Method for preparing DC vaccine based on exosome derived from liver cancer organoid

Abstract

The invention provides a method for preparing a DC vaccine based on exosomes from liver cancer organoid sources. The method comprises the following steps: S1, constructing a liver cancer organoid model; s2, extracting an exosome from the liver cancer organ model; s3, culturing DC cells of the liver cancer patient; s4, co-culturing the exosome obtained in the step S2 and the DC cells cultured in the step S3, and then harvesting mature DC cells. Exosome components containing liver cancer tumor antigen information are extracted and then added into a DC cell culture medium for antigen loading, and finally mixed immune cells activated by DC cells are added into autologous liver cancer organs of a patient, so that specific killing of the liver cancer organs can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor vaccines, and in particular to a method for preparing DC vaccines based on exosomes derived from liver cancer organoids. Background Art

[0002] Tumor diseases are currently a global health threat to humanity. Changing the plight of fear of cancer and despair of having cancer is a problem that needs to be solved urgently worldwide. According to the epidemiological report released by the World Health Organization's International Cancer Center in 2020, there are approximately 19.3 million new cases of cancer and nearly 10 million cancer deaths each year worldwide. It is estimated that by 2040, the global burden will reach 28.4 million cases, an increase of 47% from 2020.

[0003] Immunotherapy has fundamentally changed the treatment status of tumor diseases. From the development of immunomodulatory factors such as thymosin to the widespread use of immune-regulatory inhibitors such as PD-1 inhibitors, including the creation of adoptive immune cell therapy and tumor vaccines, the goal of tumor cure has gradually changed from being out of reach to being full of hope. Tumor vaccines are a type of product that achieves therapeutic effects such as inhibiting or killing tumor cells, clearing tiny residual lesions or precancerous lesions, and establishing lasting anti-tumor memory by inducing or enhancing the body's specific active immune response to tumor antigens. In October 2022, the Drug Evaluation Center of the National Medical Products Administration issued the "Technical Guidelines for Clinical Trials of Tumor Therapeutic Vaccines (Draft for Comments)". In 2023 and 2024, a number of tumor vaccine products have successively obtained new drug clinical trial authorization (IND). In 2024, the Drug Evaluation Center of the National Medical Products Administration once again issued the "Technical Guidelines for Non-clinical Research of Tumor Therapeutic Vaccines (Draft for Comments)" to vigorously promote the research and development of tumor vaccines. At present, clinical research results at home and abroad have shown that tumor vaccine therapy can significantly activate the immune status of tumor patients in a variety of solid tumors, and the response rate is better than that of immunosuppressant therapy. Tumor vaccine therapy has the characteristics of specifically activating anti-tumor immunity and forming persistent immune memory, which is different from adoptive cell therapy such as CAR-T and immunosuppressant therapy targeting immune cell function. It is one of the most promising immunotherapy strategies.

[0004] The key to the effectiveness of tumor vaccines lies in the acquisition of tumor antigens. According to the specificity of tumor antigens, tumor antigens can be divided into tumor-associated antigens (TAA) and tumor-specific antigens (TSA). The acquisition of tumor antigens can be divided into four main forms: tumor tissue lysate, DNA, RNA, and protein peptides. First of all, personalized tumor vaccines prepared based on TAA obtained from tumor tissue lysates are the most commonly used tumor vaccine therapy in clinical practice, and have shown good safety and effectiveness in clinical studies of malignant tumors such as glioma, ovarian cancer, and melanoma. Despite this, the main component of tumor tissue lysates is TAA, and the abundance of tumor-specific antigens is often not ideal, resulting in inconsistent immunogenicity. Some patients can obtain good immune activation, while some patients cannot achieve immune activation, and may even induce nonspecific immune responses. In recent years, with the improvement of antigen prediction methods and the advancement of sequencing technology, tumor-specific antigens have gradually been identified. The expression of specific tumor antigens in the form of DNA, RNA, and protein peptides is the main direction of tumor vaccine research and development, which provides good technical support for tumor vaccine research and development. However, solid tumors such as liver cancer and pancreatic cancer are highly heterogeneous, and the accuracy of antigen prediction is currently not ideal. Specific antigens with high immunogenicity and universality among the population are difficult to screen. Therefore, no matter what method is used to express specific antigens, it is still some distance away from the clinical application of cancer tumor vaccines.

