A method for inducing the application of pluripotent stem cell-derived exosomes in a DC vaccine

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses a method for applying induced pluripotent stem cell-derived exosomes in a DC vaccine. The application provides a preparation method of a tumor vaccine, which comprises the following steps: using induced pluripotent stem cell exosomes to sensitize DC cells to obtain the tumor vaccine. The induced pluripotent stem cell-derived exosomes provided by the application have the characteristics of uniform particle size, low toxicity, good cell compatibility, high yield and the like. The DC vaccine provided by the application has the abilities of tumor treatment, tumor prevention and tumor metastasis inhibition, and has low toxicity, and is a simple, practical and applicable technology and method. The application solves the problems of tumorigenicity, large volume, interception by lung and other tissues, difficulty in being taken up by DC, only tumor prevention ability and the like of the existing induced pluripotent stem cell vaccine, and solves the problem of lack of antigen source in the construction of the DC vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for applying induced pluripotent stem cell-derived exosome antigens in DC vaccines. Background Technology

[0002] Cancer is a major global health problem and a leading cause of death. Despite significant advances in many treatments over the past two decades, leading to a decrease in mortality rates for some types of cancer, the total number of cancer cases and related mortality rates are expected to continue rising in the foreseeable future. It is estimated that by 2040, there will be over 29 million new cancer cases globally, and cancer-related deaths will account for one-sixth of all deaths. The lack of effective cancer prevention strategies and treatments for cancer patients significantly hinders the goal of conquering cancer. Therefore, the academic community is actively searching for effective cancer treatments, including surgery, radiotherapy, chemotherapy, and immunotherapy. Significant progress has been made in the field of stem cell-based tumor vaccines.

[0003] In 1906, Georg, an assistant to biochemist Paul Ehrlich, The first observation was that inoculation of mice with fetal tissue led to rejection of transplanted tumors. Subsequent extensive preclinical studies have shown that inoculation with embryonic material can prevent not only the growth of transplantable tumors but also tumor formation induced by viruses and chemicals. However, ethical constraints related to embryonic stem cells (ESCs), the reduction in the availability of matching cancer antigens, and the unwanted immune responses arising from HLA incompatibility in allogeneic vaccine settings limit its clinical application. This has prompted the academic community to search for more suitable stem cell-derived anti-tumor vaccines.

[0004] In 2006, Shinya Yamanaka et al. introduced four transcription factors (Oct4, Sox2, Klf4, and cMyc) into mouse fibroblasts via retroviral vectors, transforming them into pluripotent stem cells with morphology and function similar to embryonic stem cells, termed induced pluripotent stem cells (iPSCs). This sparked researchers' interest in using iPSCs as tumor vaccines. In 2018, Joseph Wu and colleagues reported in Cell Stem Cell that autologous somatic cell-induced iPSCs could be used to prepare effective prophylactic and adjuvant vaccines to control tumor growth in syngeneic mouse mesothelioma, as well as breast and skin cancer models. iPSCs have the advantages of easy availability and patient targeting, and fewer ethical concerns than ESCs. However, some intractable problems remain. First, the shared gene expression profiles between iPSCs and cancer cells provide a basis for teratoma formation; despite pre-injection irradiation, tumorigenic risks still exist, leading to serious safety concerns. Secondly, iPSCs, as cell-based vaccines, are relatively large and easily intercepted, making them difficult for dendritic cells (DCs) to phagocytose and present antigens. Furthermore, no therapeutic effect of iPSC vaccines on tumors has been observed, suggesting that established tumors may possess an immunosuppressive microenvironment. Therefore, there is a need to develop a safer iPSC vaccine with both preventative and therapeutic effects as an alternative strategy.

[0005] Currently, exosome-based dendritic cell (DC) vaccines have shown outstanding performance in various cancer immunotherapies. In 2001, Wolfers et al. first discovered that exosomes derived from solid tumors (such as colon and breast cancer), when transported to dendritic cells, lead to the activation of T cell-mediated immune responses, ultimately resulting in tumor rejection. Furthermore, the application of exosomes loaded onto bone marrow-derived dendritic cells (BMDCs) in vitro showed good therapeutic effects against established tumors. In 2002, Andre et al. found that exosomes isolated from malignant ascites in patients constituted a source of tumor antigens, which could be transferred to DCs, whereupon DCs induced the differentiation and expansion of tumor-specific cytotoxic T lymphocytes, thereby generating an anti-tumor response. In 2019, Yaddanapudi et al. discovered that embryonic stem cells carrying GM-CSF could effectively prevent various cancers (colon cancer, lung cancer, melanoma, and breast cancer). To date, tumor exosome vaccines have been extensively explored and researched, but the role of iPSC exosomes in tumor prevention and treatment has not yet been reported. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for applying induced pluripotent stem cell-derived exosome antigens in DC vaccines.

