Efficient induction culture kit for DC cells and induction culture method thereof

Abstract

The invention provides an efficient induction culture kit for DC cells and an induction culture method thereof, and belongs to the technical field of cell biology. The kit comprises a reagent A and a reagent B, wherein the reagent A is a GM-CSF solution, an IL-4 solution, an IL-1beta solution, an AK1 solution, an ACK1 solution, an MLK3 solution, a PLD1 solution, a type II cytoskeleton 1 solution and a type I cytoskeleton 16 solution; the reagent B comprises a PD-L1 protein solution, a CTLA-4 protein solution, a TIM-3 protein solution, an LAG-3 protein solution, a CD44 protein solution, a CEA protein solution, a neuron-specific enolase solution and a squamous cell carcinoma related antigen protein solution. According to the kit, the cell culture time is shortened, the induced maturation rate and the cell viability of the DC cells are improved, and the killing activity of the T cells on the lung cancer cells is enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and particularly relates to a kit for highly efficient induction and culture of dendritic cells (DC cells) and an induction and culture method thereof. Background Art

[0002] Cell biological immunotherapy is a treatment method that combats diseases by modifying or enhancing the functions of the body's own immune cells, which can induce long-term immune memory and reduce recurrence. It mainly includes CAR-T cell therapy, TCR-T cell therapy, dendritic cell (DC) vaccine, NK cell therapy, tumor-infiltrating lymphocyte (TIL) therapy, etc. Among them, the DC vaccine activates T cells after in vitro culture of DC cells and loading of tumor antigens. DC is the most powerful antigen-presenting cell (APC) currently discovered. Compared with other APCs (such as monocytes, macrophages, B cells, etc.) that can only stimulate activated or memory T cells, DC is also the only APC that can significantly stimulate the proliferation of naive T cells (Tn). Therefore, as the initiator of the body's adaptive T cell immune response, DC plays an extremely important role in tumor immunity.

[0003] Currently, common methods for inducing and culturing DC cells mainly involve using cytokines such as GM-CSF and IL-4 to induce the differentiation of monocytes into DC cells. Although a sufficient number of mature DC cells can be obtained, there are still many limitations. For example, the maturity and functional stability of the induced and cultured DC cells are insufficient, and they cannot effectively activate T cells, or the T cells activated by them have insufficient killing activity against tumor cells, affecting the effect of immunotherapy; secondly, the culture period is relatively long, which not only increases the culture cost, but also may lead to contamination of the cells during the culture process or other adverse changes; moreover, the cell yield is unstable, and there may be significant differences in the induction and culture results of different batches, making it difficult to ensure that sufficient numbers and qualities of DC cells can be obtained each time, which limits the application of DC cells in clinical practice.

[0004] Based on this, there is an urgent need for a kit that can highly efficiently induce DC cells. Summary of the Invention

[0005] Aiming at the defects of the prior art, the present invention provides a kit for highly efficient DC cells and an induction and culture method thereof. The kit not only has a short culture period, but also the DC cells induced and cultured with it can enhance the killing activity of T cells against lung cancer cells.

[0006] To achieve the above object, the present invention adopts the following technical solutions to solve the technical problems:

[0007] In a first aspect, the present invention provides a kit for highly efficient induction and culture of dendritic cells (DC cells), and the kit includes reagent A and reagent B;

[0008] Among them, reagent A is a GM-CSF solution, an IL-4 solution, an IL-1β solution, an AK1 solution, an ACK1 solution, an MLK3 solution, a PLD1 solution, a cytoskeleton type II 1 solution, and a cytoskeleton type I 16 solution;

[0009] Reagent B includes a PD-L1 protein solution, a CTLA-4 protein solution, a TIM-3 protein solution, a LAG-3 protein solution, a CD44 protein solution, a CEA protein solution, a neuron-specific enolase solution, and a squamous cell carcinoma-related antigen protein solution.