[0005] Therefore, from the perspective of clinical application, how to obtain a stable source of tumor tissue and increase the abundance of TSA in tumor-related antigens is a more practical method to promote the application of tumor vaccines. The primary difficulty in using tumor tissue lysates to prepare personalized tumor vaccines is the acquisition of high-quality fresh tissue specimens. During clinical surgery, there is a large degree of uncertainty in the size, transit time, and degree of necrosis of tumor tissue, which greatly affects the preparation of tumor vaccines. In recent years, the development of tumor organoid technology has provided ideas for solving this problem. Organoids are a 3D cultured cell model that can simulate the growth characteristics of human tissues in structure and function. The characteristics of organoids are that they have the ability to proliferate and differentiate, can be cultured for a long time, and can be stably passaged. Their cellular and non-cellular components can also simulate the vascular microenvironment and immune microenvironment. The advantages of organoids in the development of tumor vaccines are: first, organoids can retain about 90% of the patient's genetic information; second, the amount of organoids used is small, and the amount of fresh tissue punctured can be continuously amplified. Compared with tumor tissue lysates, there is no limit on the number, and the culture cycle is shorter than that of animal experiments, and it can be used as a long-term and stable source of tumor antigens; third, organoids can also be co-cultured with immune cells to construct an immune microenvironment of tumor-infiltrating lymphocytes that simulates the human environment, which is a good model for guiding patient immunotherapy. Studies have shown that in the application of tumor neoantigen screening, the tumor neoantigen match rate of organoids and their source tissues is 92.8%. Therefore, we use liver cancer organoids as the preparation of personalized tumor vaccines. On the one hand, it can solve the practical problem of specimen acquisition in clinical practice and provide a stable source of antigens; on the other hand, the organoid model can also be used in subsequent immune killing verification experiments, which is a good supplement to animal experiments in terms of the effectiveness and safety evaluation of preclinical transformation.

[0006] Based on the construction of liver cancer organoids, this method optimizes the presentation carrier of tumor antigens. Different from DNA, RNA, protein peptides, the role of exosomes as tumor antigen presentation carriers has gradually been revealed. Exosomes are an extracellular nano-scale vesicle structure that is rich in protein, lipid, and nucleic acid information. Although the diameter is only 30-150nm, its biomembrane structure components can prevent it from being degraded by proteases in the liquid environment between cells, completing efficient and extensive intercellular communication. Exosomes can mediate complex signal transduction between a variety of cells including tumor cells, fibroblasts, immune cells, and vascular endothelial cells, regulate the tumor microenvironment, and affect the occurrence, proliferation, and drug resistance of tumor cells. They are currently popular signal presentation tools in the field of tumors. The results of high-quality foreign papers show that compared with tumor cell lysates, the use of exosomes secreted by tumor cells of the same mass can more significantly activate the immune killing effect of effector T cells, suggesting that exosomes can enrich tumor antigens with higher purity. Therefore, we believe that the use of exosomes to enrich tumor antigens can improve the purity of tumor-specific antigens TSA.

[0007] DC cells are the only professional antigen-presenting cells in the human body and are the most effective and safest antigen carriers for antigen activation. Regardless of whether it is mRNA, DNA, protein peptides or exosomes, they all need to be presented to effector T cells and helper T cells through DC cells to complete the immune activation process. Sipuleucel-T, the world's first cell immunotherapy drug for solid tumors, uses DC cells as carriers of tumor vaccines. In China, a number of DC cell products have also been approved for IND, which fully demonstrates its application prospects. From a safety perspective, compared with adoptive cell therapies such as CAR-T and NK cells, the dosage of DC cells is only 10 6 ~10 7 The number of cells is much lower than that of CAR-T and NK cells. 9 ~10 10 Safety risks caused by the range of cell numbers. From the perspective of effectiveness, DC cells have the advantage of inducing the formation of memory T cells, which is not available in NK and T cell therapies. Therefore, this application uses autologous DC cells induced by the peripheral blood of liver cancer patients as the carrier of tumor vaccine, and finally completes the preparation of personalized DC cell vaccine for liver cancer. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing DC vaccines based on exosomes derived from liver cancer organoids, which is specifically achieved through the following technical solutions:

[0009] The present invention provides a method for preparing a DC vaccine based on exosomes derived from liver cancer organoids, comprising the following steps:

[0010] S1. Construction of liver cancer organoid model;

[0011] S2, extracting exosomes from liver cancer organoid model;

[0012] S3, culturing DC cells from patients with liver cancer;

[0013] S4. Co-culturing the exosomes obtained in step S2 and the DC cells cultured in step S3, and then harvesting the mature DC cells.

[0014] Optionally or preferably, when constructing a liver cancer organoid model, the following steps are included:

[0015] S11. Take fresh tumor tissue from a liver cancer patient, cut it into several pieces, and add it into pre-cooled PBS;

[0016] S12, disinfecting the tissue obtained in step S11, centrifuging with PBS and washing until clear;

[0017] S13, adding collagenase to the tissue fluid obtained in step S12, and chopping the tissue into pieces less than 1 mm 3 , digested at 37°C, then added PBS to terminate the reaction and wash;

[0018] S14, adding DNase to the tissue fluid obtained in step S13, digesting at 37° C., and then adding PBS to terminate the reaction and wash;

[0019] S15. Resuspend the digested tumor tissue with PBS, mix gently and filter with a mesh; incubate the collected cell suspension for 5 minutes and discard the supernatant.

[0020] S16. Resuspend the cell pellet obtained in S15 with pre-cooled matrix gel, quickly spread it on the well plate at low temperature, and incubate it at 37°C after inoculation;

[0021] S17. Add complete culture medium to each well, then change the medium every 2-3 days depending on the cell number, and subculture every 7-10 days.

[0022] Optionally or preferably, the complete culture medium comprises: DMEM / F-1, B-27, N-2, nicotinamide, N-acetyl-L-cysteine, [Leu15]-gastrin, forskolin, A83-01, EGF, FGF10, HGF, RSpo1-conditioned medium, and Wnt3a-conditioned medium.

[0023] Optionally or preferably, when extracting exosomes from the liver cancer organoid model, the following steps are included:

[0024] S21. When changing the medium of the liver cancer organoid model, collect the liver cancer organoid supernatant;

[0025] S22, centrifuging the liver cancer organoid supernatant collected in step S21 to remove cells and cell debris;

[0026] S23, taking the supernatant after centrifugation in step S22 and centrifuging it again to remove cell debris and liposomes;

[0027] S24, taking the supernatant after centrifugation in step S23, and transferring it to an ultracentrifuge for ultracentrifugation;

[0028] S25. After ultracentrifugation, the exosomes aggregated into a precipitate at the bottom of the ultracentrifuge tube. The supernatant was removed, resuspended with PBS, and ultracentrifuged again to remove the culture medium components.

[0029] S26. Remove the supernatant again to obtain the exosome precipitate, which is then placed in a cryopreservation tube for low-temperature storage.

[0030] Optionally or preferably, when culturing DC cells from liver cancer patients, the following steps are included:

[0031] S31. Collect peripheral venous blood from liver cancer patients into sterile centrifuge tubes and add an equal volume of PBS for dilution;

[0032] S32, after mixing the mixture obtained in step S31, slowly adding the mixture to the surface of the lymphocyte separation solution, and separating the mixture by density gradient centrifugation;

[0033] S33. After centrifugation, collect the buffy coat cells, add PBS and mix well, discard the supernatant by centrifugation, repeat this step and wash once more to obtain PBMCs;

[0034] S34, resuspending the PBMC obtained in step S34 in a culture medium containing autologous human serum, inoculating the culture bottles, and culturing them in an incubator;

[0035] S35. After culturing for 24 hours, collect the non-adherent cells in the supernatant of the culture flask in step S34 for subsequent co-culture, then add 1640 culture medium containing 5% autologous human serum, GM-CSF, and IL-4 into the flask, and continue culturing for 5 to 7 days in a 5% CO2, 37°C incubator to induce the formation of DC cells.

