Culture medium composition and culture method for culturing dendritic cells

Abstract

The invention discloses a culture medium composition for culturing dendritic cells and a culture method, and belongs to the field of biological medicines. Aiming at the problems of long induced differentiation period and low purity of the existing DC, the invention provides the culture medium composition for culturing the dendritic cells, the culture medium composition comprises a basic culture medium, an induction culture medium, a maturation culture medium and a function strengthening culture medium, the induction culture medium comprises the basic culture medium, and the maturation culture medium comprises a mature culture medium and a function strengthening culture medium; the GM-CSF, the IL-4, the astragalus polysaccharide and the rapamycin are added into the basic culture medium; the mature culture medium comprises a basic culture medium, and TNF-alpha, IFN-gamma, ganoderic acid and Poly (I: C) which are added into the basic culture medium, the function-enhanced culture medium comprises a basic culture medium, and IL-10, IL-12 and vitamin D3 which are added into the basic culture medium. The culture medium in each stage is combined with the synergistic effect of specific cell factors and natural plant extracts, so that high-purity and high-activity induction of the DC is realized, and the DC has the capability of resisting tumor microenvironment inhibition.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to a culture medium composition and culture method for culturing dendritic cells. Background Technology

[0002] Dendritic cells (DCs) are the most potent antigen-presenting cells in the human body, playing a vital role in tumor immunotherapy and vaccine development. Currently, in vitro induction of DCs often employs a combination of GM-CSF and IL-4, but this method suffers from several drawbacks: low induction efficiency (limited yield, typically around 30%), immature function (insufficient expression of co-stimulatory molecules like CD80, CD86, and CD40 on the induced DC surface, resulting in weak antigen-presenting ability), serum dependence (using FBS or human serum, leading to batch-to-batch variations and pathogen contamination risks), and inability to withstand immunosuppressive microenvironments (factors like TGF-β and IL-10 in the tumor microenvironment inhibit DC function). While existing techniques have attempted to add TNF-α and PGE2 to promote DC maturation, these methods still suffer from high costs, significant side effects, and long induction cycles.

[0003] Corresponding improvements have been made to address the above problems. For example, Chinese patent application CN201610088092.2, published on April 13, 2016, discloses a method for culturing dendritic (DC) cells and a culture medium. The culture medium contains recombinant human transferrin, recombinant human insulin, GM-CSF, IL-4, IL-15, progesterone, human serum albumin, and a basal culture medium. The drawback of this patent is that progesterone is a reproductive hormone known in immunology to have immunosuppressive properties; therefore, progesterone may inhibit the maturation and activation of DC cells, leading to decreased DC cell function.

[0004] For example, Chinese patent application CN202311274459.6, published on January 2, 2024, discloses a DC cell culture medium and its culture method. Specifically, it includes: DMEM / F12 medium, recombinant human transferrin, GM-CSF, IL-4, IL-15, progesterone, human serum albumin, palmitic acid, ethanolamine, astragalus polysaccharide, ephedra polysaccharide, and the remainder being water. The drawback of this patent is that although it can obtain high-purity DC cells, the culture period is long, increasing the possibility of cell aging or functional decline in vitro. Summary of the Invention

[0005] 1. The problem to be solved To address the problems of long differentiation cycles and low purity in existing dendritic cell (DC) induction methods, this invention provides a culture medium combination and method for culturing dendritic cells. The culture media at each stage of this invention combine the synergistic effects of specific cytokines and natural plant extracts to achieve high purity and high activity induction of DCs, and also possesses anti-tumor microenvironment inhibitory capabilities.

[0006] 2. Technical Solution To solve the above problems, the present invention adopts the following technical solution.

[0007] A culture medium combination for culturing dendritic cells includes a basal medium, an induction medium, a maturation medium, and a functional enhancement medium. The induction medium includes a basal medium, and GM-CSF, IL-4, astragalus polysaccharide and rapamycin added to the basal medium; The mature culture medium includes a basal culture medium, and TNF-α, IFN-γ, ganoderic acid and Poly(I:C) added to the basal culture medium; The functional enhancement culture medium includes a basal culture medium, and IL-10, IL-12 and vitamin D3 added to the basal culture medium.

[0008] Furthermore, the basal culture medium includes a mixed culture medium of X-VIVO15 medium and AIM-V medium, and the mixed culture medium contains 200-500 μg / mL recombinant human serum albumin, 2-10 μg / mL recombinant human transferrin and 2-8 μg / mL recombinant human insulin.

