efficacy of tumor organoid immune co-culture established by this invention can be used to assess the efficacy and toxic side effects of immunotherapeutic drugs targeting tumors, help screen potentially effective drugs and optimize treatment plans. Through this organ immune co-culture system, we can better understand the response of different patients to drugs, providing a basis for personalized medicine and precision treatment.

[0011] Therefore, in a first aspect, the present invention provides a serum-free culture medium for co-incubating tumor organoids and immune cells.

[0012] According to an embodiment of the present invention, the tumor organoid and immune cell co-incubation medium comprises a basal medium, a ROCK inhibitor, N-acetylcysteine, nicotinamide, a p38 Mark inhibitor, EGF, R-Spondin1, Noggin, IL-2, IL-7, IL-8, IL-12, cholesterol, and antibiotics. The basal medium is selected from Advance DMEM / F12. The tumor organoid and immune cell co-incubation medium according to an embodiment of the present invention can effectively culture tumor organoids and immune cells, obtaining a tumor organoid immunotherapy co-culture system. This allows for further evaluation of the efficacy and toxicity of immunotherapies targeting tumors, playing an important role in clinical treatment.

[0013] According to a specific embodiment of the present invention, the ROCK inhibitor is selected from Y27632.

[0014] According to a specific embodiment of the present invention, the working concentration of Y27632 is 2-20 μM.

[0015] According to a specific embodiment of the present invention, the working concentration of N-acetylcysteine ​​is 1M.

[0016] According to a specific embodiment of the present invention, the working concentration of the nicotinamide is 2.5M.

[0017] According to a specific embodiment of the present invention, the p38 MARK inhibitor is selected from SB202190, and the working concentration of SB202190 is 20 mM.

[0018] According to a specific embodiment of the present invention, the working concentration of the EGF is 100 μg / mL.

[0019] According to a specific embodiment of the present invention, the working concentration of R-Spondin1 is 1 mg / mL.

[0020] According to a specific embodiment of the present invention, the working concentration of Noggin is 100 μg / mL.

[0021] According to a specific embodiment of the present invention, the working concentration of IL-2 is 100 μg / mL.

[0022] According to a specific embodiment of the present invention, the working concentration of IL-7 is 50 μg / mL.

[0023] According to a specific embodiment of the present invention, the working concentration of IL-8 is 20 μg / mL.

[0024] According to a specific embodiment of the present invention, the working concentration of IL-12 is 10 μg / mL.

[0025] According to a specific embodiment of the present invention, the working concentration of the cholesterol is 10 μg / mL.

[0026] According to a specific embodiment of the present invention, the culture medium for co-incubating tumor organoids and immune cells further includes antibiotics. The addition of antibiotics is primarily for antibacterial purposes. It should be noted that there are no particular limitations on the types of antibiotics added to the culture medium; any antibiotic that has an antibacterial effect during cell culture is covered within the scope of protection of this invention.

[0027] According to a specific embodiment of the present invention, the antibiotic is selected from at least one of penicillin, streptomycin, amphotericin B, and gentamicin.

[0028] According to a specific embodiment of the present invention, the working concentration of the antibiotic is 50 μg / mL to 100 μg / mL.

[0029] According to an embodiment of the present invention, the tumor organoids include any one of the following: head and neck squamous cell carcinoma, head and neck adenocarcinoma, esophageal cancer, lung adenocarcinoma, liver cancer, bile duct cancer, gastric cancer, cardia cancer, breast cancer, and colorectal cancer organoids;

[0030] The immune cells include any one of the following: human PBMCs, T lymphocytes, NK lymphocytes, CAR-T cells, CAR-NK cells, and TILs cells.

[0031] According to an embodiment of the present invention, the tumor organoids are lung adenocarcinoma, colon cancer, and gastric cancer organoids, and the immune cells are human PBMCs.

[0032] A second aspect of the present invention provides an immune microenvironment system for tumor organoids. According to an embodiment of the present invention, the immune microenvironment system comprises:

[0033] Single-cell tumor organoids, immune cells, and the co-incubation culture medium for tumor organoids and immune cells as described in the first aspect.

[0034] In this process, the single cells of the tumor organoids and the immune cells are co-cultured in the tumor organoid and immune cell co-incubation medium.

[0035] The tumor organoid immunotherapy co-culture method established in this invention can be used to evaluate the efficacy and toxicity of immunotherapies targeting tumors, helping to screen potentially effective drugs and optimize treatment regimens. Through this organoid immunotherapy co-culture system, we can better understand the responses of different patients to drugs, providing a basis for personalized medicine and precision treatment. The immunotherapy co-culture model established in this invention has broad application prospects in drug research, personalized medicine, and tumor immunotherapy, providing an important experimental platform and guidance for tumor research and treatment, and possessing significant social, economic, and scientific value.

[0036] According to an embodiment of the present invention, in the tumor organoid immune microenvironment system, the ratio of single cells of the tumor organoid to immune cells is 1:(5-10), preferably, the ratio of single cells of the tumor organoid to immune cells is 1:10, and an excessive number of immune cells does not significantly increase the number of tumor cell deaths.

[0037] According to an embodiment of the present invention, the single cells of the tumor organoids are selected from any one of the single cells of head and neck squamous cell carcinoma, head and neck adenocarcinoma, esophageal cancer, lung adenocarcinoma, liver cancer, bile duct cancer, gastric cancer, cardia cancer, breast cancer, and colorectal cancer organoids;

[0038] The immune cells include any one of the following: human PBMCs, T lymphocytes, NK lymphocytes, CAR-T cells, CAR-NK cells, and TILs cells.

[0039] According to an embodiment of the present invention, the single cells of the tumor organoid are lung adenocarcinoma, colon cancer, and gastric cancer cells, and the immune cells are human PBMCs.

[0040] A third aspect of this invention provides a method for constructing an immune microenvironment for tumor organoids. According to an embodiment of the invention, the construction method includes:

[0041] (1) Obtain single cells of tumor organoids;

[0042] (2) The single cells of the tumor organoids are mixed with immune cells and co-cultured in the tumor organoid and immune cell co-incubation medium described in the first aspect.

[0043] Wherein, the single-cell and immune cells of the tumor organoids are the single-cell and immune cells of the tumor organoids in the tumor organoid immune microenvironment system described in the second aspect.

[0044] When the single cells of the tumor organoids are mixed with the immune cells, the inoculation ratio is 1:(5-10).

[0045] The tumor organoid immune microenvironment system constructed using the method of this invention can simulate the immune microenvironment in vivo, providing more realistic physiological conditions and facilitating the study of the interaction between immune cells and tumor cells and their response to immunotherapy drugs.

[0046] A fourth aspect of this invention provides a method for evaluating the efficacy of tumor organoid immunotherapy through co-culture. According to an embodiment of this invention, the method includes:

[0047] 1) The test drug is added to the tumor organoid immune microenvironment system described in the second aspect for one incubation;

[0048] 2) Add cell marker antibodies to the system after the first incubation in step 1) and perform a second incubation;

[0049] 3) Fluorescence detection was performed on the system after the second incubation. The drug sensitivity was evaluated by comparing the changes in the fluorescence intensity of Caspase 3 / 7 in tumor cells of the experimental group and the control group. The experimental group consisted of cells treated with the test drug, while the control group consisted of cells not treated with the test drug.

[0050] Currently, there is no suitable universal culture medium for co-culturing tumor organoids and immune cells, and existing methods for evaluating co-culture of immune cells suffer from drawbacks such as difficulties in standardization and calibration, and high time and cost. Therefore, the tumor organoid-immune cell co-incubation culture medium, the construction of the tumor organoid immune microenvironment, and the method for evaluating drug efficacy provided by this invention offer powerful tools for drug development and the evaluation of co-culture of organoids and immune cells.

[0051] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0052] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0053] Figure 1 is a bright-field diagram of the immune co-culture system constructed according to an embodiment of the present invention;

[0054] Figure 2 is a graph of fluorescence staining detection results according to an embodiment of the present invention and statistical analysis data of Caspase 3 / 7 fluorescence intensity. In the bar chart on the right, the three bars at the 5:1 and 10:1 ratios represent PDO, PDO+PBMC, and PDO+PBMC+Pembrolizumab, respectively. Detailed Implementation

[0055] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0056] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0057] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0058] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0059] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0060] According to a specific embodiment of the present invention, the present invention provides a serum-free culture medium for co-incubating tumor organoids and immune cells.

[0061] The tumor organoid and immune cell co-incubation medium includes basal medium, ROCK inhibitor, N-acetylcysteine, nicotinamide, p38 Mark inhibitor, EGF, R-Spondin1, Noggin, IL-2, IL-7, IL-8, IL-12, cholesterol, and antibiotics. The basal medium is selected from advance DMEM / F12.

[0062] According to a specific embodiment of the present invention, the ROCK inhibitor is selected from Y27632.

[0063] According to a specific embodiment of the present invention, the working concentration of Y27632 is 2-20 μM. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 μM or any range between two of these values.

[0064] According to a specific embodiment of the present invention, the working concentration of N-acetylcysteine ​​is 1M.

[0065] According to a specific embodiment of the present invention, the working concentration of the nicotinamide is 2.5M.

[0066] According to a specific embodiment of the present invention, the p38 MARK inhibitor is selected from SB202190, and the working concentration of SB202190 is 20 mM.

[0067] According to a specific embodiment of the present invention, the working concentration of the EGF is 100 μg / mL.

[0068] According to a specific embodiment of the present invention, the working concentration of R-Spondin1 is 1 mg / mL.

[0069] According to a specific embodiment of the present invention, the working concentration of Noggin is 100 μg / mL.

[0070] According to a specific embodiment of the present invention, the working concentration of IL-2 is 100 μg / mL.

[0071] According to a specific embodiment of the present invention, the working concentration of IL-7 is 50 μg / mL.

[0072] According to a specific embodiment of the present invention, the working concentration of IL-8 is 20 μg / mL.

[0073] According to a specific embodiment of the present invention, the working concentration of IL-12 is 10 μg / mL.

[0074] According to a specific embodiment of the present invention, the working concentration of the cholesterol is 10 μg / mL.

[0075] According to a specific embodiment of the present invention, the culture medium for co-incubating tumor organoids and immune cells further includes antibiotics. The addition of antibiotics is primarily for antibacterial purposes. It should be noted that there are no particular limitations on the types of antibiotics added to the culture medium; any antibiotic that has an antibacterial effect during cell culture is covered within the scope of protection of this invention.

[0076] According to a specific embodiment of the present invention, the antibiotic is selected from at least one of penicillin, streptomycin, amphotericin B, and gentamicin.

[0077] According to a specific embodiment of the present invention, the working concentration of the antibiotic is 50 μg / mL to 100 μg / mL. For example, 50, 52, 55, 57, 60, 62, 65, 67, 70, 72, 75, 77, 80, 82, 85, 87, 90, 92, 95, 97, 100 μg / mL or a range between any two of these values.

[0078] According to a specific embodiment of the present invention, the tumor organoids include, but are not limited to, any one of the following: head and neck squamous cell carcinoma, head and neck adenocarcinoma, esophageal cancer, lung adenocarcinoma, liver cancer, bile duct cancer, gastric cancer, cardia cancer, breast cancer, and colorectal cancer organoids;

[0079] The immune cells include any one of the following: human PBMCs, T lymphocytes, NK lymphocytes, CAR-T cells, CAR-NK cells, and TILs cells.

[0080] According to a specific embodiment of the present invention, the tumor organoids are lung adenocarcinoma, colon cancer, and gastric cancer organoids, and the immune cells are human PBMCs.

[0081] According to a specific embodiment of the present invention, the present invention also provides a tumor organoid immune microenvironment system, the immune microenvironment system comprising:

[0082] Single-cell tumor organoids, immune cells, and the aforementioned co-incubation culture medium for tumor organoids and immune cells.

[0083] In this process, the single cells of the tumor organoids and the immune cells are co-cultured in the tumor organoid and immune cell co-incubation medium.

[0084] According to a specific embodiment of the present invention, in the tumor organoid immune microenvironment system, the ratio of single cells of the tumor organoid to immune cells is 1:(5-10). For example, the ratio of single cells of the tumor organoid to immune cells can be 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10, or any range between two of these ratios.

[0085] According to a specific embodiment of the present invention, the single cells of the tumor organoids are selected from any one of the single cells of head and neck squamous cell carcinoma, head and neck adenocarcinoma, esophageal cancer, lung adenocarcinoma, liver cancer, bile duct cancer, gastric cancer, cardia cancer, breast cancer, and colorectal cancer organoids.

[0086] The immune cells include any one of the following: human PBMCs, T lymphocytes, NK lymphocytes, CAR-T cells, CAR-NK cells, and TILs cells.

[0087] According to a specific embodiment of the present invention, the single cells of the tumor organoid are lung adenocarcinoma, colon cancer, and gastric cancer cells, and the immune cells are human PBMCs.

[0088] According to a specific embodiment of the present invention, the single cells of the tumor organoids can be obtained by digestion with organoid passage digestion solution at 37°C and 5% CO2 for 5-30 minutes, or by other methods. Furthermore, the organoids can be observed under a microscope to determine whether they have divided into single cells.

[0089] According to a specific embodiment of the present invention, the tumor organoids are obtained, including but not limited to, through the following means:

[0090] Tissue analogs with a certain spatial structure are formed by using adult tumor stem cells in the patient's body and culturing them in three dimensions (3D) in vitro using matrix gel.

[0091] According to a specific embodiment of the present invention, the present invention provides a method for constructing an immune microenvironment of tumor organoids, the method comprising:

[0092] (1) Obtain single cells of tumor organoids;

[0093] (2) Mix the single cells of the tumor organoids with immune cells and co-culture them in the aforementioned tumor organoid and immune cell co-incubation medium.

[0094] The single-cell and immune cells of the tumor organoids mentioned above refer to the single-cell and immune cells of the tumor organoids in the aforementioned tumor organoid immune microenvironment system.

[0095] When the tumor organoid single cells are mixed with the immune cells, the inoculation ratio is 1:(5-10). For example, the ratio of the tumor organoid single cells to the immune cells can be 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10, or any range between two of these ratios.

[0096] According to a specific embodiment of the present invention, the single cells of the tumor organoids can be obtained by digestion with organoid passage digestion solution at 37°C and 5% CO2 for 5-30 minutes, or by other methods. Furthermore, the organoids can be observed under a microscope to determine whether they have divided into single cells.

[0097] According to an embodiment of the present invention, the mixed cells obtained by mixing single cells of the tumor organoids with immune cells can be seeded on a low-absorption plate for suspension culture.

[0098] According to a specific embodiment of the present invention, a method for constructing an immune co-culture system for tumor organoids is proposed. The method includes: mixing tumor organoid single cells and PBMCs at a ratio of 1:(5-10) and seeding them into a 96-well low-absorption plate. For example, the ratio of tumor organoid single cells to PBMCs can be 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10, or any range between two of these ratios.

[0099] According to a specific embodiment of the present invention, the present invention provides a method for evaluating the efficacy of tumor organoid immunotherapy co-culture, the method comprising:

[0100] 1) The test drug was added to the tumor organoid immune microenvironment system described above for one incubation;

[0101] 2) Add cell marker antibodies to the system after the first incubation in step 1) and perform a second incubation;

[0102] 3) Perform fluorescence detection on the system after the second incubation, and evaluate the efficacy of the test drug based on the changes in the fluorescence intensity of Caspase3 / 7 in the tumor cells of the experimental group and the control group.

[0103] According to a specific embodiment of the present invention, the test drug includes, but is not limited to, monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, and small molecule inhibitors.

[0104] According to a specific embodiment of the present invention, the reagents and consumables used in the method include: cell staining buffer, CellTrace... TM Blue staining solution, CellTrace TM Red CMTPX staining solution, 4% paraformaldehyde, antibody incubation solution, etc.

[0105] Immunofluorescence was used to compare the changes in the fluorescence intensity of Caspase 3 / 7 in tumor cells of the experimental group and the control group to evaluate drug sensitivity.

[0106] According to a specific embodiment of the present invention, the cell staining buffer comprises: fetal bovine serum and phosphate buffer.

[0107] According to a specific embodiment of the present invention, the volume percentage concentration of the fetal bovine serum in the cell staining buffer is 0.5%-10%, for example: 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, or any range between any two of these values.

[0108] According to a specific embodiment of the present invention, the pH value of the phosphate buffer solution is 7-8.