[0036] Optionally or preferably, when co-culturing exosomes and DC cells, the DC cells are replaced with 1640 culture medium containing 5% autologous human serum, TNF-α, PGE2, IL-6 and IL-1β, and 40-100ug / ml of organoid exosomes are added, and the cells are cultured in a 5% CO2, 37°C incubator for 2 to 3 days for antigen loading, and mature DC cells are harvested.

[0037] Based on the above technical solution, the following technical effects can be produced:

[0038] The present invention constructs a liver cancer organoid model, extracts exosome components containing liver cancer tumor antigen information, adds them into DC cell culture medium for antigen loading, and finally harvests mature DC cells, thereby achieving specific killing of liver cancer organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0040] Figure 1 The results of organoid microscopy in 3 patients with liver cancer;

[0041] Figure 2 This is the result of exosome nanoflow cytometry detection;

[0042] Figure 3 The results of electron microscopic observation of exosomes;

[0043] Figure 4 This is the test result of exosome protein markers;

[0044] Figure 5 These are the results of the personalized DC cell vaccine organoid killing experiment. DETAILED DESCRIPTION

[0045] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] The present invention provides a method for preparing a DC vaccine based on exosomes derived from liver cancer organoids, comprising the following steps:

[0048] S1. Construction of liver cancer organoid model, including:

[0049] S11, take 8cm from liver cancer patients 3 Cut the above fresh tumor tissue into several pieces, add them into pre-cooled 4℃ PBS, keep the temperature low and transfer them into the biosafety cabinet within 20 minutes;

[0050] S12, immersing the tissue obtained in step S11 in chlorhexidine for 15 minutes for disinfection, centrifuging with PBS and washing until clear;

[0051] S13, add collagenase to the tissue fluid obtained in step S12, and cut the tissue into pieces of 0.5-1 mm 3 , at 37°C for 30 minutes, and then PBS was added to terminate the reaction and wash; wherein the volume ratio of the added collagenase to PBS was 1:2;

[0052] S14, adding DNA enzyme to the tissue fluid obtained in step S13, digesting at 37° C. for 10 minutes, and then adding PBS to terminate the reaction and wash; wherein the volume ratio of the added DNA enzyme to PBS is 1:4;

[0053] S15. Resuspend the digested tumor tissue with 10 ml PBS, mix gently and filter with a 100 um mesh; centrifuge the collected cell suspension at 300 g for 5 minutes and discard the supernatant;

[0054] S16, the cell pellet obtained in S15 was centrifuged at 2×10 6 / ml density was resuspended with pre-cooled matrix gel and quickly spread on the well plate at low temperature, 50ul per well of the 24-well plate, and incubated at 37℃ for 30min after inoculation;

[0055] S16. Add 500ul of complete culture medium to each well, wherein the complete culture medium includes: DMEM / F-1, B-27, N-2, nicotinamide, N-acetyl-L-cysteine, [Leu15]-gastrin, forskolin, A83-01, EGF, FGF10, HGF, RSpo1-conditioned medium, Wnt3a-conditioned medium; then change the medium every 2-3 days according to the number of cells, and passage every 7-10 days.

[0056] S2. Extracting exosomes from liver cancer organoid model, specifically including the following steps:

[0057] S21. When changing the medium of the liver cancer organoid model, collect the liver cancer organoid supernatant, collect 50 ml of culture supernatant to extract exosomes;

[0058] S22, transfer the clear liquid to a centrifuge tube, place the centrifuge tube in a high-speed refrigerated centrifuge, and centrifuge at 1000g / min for 10 minutes to remove cells and cell debris; use a 10ml pipette to aspirate the supernatant and transfer it to a new 50ml centrifuge tube;

[0059] S23, centrifuge again at 10000 g / min for 30 min to remove cell debris and liposomes;

[0060] S24, use a 20ml empty needle to draw the supernatant and transfer it to a 38ml UC open tube for packaging. TM XPN ultracentrifuge, set parameters rotor: SW32 Ti, ultracentrifugation at 100,000 × g for 70 min;

[0061] S25. After ultracentrifugation, the exosomes aggregated into a precipitate at the bottom of the ultracentrifuge tube. The supernatant was removed and resuspended with 4°C precooled PBS. Ultracentrifugation was repeated for 70 min to remove the culture medium components.