[0007] The primary objective of this invention is to provide a method for preparing a tumor vaccine, comprising the following steps: sensitizing DC cells with induced pluripotent stem cell exosomes to obtain the tumor vaccine.

[0008] Preferably, when using induced pluripotent stem cell exosomes to sensitize DC cells, the total protein concentration of the induced pluripotent stem cell exosomes is 10-100 μg / mL.

[0009] More preferably, when using induced pluripotent stem cell exosomes to sensitize DC cells, the total protein concentration of the induced pluripotent stem cell exosomes can specifically be 80 μg / mL.

[0010] The method for sensitizing DC cells using induced pluripotent stem cell exosomes specifically includes the following steps: adding induced pluripotent stem cell exosomes to the DC cell culture system and culturing for 24 hours.

[0011] The culture system is a serum-free induced pluripotent stem cell culture medium.

[0012] The second objective of this invention is to provide a method for preparing the above-mentioned induced pluripotent stem cell exosomes, comprising the following steps: culturing induced pluripotent stem cells in cell culture medium, and extracting the induced pluripotent stem cell exosomes from the supernatant of the induced pluripotent stem cell culture medium; wherein the cell culture medium is a serum-free cell culture medium.

[0013] The extraction of induced pluripotent stem cell exosomes from the supernatant of the induced pluripotent stem cell culture medium was achieved by ultracentrifugation.

[0014] The method for preparing induced pluripotent stem cell exosomes specifically includes the following steps:

[0015] S1. Induced pluripotent stem cells were cultured in cell culture medium. The supernatant of the culture medium was collected. First, the cells and cell debris were removed by low-speed centrifugation at 800g. Then, the supernatant was collected after centrifugation at 10000g. The supernatant was then centrifuged again at 4℃ and 100000g for 1.5h.

[0016] S2. After completing step S1, resuspend the precipitate with PBS and then centrifuge at 10000g and 4℃ for 1.5h.

[0017] S3. After completing step S2, resuspend the precipitate with PBS to obtain induced pluripotent stem cell exosomes.

[0018] Preferably, the cell culture supernatant in step S1 is specifically the culture supernatant of induced pluripotent stem cells cultured for 24 hours.

[0019] Preferably, the induced pluripotent stem cells are of mouse or human origin.

[0020] Preferably, the tumor vaccine is a melanoma vaccine.

[0021] A third objective of this invention is to provide a tumor vaccine prepared using the above method.

[0022] This invention also provides a product whose active ingredient is dendritic cells (DCs) sensitized to pluripotent stem cell exosomes; the uses of the product include:

[0023] (a) Promotes T cell proliferation;

[0024] (b) Inducing T lymphocytes to secrete IFN-γ;

[0025] (c) Kills tumor cells.

[0026] The present invention also provides the application of the induced pluripotent stem cell exosomes or DC cells sensitized by induced pluripotent stem cell exosomes in the preparation of tumor vaccines.

[0027] This invention also provides the application of the induced pluripotent stem cell exosomes or DC cells sensitized by induced pluripotent stem cell exosomes in the preparation of products; the uses of the products include:

[0028] (a) Promotes T cell proliferation;

[0029] (b) Inducing T lymphocytes to secrete IFN-γ;

[0030] (c) Kills tumor cells.

[0031] The present invention also provides a kit comprising induced pluripotent stem cell exosomes and dendritic cells; the uses of the kit include:

[0032] (a) Promotes T cell proliferation;

[0033] (b) Inducing T lymphocytes to secrete IFN-γ;

[0034] (c) Kills tumor cells.

[0035] The specific method for preparing any of the above-mentioned DC cells can be as follows:

[0036] (1) Mice were euthanized by cervical dislocation, and the tibia and femur of C57BL / 6 mice were taken and bone marrow progenitor cells were collected.

[0037] (2) After incubation at 37°C and 5% CO2 for 4 hours, non-adherent cells were removed by rinsing with phosphate-buffered saline (PBS);

[0038] (3) Adherent cells were cultured in RPMI 1640 medium containing 500 U / mL IL-4 and 1000 U / mL GM-CSF to induce them to differentiate into immature DCs;

[0039] (4) Add 1 μg / mL of LPS on the 8th day of induction to induce DC maturation.