[0009] Furthermore, the induction medium contains GM-CSF: 50~100 ng / mL; IL-4: 20~70 ng / mL; Astragalus polysaccharide: 20~80 μg / mL; Rapamycin: 2~5 nM.

[0010] Furthermore, the mature culture medium contains TNF-α: 10~30 ng / mL; IFN-γ: 10~50 ng / mL; ganoderic acid: 10~80 μg / mL; Poly(I:C): 1~30 μg / mL.

[0011] Furthermore, the functional enhancement culture medium contains IL-10: 2~10 ng / mL; IL-12: 1~30 ng / mL; and vitamin D3: 1~30 nM.

[0012] A culture method using a culture medium combination for culturing dendritic cells as described in any of the above technical solutions includes the following steps: S1: Induction culture: Mononuclear cells are seeded in induction medium and cultured for 2-4 days to obtain induced cultured cells; S2: Maturation culture: Induced culture cells are seeded into maturation medium and cultured for 2-4 days to obtain mature culture cells; S3: Functional enhancement culture: Inoculate mature cultured cells in functional enhancement medium and culture for 2-4 days.

[0013] Furthermore, before step S1, step S0 is included: isolation of monocytes; specifically, it includes the following steps: S01: PBMCs were obtained from peripheral blood using density gradient centrifugation. S02: Resuspend PBMCs in PBS medium containing 2% human serum albumin and adjust the cell density to 5 × 10⁶ cells / year. 6 cells / mL; S03: Seed the cell suspension into cell culture flasks or dishes coated with poly-L-lysine and incubate at 37°C in a 5% CO2 incubator for 2 hours; S04: After incubation, repeatedly pipet and aspirate the supernatant to remove non-adherent lymphocytes.

[0014] Furthermore, in step S03, the inoculation density is (1~2)×10⁻⁶. 6 pcs / cm 2 It also includes step S05: washing adherent cells 2-3 times with pre-warmed PBS culture medium to thoroughly remove any remaining non-adherent cells.

[0015] Furthermore, in step S2, when culturing in the mature culture medium, half of the mature culture medium is replaced every 24 hours.

[0016] 3. Beneficial effects (1) In the induction culture medium of the present invention, the synergistic use of Astragalus polysaccharide and rapamycin enhances the directed differentiation of DC precursor cells and improves the differentiation yield of DC cells; in the maturation culture medium, the combined use of ganoderic acid and Poly(I:C) enhances the anti-tumor microenvironment ability of DC cells and improves the antigen presentation effect of DC cells; in the functional enhancement culture medium, the phased addition of IL-10 and vitamin D3 regulates the immune tolerance and activation balance of DC cells and improves the antigen presentation effect of DC cells on T cells; the entire culture medium combination achieves high purity and high activity induction of DC cells and has the ability to inhibit the anti-tumor microenvironment. (2) The two culture media used in the basic culture media of this invention are both serum-free and have clearly defined chemical compositions. By mixing complementary components, a culture medium with superior nutrition, support and stability is created to meet the needs of specific cells in both rapid expansion and function maintenance. The addition of recombinant human serum albumin, recombinant human transferrin and recombinant human insulin simulates the functions of serum in nutrient transport, delivery of essential trace elements and growth signal stimulation, thereby completely replacing serum, completely avoiding animal-derived components, and meeting clinical-grade production standards. (3) The present invention limits the content of each raw material in the induction culture medium, maturation culture medium and functional enhancement culture medium. During the induction of DC cells, each component must be controlled at an appropriate concentration in order to achieve a synergistic effect. If the concentration is below the given range, the effect of inducing differentiation will not be achieved. If the concentration is above the range, it will lead to apoptosis of cells due to excessive differentiation, decreased cell activity, decreased induction yield and decreased antigen presentation ability, etc. For example, the role of TNF-α is to promote DC maturation, and the role of IFN-γ is to enhance antigen presentation ability. Excessive addition will lead to excessive differentiation of DC cells. (4) The culture method of the present invention achieves high induction efficiency by using a three-stage induction strategy combined with the synergistic effect of specific cytokines and natural plant extracts: DC yield is increased to more than 1.5 times that of the traditional method (yield is about 40~80%); high functional maturity: the expression of co-stimulatory molecules is significantly improved and the antigen presentation ability is strong; and resistance to inhibitory microenvironment: resistance to inhibitory factors such as TGF-β; finally, high purity and high activity induction of DC are achieved, and it has the ability to inhibit the tumor microenvironment. (5) The mononuclear cell separation and adhesion method in this invention does not require expensive magnetic bead sorting reagents and special equipment, which significantly reduces the cost of initial cell separation and is more suitable for basic research or small-scale clinical preparation. It is also simple to operate, easy to master and implement, and has low technical barriers. At the same time, the separated cells have high purity, usually >90%, and the cells obtained by the adhesion method can be directly used for subsequent induction culture, with a smooth process. Attached Figure Description

[0017] Figure 1 This is a flow cytometry comparison diagram of Example 1; Figure 2 This is a flow cytometry comparison diagram of Example 2; Figure 3 This is a comparison diagram of the mixed lymphocyte reactions (verifying the antigen presentation effect of induced DC cells) in Examples 1 and 2. Detailed Implementation

[0018] The present invention will now be further described with reference to specific embodiments and accompanying drawings.

[0019] A culture medium combination for culturing dendritic cells includes a basal medium, an induction medium, a maturation medium, and a functional enhancement medium. The induction medium includes a basal medium, and GM-CSF, IL-4, astragalus polysaccharide and rapamycin added to the basal medium; The mature culture medium includes a basal culture medium, and TNF-α, IFN-γ, ganoderic acid and Poly(I:C) added to the basal culture medium; The functional enhancement culture medium includes a basal culture medium, and IL-10, IL-12 and vitamin D3 added to the basal culture medium.

[0020] In this embodiment, different culture media are provided at different stages. The staged culture media precisely provide the key signals required at each stage, avoiding signal conflicts and timing disorders. Moreover, the objectives of each stage are clear: induction culture medium: the core is to expand the quantity and obtain a large number of high-purity DC precursors; maturation culture medium: the core is to activate the function and endow DCs with strong antigen presentation and co-stimulatory capabilities; functional enhancement culture medium: the core is to optimize the quality and fine-tune the immune characteristics of DCs to make them more suitable for the anti-tumor environment, ensuring that the final DC product is not only sufficient in quantity, but also powerful and stable in function.

[0021] Secondly, different culture media combined with the synergistic effects of specific cytokines and natural plant extracts: in the induction culture medium, the synergistic use of astragalus polysaccharide and rapamycin enhances the directed differentiation of DC precursor cells and increases the differentiation yield of DC cells; in the maturation culture medium, the combined use of ganoderic acid and Poly(I:C) enhances the anti-tumor microenvironment ability of DC cells and improves the antigen presentation effect of DC cells; in the functional enhancement culture medium, the phased addition of IL-10 and vitamin D3 regulates the immune tolerance and activation balance of DC cells and improves the antigen presentation effect of DC cells on T cells; the entire culture medium combination achieves high-purity and high-activity induction of DC cells and possesses the ability to inhibit the anti-tumor microenvironment.

[0022] In one specific embodiment, the basal culture medium includes a mixed culture medium of X-VIVO15 medium and AIM-V medium, and the mixed culture medium contains 200-500 μg / mL recombinant human serum albumin, 2-10 μg / mL recombinant human transferrin and 2-8 μg / mL recombinant human insulin.

[0023] Specifically, this embodiment achieves a serum-free basal culture medium system, thereby completely avoiding animal-derived components and meeting clinical-grade production standards. Because it is serum-free, functional factors capable of achieving serum-like functions are added, maximizing support for high-rate, high-quality, and high-functionality expansion of specific cells under absolute safety conditions (no pathogen risk, no batch-to-batch variation).

[0024] In one specific embodiment, the induction medium contains GM-CSF: 50-100 ng / mL; IL-4: 20-70 ng / mL; Astragalus polysaccharide: 20-80 μg / mL; and rapamycin: 2-5 nM. GM-CSF and IL-4 serve as the basic driving axis for DC differentiation. Astragalus polysaccharide provides an early and potent activation signal through the TLR4 / NF-κB pathway, forming cross-stimulation with the basic pathway, significantly amplifying the differentiation signal and improving induction efficiency. Simultaneously, a low dose of rapamycin is introduced to moderately inhibit the activation of protective autophagy through the mTOR pathway, metabolically reprogramming and quality-controlled the differentiation process. The synergistic effect of these three components ensures that monocytes can differentiate into functionally complete immature DCs efficiently, synchronously, and with high quality, laying a solid foundation for subsequent maturation and functional enhancement stages.