[0010] In some preferred embodiments, the concentration of the GM-CSF solution is 500 - 600 ng / mL, the concentration of the IL-4 solution is 1000 - 3000 U / mL, the concentration of the IL-1β solution is 5000 - 7000 IU / mL, the concentration of the AK1 solution is 50 - 200 ng / mL, the concentration of the ACK1 solution is 50 - 200 ng / mL, the concentration of the MLK3 solution is 30 - 70 ng / mL, the concentration of the PLD1 solution is 30 - 70 ng / mL, the concentration of the cytoskeleton type II 1 solution is 50 - 200 ng / mL, and the concentration of the cytoskeleton type I 16 solution is 50 - 200 ng / mL.

[0011] In some preferred embodiments, the volume ratio of the GM-CSF solution, the IL-4 solution, the IL-1β solution, the AK1 solution, the ACK1 solution, the MLK3 solution, the PLD1 solution, the cytoskeleton type II 1, and the cytoskeleton type I 16 solution is (3 - 7):(1 - 3):(0.5 - 1.5):(1 - 3):(0.5 - 1.5):(0.5 - 1.5):(1 - 3):(2 - 4):(1 - 3).

[0012] In some preferred embodiments, the concentration of the PD-L1 protein solution is 200 - 400 ng / mL, the concentration of the CTLA-4 protein solution is 50 - 200 ng / mL, the concentration of the TIM-3 protein solution is 50 - 200 ng / mL, the concentration of the LAG-3 protein solution is 50 - 200 ng / mL, the concentration of the CD44 protein solution is 300 - 500 ng / mL, the concentration of the CEA protein solution is 300 - 700 ng / mL, the concentration of the neuron-specific enolase solution is 100 - 200 ng / mL, and the concentration of the squamous cell carcinoma-related antigen protein solution is 100 - 200 ng / mL.

[0013] In some of these preferred embodiments, the volume ratio of the PD-L1 protein solution, CTLA-4 protein solution, TIM-3 protein solution, LAG-3 protein solution, CD44 protein solution, CEA protein solution, neuron-specific enolase solution, and squamous cell carcinoma-related antigen protein solution is (1-3):(0.5-1.5):(0.5-1.5):(0.5-1.5):(2-4):(1-3):(0.5-1.5):1.

[0014] In a second aspect, the present invention provides a method for inducing and culturing DC cells using the above-mentioned high-efficiency induction and culture kit for DC cells, comprising the following steps:

[0015] S1: Inoculate PBMC in a 1640 medium for culture, and then discard the suspended cells. The adherent cells obtained are DC cells;

[0016] S2: Add serum-free RPMI 1640 medium and reagent A to the DC cells and continue culturing;

[0017] S3: Change the medium once on the next day and supplement with reagent A to continue culturing;

[0018] S4: On the third day, add reagent B to continue culturing;

[0019] S4: On the fourth day, collect the mature DC cells.

[0020] In some of these preferred embodiments, the seeding density of the PBMC is 1×10 7 cells / mL.

[0021] In some of these preferred embodiments, the culture conditions are 5% CO2 and 37°C.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a high-efficiency induction and culture kit for DC cells, which not only effectively improves the specific expression of DC cells, enhances the expression of related cytokines in the culture supernatant of DC cells, but also shortens the cell culture time, increases the induction and maturation rate and cell activity of DC cells, and enhances the killing activity of T cells against lung cancer cells, providing a new idea for the induction and culture of DC cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 It is a micrograph taken at 100x magnification of DC cells induced and cultured by the highly efficient induction culture kit in Example 1 of the present invention.

[0026] Figure 2 It is a micrograph taken at 100x magnification of DC cells induced and cultured by the highly efficient induction culture kit in Comparative Example 1 of the present invention.

[0027] Figure 3 It is a micrograph taken at 100x magnification of DC cells induced and cultured by the highly efficient induction culture kit in Comparative Example 2 of the present invention.

[0028] Figure 4 It is a graph showing the detection result of the IL-12 content secreted by DC cells of the present invention.

[0029] Figure 5 It is a graph showing the detection result of the killing activity of T cells presented by DC mature cells of the present invention against lung cancer cells. Detailed Embodiments

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0032] In the present invention, the test methods used are all conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0033] In the present invention, the cell culture conditions are 5% CO2 and 37°C.

[0034] In the present invention, the preparation of PBMC (peripheral blood mononuclear cells) specifically includes the following steps:

[0035] 1. Collect 50 mL of human peripheral venous blood as a blood sample. After centrifuging at 3000 rpm for 30 min, separate the upper plasma and lower blood cells. First, inactivate the upper plasma in a 56 °C water bath for 40 min, then centrifuge at 3000 rpm for 20 min at 4 °C, and then remove the lower precipitate to obtain inactivated plasma, which is stored in a 4 °C refrigerator for later use;

[0036] 2. Dilute the lower blood cells with physiological saline to 50 mL, gently mix up and down, then prepare 2 lymphocyte separation tubes each containing 5 mL of Ficoll lymphocyte separation solution, and then evenly add the blood cell and physiological saline mixture to the 2 separation tubes. After centrifuging at 3000 rpm for 20 min, the liquid layers. Take the white cloudy liquid layer and collect it into a new 50 mL centrifuge tube to obtain a PBMC suspension;

[0037] 3. After centrifuging the PBMC suspension at 2000 rpm for 10 min, discard the supernatant; add 30 mL of physiological saline to the cell precipitate for washing. After centrifuging at 2000 rpm for 10 min, discard the supernatant, and repeat the washing 3 times to obtain PBMC.

[0038] Example 1

[0039] A method for highly efficient induction and culture of DC cells specifically includes the following steps:

[0040] 1. Inoculate PBMC at 1×10 7 cells / mL into 20 mL of serum-free RPMI 1640 medium and culture in a cell incubator for 2 h. The adherent cells obtained are DC cells, and the suspended cells are mainly T lymphocytes.

[0041] 2. Induce and differentiate DC cells:

[0042] (1) Remove the non-adherent cells, add 50 mL of serum-free RPMI 1640 medium and 3 mL of highly efficient inducer to the DC cells, and culture in a cell incubator;

[0043] Among them, the highly efficient inducer includes GM-CSF solution at 550 ng / mL, IL-4 solution at 2000 U / mL, IL-1β solution at 6000 IU / mL, AK1 solution at 100 ng / mL, ACK1 solution at 100 ng / mL, MLK3 solution at 50 ng / mL, PLD1 solution at 50 ng / mL, type II cytoskeleton 1 solution at 100 ng / mL, type I cytoskeleton 16 solution at 100 ng / mL;

[0044] The volume ratio of the GM-CSF solution, IL-4 solution, IL-1β solution, AK1 solution, ACK1 solution, MLK3 solution, PLD1 solution, Type II cytoskeleton 1, and Type I cytoskeleton 16 solution is 5:2:1:2:1:1:2:3:2.

[0045] (2) Replace half of the culture medium once on the second day and supplement with the high-efficiency inducer.

[0046] (3) On the third day, add 3 mL of the maturation activator and continue culturing in the cell incubator. Among them, the maturation activator includes a PD-L1 protein solution at 300 ng / mL, a CTLA-4 protein solution at 100 ng / mL, a TIM-3 protein solution at 100 ng / mL, a LAG-3 protein solution at 100 ng / mL, a CD44 protein solution at 400 ng / mL, a CEA protein solution at 500 ng / mL, a neuron-specific enolase solution at 150 ng / mL (Aimix Biotechnology Co., Ltd., 1 mg / ml solution in 20 mM Tris-HCl (pH 7.5) containing 100 mM KCl and 5 mM MgSO4), and a squamous cell carcinoma-related antigen protein solution at 150 ng / mL (Fisher Scientific, 0.1 mg). The volume ratio of the solutions is 2:1:1:1:3:2:1:1.

[0047] (4) On the 4th day, collect the mature DC cells.

[0048] As Figure 1 shown, the suspended cells are the mature DC cells obtained by culturing, indicating that the kit and induction method of this example can obtain a relatively large number of mature DC cells and can significantly improve the proliferation rate of DC cells.

[0049] Example 2

[0050] A method for highly efficient induction and culture of DC cells specifically includes the following steps:

[0051] 1. Inoculate PBMC at 1×10 7 cells / mL into 20 mL of serum-free RPMI 1640 medium and culture in a cell incubator for 2 h. The adherent cells obtained are DC cells, and the suspended cells are mainly T lymphocytes.