[0040] Compared with the prior art, the beneficial effects of the present invention are:

[0041] This invention utilizes exosomes from induced pluripotent stem cell (iPSC) culture supernatants as DC antigens for the construction of anti-tumor DC vaccines, providing a new approach for developing highly efficient DC vaccines. On one hand, the exosome lipid membrane structure enhances cell entry efficiency; on the other hand, the exosomes in the culture supernatant can carry tumor-associated antigens. Therefore, exosomes from iPSC supernatants significantly improve the activation efficiency of BMDCs in vitro, thereby greatly enhancing the body's anti-tumor immune response. Furthermore, the DC vaccine constructed from exosomes secreted from iPSC supernatants can effectively stimulate T cell proliferation and enhance the cytotoxic effect of CTLs. After activating T cells with exosome-sensitized DCs from iPSC culture supernatants, the T cells were added to tumor cells, and at an effector-to-target ratio of 20:1, they effectively killed tumor cells within 8 hours.

[0042] The induced pluripotent stem cell supernatant exosomes provided by this invention have uniform particle size, low toxicity, good cell compatibility, and are easy to handle and apply in clinical tumor immunotherapy. It is a simple, easy-to-implement, and practical application technology and method. It solves the problems of existing induced pluripotent stem cell vaccines, such as tumorigenicity, large size making them easily intercepted by tissues like the lungs and difficult for dendritic cells (DCs) to take up, and only having tumor prevention capabilities. It also solves the problem of insufficient antigen sources in DC vaccine construction. Attached Figure Description

[0043] Figure 1 A schematic diagram illustrating the principle of a DC vaccine that induces sensitization of pluripotent stem cell exosomes.

[0044] Figure 2 Flowchart for the preparation of mouse-derived induced pluripotent stem cells

[0045] Figure 3 Flowchart for the preparation of human induced pluripotent stem cells

[0046] Figure 4 The NTA results for induced pluripotent stem cell exosomes are shown in the figure.

[0047] Figure 5 Electron micrograph of exosomes from induced pluripotent stem cells.

[0048] Figure 6This is a Western blot image showing the results of the Western blot validation of induced pluripotent stem cell exosomes.

[0049] Figure 7 The activation effect of DCs on T lymphocytes by exosomes derived from pluripotent stem cell culture supernatant.

[0050] Figure 8 A graph showing the analysis of IFN-γ and IL-2 secretion by DC-induced T lymphocytes after activation of exosomes derived from pluripotent stem cell culture supernatant.

[0051] Figure 9 Figure 1 shows the in vitro killing effect of T cells activated by DCs sensitized to pluripotent stem cells (pluripotent stem cells) on tumor cells and the in vitro killing effect of T cells activated by DCs sensitized to tumor cell exosomes on induced pluripotent stem cells.

[0052] Figure 10 The in vivo prevention effect of DCs induced by pluripotent stem cell exosome activation on tumor cells.

[0053] Figure 11 The in vivo therapeutic effect of DCs (dendrones) induced by pluripotent stem cell exosome activation on tumor cells.

[0054] Figure 12 Figure showing the biosafety results of DCs used to induce activation of pluripotent stem cell exosomes. Detailed Implementation

[0055] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0056] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0057] In the quantitative experiments in the following examples, at least three replicate experiments were set up, and the average value of the results was taken.

[0058] The preparation method of induced pluripotent stem cell exosomes in the following examples is as follows: take the cell culture supernatant, centrifuge at 800g for 5 minutes, centrifuge at 2000g for 10 minutes, and finally centrifuge at 100000g at 4℃ for 90 minutes, and reselect the precipitate after centrifugation with PBS.

[0059] Example 1: Preparation of exosomes from induced pluripotent stem cell culture supernatant

[0060] I. Preparation of mouse-derived induced pluripotent stem cells

[0061] (1) Day -2: Resuscitate mouse embryonic fibroblasts from liquid nitrogen and plate them (or use freshly extracted mouse embryonic fibroblasts for direct infection on day 0).

[0062] (2) Day 0: 5 x 10 5 MEF cells were taken from a -80°C freezer and infected with Sendai virus carrying four reprogramming factors (Oct4, c-Myc, Sox2, Klf4) at an MOI of 5:1.

[0063] (3) Days 1-6: Change the medium daily with fresh MEF medium.

[0064] (4) Day 7: Coat the culture dish with 2% gelatin, digest the MEF and transfer it to the 2% gelatin coated culture dish.

[0065] (5) Day 8: Change the medium with a completely mouse-derived induced pluripotent stem cell culture medium.

[0066] (6) Days 9-16: Change the medium daily with complete mouse-derived induced pluripotent stem cell culture medium and observe cell morphology. If large clones are formed or clones are about to grow, manually pick single clones and transfer them to a new culture plate coated with 2% gelatin.

[0067] Analysis results as follows Figure 2 As shown in the figure. The results indicate that MEF-induced mouse iPSC monoclonal cells were successfully produced, purified, and exhibited classic embryonic stem cell morphology.

[0068] II. Preparation of Human Induced Pluripotent Stem Cells

[0069] (1) Day -4: Peripheral blood mononuclear cells were resuscitated from liquid nitrogen and plated (or freshly extracted peripheral blood mononuclear cells were directly infected on day 0).

[0070] (2) Day -3 to Day -1: Use 0.5 mL of fresh whole stempro TM Replace half of the medium with -34 (Gibico, USA) medium.

[0071] (3) Day 0: 5 x 10 5 PBMC cells were infected with Sendai virus carrying four reprogramming factors (Oct4, c-Myc, Sox2, Klf4) at an MOI of 5:1 after being taken from a -80°C freezer.

[0072] (4) Day 1: Use fresh, whole stempro TMThe medium was changed at -34°C (Gibico, USA) to remove Sendai virus.

[0073] (5) Day 3: Inoculated cells were seeded into matrix gel-coated culture dishes and cultured with Stempro, a cytokine-free medium. TM Cultured in -34°C (Gibico, USA) medium.

[0074] (6) Days 4-6: Use cytokine-free stempro every other day TM Change the medium at -34°C (Gibico, USA).

[0075] (7) Day 7: Begin transitioning to Stemflex medium, adding half of the Stemflex medium without cytokines. TM Replace the -34 (Gibico, USA) medium with Stemflex (Gibico, USA) medium.

[0076] (8) Day 8: Replace the entire medium with Stemflex (Gibico, USA) medium to end the transition and continue culturing cells on matrix gel-coated culture dishes.

[0077] (9) Days 9–28: Change the medium daily with fresh Stemflex medium (Gibico, USA) and monitor for the appearance of induced pluripotent stem cell clones in the culture vessel. If large clones are formed or clones are about to grow, manually pick single clones and transfer them to new culture plates coated with matrix gel.

[0078] Analysis results as follows Figure 3 As shown in the figure. The results indicate that human iPSC monoclonal cells induced by PBMCs were successfully generated, purified, and exhibited classic embryonic stem cell morphology.

[0079] III. Obtaining the supernatant from induced pluripotent stem cell culture

[0080] Induced pluripotent stem cells were cultured in serum-free medium, and the cell culture supernatant was collected every 24 hours.

[0081] IV. Extraction of exosomes from cell culture supernatant using ultracentrifugation

[0082] 1. Remove cells and cell debris from the cell culture supernatant obtained in step 2 by centrifugation at 800g, then centrifuge at 10000g, filter through a 0.22μm filter membrane, collect the filtered supernatant in an ultracentrifuge tube, and centrifuge at 100000g at 4℃ for 1.5h.

[0083] 2. After completing step 1, discard the supernatant, wash the precipitate with PBS at 4°C, and rinse the bottom precipitate for more than 2 minutes to obtain a solution containing exosomes.

[0084] 3. Inject the exosome-containing solution obtained in step 2 into an ultracentrifuge tube, and then centrifuge at 100,000 g and 4°C for 1.5 h. After centrifugation, cut off the seal, discard the supernatant, collect the exosomes with PBS, and obtain an exosome suspension, which is then stored at -80°C for later use.

[0085] 4. Use a particle size analyzer to measure the particle size of exosomes.

[0086] (1) Take an EP tube and pipette 5 μL of exosomes into the EP tube. Then, pipette 995 μL of PBS to dilute the exosomes. Generally, dilute the exosomes to a concentration of 2 x 10⁻⁶. 8 / mL to 1x10 9 / mL is preferred.

[0087] (2) Add the exosomes into the NanoSight sample chamber and adjust the instrument parameters. Measure each set of data at least 3 times, for at least 40 seconds each time, until the exosomes appear as sharp spots.

[0088] (3) Repeat the test multiple times, and record no less than 900 nanoparticles each time.

[0089] Analysis results as follows Figure 4 As shown in the figure. The results indicate that the exosome particle size distribution is between 30-150 nm and is relatively uniform.

[0090] 5. Observation of exosome morphology using transmission electron microscopy

[0091] (1) Resuspend the exosomes in double-distilled water, and then drop 10 μL of the exosomes onto a Formvar carbon-coated grid.

[0092] (2) Wait 5 minutes for it to air dry naturally, then use filter paper to absorb the excess exosomes.

[0093] (3) Then, negative staining of exosomes was performed for 2 minutes using 2% uranyl acetate aqueous solution, and excess solution was removed.

[0094] (4) After drying, use a transmission electron microscope to take electron micrographs at 120kV.

[0095] Analysis results as follows Figure 5 As shown in the figure. The results show that the exosomes are elliptical in shape and have a complete double membrane structure.

[0096] 6. Western blot detection of exosome surface markers

[0097] Exosomes are secreted by cells under normal and pathological conditions and contain various membrane proteins and cytoplasmic proteins. Common exosome-specific markers include membrane proteins such as CD9, CD63, and CD81.

[0098] (1) Protein quantification: Protein quantification was performed using the BCA method for exosomes or whole-cell lysates. In short, first, 60 μL of BCA protein standard and 540 μL of double-distilled water were mixed to prepare standards of different concentrations. Then, 50 μL of protein standards of different concentrations or diluted samples were added to each well of a 96-well plate. Next, 150 μL of the BCA reagent mixture was added to each well and incubated at 37°C for 30 minutes. Finally, the samples were detected at 560 nm using a spectrophotometer and the results were calculated.

[0099] (2) Gel preparation and electrophoresis: The concentrations of the separating gel and the compression gel are determined by the specific molecular weight of the target protein. After diluting the sample to an appropriate concentration using lysis buffer, mix it with the loading buffer at a ratio of 3:1, and then heat at 95°C for 10 minutes to denature it. After loading the sample, perform electrophoresis at a constant voltage of 80V. Once the protein markers separate electrophoretic bands of different sizes, adjust the voltage to a constant voltage of 120V for separating gel electrophoresis until the dye front reaches the bottom of the gel.

[0100] (3) Electroporation of proteins: In order to transfer the target protein on SDS-PAGE to the PVDF membrane, electroporation was performed for 2.5 hours at a constant current of 250mA in pre-cooled electroporation buffer.

[0101] (4) Blocking, incubation, and imaging: After electroblotting, the membrane was blocked with 5% BSA. Then, the sheared membrane was incubated overnight at 4°C with the corresponding primary antibody; the sheared membrane was washed three times with TBST (5 minutes each time), followed by incubation with the corresponding secondary antibody (room temperature, 2 hours), and then washed. Finally, the target protein was imaged using ECL chemical imaging.

[0102] Analysis results as follows Figure 6 As shown in the figure. The results indicate that iPSC exosomes express exosome markers such as HSP70 and TSG101 (cytoplasmic proteins), CD63 and CD81 (transmembrane proteins).

[0103] Example 2: Application of induced pluripotent stem cell exosomes in DC vaccines

[0104] I. Activation of DCs by Exosomes of Induced Pluripotent Stem Cells

[0105] 1. Induction and culture of human peripheral blood-derived dendritic cells (DCs)

[0106] (1) Collect 25 mL of peripheral blood from healthy volunteers. Mix the peripheral blood with a pipette, centrifuge at 800 g for 5 min. The upper layer is a light yellow plasma layer, and the remaining liquid is concentrated blood cells.

[0107] (2) The upper plasma obtained in step (1) is placed into a centrifuge tube, sealed with sealing film, and inactivated in a water bath at 56°C for 30 minutes. After centrifugation, the supernatant is collected for later use.

[0108] (3) Take the blood cells obtained in step (1) and slowly transfer the diluted blood cells into a centrifuge tube containing lymphocyte separation medium (GE, USA) at a depth of 1 cm from the separation medium slant. Centrifuge the transferred blood cells at 800g for 15 min. Aspirate the plasma dilution above the white membrane layer into the waste liquid tube; then aspirate the white membrane layer (mononuclear cells) in the centrifuge tube, transfer it to a new centrifuge tube, centrifuge at 800g for 5 min, discard the supernatant, and collect the cell pellet.