[0025] In one specific embodiment, the maturation culture medium contains TNF-α: 10-30 ng / mL; IFN-γ: 10-50 ng / mL; ganoderic acid: 10-80 μg / mL; and Poly(I:C): 1-30 μg / mL. TNF-α and IFN-γ, as classical signaling axes, synergistically upregulate the expression of MHC and co-stimulatory molecules, laying the foundation for DC maturation. Poly(I:C) simulates viral invasion through the TLR3 pathway, inducing a type I interferon storm, strongly amplifying the maturation signal, and significantly enhancing the cross-presentation capacity of DCs. Crucially, ganoderic acid antagonizes the signal transduction of TGF-β, a key inhibitor of the tumor microenvironment, thus 'unblocking' DC maturation and synergistically increasing the level of co-stimulatory molecules. These four components respectively act as 'initiating signals,' 'enhancing signals,' 'amplifying signals,' and 'defense enhancers,' jointly ensuring that the obtained DC phenotype is fully mature, functionally robust, and capable of resisting the immunosuppressive microenvironment.

[0026] In one specific embodiment, the functional enhancement culture medium contains IL-10: 2~10 ng / mL; IL-12: 1~30 ng / mL; and Vitamin D3: 1~30 nM. By using IL-10, Vitamin D3, and IL-12 in combination, precise regulation of DC function is achieved. Specifically, IL-10 and Vitamin D3 work synergistically to induce a stable 'semi-tolerant' phenotype in DCs through the STAT3 and VDR pathways, significantly enhancing their lymph node homing ability and improving their adaptability and application safety in an inhibitory microenvironment. Simultaneously, IL-12, as a key Th1 polarizing factor, effectively balances the inhibitory tendencies of the former two, ensuring that the immune activation and T-cell driving capacity of the DC core are not weakened.

[0027] A culture method using a culture medium combination for culturing dendritic cells as described in any of the above technical solutions includes the following steps: S1: Induction culture: Mononuclear cells are seeded in induction medium and cultured for 2-4 days to obtain induced cultured cells; S2: Maturation culture: Induced culture cells are seeded into maturation medium and cultured for 2-4 days to obtain mature culture cells; S3: Functional enhancement culture: Inoculate mature cultured cells in functional enhancement medium and culture for 2-4 days.

[0028] The culture method in this embodiment employs a three-stage induction strategy, with each stage typically involving approximately 3 days of culture. By combining the synergistic effects of specific cytokines and natural plant extracts, high induction efficiency is achieved: DC yield is increased to more than 1.5 times that of traditional methods (yield of approximately 40-80%); high functional maturity is achieved: co-stimulatory molecule expression is significantly enhanced, and antigen presentation ability is strong; and resistance to inhibitory microenvironment is achieved: resistance to inhibitory factors such as TGF-β is enhanced; ultimately, high-purity and high-activity induction of DCs is achieved, along with the ability to inhibit the tumor microenvironment.

[0029] In one specific embodiment, step S0 is included before step S1: isolation of monocytes; specifically including the following steps: S01: PBMCs were obtained from peripheral blood using density gradient centrifugation. S02: Resuspend PBMCs in PBS medium containing 2% human serum albumin and adjust the cell density to 5 × 10⁶ cells / year. 6 cells / mL; S03: Seed the cell suspension into cell culture flasks or dishes coated with poly-L-lysine and incubate at 37°C in a 5% CO2 incubator for 2 hours; S04: After incubation, repeatedly pipet and aspirate the supernatant to remove non-adherent lymphocytes.

[0030] It is particularly worth noting that the cell separation method used in this embodiment is the adherence method. The adherence method does not require expensive magnetic bead sorting reagents and special equipment, which significantly reduces the cost of initial cell separation. Moreover, the purity of mononuclear cells separated by the adherence method is usually >90%, which can achieve a purity effect that is basically the same as that of magnetic bead sorting.

[0031] In one specific embodiment, the inoculation density in step S03 is (1~2)×10 6 pcs / cm 2， It's worth noting that using this density for monocyte isolation ensures that most monocytes are captured and that the cell density is appropriate for subsequent cell induction. The procedure also includes step S05: washing adherent cells 2-3 times with pre-warmed PBS to thoroughly remove any remaining non-adherent cells. Step S05, through physical washing, further enhances the initial purity of the monocytes, providing a fundamental guarantee for obtaining functionally superior, phenotypically consistent, and reproducible dendritic cells.

[0032] In one specific embodiment, during step S2, when culturing in the mature culture medium, half of the mature culture medium is replaced every 24 hours.

[0033] To further facilitate understanding of the solution in this application, the following examples are provided: Example 1 A method for culturing DC cells includes the following steps: 1. Prepare serum-free basal medium: Mix X-VIVO™15 and AIM-V® medium at a volume ratio of 2:1, and add 300 μg / mL recombinant human serum albumin (rHSA), 5 μg / mL recombinant human transferrin and 4 μg / mL recombinant human insulin; 2. Cultivation process: 2.1 Mononuclear cell isolation: PBMCs were isolated from peripheral blood using density gradient centrifugation. Resuspend PBMCs in PBS or basal medium containing 2% human serum albumin (HSA) and adjust the cell density to 5 × 10⁶ cells / day. 6 cells / mL; Seed the cell suspension into cell culture flasks or dishes coated with poly-L-lysine (seeding density 1×10⁻⁶). 6 pcs / cm 2 Incubate at 37°C in a 5% CO2 incubator for 2 hours; Monocyte adhesion: Monocytes adhere to the surface of the culture dish due to their adhesive properties, while lymphocytes are suspended in the culture medium; After incubation, gently blow and aspirate the supernatant multiple times to remove non-adherent lymphocytes (mainly T cells, B cells, and NK cells). Gently wash adherent cells twice with pre-warmed PBS or basal culture medium to thoroughly remove any remaining non-adherent cells.

[0034] Three-stage induction culture: Phase 1 (0-3 days): Induction medium was prepared by adding 80 ng / mL GM-CSF, 50 ng / mL IL-4, 40 μg / mL Astragalus polysaccharide and 3 nM rapamycin to serum-free basal medium. The medium was then added to culture dishes with attached mononuclear cells and incubated statically at 37°C in a 5% CO2 incubator. Phase Two (Days 4-6): Prepare maturation medium using serum-free basal medium supplemented with 20 ng / mL TNF-α, 15 ng / mL IFN-γ, 25 μg / mL ganoderic acid, and 10 μg / mL Poly(I:C). Gently collect the medium and suspended cells, centrifuge to remove the old medium. Resuspend the cells in the maturation medium and re-add them to culture dishes. Incubate at 37°C in a 5% CO2 incubator. Change half the medium every 24 hours (gently remove half of the medium and add the same volume of fresh maturation medium, being careful not to aspirate the cells). Phase Three (7-9 days): Prepare a serum-free basal medium by adding 5 ng / mL IL-10, 10 ng / mL IL-12, and 10 nM vitamin D3 to prepare an enhanced medium. Gently collect the medium and suspended cells, and centrifuge to remove the old medium. Resuspend the cells in the enhanced medium, then add them back to the culture dish and incubate at 37°C in a 5% CO2 incubator for 48 hours.

[0035] Harvesting and Quality Inspection: like Figure 1 As shown, all cells were collected and fluorescently labeled with flow cytometry direct-labeled antibodies. The positive rates of CD14, CD83, CD209, and CD11C were detected by flow cytometry. The quality control requirements were met if CD14 ≤ 5%, CD83 ≥ 90%, CD209 ≥ 90%, and CD11C ≥ 90%.

[0036] Example 2 Based on the same principles as Example 1, except that no ganoderic acid is added in the second stage of the three-stage induction culture, the rest is exactly the same as Example 1.

[0037] Harvesting and Quality Inspection: like Figure 2As shown, all cells were collected and fluorescently labeled with flow cytometry direct-labeled antibodies. The positive rates of CD14, CD83, CD209 and CD11C were detected by flow cytometry. CD14 ≤ 5%, CD83 ≥ 50% (low expression, not meeting quality control requirements), CD209 ≥ 90% and CD11C ≥ 90% did not meet quality control requirements. Meanwhile, the antigen presentation effects of the induced DC cells verified in Examples 1 and 2 are as follows: Figure 3 As shown, the obtained dendritic cells (DCs) were co-incubated with allogeneic PBMCs, and the antigen-presenting capacity of the DCs was analyzed by detecting the secretion of cytokines in the supernatant. Figure 3 It can be concluded without a doubt that the DC cells cultured in this application have a more obvious antigen presentation effect, and exhibit better stimulation effect when co-incubated with PBMC cells from multiple allogeneic sources, with a higher secretion of human IFN-γ.

[0038] Example 3 Similar to Example 1, except that different concentrations of IFN-γ were added in stage two of the three-stage induction culture: 5 ng / mL, 15 ng / mL, 45 ng / mL, and 135 ng / mL. Everything else was identical to Example 1. The results are shown in Table 1. Table 1. Schematic diagram of structures added at different IFN-γ concentrations

[0039] At the start of induction, each group was treated with the same 15M PBMCs, with these Donor monocytes comprising 31.36% of the total. If all monocytes were captured, the total number of monocytes in each group would be approximately 4.70E+06. Table 1 shows the total number of viable cells harvested, cell viability, and induction yield after induction with different concentrations of IFN-γ. While adding 5 ng / mL IFN-γ resulted in a higher induction yield, some cells remained undifferentiated, failing to achieve the desired induction effect. Adding 135 ng / mL IFN-γ resulted in lower overall cell viability and induction yield, indicating over-induction. The optimal cell viability and induction yield were achieved with 15 ng / mL and 45 ng / mL IFN-γ.

[0040] The examples described herein are merely preferred embodiments of the invention and are not intended to limit the concept and scope of the invention. Any modifications and improvements made by those skilled in the art to the technical solutions of the invention without departing from the design concept of the invention should fall within the protection scope of the invention.

Claims

1. A culture medium composition for culturing dendritic cells, characterized in that: This includes basal culture medium, induction culture medium, maturation culture medium, and functional enhancement culture medium; The induction medium includes a basal medium, and GM-CSF, IL-4, astragalus polysaccharide and rapamycin added to the basal medium; The mature culture medium includes a basal culture medium, and TNF-α, IFN-γ, ganoderic acid and Poly(I:C) added to the basal culture medium; The functional enhancement culture medium includes a basal culture medium, and IL-10, IL-12 and vitamin D3 added to the basal culture medium.

2. The culture medium composition for culturing dendritic cells according to claim 1, characterized in that: The basal culture medium includes a mixture of X-VIVO15 medium and AIM-V medium, and the mixture contains 200-500 μg / mL recombinant human serum albumin, 2-10 μg / mL recombinant human transferrin and 2-8 μg / mL recombinant human insulin.

3. A culture medium composition for culturing dendritic cells according to claim 1 or 2, characterized in that: The induction medium contained GM-CSF: 50-100 ng / mL; IL-4: 20-70 ng / mL; Astragalus polysaccharide: 20-80 μg / mL; and Rapamycin: 2-5 nM.

4. A culture medium composition for culturing dendritic cells according to claim 1, characterized in that: The mature culture medium contained TNF-α: 10~30 ng / mL; IFN-γ: 10~50 ng / mL; ganoderic acid: 10~80 μg / mL; and Poly(I:C): 1~30 μg / mL.

5. A culture medium composition for culturing dendritic cells according to claim 1, characterized in that: The functional enhancement culture medium contains IL-10: 2~10 ng / mL; IL-12: 1~30 ng / mL; and vitamin D3: 1~30 nM.

6. A method for culturing dendritic cells using the culture medium combination for culturing dendritic cells as described in any one of claims 1-5, characterized in that: Includes the following steps: S1: Induction culture: Mononuclear cells are seeded in induction medium and cultured for 2-4 days to obtain induced cultured cells; S2: Maturation culture: Induced culture cells are seeded into maturation medium and cultured for 2-4 days to obtain mature culture cells; S3: Functional enhancement culture: Inoculate mature cultured cells in functional enhancement medium and culture for 2-4 days.

7. The method for culturing dendritic cells according to claim 6, characterized in that: Before step S1, step S0, the isolation of monocytes, is included; specifically, the following steps are included: S01: PBMCs were obtained from peripheral blood using density gradient centrifugation. S02: Resuspend PBMCs in PBS medium containing 2% human serum albumin and adjust the cell density to 5 × 10⁶ cells / year. 6 cells / mL; S03: Seed the cell suspension into cell culture flasks or dishes coated with poly-L-lysine and incubate at 37°C in a 5% CO2 incubator for 2 hours; S04: After incubation, repeatedly pipet and aspirate the supernatant to remove non-adherent lymphocytes.

8. The method for culturing dendritic cells according to claim 7, characterized in that: In step S03, the inoculation density is (1~2)×10⁻⁶. 6 pcs / cm 2 It also includes step S05: washing adherent cells 2-3 times with pre-warmed PBS culture medium to thoroughly remove any remaining non-adherent cells.

9. A method for culturing dendritic cells according to claim 6, characterized in that: In step S2, when culturing in the mature culture medium, half of the mature culture medium is replaced every 24 hours.

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