[0109] According to a specific embodiment of the present invention, the CellTrace TM Red CMTPX staining solution includes: CellTrace TM Red CMTPX dye, cell staining buffer.

[0110] According to a specific embodiment of the present invention, the CellTrace TM Blue staining solution includes: CellTrace TM Blue dye, cell staining buffer.

[0111] According to a specific embodiment of the present invention, the antibody incubation solution includes: Caspase 3 / 7 fluorescent antibody and cell staining buffer.

[0112] According to a specific embodiment of the present invention, the fluorescent antibody has a volume percentage concentration of 0.5%-5% in the cell staining buffer, for example: 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or any range between two of these values.

[0113] According to a specific embodiment of the present invention, the method for evaluating the efficacy of immunoco-culture mainly compares the fluorescence intensity of Caspase3 / 7 in tumor cells of each group.

[0114] According to a specific embodiment of the present invention, the immunoco-culture efficacy evaluation method analyzes the fluorescence intensity of Caspase 3 / 7 in tumor cells by fluorescence staining.

[0115] According to a specific embodiment of the present invention, tumor organoids and PBMCs are co-cultured in vitro with the immune checkpoint inhibitor PD-1 for 2 days, and the fluorescence intensity of Caspase3 / 7 in tumor cells is obtained by immunofluorescence technology to evaluate the therapeutic effect of the immune checkpoint inhibitor PD-1 drug.

[0116] Immunofluorescence technology is based on the specificity of antigen-antibody reactions. A specific fluorescent reaction will only occur under a fluorescence microscope when the antigen binds to the corresponding antibody, thus ensuring the accuracy of the detection results. Furthermore, it can eliminate interference from other non-specific substances, improving the sensitivity and accuracy of the detection. Therefore, the method for evaluating the efficacy of tumor organoid immune microenvironment provided by this invention has significant scientific and social value.

[0117] This invention proposes a method for evaluating the efficacy of tumor organoid immunotherapy co-culture. The method uses immunofluorescence to detect the fluorescence intensity of Caspase 3 / 7 in tumor cells to determine the immunogenicity. The method includes single-cell preparation of tumor organoids, co-inoculation of tumor organoids and immune cells, incubation of tumor organoids and immune cells with fluorescent antibodies, and fluorescence microscopy imaging. The reagents and consumables include: complete culture medium for tumor organoids, complete culture medium for immune cells, co-incubation medium for tumor organoids and immune cells, low-adsorption 96-well plates, cell staining buffer, and CellTrace. TM Blue staining solution, CellTrace TM Red CMTPX staining solution and Caspase 3 / 7 antibody. The tumor organoid immune microenvironment system constructed using the method of this invention is stable and highly reproducible, and can be used for in vitro efficacy evaluation of drugs targeting the tumor immune microenvironment.

[0118] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0119] The reagents and their catalog numbers used in the following examples are shown in Table 1.

[0120] Table 1. Reagent Sources

[0121] Example 1: Construction of an immune co-culture system for tumor organoids with different cell ratios

[0122] 1. Organoid resuscitation: Preheat the basal culture medium (purchased from bioGenous, B213152) required for organoid resuscitation at 37°C. Rapidly thaw the cryovials in a 37°C water bath. Stop the water bath immediately when only a small amount of ice remains in the cryovials and transfer them to a clean work surface. Transfer the cryopreservation suspension to centrifuge tubes and slowly add 5-10 times the volume of preheated basal culture medium, mixing gently. Centrifuge the resulting organoid suspension (horizontal rotor, 150-300g, 3 min), discard the supernatant, and resuspend the organoid pellet in basal culture medium. Centrifuge the organoid suspension obtained in the above steps (horizontal centrifuge rotor, 150-300g, 3min), discard the supernatant to obtain organoids, encapsulate the organoids with matrix gel and seed them into 24-well plates at a cell density of 100-1000 cells / μL, incubate the culture plate in a 37℃ incubator for 20min, and add 500μL of tumor organoid complete culture medium to each well for culture.

[0123] 2. Organoid Collection: This experiment is performed when tumor organoids grow to approximately 100 μm. While retaining the original culture medium in the well plate (or removing the culture medium and adding an equal volume of basal culture medium), gently scrape (or pipette) the mixture of matrix gel and organoids using the tip of a pipette, transfer it to a 1.5 mL or 15 mL centrifuge tube, and pipette 5-10 times to separate the organoids from the matrix gel. Centrifuge at 100-300 g for 3 min.

[0124] 3. Organoid staining: Discard the supernatant obtained in the previous step, and add 100 μL of pre-prepared CellTrace. TM Red CMTPX staining working solution was incubated at 37°C for 30 min. After incubation, an appropriate volume of cell staining buffer was added to wash the cells and stop staining immediately (Note: Cell staining buffer is PBS + 2% fetal bovine serum).

[0125] 4. PBMC cell staining: After centrifuging and collecting PBMC cells, discard the supernatant and add 100 μL of pre-prepared CellTrace staining solution. TM Incubate the blue staining working solution in a 37°C incubator for 30 minutes. After incubation, add an appropriate volume of cell staining buffer and wash immediately to stop staining.

[0126] 5. Tumor cell collection and counting: Tumor organoids were collected by centrifugation, resuspended in a medium co-incubated with tumor cells and immune cells, and 20 μL was used for single-cell preparation and counting to obtain the number of tumor organoids.

[0127] 6. PBMC cell collection and counting: Collect stained PBMC cells by centrifugation, resuspend them in co-culture complete medium and count them to obtain the number of stained PBMCs.

[0128] 7. Inoculation of tumor organoids and immune cells: Mix tumor organoids with PBMC cells at ratios of 1:5 and 1:10, and inoculate 200 μL of cell suspension into each well of a 96-well plate.

[0129] The experimental results on the third day of culture are shown in Figure 1. The figure shows the bright field situation of tumor cells and PBMCs co-cultured. PBMCs aggregated around the tumor organoids, and the tumor organoids showed lysis.

[0130] 8. Cell fixation: After 72 h of co-culture, remove 100 μL of culture medium from each well, add 4% paraformaldehyde and fix at 4°C for 30 min, then wash twice.

[0131] 9. Antibody incubation: Add 100 μL of Caspase 3 / 7 (1:50) antibody dilution to each well and incubate at room temperature for 30 min;

[0132] 10. Fluorescence Imaging: Select an appropriate fluorescence channel for fluorescence imaging.

[0133] 11. Immunofluorescence detection, as shown in Figure 2, tumor organoids are illuminated by red fluorescence (CellTrace). TM Red CMTPX labeled, PBMC cells were stained with blue fluorescence (CellTrace). TM The activity of tumor organoids was evaluated by detecting the expression intensity of green fluorescence (Caspase 3 / 7) in different groups of organoids labeled with Blue. The figure shows that the fluorescence intensity of Caspase 3 / 7 in tumor organoids significantly increased in different cell ratios of the PD-1 inhibitor groups, indicating a significant effect of the PD-1 inhibitor. However, the fluorescence intensity of Caspase 3 / 7 at ratios of 1:5 and 1:10 was not significantly different, indicating that an excessive number of immune cells does not significantly increase the number of tumor cell deaths. Based on the preliminary experimental results, the optimal cell ratio should be determined. In this study, the optimal cell ratio for co-culturing tumor organoids and PBMCs with PD-1 inhibitors was 1:10.

[0134] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0135] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A culture medium for co-incubating tumor organoids and immune cells, characterized in that, The tumor organoid and immune cell co-incubation medium includes basal medium, ROCK inhibitor, N-acetylcysteine, nicotinamide, p38 Mark inhibitor, EGF, R-Spondin1, Noggin, IL-2, IL-7, IL-8, IL-12, cholesterol, and antibiotics. The basal medium is selected from advance DMEM / F12.

2. The culture medium according to claim 1, characterized in that, The ROCK inhibitor is selected from Y27632, and the working concentration of Y27632 is 2-20 μM.

3. The culture medium according to claim 1, characterized in that, The working concentration of the N-acetylcysteine ​​is 1M.

4. The culture medium according to claim 1, characterized in that, The working concentration of the nicotinamide is 2.5M.

5. The culture medium according to claim 1, characterized in that, The p38 MARK inhibitor is selected from SB202190, and the working concentration of SB202190 is 20 mM.