[0109] (4) Resuspend the cells obtained in step (3) in serum-free AIM-V (Gibco, USA) medium without added cytokines and adjust the cell density to 2 × 10⁶ cells / year. 6 Inoculate one well per well in a 24-well culture plate and incubate aseptically for 2 hours at 37°C and saturated humidity with 5% CO2.

[0110] (5) After completing step (4), collect the suspended cells and culture medium in new 15 mL centrifuge tubes. Add 1 mL of AIM-V (Gibco, USA) medium supplemented with GM-CSF (Peprotech, USA) and IL-4 (Peprotech, USA) cytokines to each well of a 24-well plate after removing the suspended cells and medium. Incubate aseptically for 2 days at 37°C with 5% CO2 and saturated humidity. On day 3, use a pipette to remove 500 μL of the old medium, being careful not to insert the pipette tip too deep into the bottom of the plate to avoid losing too many cells. Replenish with 500 μL of fresh AIM-V (Gibco, USA) medium containing GM-CSF and IL-4 cytokines and continue culturing. On day 5, observe the cells under a microscope to see a distinct dendritic morphology. Collect semi-suspended cells and test the expression rate of the DC surface marker CD11c. If the expression rate is greater than 90%, the DC cell induction culture is complete.

[0111] 2. Induction of pluripotent stem cell exosome-sensitized DCs: The DCs prepared in step 1 were grouped as follows: Exosome group (iPSC-EXO): The exosome suspension prepared in Example 1 (total exosome protein concentration of 80 μg / mL) was added to the culture system. Untreated group (Medium): No treatment was performed. Cells and culture supernatants from each group were collected on day 2 for subsequent detection.

[0112] 3. Flow Cytometry Detection of DC Surface Marker Expression: DC cells in each group were washed twice with PBS containing 1% BSA, centrifuged at 1200 rpm for 5 min each time, and collected for analysis. PE-labeled CD11c (Biolegend, USA), APC-labeled CD80 (Biolegend, USA), FITC-labeled CD83 (Biolegend, USA), APC-labeled CD86 (Biolegend, USA), and PE-labeled CD14 (Biolegend, USA) were added, and the mixture was thoroughly vortexed and incubated at 4°C in the dark for 20 min. After incubation, the cells were washed twice with 1 mL of 4°C pre-chilled PBS (Hyclone, USA) containing 1% BSA (Sigma, USA), centrifuged at 1400 rpm for 5 min each time to remove unbound antibodies. The cells were resuspended in 300 μL of 4°C pre-chilled PBS containing 1% BSA, and the expression of surface molecules was analyzed by flow cytometry.

[0113] II. Activation of T lymphocytes by DCs activated from exosomes derived from induced pluripotent stem cell culture supernatant

[0114] 1. Activation effect of DCs induced by exosomes of pluripotent stem cells on T lymphocytes: Mononuclear cells were isolated from human peripheral blood using density gradient centrifugation. After 2 hours of adhesion, the suspended cells were harvested as T lymphocytes. The experiment was divided into three groups: Experimental group: Exosome-derived DCs (iPSC-EXO) collected in step 1 were cultured with T lymphocytes at a ratio of 1:10. On day 1, 1000 U / mL of IL-2 cytokine was added. On day 3, the medium was replaced by half and the above cytokine was added. On day 4, the cell supernatant and cells were collected. DC group: Blank DCs were cultured with T lymphocytes at a ratio of 1:10. On day 1, 1000 U / mL of IL-2 cytokine was added. On day 3, the medium was replaced by half and the above cytokine was added. On day 4, the cell supernatant and cells were collected. Blank control group: The same amount of T cells as the two groups above were added to the same amount of PBS. On day 1, 1000 U / mL of IL-2 cytokine was added. On day 3, half the amount of medium was replaced and the above cytokine was added. On day 4, the cell supernatant and cells were collected.

[0115] Blank group: The untreated group (Medium) collected in step 1, 2, was added to cultured T lymphocytes. On day 1, 1000 U / mL of IL-2 cytokine was added. On day 2, the medium was changed by half and the above cytokine was added. On day 3, the cell supernatant and cells were collected.

[0116] After the above treatment, the cell proliferation of each group was detected using the CCK8 assay kit.

[0117] Results analysis: such as Figure 7 As shown, compared with the blank control and blank DC group, T cells in the iPSC exosome sensitized DC group showed significant proliferation.

[0118] 2. ELISA detection of IFN-γ content in culture supernatant

[0119] The cell supernatants collected from each group in step 1 for 24 hours were analyzed using ELISA to detect the IFN-γ and IL-2 levels. Blank wells (blank control wells without sample or enzyme-labeled reagent, all other steps were the same), standard wells, and sample wells were prepared. Antibody was added to each well at 100 μL and incubated overnight at 4°C. The 96-well plate was washed four times with 0.5% Tween-20 PBS, the last time being gently patted dry. 200 μL of buffer diluent was added to each well, and the plate was blocked at room temperature for 1 hour. The diluent was removed, and the 96-well plate was washed four times with 0.5% Tween-20 PBS, the last time being gently patted dry. 100 μL of standard and DC supernatant from each group were added to each well, and the plate was incubated at room temperature for 2 hours.

[0120] After incubation, remove the liquid from the well plate, wash the 96-well plate four times with 0.5% Tween-20 PBS, and pat dry the plate as much as possible after the last wash. Add 100 μL of detection antibody to each well and incubate at room temperature for 1 hour. After incubation, remove the liquid from the well plate, wash the 96-well plate four times with 0.5% Tween-20 PBS, and pat dry the plate as much as possible after the last wash. Add 100 μL of HPR-labeled avidin to each well and incubate at room temperature in the dark for 30 minutes. After incubation, remove the liquid from the well plate, wash the 96-well plate five times with 0.5% Tween-20 PBS, and pat dry the plate as much as possible after the last wash. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 15 minutes. After incubation, 100 μL of 1 mol / L H2SO4 was added to each well to stop the color development. The absorbance (OD value) at 450 nm was measured using a microplate reader, and the concentration of cytokines in the sample was calculated.

[0121] Results analysis: such as Figure 8 As shown, compared with the blank control and blank DC group, the levels of IFNγ and IL-2 in the culture supernatant of T cells in the iPSC exosome sensitized DC group were significantly increased, suggesting that T cells were activated by iPSC exosome sensitized DC.

[0122] 3. The in vitro killing effect of T cells stimulated by DCs activated by pluripotent stem cell exosomes on tumor cells.

[0123] B16F10 cells were fed at a rate of 1×10 5The cells were resuspended at a concentration of 1 / mL in 96-well culture plates and cultured at 37°C, 5% CO2, and saturated humidity for 8 hours. Then, the plates were washed once with 37°C medium to remove any non-adherent cells.

[0124] Splenic T lymphocytes from individuals vaccinated with DC+EXO vaccine and from the control group were mixed with B16F10, LLC, 4T1, and MC38 cells at effector-to-target ratios of 5:1, 10:1, and 20:1 and cultured at 37°C, 5% CO2, and saturated humidity for 8 hours.

[0125] Using the same method, DC vaccines sensitized with 80ug / ml B16-F10 exosomes and controls were administered. Splenic T cells from each group were then incubated with miPSCs and hiPSCs at effector-target ratios of 5:1, 10:1, and 20:1 for 8 hours.

[0126] The killing effect of each group was detected by the lactate dehydrogenase method, and the OD value at a wavelength of 495 nm was measured by an ELISA reader. Cell kill rate was calculated as follows: Cell kill rate (%) = (OD value of experimental group - OD value of minimum kill control group) / (OD value of maximum kill control group - OD value of minimum kill control group) × 100%.

[0127] The results are as follows Figure 9 As shown. The results indicated that after co-culturing effector cells and target cells stimulated by activated DCs in different treatment groups at different effector-to-target ratios (20:1, 10:1, and 5:1) for 8 hours, a significant cytotoxic killing response could be observed. When the effector-to-target ratio was 20:1, the DC+mEXO-induced cytotoxicity against B16-F10 cells was 53.0±0.7% (…). Figure 9 a) LLC cells (40.0±1.9%) Figure 9 b) 4T1 cells (30.1±1.8%) Figure 9 c) and MC38 cells (23.5 ± 0.6%) Figure 9 d) Lymphotoxic response, and DC+hEXO-induced effects on B16F10 cells (49.3±0.3%) Figure 9 a) LLC cells (38.9±0.4%) Figure 9 b) Specific killing responses were observed in 4T1 cells (23.6±1.1%) and MC38 cells (21.5±0.4%) compared to PBS (23.5±0.6%), showing greater effectiveness than in the DC and PBS groups. To test whether this killing effect was based on shared antigenic epitopes between iPSCs and cancer cells, we prepared a DC vaccine sensitized with B16-F10 exosomes (DC+BEXO). Mice were then inoculated with DC+BEXO using the same protocol; DC+BEXO also induced specific killing responses against miPSCs (54.4±1.0%). Figure 9e) and hiPSC (45.0 ± 0.9%) Figure 9 f) Lymphotoxicity. These results indicate that both DC+hEXO and DC+mEXO possess broad-spectrum antitumor activity based on shared antigenic epitopes, particularly against melanoma.

[0128] 4. The in vivo preventive and therapeutic effects of induced pluripotent stem cell exosome-activated dendritic cells (DCs) on tumor cells.

[0129] Mice were administered a DC vaccine loaded with iPSC exosomes four times consecutively, with each dose given weekly. Seven days after the last vaccination, 1×10⁻⁶ mmol / L of the vaccine was administered subcutaneously. 6 Melanoma cells were used to observe melanoma growth. The preventive effect of a DC vaccine loaded with iPSC exosomes on melanoma was verified.

[0130] The results are as follows Figure 10 As shown in the figure, mice were treated with different groups and then inoculated with B16 cells. The results showed that tumor growth was significantly inhibited in the DC+EXO group, and the tumors were 3-4 times smaller than those in the PBS group and the DC group.

[0131] 1×10 was administered subcutaneously to the back beforehand. 6 Mouse melanoma cells were injected intratumorally with a DC vaccine loaded with iPSC exosomes 5 days later (once every 3 days for a total of 4 times) to test its therapeutic effect on tumors.

[0132] The results are as follows Figure 11 The study showed that mice were first inoculated with B16 cells, and then treated using the methods described in the respective treatment groups. The results indicated that tumor growth was significantly inhibited in the DC+mEXO group, with tumors being 1-3 times smaller than those in the PBS and DC groups.

[0133] 5. Biosafety of DCs induced to activate pluripotent stem cell exosomes

[0134] To verify the biosafety of DC+EXO, we continuously monitored the body weight of mice. After 12 weeks of observation, there was no significant difference in body weight between different groups. Figure 12 a). Furthermore, after co-culturing with DC+EXO, we analyzed the activity of human L02 normal hepatocytes and HK2 normal renal proximal tubular epithelial cells using CCK8 assay. No significant differences were found between different concentration groups, indicating that DC+EXO does not alter the activity of these normal hepatic and renal proximal tubular epithelial cell lines. Figure 12 b). In addition, we measured the levels of alkaline phosphatase (ALP) and alanine aminotransferase (ALT), which are indicators of liver damage. Figure 12 c, 12d). We also measured creatinine (Cre) and blood urea nitrogen (BUN) levels, which are indicators of liver damage. Figure 12e, 12f). Simultaneously monitor white blood cells (WBC), red blood cells (RBC), and platelets (PLT) (e, 12f). Figure 12 g, 12h, 12i). There was no significant difference between the DC+EXO treatment group and the control group. Finally, we prepared HE sections of the inoculated and control mice (g, 12h, 12i). Figure 12 j). The results showed that the DC vaccine had no significant toxicity to the viscera of mice. In conclusion, the DC+EXO vaccine demonstrated good biocompatibility in vivo.

[0135] Obviously, the specific implementation schemes described above are merely a further detailed explanation of the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above descriptions are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a melanoma vaccine, characterized by, The method comprises the following steps: using induced pluripotent stem cell exosomes to sensitize DC cells to obtain the melanoma vaccine; when the induced pluripotent stem cell exosomes are used to sensitize the DC cells, the total protein concentration of the induced pluripotent stem cell exosomes is 80 μg / mL; and the induced pluripotent stem cells are induced from mouse embryonic fibroblasts or human peripheral blood mononuclear cells.

2. The method for preparing a melanoma vaccine according to claim 1, characterized in that, The induced pluripotent stem cell exosomes are obtained by the following method: culturing induced pluripotent stem cells in a cell culture medium, and extracting the induced pluripotent stem cell exosomes from the supernatant of the induced pluripotent stem cell culture medium; and the cell culture medium is a serum-free cell culture medium.

3. The melanoma vaccine prepared by the method in any one of claims 1-2.

4. Use of induced pluripotent stem cell exosome sensitized DC cells in the preparation of a melanoma vaccine, characterized in that, The method comprises the following steps: using induced pluripotent stem cell exosomes to sensitize DC cells to obtain the melanoma vaccine; when the induced pluripotent stem cell exosomes are used to sensitize the DC cells, the total protein concentration of the induced pluripotent stem cell exosomes is 80 μg / mL; and the induced pluripotent stem cells are induced from mouse embryonic fibroblasts or human peripheral blood mononuclear cells.

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