[0062] S26. Remove the supernatant again to obtain the exosome precipitate, resuspend it with 100uL 1×PBS, and use a 200uL pipette tip to continuously and gently blow along the bottom wall of the tube. Take 5uL for BCA protein concentration determination and store it in a cryogenic tube at -80℃.

[0063] S3. Cultivating DC cells from liver cancer patients, comprising the following steps:

[0064] S31. Collect peripheral venous blood (heparin anticoagulated) from liver cancer patients into sterile centrifuge tubes and add an equal volume of PBS for dilution;

[0065] S32, after mixing the mixture obtained in step S31, slowly add the mixture to the surface of the lymphocyte separation solution, separate the mixture by density gradient centrifugation, set the centrifugation parameters to 400×g, centrifuge at room temperature for 30 min, and adjust the speed to the lowest setting;

[0066] S33. After centrifugation, collect the buffy coat cells, add PBS and mix well, centrifuge at 400×g for 10 min, discard the supernatant, repeat this step and wash once more to obtain PBMCs;

[0067] S34, resuspending the PBMC obtained in step S34 in 1640 culture medium containing 5% autologous human serum, inoculating into a culture bottle, and culturing in a 5% CO2, 37°C incubator;

[0068] S35. After culturing for 24 hours, collect the non-adherent cells in the supernatant of the culture flask in step S34 for subsequent co-culture, then add 1640 culture medium containing 5% autologous human serum, GM-CSF, and IL-4 into the flask, and continue culturing for 5 to 7 days in a 5% CO2, 37°C incubator to induce the formation of DC cells.

[0069] S4. The obtained cells were changed to 1640 culture medium containing 5% autologous human serum, TNF-α, PGE2, IL-6 and IL-1β, and 40ug / ml of organoid exosomes were added. The cells were cultured in a 5% CO2, 37°C incubator for 2 to 3 days for antigen loading, and mature DC cells were harvested.

[0070] In order to illustrate the beneficial effects of the present invention, fresh tumor tissues and adjacent tissues from 3 liver cancer patients were selected to construct the liver cancer organoid model described in the present invention. Figure 1 Shown are the results of microscopic observation of organoids from three patients with liver cancer.

[0071] The supernatant of liver cancer organoids and liver cancer adjacent tissue organoids was collected to separate and extract exosomes, such as Figure 2 Nanoflow cytometry results showed that the average particle size was 67.77 nm, the particle concentration was 1.33E+10 Particles / mL, and the protein concentration detected by BCA was between 895.45 ng / ul and 4344.64 ng / ul;

[0072] Take 10ul of sample and fix it, then negatively stain it with uranyl acetate. Figure 3 Electron microscopic observations showed that exosomes were typical spherical or bowl-shaped vesicles with a particle size of 30-100 nm;

[0073] like Figure 4 The results of Western blot detection showed that the exosome positive protein markers Tsg101 / Alix / CD9 were expressed, while the exosome negative protein marker Calnexin was not expressed.

[0074] Mature DC cells were isolated and induced by culture from the patient's peripheral blood. During the construction of liver cancer organoids, the exosome components containing tumor antigen information were extracted from the culture medium. Then, the exosomes from liver cancer organoids, the exosomes from paracancerous organoids, liver cancer organoid lysates, and paracancerous organoid lysates were added to the DC cell culture medium for antigen loading. Finally, the mixed immune cells activated by DC cells were added to the patient's autologous liver cancer organoids at a ratio of 10:1. At the same time, Casepase3 / 7 fluorescent markers were used for staining to observe the changes in fluorescence signals in real time. Figure 5 As shown, the results suggested that both exosomes derived from liver cancer organoids and liver cancer organoid lysates showed immunogenicity, and activated effector T cells achieved specific killing of liver cancer organoids.

[0075] The above results suggest that exosomes derived from liver cancer organoids can effectively enrich tumor antigen components, and DC cell vaccines based on liver cancer organoid exosomes loaded with antigens can significantly activate specific anti-tumor immune responses.

[0076] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing a DC vaccine based on exosomes derived from liver cancer organoids, characterized in that: The following steps are involved: S1. Construction of liver cancer organoid model; S2, extracting exosomes from liver cancer organoid model; S3, culturing DC cells from patients with liver cancer; S4. Co-culturing the exosomes obtained in step S2 and the DC cells cultured in step S3, and then harvesting the mature DC cells.