6. The culture medium according to claim 1, characterized in that, The working concentration of EGF is 100 μg / mL.

7. The culture medium according to claim 1, characterized in that, The working concentration of R-Spondin1 is 1 mg / mL.

8. The culture medium according to claim 1, characterized in that, The working concentration of Noggin is 100 μg / mL.

9. The culture medium according to claim 1, characterized in that, The working concentration of IL-2 is 100 μg / mL.

10. The culture medium according to claim 1, characterized in that, The working concentration of IL-7 is 50 μg / mL.

11. The culture medium according to claim 1, characterized in that, The working concentration of IL-8 is 20 μg / mL.

12. The culture medium according to claim 1, characterized in that, The working concentration of IL-12 is 10 μg / mL.

13. The culture medium according to claim 1, characterized in that, The working concentration of cholesterol is 10 μg / mL.

14. The culture medium according to claim 1, characterized in that, The culture medium for co-incubating tumor organoids and immune cells further includes antibiotics.

15. The culture medium according to claim 14, characterized in that, The antibiotic is selected from at least one of penicillin, streptomycin, amphotericin B, and gentamicin.

16. The culture medium according to claim 14, characterized in that, The working concentration of the antibiotic is 50 μg / mL to 100 μg / mL.

17. The culture medium according to claim 1, characterized in that, The tumor organoids include any one of the following: head and neck squamous cell carcinoma, head and neck adenocarcinoma, esophageal cancer, lung adenocarcinoma, liver cancer, bile duct cancer, gastric cancer, cardia cancer, breast cancer, and colorectal cancer organoids. The immune cells include those selected from human peripheral blood mononuclear cells, T lymphocytes, NK lymphocytes, and CAR-T cells. Either CAR-NK cells or TILs cells.

18. A tumor organoid immune microenvironment system, characterized in that, include: Single-cell tumor organoids, immune cells, and a culture medium for co-incubating tumor organoids and immune cells as described in any one of claims 1-17; In this process, the single cells of the tumor organoids and the immune cells are co-cultured in the tumor organoid and immune cell co-incubation medium.

19. The tumor organoid immune microenvironment system according to claim 18, characterized in that, In the tumor organoid immune microenvironment system, the ratio of single cells of the tumor organoid to immune cells is 1:(5-10).

20. The tumor organoid immune microenvironment system according to claim 18 or 19, characterized in that, The single cells of the tumor organoids are selected from any one of the single cells of head and neck squamous cell carcinoma, head and neck adenocarcinoma, esophageal cancer, lung adenocarcinoma, liver cancer, bile duct cancer, gastric cancer, cardia cancer, breast cancer, and colorectal cancer organoids. The immune cells include any one of the following: human PBMCs, T lymphocytes, NK lymphocytes, CAR-T cells, CAR-NK cells, and TILs cells.

21. A method for constructing an immune microenvironment for tumor organoids, characterized in that, include: (1) Obtain single cells of tumor organoids; (2) Mix the single cells of the tumor organoids with immune cells and co-culture them in the tumor organoid and immune cell co-incubation medium according to any one of claims 1-17. Wherein, the single-cell and immune cells of the tumor organoids are the single-cell and immune cells of the tumor organoids in the tumor organoid immune microenvironment system according to any one of claims 18-20. When the single cells of the tumor organoids are mixed with the immune cells, the inoculation ratio is 1:(5-10).

22. A method for screening antitumor drugs, characterized in that, include: The drug to be screened is mixed with the tumor organoid immune microenvironment system according to any one of claims 18-20, and the number of live cells in the tumor organoids before and after mixing is compared, wherein the reduction in the number of live cells in the tumor organoids after mixing is an indicator of the effectiveness of the drug to be screened as an anti-tumor drug.

23. A method for evaluating the efficacy of tumor organoid immunotherapy co-culture, characterized in that, include: 1) The test drug is added to the tumor organoid immune microenvironment system according to any one of claims 18-20 for one incubation; 2) Add cell marker antibodies to the system after the first incubation in step 1) and perform a second incubation; 3) Fluorescence detection was performed on the system after the second incubation. The sensitivity of the drug was evaluated by comparing the changes in the fluorescence intensity of Caspase3 / 7 in tumor cells of the experimental group and the control group. The experimental group was the group with the test drug added, and the control group was the group without the test drug added.

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