Culture method and application of macrophage

Abstract

The application provides a culture method of macrophages and application thereof, and the culture method comprises the following steps: using human PBMCs, adding an induction culture medium to induce differentiation into macrophages, wherein the induction culture medium comprises a basic culture medium, serum, M-CSF and 5-15 muM norcantharidin, wherein liquid supplementing or replacing is performed every 2-3 days; 100 U / mL IFN-gamma is added to the induction culture medium on the fifth day to induce macrophages; the macrophages are continuously cultured for 2 days, and corresponding macrophages are obtained; the cells and supernatant are obtained by centrifugation, and the cells and supernatant are detected. By using the method, M1 polarization of macrophages can be inhibited, and a new application prospect of anti-inflammation of macrophages is provided.

Description

Macrophage culture methods and applications Technical Field

[0001] This invention relates to the field of cell culture technology, and more particularly to a culture medium for macrophages and its applications. Background Technology

[0002] Macrophages, as core effector cells of the body's innate immune system, play a crucial role in immune defense, regulation of inflammatory responses, tissue repair, and the development and progression of diseases. Macrophages can be divided into the M1 phenotype, which has pro-inflammatory and anti-tumor effects, and the M2 phenotype, which has anti-inflammatory and pro-tumor progression effects.

[0003] However, in actual disease treatment scenarios, overactivated M1 macrophages often lead to uncontrolled inflammatory responses, causing tissue damage and even exacerbating disease progression. For example, in inflammatory diseases such as rheumatoid arthritis and acute lung injury, the massive infiltration and overactivation of M1 macrophages are important reasons for disease deterioration. Therefore, discovering drugs or active ingredients that can effectively inhibit the M1 macrophage phenotype activation would provide new treatment strategies and interventions for these inflammatory-related diseases. Summary of the Invention

[0004] Therefore, the present invention aims to solve the problem of macrophage culture activating to the M1 type in the prior art.

[0005] This invention provides a method for culturing macrophages using human PBMCs, which, when cultured under specific conditions, can inhibit M1 type polarization of macrophages.

[0006] Specifically, the first aspect of the present invention provides a method for culturing macrophages, comprising the following steps: using human PBMCs, adding induction culture medium to induce differentiation into macrophages, wherein the induction culture medium includes basal culture medium, serum, M-CSF and 5-15 μM norepinephrine, wherein the medium is replenished or changed every 2-3 days;

[0007] On day 5, 100 U / mL of IFN-γ was added to the induction medium to induce macrophages;

[0008] Continue culturing for 2 days to obtain the corresponding macrophages. Centrifuge to collect the cells and supernatant for analysis.

[0009] In this invention, the corresponding human PBMCs can be obtained by pre-collecting, frozen, and thawed cells, and then resuspended and seeded before being added to the induction medium. Specifically, the human PBMCs are thawed, resuspended in basal medium, and inoculated at a concentration of 0.5~0.75×10⁻⁶. 7Cells were seeded at 1 / mL and 2mL / well in 6-well plates. After adhering and culturing for 2 hours, non-adhering cells were removed.

[0010] Specifically, in this invention, human PBMCs are used. Compared with animal-derived PBMCs, the gene expression, signaling pathways, surface markers, and cytokine profiles of the macrophages obtained are completely consistent with human diseases and physiological states. They can be used to study individualized immune responses or disease susceptibility, providing a more direct and reliable standard for subsequent drug development and precision medicine, and ensuring subsequent clinical applications.

[0011] The induction medium includes basal medium, serum, M-CSF, and 5-15 μM norcantharidin. The basal medium and serum provide essential nutrients, vitamins, amino acids, and inorganic salts necessary for cell growth, proliferation, and differentiation. Serum provides additional growth factors, hormones, and adhesion factors, creating a stable microenvironment for cell survival and differentiation.

[0012] M-CSF is a macrophage colony-stimulating factor, a key cytokine for inducing monocyte differentiation into macrophages. By binding to its receptor, it activates downstream signaling pathways, driving monocyte adhesion, morphological changes, and the expression of macrophage-specific markers. It is a core inducer of macrophage differentiation and the foundation for obtaining high-purity, high-yield macrophages.

[0013] Crucially, the inventors of this invention discovered that adding norcantharidin before adding IFN-γ can inhibit its subsequent M1 polarization. Norcantharidin is used in this system as an early inhibitor of M1 polarization, its core function being to intervene and inhibit the M1 polarization pathway early in differentiation, thereby affecting macrophage M1 polarization. Specifically, norcantharidin and M-CSF are added during the early differentiation stage for the culture of human PBMCs.

[0014] During the initial induction culture, the medium is replenished or replaced every 2-3 days to maintain a stable concentration of norcantharidin, ensuring the persistence and stability of the inhibitory effect during the 7-day culture period. This also provides a stable culture environment for the long differentiation process, ensuring healthy cell growth.

[0015] On day 5, 100 U / mL of IFN-γ was added as polarization induction pressure.

[0016] In an optional embodiment of the first aspect of the present invention, the concentration of M-CSF is 50-200 ng / mL.

[0017] In this implementation, the concentration of M-CSF ranges from 50 to 200 ng / mL to ensure that it can provide a sufficiently strong signal stimulus to drive most monocytes to initiate the differentiation process. At the same time, while ensuring maximum differentiation efficiency, it avoids cost waste caused by excessive addition of cytokines and prevents unexpected cell responses or functional deviations that may be caused by excessively strong signals.

[0018] In an optional embodiment of the first aspect of the present invention, the amount of norcantharidin added is 5-10 μM.

[0019] In an optional embodiment of the first aspect of the present invention, the amount of norcantharidin added is 5 μM.

[0020] In one optional embodiment of the first aspect of the present invention, the amount of norcantharidin added is 10 μM.

[0021] In a preferred experimental design of this invention, the amount of norcanthate added is 5-10 μM. This concentration range has been shown to effectively interfere with the IFN-γ-mediated M1 polarization signaling pathway. 5 μM was determined to be the most preferred concentration. Under this preferred concentration, macrophages cultured using the method of this invention showed significantly more characteristic M1 depolarization compared to the control group induced by M-CSF and IFN-γ without norcanthate. At 10 μM, all groups showed inhibition of M1 polarization, but the inhibitory effect was relatively mild.

[0022] In an optional embodiment of the first aspect of the present invention, the serum is selected from autologous plasma, serum substitutes, and fetal bovine serum.

[0023] The serum is preferably autologous plasma, which is derived from the human PBMC donor. It can completely eliminate heterologous immune responses and batch-to-batch differences, and provides a factor composition that is closest to the donor's internal environment.

[0024] In an optional embodiment of the first aspect of the present invention, the basal culture medium is AIM-V or KBM581. Preferably, the culture medium is AIM-V medium.

[0025] In an optional embodiment of the first aspect of the present invention, the detection includes one or more of the following: detection of M1 type markers CD40, CD64, CCR7 expression, mitochondrial membrane potential JC-1 staining, and phagocytic function.

[0026] Specifically, the present invention also performs polarity detection on cultured macrophages to determine whether they inhibit M1 polarization.

[0027] The study included the detection of expression levels of CD40, CD64, and CCR7, all of which are classic characteristic surface markers of M1 macrophages. CD40, belonging to the TNF receptor superfamily, is highly expressed on the surface of activated immune cells. Its binding to its ligand CD40L is a key co-stimulatory signal amplifying pro-inflammatory immune responses and enhancing antigen presentation. CD64 is a high-affinity immunoglobulin G (IgG) Fc receptor. M1 macrophages highly express CD64, mediating antibody-dependent cytotoxicity (ADCC) and phagocytosis, making it a crucial molecule for their potent effector functions. CCR7 is a chemokine receptor that guides cell migration to lymphoid tissues. CCR7 expression on M1 macrophages is key to their antigen presentation function, interaction with T cells, and initiation of adaptive immunity.

[0028] JC-1 staining was used to detect mitochondrial membrane potential. JC-1 is a fluorescent probe that exists in different forms depending on the mitochondrial membrane potential and is used to assess cellular energy metabolism. M1 macrophages mainly rely on rapid glycolysis for energy, and their mitochondrial function is usually inhibited to some extent, manifested as a decrease in mitochondrial membrane potential.

[0029] Phagocytosis is a core and fundamental function of macrophages, but the phagocytic targets and efficiency can differ under different polarization states. M1 macrophages typically exhibit strong phagocytic activity towards opsonized particles (such as IgG-coated microspheres) or pathogens. Phagocytic capacity can be quantified by detecting intracellular fluorescence intensity using flow cytometry or fluorescence microscopy after co-incubation with fluorescently labeled microspheres (such as FITC-dextran or pHrodo-labeled bacterial / zymosan particles).

[0030] As a second aspect of the invention, protection is also claimed for the use of a culture medium as described in any of the first aspects of the invention in macrophage culture.

[0031] Specifically, a second aspect of the present invention claims protection for specific industrial and laboratory applications for inhibiting M1 type polarization of macrophages. These applications include, but are not limited to, the following:

[0032] This medium is used to prepare macrophages with suppressed M1 polarization, enabling the routine in vitro induction of macrophage products with weakened M1 characteristics and anti-inflammatory or immunomodulatory tendencies. Furthermore, it can be used to establish in vitro cell models for inflammatory disease research. By culturing PBMCs from patients or disease-prone animal models in this medium, a macrophage model with M1 response deficiency or suppressed inflammatory response can be constructed. This model is suitable for studying the mechanisms of macrophage dysfunction in chronic inflammation, such as rheumatoid arthritis, atherosclerosis, and metabolic inflammation, or for assessing the plasticity of immune cells in disease states. Alternatively, it can be used to screen and evaluate substances with anti-M1 polarization activity and for the research and development of immunomodulatory therapies.

[0033] Beneficial effects:

[0034] This invention provides a method for culturing macrophages. Adding norcantharidin before adding IFN-γ can inhibit the subsequent polarization of macrophages to the M1 type. