CN113502268A

CN113502268A - Preparation method of dendritic cells

Info

Publication number: CN113502268A
Application number: CN202110832946.4A
Authority: CN
Inventors: 李景圆; 张宇; 张月; 荣耀星; 于艳秋
Original assignee: Shenyang Cell Therapy Engineering Technology Research And Development Center Co ltd
Current assignee: Shenyang Cell Therapy Engineering Technology Research And Development Center Co ltd
Priority date: 2021-07-22
Filing date: 2021-07-22
Publication date: 2021-10-15

Abstract

The invention discloses a preparation method of dendritic cells, which comprises the following steps: separating human mononuclear cells; and (II) under the condition suitable for cell growth and proliferation, inducing and preparing DCregs cells, wherein the DCregs cells are specifically prepared as follows: (1) inoculation: adherent culture of human mononuclear cells with a basic culture medium, screening adherent cells, and adding a DC culture medium; (2) on the 3 rd day of culture, supplementing a DC culture medium; (3) culturing on day 5, adding mesenchymal stem cell exosomes, vEGF, TGF-beta and IL-10 to the culture; (4) and culturing for 7 days to obtain the DCregs cells. The DCreg cells prepared by the method can be used for living body transplantation and assisted reproduction technology. The dendritic cells obtained by the method have low MFI values of HLA-DR +, CD80+ and CD83+, low HLA-DR indicates weak rejection reaction, and the low CD83 and T cell costimulatory molecule CD80 cause low reaction of induced allogeneic CD4+ and CD8+ T cells.

Description

Preparation method of dendritic cells

Technical Field

The invention relates to a preparation method of dendritic cells, belonging to the technical field of immune cells.

Background

Regulatory dendritic cells (dcregs) are a subset of DCs with negative immune regulation. DCreg can induce the immune tolerance of the organism through different mechanisms, and plays an important role in different fields of organ transplantation, autoimmune diseases and the like.

After a patient is subjected to a transplant operation, rejection reactions of different degrees may occur, an immunosuppressant can be clinically used for relieving the rejection reactions and prolonging the function of transplantation, some rejection reactions are strong, and severe rejection reactions can cause transplant failure and cause great damage to the body of the patient. Donor-derived regulatory dendritic cells (dcregs) have been shown to promote long-term graft survival in rodents, and to suppress allograft rejection in non-human primates and balance the immune response based on robust preclinical data. At present, the safety and curative effect test of DCreg from human donor in living body transplantation is carried out, and the DCreg cells have the tendency of being applied to clinic, so the invention researches an induction preparation method of the DCreg cells and carries out early-stage research on clinical application.

The Chinese invention patent CN 105886471A discloses a preparation method of a novel regulatory dendritic cell and a tumor immunotherapy method combining DC-CIK. Isolating mammalian mesenchymal stem cells and hematopoietic stem cells; co-culturing the obtained mammalian mesenchymal stem cells and hematopoietic stem cells under conditions suitable for cell growth and proliferation; after co-culturing for 1 to 7 days, the obtained novel regulatory dendritic cells are recovered and identified from the suspension culture solution. The novel regulatory dendritic cells are transformed into mature regulatory dendritic cells under the induction of CpGODN (CpG oligonucleotide) or an inducer with the same induction function, wherein the expression level of irf4+ and irf8+ is increased, and the cells have an immunoregulation function. The novel regulatory dendritic cells can inhibit tumor growth and can be combined with DC-CIK to carry out tumor immunotherapy. In the patent, in the preparation method of the regulatory dendritic cells, expression levels of irf4+ and irf8+ are increased and the regulatory dendritic cells have an immune regulation function, irf4+ is a marker of cDC2, irf8+ is a marker of cDC1, and irf4+ and irf8+ are not mentioned as markers of the regulatory dendritic cells in the literature. The patent mentions that the novel regulatory dendritic cell is capable of inhibiting tumor growth, a negatively regulated cell.

Disclosure of Invention

In view of the above prior art, the present invention provides a method for preparing dendritic cells.

The invention is realized by the following technical scheme:

a method for preparing dendritic cells, comprising the following steps:

separating human mononuclear cells;

and (II) under the condition suitable for cell growth and proliferation, inducing and preparing DCreg cells, wherein the DCreg cells are specifically prepared as follows:

(1) inoculation: carrying out adherent culture on the human body mononuclear cells obtained in the step (one) for 2 hours by using a basic culture medium, screening adherent cells, and adding a DC culture medium; the DC culture medium consists of a basic culture medium, recombinant human GM-CSF and recombinant human IL-4, wherein the final concentration of the recombinant human GM-CSF is 500-1000 IU/ml, and the final concentration of the recombinant human IL-4 is 800-1000 IU/ml;

(2) on the 3 rd day of culture, supplementing a DC culture medium;

(3) on the 5 th day of culture, adding 5-12 ug/mL of mesenchymal stem cell exosome, 50-200 ng/mL of vEGF, 5-12 ng/mL of TGF-beta and 300-800 IU/mL of IL-10 into the culture;

(4) and culturing for 7 days to obtain DCreg cells, and performing phenotype and function analysis.

Further, the basic culture medium is selected from KBM581 culture medium. The KBM581 culture medium is a commercial culture medium in the prior art, and comprises the components of glucose, biotin, pantothenic acid, nicotinamide, inositol, folic acid, albumin, B vitamins and more than 20 amino acids.

Further, the final concentration of the recombinant human GM-CSF is 800 IU/ml.

Further, the final concentration of the recombinant human IL-4 is 1000 IU/ml.

Further, the concentration of the mesenchymal stem cell exosomes is 10 ug/ml.

Further, the concentration of vEGF is 100 ng/ml.

Further, the concentration of the TGF-beta is 10 ng/ml.

Further, the concentration of IL-10 is 600 IU/mL.

The DCreg cells prepared by the method can be used for living body transplantation and can also be applied to an assisted reproduction technology, 14-28 days before the embryo implantation is treated by the test-tube infant, the DCreg cells are induced by extracting maternal mononuclear cells, and the DCreg cells of the maternal are stimulated by using the PBMC cell membrane vesicles of the father or the third party, so that the proliferation reactivity of CD4+ cells and CD8+ cells is reduced, the implantation rate of the embryo is improved, and the embryo planting environment is improved.

The dendritic cell preparation method of the invention adds vascular endothelial growth factor (vEGF) and umbilical cord mesenchymal stem cell exosome on the basis of IL-10 and TGF-beta (in the prior art, some documents use vitamin D3, IL-10, IL-35 and TGF-beta to induce DCreg cells, IL-10, IL-35 and TGF-beta are immunosuppressive cytokines, induce different Treg cells and secretion factors thereof, and inhibit the activity of Th1 cells and Th17 cells). Vascular endothelial growth factor (vEGF) belongs to a platelet-derived growth factor superfamily, is the most important member of the platelet-derived growth factors, stimulates the proliferation and survival of endothelial cells, can induce angiogenesis, and improves the permeability of capillaries. The formation of the vascular system is closely related to the growth of the tumor, the vEGF content in the microenvironment of the tumor patient is higher than that of a normal healthy population, and the exogenous vEGF influences the maturation of DC cells and promotes the formation of DCreg (the effect is discovered by the invention). Exosomes are extracellular vesicles of nanometer size (40-150 nm), have lipid bilayer membranes, are released by various cells, are part of an intercellular communication system, and carry RNA and proteins. The umbilical cord stem cell-derived exosome (the exosome plays a role in the invention in playing an immunoregulation function, promoting the formation of DCreg and indirectly promoting the proliferation of Treg cells) repairing regenerated tissues, inhibiting inflammatory reaction, regulating the immune cell recovery function of an organism and regulating an inflammatory state.

The dendritic cells (DCreg) obtained by the method of the invention are induced and prepared to be in phase with mature DC cellsIn contrast, the MFI values for HLA-DR +, CD80+, and CD83+ were all lower, with low HLA-DR indicating a weaker rejection response, and lower CD83 and the T cell costimulatory molecule CD80 causing lower allogenic CD4+ and CD8+ T cell responses. The CSFE proliferation reaction experiment shows that: compared with immature DCreg cells, after the dendritic cells (Dcreg) obtained by induction preparation by the method are mixed with CFSE-labeled PBMC cells, CD4+ cells and CD8+ cells are more slowly proliferated, and after induction, CD3+ cells are more slowly proliferated⁺CD4⁺CD25^hi CD127^lowFoxp3⁺The proportion of Treg cells becomes high, so that the host tolerance can be improved, and the transplantation success rate is further improved.

The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the meaning of the present invention will prevail.

Drawings

FIG. 1: HLA-DR + cell ratio, CD 14-cell ratio, CD1a + cell ratio, CD80+ cell ratio, CD83+ cell ratio, wherein a: HLA-DR + cell ratio; b: CD 14-cell ratio; c: CD1a + cell fraction; d: CD80+ cell proportion; e: CD83+ cell fraction.

FIG. 2: schematic comparison of the average intensity of fluorescence of HLA-DR, CD80, CD83 surface molecules of DCreg cells (from Experimental group 1) and mature DCs (from Experimental group 3), wherein, in the upper row: DCreg cells (from experimental group 1); and (3) lower row: mature DCs (from Experimental group 3).

FIG. 3: comparison of the average intensity of the fluorescence of the surface molecules of CD80 from immature DC (obtained from experiment group 2), mature DC (obtained from experiment group 3) and DCreg cells (obtained from experiment group 1) shows that the MFI value of Dcreg cells is lower than that of CD80 from mature DC cells and higher than that of CD80 from immature DC cells, indicating that CD4+ and CD8+ T cells are less reactive.

FIG. 4: schematic representation of the proportion of CD4+ and CD8+ T cells before mixing immature DCs (from experiment 2), DCreg cells (from experiment 1), mature DCs (from experiment 3) and CFSE-labeled PBMC cells.

FIG. 5: schematic representation of the proportion of CD4+ and CD8+ T cells after mixing immature DCs (from experiment 2), DCreg cells (from experiment 1), mature DCs (from experiment 3) with CFSE-labeled PBMC cells and co-culturing for 5 days.

FIG. 6: comparison of CD4+ and CD8+ cell proliferation after mixing immature DCs (from experiment 2), DCreg cells (from experiment 1), and mature DCs (from experiment 3) with CFSE-labeled PBMC cells and co-culturing for 5 days is shown.

FIG. 7: CD3+ CD4+ CD25 before and after induction of DCreg cells (from Experimental group 1)^hiSchematic representation of the T cell ratio of (a).

Detailed Description

The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.

The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.

Example 1 preparation of dendritic cells

Reagent and consumable

(1) Main experimental reagent

Reagent: KBM581 Medium (corning Co.), Glutamine (Gibco Co.), human lymphocyte isolate (Ficoll, Tianjin ocean biologicals).

Cytokines: IL-4, GM-CSF, TGF- β, vEGF, IL-10, TNF- α, IL-6, IL-1 β, and prostaglandin E2 (all available from Peprotech corporation), human recombinant IL-2 (Sansheng pharmaceuticals);

antibody: HLA-DR antibody, CD14, CD1a, CD80 and CD83 antibodies (all purchased from BD Co.), CFSE proliferation kit (Union).

(2) Relevant consumables of main experiment

15ml centrifuge tube, 50ml centrifuge tube, 10ml pipette, 25ml pipette, T-25cm²、T-75cm²Cell culture flasks (Corning), 24-well plates, 96-well concave bottom plates (Corning).

Second, the experimental procedure

The method comprises the following steps of (I) isolating human mononuclear cells (PBMC), wherein the PBMC comprises the following steps:

(1) aseptically collecting about 100ml of peripheral blood of healthy volunteers in a blood collecting bag of 100ml of anticoagulant;

(2) separating plasma: 840g, centrifuging for 10min, room temperature, increasing speed by 9, decreasing speed by 5 (increasing speed by 9, decreasing speed by 5' is the lifting speed of the centrifuge);

(3) plasma inactivation: inactivating in 56 deg.C constant temperature water bath for 30min, placing into-20 deg.C, cold precipitating for 10min at 2000g, centrifuging for 15min, 4 deg.C, increasing speed to 9, and decreasing speed to 9;

(4) diluting: diluting with 0.9% physiological saline to remove blood plasma, and mixing to obtain 30ml blood;

(5) paving a sample: slowly adding the upper layer of the human lymphocyte separation solution at a constant speed by a ratio of 2:1 for 20min at 935g, at room temperature, at an ascending speed of 1 and at a descending speed of 1;

(6) extracting mononuclear cells: after centrifugation, obvious layering appears in the centrifugal tube, and a mononuclear cell layer (leucocyte layer) is absorbed;

(7) the first washing: mixing normal saline and mononuclear cells at a ratio of 1:1, 935g for 7min at room temperature, increasing the speed by 9 percent, and decreasing the speed by 9 percent;

(8) and (3) second washing: washed with physiological saline, 336g, 8min, room temperature, 9 rising speed and 9 falling speed.

(1) inoculation: the human mononuclear cells obtained in the step (one) are cultured by using KBM581 medium at 1 x 10^6/cm²The cell density of (A) was inoculated in a T25 flask, 5% CO₂Incubating in a constant temperature incubator at 37 deg.C for 2h, and collecting the upper layerRemoving adherent cells, and adding 5ml of DC culture medium;

the DC culture medium consists of a basic culture medium, GM-CSF and IL-4, wherein the final concentration of the GM-CSF is 800IU/ml, and the final concentration of the IL-4 is 1000 IU/ml;

(2) on the 3 rd day of culture, 5ml of DC culture medium is supplemented;

(3) on day 5 of culture, the cultures were divided into three experimental groups:

experimental group 1:10 ug/ml (final concentration, the same below) of mesenchymal stem cell exosomes were added to the culture { umbilical cord mesenchymal stem cell exosomes were obtained by continuous differential centrifugation, filtration, washing and enrichment of umbilical cord mesenchymal stem cell culture fluid cultured in this laboratory, as follows: umbilical cord mesenchymal stem cells are derived from Wharton's jelly prepared by donating umbilical cord tissues, and are cultured by attaching mesenchymal stem cell culture Medium (the culture Medium is the conventional culture Medium in the prior art, and the culture Medium is MSC NutriStem XF basic Medium purchased from BI company, the product number is 05-200-1A) and 10% serum substitute, and the culture Medium is saturated with 5% CO at 37 ℃ and 5% of saturated humidity₂Subculturing to 3 rd generation in the incubator to obtain 50ml cell culture supernatant, and removing cell by discarding precipitate at 300g × 10min and 2000g × 20min at 4 deg.C; then 10000g multiplied by 30min, abandoning the sediment, removing cell fragments and the like, then 10000g multiplied by 60min, abandoning the supernatant, finally obtaining the sediment as the exosome, resuspending the sediment by 50mL of PBS solution, mixing, then centrifuging by 10000g multiplied by 60min, suspending the sediment by 5mL of PBS solution, determining the protein concentration by BCA method, subpackaging the purified exosome in an EP tube, and placing the tube in a refrigerator at minus 80 ℃ for storage, 100ng/mL vEGF, 10ng/mL TGF-beta and 600IU/mL IL-10;

experimental group 2: adding 10ng/mL TGF-beta and IL-10 to the culture at a final concentration of 600 IU/mL;

experimental group 3: adding 10ng/ml TNF- α, 20ng/ml IL-6, 10ng/ml IL-1 β, and 1.0uM prostaglandin E2 to the culture to form a pro-inflammatory cytokine mixture, inducing maturation of cultured monocyte-derived DC cells;

(4) on the 7 th day of culture, 336g, 8min, the culture solution is centrifuged to remove the DC culture solution at the rising speed of 9 and the falling speed of 9, and the DC culture solution is counted for phenotypic and functional analysis.

(III) DCreg cell phenotypic and functional analysis

(1) DCreg cell identification

Firstly, transferring 1ml of DCreg cell suspension into a 1.5ml centrifuge tube, centrifuging for 5 minutes at the temperature of 4 ℃ at 300g, and carefully sucking off a supernatant;

② washing the cells with a proper amount of PBS, centrifuging for 5 minutes at 4 ℃ at 300g, carefully sucking the supernatant;

③ resuspending the cells with precooled PBS and adjusting the final concentration of the cells to 1X 10⁷cells/ml, lightly blowing, beating and uniformly mixing;

taking 100 mu l of cell suspension as a blank control group, taking 100 mu l of cell suspension as a parallel control group, taking 200 mu l of cell suspension as an experimental group, adding APC-HLA-DR, PerCP-Cy5.5-CD14, PE-CD1a, PE-Cy7-CD80 and FITC-CD83 antibodies (all purchased from BD company), and incubating for 30-40 minutes at 4 ℃;

adding a proper amount of PBS to clean cells, centrifuging for 5 minutes at the temperature of 4 ℃ at 300g, and carefully sucking off the supernatant;

sixthly, resuspending the cells by 500 mu l of PBS, and detecting on a machine.

(2) Flow cytometry collection

Firstly, an SA3800 full-spectrum flow cytometry analyzer is started in advance to carry out preheating and equipment self-checking;

secondly, new experiment: selecting a 'Preparation' guide label, entering an Experiment Preparation interface, clicking an 'Experiment Template' button, selecting a 'Blank Template', inputting naming information in a Name text box, and clicking 'Create Experiment', thereby creating a new Experiment Template;

putting the Sample-S tube, clicking 'Preview', setting a 'Fluorescence PMT Voltage' value to enable the maximum Intensity _ H value to be close to 105, and clicking 'Stop' to unload the Sample-S tube;

put into Unsiteined sample tube, click "Preview", set other parameters, do not use FSC to set threshold value when absolute count is carried out!

Collecting cells of each group: and putting the sample tube to be tested, clicking 'Preview', and clicking 'Acquire' to collect the sample tube when the Flow condition state is changed into Stable. The same set of experiments must use the same parameters after collection begins, and if changed, all are collected again.

(3) Streaming results analysis

Adding the Marker and the corresponding fluorescent signal;

importing a corresponding fluorescence curve in a database, and establishing a positive control tube to establish a curve for a new fluorescence signal;

selecting an Unstabained group, trapping a main cell group, setting the main cell group as an A gate, setting FSC-H/FSC-A, displaying cells in the A gate, and trapping a single cell group B;

and fourthly, setting side angle scattering SSC-A/HLA-DR, displaying cells in a B gate, and setting a C gate: including all HLA-DR non-negative cells;

setting SSC-A/CD14 to show the cell in C gate, setting L gate: including all CD14 non-positive cells;

sixthly, setting SSC-A/CD1a, showing the cells in the L gate, setting O: including all CD1a non-negative cells;

and (c) setting SSC-A/CD80, displaying the cell in the L gate, setting N: including all CD80 non-negative cells;

setting SSC-A/CD83, displaying cell in L gate, setting M: including all CD83 non-negative cells;

and ninthly, saving data, and cleaning and closing the instrument.

(4) CFSE proliferative response

Eighthly, preparing a CFSE working solution;

collecting PBMC cells, and washing the PBMC cells once by using PBS;

③ incubating the CFSE working solution at 37 ℃ for 15min, and adding serum to terminate the reaction;

centrifugally washing the solution twice by PBS (phosphate buffer solution), and washing away the CFSE molecules adhered to the surface;

resuspending KBM581 culture medium, culturing DCreg cells obtained from experiment group 1, DCreg cells obtained from experiment group 2, and mature DC (mDC) obtained from experiment group 3 in complete culture medium (adding IL-2 with final concentration of 500IU/ml based on basal medium) at a ratio of 1:10(DC: PBMC) with CFSE-labeled PBMC for 5 days;

sixthly, analyzing the state of the T cells by flow cytometry.

Each set of experiments was repeated three times.

Third, experimental results

As shown in fig. 1, in the mDC analysis, after removing the effect of lymphocytes and cell debris from the sample, the HLA-DR + cell fraction was 95.31%, with a CD 14-cell fraction of 94.60%, and the population was further analyzed to have a CD1a + cell fraction of 61.81%, a CD80+ cell fraction of 98.36%, and a CD83+ cell fraction of 99.00%. The mature DC obtained by the method has high maturity, the proportion of the co-stimulatory factor CD80 is high, and the antigen can be effectively presented to T cells.

As shown in FIG. 2, the average intensity of fluorescence of surface molecules of HLA-DR, CD80 and CD83 of DCreg cells (obtained from experiment group 1) and mature DCs (obtained from experiment group 3) is compared: the obtained Dcreg cells in experimental group 1 produced lower MFI values for HLA-DR +, CD80+ and CD83+ than mature DC cells, low HLA-DR indicates weakened rejection response, and low CD83 and T cell costimulatory molecule CD80 cause weakened allogeneic CD4+ and CD8+ T cell responses (86% and 94% in the figure indicate the ratio of DCreg cells circled in the flow chart and mature DC to the total cells analyzed).

As shown in FIG. 3, the comparison of the average intensity of fluorescence of CD80 surface molecules in immature DCs (obtained from experiment 2), mature DCs (obtained from experiment 3) and DCreg cells (obtained from experiment 1) is shown: the lower MFI value of Dcreg cells compared to mature DC cells CD80 and higher MFI value than that of immature DC cells CD80, implying low reactivity of CD4+ and CD8+ T cells.

As shown in fig. 4 and 5, the proportion of CD4+ T cells before mixing immature DCs (obtained in experimental group 2), DCreg cells (obtained in experimental group 1), and mature DCs (obtained in experimental group 3) with CFSE-labeled PBMC cells was 0.73%, the proportion of CD8+ T cells was 1.47%, and the proportion of CD4+ cells after mixing and co-culturing for 5 days was 2.3%, 1.35%, and 14.8%, respectively; the proportion of CD8+ T cells was 2.1%, 1.86% and 12.9%, respectively.

As shown in fig. 6, a comparison of proliferation of CD4+ cells and CD8+ cells after mixing immature DCs (from experimental group 2), DCreg cells (from experimental group 1), and mature DCs (from experimental group 3) with CFSE-labeled PBMC cells and co-culturing for 5 days shows that DCreg cells added with vfgf proliferated more slowly than conventional DCreg cells mixed with CFSE-labeled PBMC cells, CD4+ cells and CD8+ cells.

As shown in FIG. 7, CD3+ CD4+ CD25 before and after induction of DCreg cells (obtained from Experimental group 1)^hiSchematic T cell ratio of (a): post-induction CD3+ CD4+ CD25^hiCD127^lowThe proportion of Fowp3+ Treg cells became high, thus improving host tolerance and consequently increasing the success rate of transplantation (21.8% of CD3+ T cells, with 80.2% of CD4+ T cells, circled as shown in the figure, further analysis of CD25^hiThe ratio was 6.35% at CD4+ CD25^hiFurther analysis on T cells gave CD127^lowThe proportion of the Fowp3+ Treg cells after induction is 82.9%; the same pre-and post-induction analysis methods were used to obtain pre-induction CD127^lowThe proportion of foxp 3+ Treg cells was 39.3%).

The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.

Claims

1. A method for producing a dendritic cell, comprising: the method comprises the following steps:

separating human mononuclear cells;

(1) inoculation: carrying out adherent culture on the human body mononuclear cells obtained in the step (one) by using a basic culture medium, screening adherent cells, and adding a DC culture medium; the DC culture medium consists of a basic culture medium, recombinant human GM-CSF and recombinant human IL-4, wherein the final concentration of the recombinant human GM-CSF is 500-1000 IU/ml, and the final concentration of the recombinant human IL-4 is 800-1000 IU/ml;

(2) on the 3 rd day of culture, supplementing a DC culture medium;

(4) and culturing for 7 days to obtain the DCreg cells.

2. The method for producing dendritic cells according to claim 1, wherein: the basic culture medium is selected from KBM581 culture medium.

3. The method for producing dendritic cells according to claim 1, wherein: the final concentration of the recombinant human GM-CSF is 800 IU/ml;

and/or: the final concentration of the recombinant human IL-4 is 1000 IU/ml.

4. The method for producing dendritic cells according to claim 1, wherein: the concentration of the mesenchymal stem cell exosome is 10 ug/ml;

and/or: the concentration of vEGF is 100 ng/ml;

and/or: the concentration of the TGF-beta is 10 ng/ml;

and/or: the concentration of the IL-10 is 600 IU/mL.

5. The method for producing dendritic cells according to claim 1, wherein: the specific mode for separating the human mononuclear cells is as follows:

(1) aseptically collecting peripheral blood 100ml in a blood collecting bag of anticoagulant;

(2) separating the plasma;

(3) plasma inactivation;

(5) paving a sample: slowly adding the blood sample into the upper layer of the human lymphocyte separation liquid at a constant speed according to the proportion of 2:1, and centrifuging;

(6) extracting mononuclear cells: after centrifugation, obvious layering appears in the centrifugal tube, and a mononuclear cell layer is absorbed;

(7) the first washing: mixing the physiological saline and the mononuclear cells, and centrifuging;

(8) and (3) second washing: washed with physiological saline and centrifuged.

6. The method for producing dendritic cells according to claim 1, wherein: the adherent culture with the basal culture medium has the specific mode that: at 1 x 10^6/cm²Cell density of (2) inoculated in a culture flask, 5% CO₂And incubating in a constant temperature incubator at 37 ℃ for 2 h.

7. A dendritic cell produced by the production method according to any one of claims 1 to 6.

8. Use of the dendritic cells of claim 7 as transplantation cells for transplantation in vivo, assisted reproduction.