Mitochondrial transplanted dendritic cells, and preparation method and application thereof

Abstract

The application discloses a kind of mitochondrion transplanted dendritic cells (DCs) and its preparation method and application, belong to cell therapy field, the mitochondrion transplanted dendritic cell includes: (Z1) free mitochondrion;And (Z2) dendritic cell;Wherein, the free mitochondrion and dendritic cell are obtained by co-incubation 12~48h the mitochondrion transplanted dendritic cell, this method is suitable for the mitochondrion transplantation of a variety of different donor cell sources, DCs can be functionally oriented construction according to actual application target.The mitochondrion transplanted dendritic cell of the application significantly enhances the function of DCs by mitochondrion transplantation, not only significantly improves its survival rate in adversity, but also promotes tumor antigen presentation capacity about five times, so as to show more excellent curative effect in antitumor immunotherapy and a variety of disease intervention.

Description

Technical Field

[0001] This invention belongs to the field of cell therapy, specifically relating to a mitochondrial transplantable dendritic cell, its preparation method, and its application. Background Technology

[0002] Dendritic cells (DCs) are powerful antigen-presenting cells that act as a bridge between innate and adaptive immunity in the immune system. When the body encounters pathogens or tumor antigens, immature DCs take up and process the antigens, mature upon stimulation (such as inflammatory signals), and then migrate to lymph nodes. There, they contact T cells and present the antigens to their T cell receptors, thereby activating naïve T cells and triggering a specific immune response. This process is crucial for the body to defend against infection, eliminate tumors, and maintain immune homeostasis. However, the application of dendritic cells in actual immune responses or immunotherapy still faces several challenges. First, in the tumor microenvironment, DCs are easily inhibited by immunosuppressive factors (such as TGF-β and IL-10), leading to impaired maturation and reduced antigen presentation and T cell activation. Second, DCs induced in vitro have short survival times and low migration efficiency in vivo, making it difficult for them to effectively enter lymphatic tissues. In addition, DCs from different individuals exhibit functional differences, and the preparation process lacks standardization, affecting their widespread application in vaccines and cell therapy.

[0003] Therefore, there is an urgent need in this field to develop methods that can enhance the activity of DCs, prolong their functional time, and improve their in vivo efficacy in order to solve the problems encountered in current research and clinical applications. Summary of the Invention

[0004] The present invention aims to develop a method that can enhance the activity of DCs, prolong their functional time and improve their in vivo efficacy, specifically relating to a mitochondrial transplantable dendritic cell and its preparation method and application.

[0005] In a first aspect of the invention, a mitochondrial transplanted dendritic cell is provided, which is obtained by co-incubating free mitochondria with dendritic cells.

[0006] In another preferred embodiment, the mitochondrial transplanted dendritic cells contain 3-60% exogenous mitochondria.

[0007] In another preferred embodiment, the dendritic cells are bone marrow-derived dendritic cells (BMDCs).

[0008] In another preferred embodiment, the proportion of exogenous mitochondria in the dendritic cells is obtained by counting the staining of exogenous and exogenous mitochondria.

[0009] In another preferred embodiment, the free mitochondria are obtained by extracting mitochondria from donor cells, wherein the donor cells are selected from the group consisting of: stem cells, immune cells, platelets, erythrocytes, glial cells, ependymal cells, neurons, fibroblasts, endothelial cells, muscle cells, epithelial cells, fibroblasts, tumor cells, keratinocytes, brown adipocytes, white adipocytes, hepatocytes, osteoblasts, osteogenic precursor cells, or combinations thereof.

[0010] In another preferred embodiment, the stem cells are selected from the group consisting of: bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, synovial membrane-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, lung mesenchymal stem cells, liver mesenchymal stem cells, skeletal muscle mesenchymal stem cells, hematopoietic stem cells, neural stem cells, epithelial stem cells, skeletal muscle stem cells, lung stem cells, intestinal stem cells, dental pulp stem cells, spermatogonia, and embryonic stem cells.

[0011] In another preferred embodiment, the immune cells are selected from the group consisting of: DCs, T cells, B cells, natural killer cells (NK cells), NKT cells, γδT cells, macrophages, neutrophils, eosinophils, and basophils.

[0012] In another preferred embodiment, the glial cells are selected from the group consisting of astrocytes, oligodendrocytes, Schwann cells, and microglia.

[0013] In another preferred embodiment, the neurons are selected from the group consisting of: cerebral cortical neurons, cerebellar neurons, brainstem neurons, spinal cord neurons, somatic neurons, autonomic neurons, ganglion neurons, retinal neurons, auditory neurons, olfactory neurons, and gustatory neurons.

[0014] In another preferred embodiment, the fibroblasts are selected from the group consisting of: lung fibroblasts, cardiac fibroblasts, renal fibroblasts, intestinal fibroblasts, tumor-associated fibroblasts, synovial fibroblasts, and skin fibroblasts.

[0015] In another preferred embodiment, the endothelial cells are selected from the group consisting of: lymphatic endothelial cells, endocardial endothelial cells, vascular endothelial cells, brain microvascular endothelial cells, alveolar capillary endothelial cells, hepatic sinusoidal endothelial cells, glomerular endothelial cells, cardiac endothelial cells, intestinal endothelial cells, intestinal microvascular endothelial cells, and ocular endothelial cells.

[0016] In another preferred embodiment, the muscle cells are selected from the group consisting of cardiomyocytes, skeletal muscle cells, and smooth muscle cells.

[0017] In another preferred embodiment, the epithelial cells are selected from the group consisting of: skin epithelial cells, nasal cavity epithelial cells, pharyngeal epithelial cells, tracheal and bronchial epithelial cells, terminal bronchial epithelial cells, alveolar epithelial cells, oral cavity epithelial cells, esophageal epithelial cells, gastric epithelial cells, small intestinal epithelial cells, large intestinal epithelial cells, liver epithelial cells, pancreatic epithelial cells, renal tubular epithelial cells, glomerular epithelial cells, ureteral epithelial cells, bladder epithelial cells, urethral epithelial cells, ureteral epithelial cells, endometrial epithelial cells, vaginal epithelial cells, penile urethral epithelial cells, salivary gland epithelial cells, thyroid epithelial cells, breast epithelial cells, corneal epithelial cells, nasal cavity epithelial cells, and taste bud epithelial cells of the tongue.

[0018] In another preferred embodiment, the fibroblasts are selected from the group consisting of: lung fibroblasts, cardiac fibroblasts, renal fibroblasts, intestinal fibroblasts, tumor-associated fibroblasts, and synovial fibroblasts.

[0019] In another preferred embodiment, the tumor cells are selected from the group consisting of: leukemia cells, pancreatic cancer cells, breast cancer cells, lung cancer cells, colorectal cancer cells, liver cancer cells, prostate cancer cells, esophageal cancer cells, ovarian cancer cells, gastric cancer cells, uterine cancer cells, bladder cancer cells, thyroid cancer cells, kidney cancer cells, melanoma cells, and lymphoma cells.

[0020] In another preferred embodiment, the donor cells for the free mitochondria are selected from the group consisting of: embryonic fibroblast cell line 3T3 cells, human embryonic kidney cell line 293T cells, human pancreatic cancer cell line PANC-1 cells, human monocytic leukemia cell line THP-1, and mouse monocytic macrophage cell line RAW 267.4 cells.

[0021] In another preferred embodiment, the mitochondrial donor cell may be a cell of different species, including: mammalian cells, bird cells, fish cells, insect cells, plant cells, microbial cells, etc.

[0022] In another preferred embodiment, the dendritic cells are selected from the group consisting of: human conventional myeloid dendritic cells, human plasmacytoid dendritic cells, or human mononuclear cell-derived dendritic cells; preferably, the dendritic cells are selected from the group consisting of: bone marrow-derived dendritic cells (BMDCs), mononuclear cell-derived dendritic cells (MoDCs), plasmacytoid dendritic cells (pDCs), Langerhans cells (LCs) or their precursor cells, isolated and enriched classical dendritic cell subsets cDC1 or cDC2, and dendritic cells directly isolated from peripheral blood (PBDCs).

[0023] In another preferred embodiment, the dendritic cells are selected from the group consisting of human peripheral blood mononuclear cells and bone marrow-derived dendritic cells (BMDCs).

[0024] In a second aspect of the invention, a method for preparing mitochondrial transplantable dendritic cells as described in the first aspect is provided, the method comprising the steps of:

[0025] Mitochondrial transplanted dendritic cells were obtained by co-incubating dendritic cells with free mitochondria.

[0026] In another preferred embodiment, during the incubation process, the ratio of the number of free mitochondria to DCs is 0.5 to 1.5:1.

[0027] In another preferred embodiment, during the incubation process, the co-incubation time of the free mitochondria and DCs is 12 to 48 hours.

[0028] In another preferred embodiment, the dendritic cells are prepared by taking peripheral blood or bone marrow and inducing differentiation to obtain dendritic cells (DCs).

[0029] In another preferred embodiment, the free mitochondria are prepared by the following method: donor cells are digested, the cell pellet is collected by centrifugation, and the free mitochondria are obtained by extraction using a mitochondrial extraction kit and centrifugation.

[0030] In another preferred embodiment, the incubation step further includes: co-incubating the free mitochondria with the dendritic cells (DCs) and then centrifuging to obtain mitochondrial transplanted dendritic cells.

[0031] In another preferred embodiment, the ratio of free mitochondria to DCs is between 0.1 and 5:1, with the number of free mitochondria calculated based on the number of donor cells.

[0032] In another preferred embodiment, the co-incubation time is 6 to 72 hours.

[0033] In another preferred embodiment, the method includes the steps of:

[0034] i) Collect peripheral blood or bone marrow and induce differentiation to obtain dendritic cells (DCs);

[0035] ii) Collect donor cells by digestion and centrifugation to collect cell pellets, then extract and collect free mitochondria using a mitochondrial extraction kit by centrifugation;

[0036] iii) After co-incubating the free mitochondria obtained in step ii) with the DCs obtained in step i), the mitochondrial transplanted dendritic cells were obtained by centrifugation;

[0037] The ratio of free mitochondria to DCs is between 0.1 and 5:1, and the number of free mitochondria is calculated based on the number of donor cells.

[0038] The co-incubation time described in step ii) is 6 to 72 hours.

[0039] In another preferred embodiment, the preparation of dendritic cells in step i) includes the following steps:

[0040] i-1) Take peripheral blood and mix it with PBS and slowly chromatographically filter it onto Ficoll solution. After centrifugation, collect the white turbid layer, then wash it with PBS to remove platelets. Place the cells in 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin and antibiotics) and incubate in an incubator. Remove the suspended cells to obtain peripheral blood mononuclear cells.

[0041] i-2) Peripheral blood mononuclear cells were cultured in 1640 complete medium containing 20 ng / mL GM-CSF and 10 ng / mL IL-4. Suspension cells were collected and centrifuged to obtain dendritic cells (DCs); or

[0042] ia) Bone marrow cells were collected, filtered, lysed, and centrifuged to obtain a cell suspension. The suspension was then cultured in 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin and iodine) containing 20 ng / mL GM-CSF and 10 ng / mL IL-4. The suspended cells were collected and centrifuged to obtain BMDCs.

[0043] In another preferred embodiment, the ratio of free mitochondria to DCs in step iii) is 0.5 to 1.5:1.

[0044] In another preferred embodiment, the co-incubation time of the free mitochondria and DCs in step iii) is 12 to 48 hours.

[0045] In a third aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0046] 1) Mitochondrial transplanted dendritic cells as described in the first aspect; and

[0047] 2) Pharmaceutically acceptable carrier.

[0048] In another preferred embodiment, the content of the mitochondrial transplanted dendritic cells in the pharmaceutical composition is 50%-100%.

[0049] In another preferred embodiment, the mitochondrial transfer efficiency of the mitochondrial transplanted dendritic cells is 50%-100%, and the transfer efficiency is quantified by the proportion of dendritic cells with successfully transferred mitochondria to the total number of dendritic cells.

[0050] In another preferred embodiment, the exogenous mitochondria in the mitochondrial transplanted dendritic cells account for 3%-60% of the total mitochondria.

[0051] In another preferred embodiment, the pharmaceutically acceptable carrier includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents.

[0052] In another preferred embodiment, the pharmaceutically acceptable carrier includes one or a combination of water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, etc.

[0053] In a fourth aspect of the invention, there is provided the use of mitochondrial transplanted dendritic cells as described in the first aspect or pharmaceutical compositions as described in the third aspect for the preparation of formulations for cancer treatment and / or immune-related diseases, wherein the formulations comprise:

[0054] (1) the mitochondrial transplanted dendritic cells described in the first aspect or the pharmaceutical composition described in the third aspect; and

[0055] (2) Medications for cancer treatment and / or immune-related diseases.

[0056] In another preferred embodiment, the cancer is selected from the group consisting of: liver cancer, colorectal cancer, prostate cancer, ovarian cancer, breast cancer, lung cancer, stomach cancer, pancreatic cancer, esophageal cancer, uterine cancer, bladder cancer, thyroid cancer, kidney cancer, glioma, meningioma, neuroblastoma, melanoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, osteosarcoma, synovial sarcoma, liposarcoma, rhabdomyosarcoma, etc.

[0057] In another preferred embodiment, the immune-related disease is selected from the group consisting of: systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, asthma, atopic dermatitis, allergic rhinitis, inflammatory bowel disease, psoriasis, atherosclerosis, congenital dendritic dysfunction, chronic granulomatous disease, acute immune rejection, and immune tolerance.

[0058] The drugs for immune-related diseases, such as those for mitochondrial transplantation dendritic cells as described in the first aspect or the drug compositions as described in the third aspect, are drugs for DC-related immunotherapy and DC-related synergistic immunotherapy.

[0059] The DCs-related immunotherapy and DCs-related synergistic immunotherapy are selected from the following groups: engineered DCs therapy, DCs synergistic antigen therapy, DCs synergistic exosome therapy, chimeric antigen receptor DCs therapy, DCs combined with immune checkpoint inhibitor therapy, DCs synergistic T-cell therapy, DCs synergistic NK-cell therapy, DCs synergistic chemotherapy therapy, DCs synergistic targeted drug therapy, DCs synergistic hormone therapy, and DCs synergistic radiotherapy therapy.

[0060] In another preferred embodiment, the drug for cancer treatment and / or immune-related diseases is a tumor antigen.

[0061] In another preferred embodiment, the tumor antigen is selected from the group consisting of inactivated MC38 cells and inactivated RM-1 cells.

[0062] The formulation of the mitochondrial transplanted dendritic cells as described in the first aspect or the pharmaceutical composition as described in the third aspect has one or more characteristics selected from the group consisting of:

[0063] (a) Dendritic cell survival rate >85% in high concentrations of LPS;

[0064] (b) Improve the maturation degree of dendritic cells and enhance their antigen presentation ability;

[0065] (c) Reduces tumor volume and enhances anti-tumor effect.

[0066] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0067] Figure 1 This is a map showing the colocalization of endogenous and exogenous mitochondria.

[0068] Figure 2 Changes in BMDC cell survival rate after mitochondrial transplantation.

[0069] Figure 3 This represents the changes in the maturation level of BMDCs after mitochondrial transplantation.

[0070] Figure 4 The transplantation efficiency of mitochondria in BMDCs.

[0071] Figure 5 Changes in tumor volume before and after treatment in MC38 tumor-bearing mice.

[0072] Figure 6 Changes in body weight before and after treatment in MC38 tumor-bearing mice. Detailed Implementation

[0073] Through extensive and in-depth research and numerous experimental screenings, the inventors have unexpectedly developed a novel mitochondrial transplantation dendritic cell (DC) method, obtained by co-incubating free mitochondria with dendritic cells. In this invention, mitochondrial transplantation dendritic cells are prepared by co-incubating a specific ratio of free mitochondria with dendritic cells under certain conditions. This method is applicable to mitochondrial transplantation from various donor cell sources, and DCs can be functionally targeted according to specific application goals. Furthermore, the mitochondrial transplantation dendritic cells of this invention significantly enhance DC function through mitochondrial transplantation. On the one hand, it improves the maturation degree of mitochondrial transplantation dendritic cells, increasing tumor antigen presentation capacity by approximately five times; on the other hand, it significantly improves survival rate in adverse environments such as high concentrations of lipopolysaccharide (LPS), achieving a survival rate >85%. It exhibits high biocompatibility and can be used in combination with drugs for cancer treatment and / or immune-related diseases, particularly in DC-related immunotherapy and synergistic immunotherapy, demonstrating superior efficacy in anti-tumor immunotherapy and intervention for various diseases, possessing significant clinical application value. Based on this, the inventors completed this invention.

[0074] Terminology Explanation

[0075] This invention relates to mitochondrial transplanted dendritic cells

[0076] Mitochondrial transfer refers to the biological process of mitochondria being transported from one cell to another. This process can be achieved through various mechanisms, including intercellular nanotubes, extracellular vesicles, cell fusion, release and uptake of free mitochondria, etc. Mitochondrial transfer plays an important role in various physiological and pathological processes, such as tissue repair, immune regulation, tumor microenvironment remodeling, and the progression of neurodegenerative diseases. By transferring functional mitochondria, donor cells can improve the metabolic state and mitochondrial function of recipient cells, and it has become an important component of intercellular metabolic interactions and signal transduction.

[0077] This invention develops a novel mitochondrial transplanted DCs, which improves the biological function of DCs by transplanting mitochondria from different sources into DCs, thereby demonstrating superior efficacy in anti-tumor immunotherapy and intervention of various diseases.

[0078] Preferably, the mitochondrial transplanted dendritic cells of the present invention are mitochondrial transplanted BMDCs.

[0079] The mitochondrial transplanted dendritic cells of the present invention comprise: (Z1) free mitochondria; and (Z2) dendritic cells; wherein the free mitochondria and dendritic cells are co-incubated for 12 to 48 hours to obtain the mitochondrial transplanted dendritic cells.

[0080] Method for preparing mitochondrial transplantable dendritic cells according to the present invention

[0081] In this invention, the mitochondrial transplanted dendritic cells are prepared through the following steps:

[0082] i) Collect peripheral blood or bone marrow and induce differentiation to obtain dendritic cells (DCs);

[0083] ii) Collect donor cells by digestion and centrifugation to collect cell pellets, then extract and collect free mitochondria using a mitochondrial extraction kit by centrifugation;

[0084] iii) After co-incubating the free mitochondria obtained in step ii) with the DCs obtained in step i), the mitochondrial transplanted dendritic cells were obtained by centrifugation;

[0085] The ratio of mitochondria to DCs is between 0.1 and 5:1, with the number of mitochondria calculated based on the number of donor cells.

[0086] The co-incubation time described in step ii) is 6 to 72 hours.

[0087] Preferably, the preparation of dendritic cells in step i) includes the following steps:

[0088] i-1) Take peripheral blood and mix it with PBS and slowly chromatographically filter it onto Ficoll solution. After centrifugation, collect the white turbid layer, then wash it with PBS to remove platelets. Place the cells in 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin and antibiotics) and incubate in an incubator. Remove the suspended cells to obtain peripheral blood mononuclear cells.

[0089] i-2) Peripheral blood mononuclear cells were cultured in 1640 complete medium containing 20 ng / mL GM-CSF and 10 ng / mL IL-4. Suspension cells were collected and centrifuged to obtain dendritic cells (DCs); or

[0090] ia) Bone marrow cells were collected, filtered, lysed, and centrifuged to obtain a cell suspension. The suspension was then cultured in 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin and iodine) containing 20 ng / mL GM-CSF and 10 ng / mL IL-4. The suspended cells were collected and centrifuged to obtain BMDCs.

[0091] Preferably, the mitochondrial transplanted dendritic cells are prepared through the following steps:

[0092] i-1) Take peripheral blood and mix it with PBS and slowly chromatographically filter it onto Ficoll solution. After centrifugation, collect the white turbid layer, then wash it with PBS to remove platelets. Place the cells in 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin and antibiotics) and incubate in an incubator. Remove the suspended cells to obtain peripheral blood mononuclear cells.

[0093] i-2) Peripheral blood mononuclear cells were cultured in 1640 complete medium containing 20 ng / mL GM-CSF and 10 ng / mL IL-4. Suspension cells were collected and centrifuged to obtain dendritic cells (DCs); or

[0094] ia) Bone marrow cells were collected, filtered, lysed, and centrifuged to obtain a cell suspension. The suspension was then cultured in 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin and iodine antibiotics) containing 20 ng / mL GM-CSF and 10 ng / mL IL-4. The suspended cells were collected and centrifuged to obtain BMDCs.

[0095] ii) Collect donor cells by digestion and centrifugation to collect cell pellets, then extract and collect free mitochondria using a mitochondrial extraction kit by centrifugation;

[0096] iii) The number of free mitochondria is calculated based on the number of donor cells. After incubating the free mitochondria with DCs at a specific ratio (0.5 to 1.5:1) for 12 to 48 hours, they are collected after centrifugation to obtain mitochondrial transplanted DCs.

[0097] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0098] 1. After mitochondrial transplantation, the survival rate in adverse environments such as high concentrations of lipopolysaccharide is significantly improved, and the maturation degree of transplanted mitochondrial dendritic cells is improved, thereby increasing the tumor antigen presentation capacity by about five times.

[0099] 2. The mitochondria and DCs used in this invention are endogenous substances. After modification, they have high biosafety and can be used in combination with drugs for cancer treatment and / or immune-related diseases. They are particularly effective in DCs-related immunotherapy and DCs-related synergistic immunotherapy, showing superior efficacy in anti-tumor immunotherapy and intervention of various diseases, and have great clinical application value.

[0100] 3. In this invention, mitochondrial transplanted dendritic cells are prepared by co-incubating a specific ratio of free mitochondria with dendritic cells under certain conditions. This method is applicable to mitochondrial transplantation from various donor cell sources. DCs can be functionally oriented according to actual application goals, and the method is simple and easy to operate.

[0101] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0102] Example 1: Preparation of Mitochondrial Transplanted DCs

[0103] (1) Collect 20-50 mL of peripheral blood from the patient, mix the peripheral blood with PBS at a 1:1 ratio, and slowly chromatography onto Ficoll solution. After centrifugation at 400 g for 30 minutes, collect the white turbid layer. Wash twice with PBS at 300 g for 10 minutes to remove platelets. Place the cells in 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin antibiotics) and incubate at 37°C in a 5% CO2 incubator for 2 hours. Remove the suspended cells to obtain peripheral blood mononuclear cells. Place the peripheral blood mononuclear cells in 1640 complete medium containing 20 ng / mL GM-CSF and 10 ng / mL IL-4 and culture for 7 days, changing half of the medium every two days. On the seventh day, collect the suspended cells and centrifuge at 500 g for 5 minutes to obtain DCs.

[0104] (2) Mouse embryonic fibroblast 3T3 cells were digested, and the cell pellet was collected and processed using a mitochondrial extraction kit (Thermo Scientific). TM Free mitochondria were obtained by extraction and centrifugation at 12000g for 15 minutes (Lot: 89874).

[0105] (3) The number of free mitochondria is calculated based on the number of donor cells. Free mitochondria are incubated with DCs in a specific ratio (0.5:1) for 12-48 hours, and then collected by centrifugation at 500g for 5 minutes to obtain mitochondrial transplanted DCs.

[0106] Example 2: Preparation of Mitochondrial Transplantation BMDCs

[0107] (1) Take femurs from 6-8 week old C57BL / 6 mice, crush them in a mortar, and collect the cells; after filtration and lysis of red blood cells, centrifuge at 500g for 5 minutes to obtain a cell suspension, and culture it in 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibodies) containing 20ng / mL GM-CSF and 10ng / mL IL-4 for 7 days, changing half of the medium every two days. On the seventh day, collect the suspended cells and centrifuge at 500g for 5 minutes to obtain BMDCs.

[0108] (2) Human embryonic kidney cell line 293T cells were digested, cell pellets were collected, and free mitochondria were obtained by extraction using a mitochondrial extraction kit and centrifugation at 12000g for 15 minutes.

[0109] (3) The number of free mitochondria is calculated based on the number of donor cells. After incubating the free mitochondria with BMDCs at a specific ratio (1:1) for 12 hours, they are collected by centrifugation at 500g for 5 minutes to obtain mitochondrial transplanted BMDCs.

[0110] Example 3: Preparation of Mitochondrial Transplanted DCs from Different Types of Donor Cells

[0111] (1) Peripheral blood DCs were obtained according to the method in Example 1.

[0112] (2) The pellets of human pancreatic cancer cell line PANC-1, human monocytic leukemia cell line THP-1, and mouse monocytic macrophage cell line RAW 267.4 were collected respectively. The free mitochondria were obtained by extraction using a mitochondrial extraction kit and centrifugation at 12000g for 15 minutes.

[0113] (3) The number of free mitochondria was calculated based on the number of donor cells. After incubating the free mitochondria with DCs at a specific ratio (1.5:1) for 48 hours, they were collected by centrifugation at 500g for 5 minutes to obtain different types of mitochondrial transplanted DCs.

[0114] Example 4: Fluorescent staining to determine the distribution of mitochondria

[0115] BMDCs were extracted according to the method in Example 2, and the BMDC cell suspension was prepared at 2×10⁻⁶. 5 BMDCs were seeded into 24-well plates at a density of 10 cells per well. First, BMDCs were stained with mitochondrial green fluorescent dye (Mito-Tracker Green) for 30 minutes; simultaneously, free mitochondria extracted from 293T cells were stained with mitochondrial deep red fluorescent dye (Mito-Tracker Deep Red 633) for 30 minutes. After staining, the free dye was removed by centrifugation, and the stained mitochondria were added to the BMDCs at a 1:1 ratio and incubated for 24 hours. The distribution of mitochondria within the cells was then observed using a confocal microscope.

[0116] Depend on Figure 1 It was observed that after the addition of exogenous mitochondria, mitochondria labeled with green and red fluorescence, respectively, could be observed within BMDCs, and these labels did not completely overlap. This demonstrates that exogenous mitochondria can enter BMDCs through incubation. The fluorescence images show that the number of exogenous mitochondria outnumbers of endogenous mitochondria by more than 50%.

[0117] The enrichment of exogenous mitochondria in BMDCs was quantified by the ratio of exogenous to exogenous mitochondria in fluorescence images, with exogenous mitochondria accounting for 30-60% of the total number of mitochondria.

[0118] Example 5: Mitochondrial transplantation efficiency

[0119] Following the method diagram of Example 2, the BMDCs cell suspension was prepared at 5 × 10⁻⁶. 5Macrophages were seeded at a density of / well in 12-well plates and incubated for 48 h with free mitochondria from 293T cells stained with mitochondrial green fluorescent dye at a 1:1 ratio. The number of mitochondria was calculated based on the number of donor cells. After incubation, macrophages were collected by centrifugation, and the fluorescence intensity of FITC channels in macrophages was measured by flow cytometry. The results are shown below. Figure 4 As shown, 85.9% of BMDCs were FITC-positive, with a transplantation efficiency of 75.9%.

[0120] Example 6: Cell viability of mitochondrial transplanted BMDCs

[0121] Cytotoxicity assay: BMDCs were extracted according to the method in Example 2. The BMDC cell suspension was seeded into 96-well plates at a density of 8000 cells per well and incubated in a 37°C incubator containing 5% CO2 for 24 h. Then, lipopolysaccharide (LPS, 10 mg / mL) and 293T-derived mitochondria (2 × 10⁻⁶ cells / mL) were added respectively. 5 ), lipopolysaccharide, and mitochondria. After 48 hours of drug treatment, the cells were washed twice with PBS, and 100 μL of CCK-8 solution was added to each well. After culturing for another 2 hours, the absorbance of each well was measured at OD450 nm using an ELISA reader. The cell viability was calculated using the following formula: Viability (%) = (Absorbance of experimental group - Absorbance of solvent control group) / (Absorbance of blank group - Absorbance of solvent control group).

[0122] from Figure 2 As can be seen, under high concentrations of LPS, the cell viability of BMDCs decreased significantly, reaching 59%. When both LPS and exogenous mitochondria were added simultaneously, the cell viability of BMDCs reached 89%, a 50% increase compared to the LPS-treated group, indicating that mitochondrial transplantation is beneficial for improving the cell viability of BMDCs. Simultaneously, when mitochondria were added alone, the cell viability of BMDCs did not change significantly, remaining at 98%, demonstrating its biocompatibility.

[0123] Example 7: Maturation level of mitochondrial transplanted BMDCs

[0124] BMDCs were extracted according to the method in Example 2, and the BMDC cell suspension was prepared at 2×10⁻⁶. 5 Cells were seeded at a density of 100 cells per well in 24-well plates and incubated for 12 hours in a 37°C incubator containing 5% CO2. Lipopolysaccharide (LPS, 1 mg / mL) and mitochondrial cells (2 × 10⁻⁶ cells / mL) were then added to the plates. 5The cells were incubated with lipopolysaccharide and mitochondria for 24 hours. After incubation, the cells were collected by centrifugation at 500g for 5 minutes, and 0.2 μL of CD11c-PerCy5.5, CD80-PE, CD86-APC, and Live-BV510 were added for staining for 30 minutes. After staining, the fluorescence intensity in the cells was detected by flow cytometry.

[0125] The results are as follows Figure 3 As shown in Table 1, after positive control LPS, the degree of maturation (CD80) of BMDCs was... + / CD86 + The maturation of BMDCs increased by 6 times after the addition of mitochondria, and the maturation degree of BMDCs also increased significantly by 5 times, proving that mitochondrial transplantation can increase the maturation of BMDCs, thereby enhancing the anti-tumor immune response.

[0126] Table 1. BMDC maturation level in each group

[0127] Grouping physiological saline Lipopolysaccharide mitochondria Lipopolysaccharide + Mitochondria Sample 1 9.5％ 54.5％ 48.4％ 60.6％ Sample 2 10.2％ 50.2％ 44.5％ 56.3％ Sample 3 8.4％ 56.8％ 40.4％ 51.0％

[0128] Example 8: Antitumor effect of THP-1 mitochondrial transplanted BMDCs combined with tumor vaccine therapy (inactivated MC38 cells)

[0129] In this embodiment, the method for preparing mitochondrial transplant BMDCs is the same as in embodiment 2, except that: in step (2), free mitochondria are extracted from THP-1 monocytic leukemia cells to prepare THP-1 mitochondrial transplant BMDCs.

[0130] 6-8 week old male C57BL / 6 mice were subcutaneously injected with 2×10⁻⁶ ppm on their backs. 6 A mouse model bearing MC38 tumor was established using murine colorectal cancer (MC38) cells. The tumor volume was increased to 100 mm². 3 Subsequently, BMDCs and mitochondrial transplanted BMDCs (2.5 × 10⁻⁶) were injected subcutaneously at the base of the tail. 6 W / mouse) and the same volume of physiological saline; 4 hours later, in mice in the BMDCs and mitochondrial transplanted BMDCs groups, tumor antigen (inactivated MC38 cells, 2.5×10) was subcutaneously injected via the tail root. 6 W / mouse). Administer the medication three times, every three days, recording tumor volume and body weight changes every three days. Volume = Length × Width 2 / 2.

[0131] The results are as follows Figure 5As shown, compared to the saline group, the tumor volume in mice in the mitochondrial transplanted BMDCs group was significantly reduced, indicating that mitochondrial transplantation effectively enhanced the anti-tumor effect of BMDCs combined with tumor vaccine therapy. Meanwhile, the mouse body weight did not change significantly, demonstrating the biocompatibility of the drug.

[0132] Example 9: Antitumor effect of THP-1 mitochondrial transplanted BMDCs combined with tumor vaccine therapy (RM-1 cells inactivated)

[0133] In this embodiment, the method for preparing mitochondrial transplant BMDCs is the same as in embodiment 2, except that: in step (2), free mitochondria are extracted from THP-1 monocytic leukemia cells to prepare THP-1 mitochondrial transplant BMDCs.

[0134] 6-8 week old male C57BL / 6 mice were subcutaneously injected with 2×10⁻⁶ ppm on their backs. 6 A mouse model bearing RM-1 tumor was established using murine prostate cancer cells (RM-1). The tumor volume was increased to 100 mm². 3 Subsequently, BMDCs and mitochondrial transplanted BMDCs (2.5 × 10⁻⁶) were injected subcutaneously at the base of the tail. 6 W / mouse) and the same volume of physiological saline; 8 hours later, in mice in the BMDCs and mitochondrial transplanted BMDCs groups, tumor antigen (inactivated RM-1 cells, 2.5×10) was subcutaneously injected via the tail root. 6 W / mouse). Administer the medication three times, every three days, recording tumor volume and body weight changes every three days. Volume = Length × Width 2 / 2. For example... Figure 6 As shown, the results indicate that the tumor volume in mice with mitochondrial transplanted BMDCs was significantly reduced compared to those with BMDCs alone, and the mice's body weight did not change significantly. This demonstrates that mitochondrial transplantation effectively enhances the anti-tumor effect of BMDCs combined with tumor vaccine therapy and has good biosafety.

[0135] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A mitochondrial transplantation dendritic cell, characterized in that, The mitochondrial transplanted dendritic cells were obtained by co-incubating free mitochondria with dendritic cells.

2. The mitochondrial transplanted dendritic cells as described in claim 1, characterized in that, The free mitochondria are obtained by extracting mitochondria from donor cells, and the donor cells are selected from the group consisting of: stem cells, immune cells, platelets, erythrocytes, glial cells, ependymal cells, neurons, fibroblasts, endothelial cells, muscle cells, epithelial cells, fibroblasts, tumor cells, keratinocytes, brown adipocytes, white adipocytes, hepatocytes, osteoblasts, osteogenic precursor cells, or combinations thereof.

3. The mitochondrial transplanted dendritic cells as described in claim 1, characterized in that, The dendritic cells are selected from the following group: human conventional myeloid dendritic cells, human plasmacytoid dendritic cells, or human mononuclear cell-derived dendritic cells; preferably, the dendritic cells are selected from the following group: bone marrow-derived dendritic cells (BMDCs), mononuclear cell-derived dendritic cells (MoDCs), plasmacytoid dendritic cells (pDCs), Langerhans cells (LCs) or their precursor cells, classical dendritic cell subsets cDC1 or cDC2 that have been isolated and enriched, and dendritic cells directly isolated from peripheral blood (PBDCs).

4. A method for preparing mitochondrial transplantable dendritic cells as described in claim 1, characterized in that, The method includes the following steps: co-incubating dendritic cells with free mitochondria to obtain mitochondrial transplanted dendritic cells.

5. The preparation method according to claim 4, characterized in that, During the incubation process, the ratio of free mitochondria to DCs is 0.5 to 1.5:

1.

6. The preparation method according to claim 4, characterized in that, During the incubation process, the co-incubation time of the free mitochondria and DCs is 12 to 48 hours.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: 1) The mitochondrial transplanted dendritic cells as described in claim 1; and 2) Pharmaceutically acceptable carrier.

8. The use of the mitochondrial transplanted dendritic cells as described in claim 1 or the pharmaceutical composition as described in claim 7, characterized in that, Preparations for the treatment of cancer and / or immune-related diseases, wherein the preparations comprise: (1) The mitochondrial transplanted dendritic cells of claim 1 or the pharmaceutical composition of claim 7; and (2) Medications for cancer treatment and / or immune-related diseases.

9. The use as described in claim 8, characterized in that, The drugs mentioned are DCs-related immunotherapies and DCs-related synergistic immunotherapies; The DCs-related immunotherapy and DCs-related synergistic immunotherapy are selected from the following groups: engineered DCs therapy, DCs synergistic antigen therapy, DCs synergistic exosome therapy, chimeric antigen receptor DCs therapy, DCs combined with immune checkpoint inhibitor therapy, DCs synergistic T-cell therapy, DCs synergistic NK-cell therapy, DCs synergistic chemotherapy therapy, DCs synergistic targeted drug therapy, DCs synergistic hormone therapy, and DCs synergistic radiotherapy therapy.

10. The use as described in claim 8, characterized in that, The formulation has one or more characteristics selected from the group consisting of: (a) Dendritic cell survival rate >85% in high concentrations of LPS; (b) Improve the maturation degree of dendritic cells and enhance their antigen presentation ability; (c) Reduces tumor volume and enhances anti-tumor effect.

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