[0052] 2. Induce and differentiate DC cells:

[0053] (1) Remove the non-adherent cells, add 50 mL of serum-free RPMI 1640 medium and 3 mL of the high-efficiency inducer to the DC cells, and culture in a cell incubator.

[0054] Among them, the highly efficient inducers include a GM-CSF solution at 500 ng / mL, an IL-4 solution at 3000 U / mL, an IL-1β solution at 7000 IU / mL, an AK1 solution at 50 ng / mL, an ACK1 solution at 50 ng / mL, an MLK3 solution at 70 ng / mL, a PLD1 solution at 30 ng / mL, a type II cytoskeleton 1 solution at 50 ng / mL, and a type I cytoskeleton 16 solution at 200 ng / mL;

[0055] The volume ratio of the GM-CSF solution, IL-4 solution, IL-1β solution, AK1 solution, ACK1 solution, MLK3 solution, PLD1 solution, type II cytoskeleton 1, and type I cytoskeleton 16 solution is 5:2:1:2:1:1:2:3:2.

[0056] (2) Replace half of the culture medium once on the second day, and supplement the highly efficient inducers;

[0057] (3) On the third day, add 3 mL of the maturation activator and continue culturing in the cell incubator; among them, the maturation activator includes a PD-L1 protein solution at 200 ng / mL, a CTLA-4 protein solution at 50 ng / mL, a TIM-3 protein solution at 200 ng / mL, a LAG-3 protein solution at 200 ng / mL, a CD44 protein solution at 300 ng / mL, a CEA protein solution at 700 ng / mL, a neuron-specific enolase solution at 100 ng / mL, and a squamous cell carcinoma-related antigen protein solution at 200 ng / mL, and the volume ratio of the solutions is 2:1:1:1:3:2:1:1;

[0058] (4) On the 4th day, collect the mature DC cells.

[0059] Example 3

[0060] A highly efficient induction and culture method for DC cells specifically includes the following steps:

[0061] 1. Inoculate PBMC at 1×10 7 cells / mL into 20 mL of serum-free RPMI 1640 medium and culture in a cell incubator for 2 h. The adherent cells obtained are DC cells, and the suspended cells are mainly T lymphocytes.

[0062] 2. Induce and differentiate DC cells:

[0063] (1) Remove the non-adherent cells, add 50 mL of serum-free RPMI 1640 medium and 3 mL of the highly efficient inducer to the DC cells, and culture in a cell incubator;

[0064] Among them, the efficient inducers include GM-CSF solution at 600 ng / mL, IL-4 solution at 1000 U / mL, IL-1β solution at 5000 IU / mL, AK1 solution at 200 ng / mL, ACK1 solution at 200 ng / mL, MLK3 solution at 30 ng / mL, PLD1 solution at 70 ng / mL, type II cytoskeleton 1 solution at 200 ng / mL, and type I cytoskeleton 16 solution at 50 ng / mL;

[0065] The volume ratio of the GM-CSF solution, IL-4 solution, IL-1β solution, AK1 solution, ACK1 solution, MLK3 solution, PLD1 solution, type II cytoskeleton 1, and type I cytoskeleton 16 solution is 5:2:1:2:1:1:2:3:2.

[0066] (2) Replace half of the culture medium once on the second day and replenish the efficient inducers;

[0067] (3) On the third day, add 3 mL of the maturation activator and continue culturing in the cell incubator; among them, the maturation activator includes PD-L1 protein solution at 400 ng / mL, CTLA-4 protein solution at 200 ng / mL, TIM-3 protein solution at 200 ng / mL, LAG-3 protein solution at 200 ng / mL, CD44 protein solution at 500 ng / mL, CEA protein solution at 300 ng / mL, neuron-specific enolase solution at 200 ng / mL, and squamous cell carcinoma-related antigen protein solution at 100 ng / mL, and the volume ratio of the solutions is 2:1:1:1:3:2:1:1;

[0068] (4) On the 4th day, collect the mature DC cells.

[0069] Comparative Example 1

[0070] The difference between Comparative Example 1 and Example 1 is that the efficient inducers are different, and the others are the same.

[0071] The efficient inducers include GM-CSF solution at 550 ng / mL, IL-4 solution at 2000 U / mL, and IL-1β solution at 6000 IU / mL, and their volume ratio is 5:2:1.

[0072] In Comparative Example 1, the DC cells obtained by culturing are as Figure 2 shown, and the number of suspended cells is very small, indicating that the proliferation rate of the DC cells in the kit of Comparative Example 1 is slower.

[0073] Comparative Example 2

[0074] The difference between Comparative Example 1 and Example 1 is that the maturation activator is different, and the others are the same.

[0075] The mature activator is a tumor polypeptide antigen solution (human non-small cell lung cancer cell A549 cell lysate) at a concentration of 25 μg / mL.

[0076] In Comparative Example 2, the DC cells obtained by culturing were as Figure 3 shown, with very few suspended cells, indicating that the proliferation rate of the DC cells in the kit of Comparative Example 2 was relatively slow.

[0077] Test Example 1

[0078] Mature DC cells can secrete high levels of IL-12. By detecting the IL-12 secreted by DC cells, the state of DC cells can be analyzed.

[0079] The culture supernatants of the DC cells induced and cultured in Example 1 and Comparative Examples 1-2 on the 4th day were collected respectively. An interleukin 12 (IL-12) ELISA detection kit was used to detect the IL-12 secreted by the mature DC cells in the culture supernatants. The specific operation was carried out according to the ELISA detection instructions, and the results are shown in Figure 4 .

[0080] As Figure 4 can be seen, compared with the kits of Comparative Examples 1-2, the content of IL-12 secreted by the mature DC cells induced and cultured in Example 1 was significantly higher than that secreted by the mature DC cells induced and cultured in the comparative examples, indicating that the kit of the present invention can effectively promote the maturation of DC cells and the expression of the related cytokine IL-12.

[0081] Test Example 2

[0082] Analyze the killing activity of the T cells presented by mature DC cells against lung cancer cells, which specifically includes the following steps:

[0083] 1. The T lymphocytes and mature DC cells in Example 1 and Comparative Examples 1-2 were co-cultured at a ratio of 20:1 respectively to enhance the induction and differentiation of T lymphocytes into cytotoxic T lymphocytes (CTL cells). IL-2 at a final concentration of 100 ng / mL, anti-CD3 monoclonal antibody at a final concentration of 100 ng / mL, and anti-CD28 monoclonal antibody at a final concentration of 100 ng / mL were added respectively. After culturing for 4 days, RPMI 1640 serum-free medium containing the above-mentioned added factors was supplemented and cultured for another 4 days to obtain highly active CTL cells with targeted tumor killing ability respectively.

[0084] 2. Lung cancer cells A549 were inoculated into a Transwell 6-well plate (1×10 5 / well), and after normal culturing for 24 h, at the same density (1×10 6The CTL cells obtained in Example 1 and Comparative Examples 1-2 above were respectively dispensed into a Transwell 6-well plate through the upper chambers (or pores). Among them, the simply cultured lung cancer cells A549 served as the negative control group (Group A), the CTL cells obtained in Example 1 dispensed into the Transwell 6-well plate served as the experimental group (Group B), the CTL cells obtained in Comparative Example 1 dispensed into the Transwell 6-well plate served as the positive control group (Group C), and the CTL cells obtained in Comparative Example 1 dispensed into the Transwell 6-well plate served as the positive control group (Group D). After co-culturing for 24 h, 48 h, and 72 h respectively, the 6-well plate was taken out, digested with trypsin, the cells were placed in a 96-well plate, CCK-8 solution (10 μL / well) was added, and the culture was continued for 1 h. The absorbance at a wavelength of 450 nm was measured with an enzyme-labeled instrument. The results are shown in Figure 5 .

[0085] It can be Figure 5 seen that the T cells activated by the DC cells induced and cultured with the kit of the present invention, when co-cultured with the lung cancer cells A549 for 24 h, 48 h, and 72 h, the growth rate of the cancer cells was inhibited and showed time-dependence. The inhibition growth rates of A549 were significantly lower than those of the negative control group A and the positive control groups C and D. This shows that the DC mature cells induced and cultured with the kit of the present invention can significantly enhance the anti-tumor activity of CTL. The kits of Examples 2-3 are the same as that of Example 1 and will not be described in detail here.

[0086] In addition, it should be understood that although this specification is described according to the embodiments, not every embodiment only contains an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An efficient induction and culture kit for DC cells, characterized in that, The kit includes Reagent A and Reagent B; Among them, Reagent A is GM-CSF solution, IL-4 solution, IL-1β solution, AK1 solution, ACK1 solution, MLK3 solution, PLD1 solution, Cytoskeleton type II 1 solution, and Cytoskeleton type I 16 solution; Reagent B includes PD-L1 protein solution, CTLA-4 protein solution, TIM-3 protein solution, LAG-3 protein solution, CD44 protein solution, CEA protein solution, neuron-specific enolase solution, and squamous cell carcinoma-related antigen protein solution.

2. The highly efficient induction and culture kit for DC cells according to claim 1, characterized in that The concentration of the GM-CSF solution is 500 - 600 ng / mL, the concentration of the IL-4 solution is 1000 - 3000 U / mL, the concentration of the IL-1β solution is 5000 - 7000 IU / mL, the concentration of the AK1 solution is 50 - 200 ng / mL, the concentration of the ACK1 solution is 50 - 200 ng / mL, the concentration of the MLK3 solution is 30 - 70 ng / mL, the concentration of the PLD1 solution is 30 - 70 ng / mL, the concentration of the Cytoskeleton type II 1 solution is 50 - 200 ng / mL, and the concentration of the Cytoskeleton type I 16 solution is 50 - 200 ng / mL.

3. The highly efficient induction and culture kit for DC cells according to claim 1, wherein The volume ratio of the GM-CSF solution, IL-4 solution, IL-1β solution, AK1 solution, ACK1 solution, MLK3 solution, PLD1 solution, Cytoskeleton type II 1, and Cytoskeleton type I 16 solution is (3 - 7):(1 - 3):(0.5 - 1.5):(1 - 3):(0.5 - 1.5):(0.5 - 1.5):(1 - 3):(2 - 4):(1 - 3).

4. The highly efficient induction and culture kit for DC cells according to claim 1, wherein The concentration of the PD-L1 protein solution is 200 - 400 ng / mL, the concentration of the CTLA-4 protein solution is 50 - 200 ng / mL, the concentration of the TIM-3 protein solution is 50 - 200 ng / mL, the concentration of the LAG-3 protein solution is 50 - 200 ng / mL, the concentration of the CD44 protein solution is 300 - 500 ng / mL, the concentration of the CEA protein solution is 300 - 700 ng / mL, the concentration of the neuron-specific enolase solution is 100 - 200 ng / mL, and the concentration of the squamous cell carcinoma-related antigen protein solution is 100 - 200 ng / mL.

5. The highly efficient induction and culture kit for DC cells according to claim 1, wherein, The volume ratio of the PD-L1 protein solution, CTLA-4 protein solution, TIM-3 protein solution, LAG-3 protein solution, CD44 protein solution, CEA protein solution, neuron-specific enolase solution, and squamous cell carcinoma-related antigen protein solution is (1 - 3):(0.5 - 1.5):(0.5 - 1.5):(0.5 - 1.5):(2 - 4):(1 - 3):(0.5 - 1.5):

1.

6. A method for inducing and culturing DC cells using the high-efficiency induction and culture kit for DC cells according to any one of claims 1-5, characterized in that, It includes the following steps: S1: Inoculate PBMC into a 1640 medium for culture, and then discard the suspended cells. The adherent cells obtained are DC cells; S2: Add serum-free RPMI 1640 medium and Reagent A to the DC cells and continue the culture; S3: Change the culture fluid once on the second day and continue the culture by adding Reagent A; S4: On the third day, add Reagent B and continue the culture; S4: On the fourth day, collect the mature DC cells.

7. The method according to claim 6, wherein The seeding density of the PBMC is 1×10 7 cells / mL.

8. The method according to claim 6, wherein The culture conditions are 5% CO2 and 37°C.

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