2. The method for preparing a DC vaccine based on exosomes derived from liver cancer organoids according to claim 1, characterized in that: When constructing a liver cancer organoid model, the following steps are included: S11. Take fresh tumor tissue from a liver cancer patient, cut it into several pieces, and add it into pre-cooled PBS; S12, disinfecting the tissue obtained in step S11, centrifuging with PBS and washing until clear; S13, adding collagenase to the tissue fluid obtained in step S12, and chopping the tissue into pieces less than 1 mm 3 , digested at 37°C, then added PBS to terminate the reaction and wash; S14, adding DNase to the tissue fluid obtained in step S13, digesting at 37° C., and then adding PBS to terminate the reaction and wash; S15. Resuspend the digested tumor tissue with PBS, mix gently and filter with a mesh; incubate the collected cell suspension for 5 minutes and discard the supernatant. S16. Resuspend the cell pellet obtained in S15 with pre-cooled matrix gel, quickly spread it on the well plate at low temperature, and incubate it at 37°C after inoculation; S17. Add complete culture medium to each well, then change the medium every 2-3 days depending on the cell number, and subculture every 7-10 days.

3. The method for preparing a DC vaccine based on exosomes derived from liver cancer organoids according to claim 2, characterized in that: The complete culture medium includes: DMEM / F-1, B-27, N-2, nicotinamide, N-acetyl-L-cysteine, [Leu15]-gastrin, forskolin, A83-01, EGF, FGF10, HGF, RSpo1-conditioned medium, and Wnt3a-conditioned medium.

4. The method for preparing a DC vaccine based on exosomes derived from liver cancer organoids according to claim 1, characterized in that: When extracting exosomes from liver cancer organoid models, the following steps are involved: S21. When changing the medium of the liver cancer organoid model, collect the liver cancer organoid supernatant; S22, centrifuging the liver cancer organoid supernatant collected in step S21 to remove cells and cell debris; S23, taking the supernatant after centrifugation in step S22 and centrifuging it again to remove cell debris and liposomes; S24, taking the supernatant after centrifugation in step S23, and transferring it to an ultracentrifuge for ultracentrifugation; S25. After ultracentrifugation, the exosomes aggregated into a precipitate at the bottom of the ultracentrifuge tube. The supernatant was removed, resuspended with PBS, and ultracentrifuged again to remove the culture medium components. S26. Remove the supernatant again to obtain the exosome precipitate, which is then placed in a cryopreservation tube for low-temperature storage.

5. The method for preparing DC vaccine based on exosomes derived from liver cancer organoids according to claim 1, characterized in that: When culturing DC cells from liver cancer patients, the following steps are included: S31. Collect peripheral venous blood from liver cancer patients into sterile centrifuge tubes and add an equal volume of PBS for dilution; S32, after mixing the mixture obtained in step S31, slowly adding the mixture to the surface of the lymphocyte separation solution, and separating the mixture by density gradient centrifugation; S33. After centrifugation, collect the buffy coat cells, add PBS and mix well, discard the supernatant by centrifugation, repeat this step and wash once more to obtain PBMCs; S34, resuspending the PBMC obtained in step S34 in a culture medium containing autologous human serum, inoculating the culture bottles, and culturing them in an incubator; S35. After culturing for 24 hours, collect the non-adherent cells in the supernatant of the culture flask in step S34 for subsequent co-culture, then add 1640 culture medium containing 5% autologous human serum, GM-CSF, and IL-4 into the flask, and continue culturing for 5 to 7 days in a 5% CO2, 37°C incubator to induce the formation of DC cells.

6. The method for preparing a DC vaccine based on exosomes derived from liver cancer organoids according to claim 1, characterized in that: When co-culturing exosomes and DC cells, DC cells were changed to 1640 culture medium containing 5% autologous human serum, TNF-α, PGE2, IL-6 and IL-1β, and 40-100ug / ml of organoid exosomes were added. The cells were cultured in a 5% CO2, 37°C incubator for 2-3 days for antigen loading, and mature DC cells were harvested.

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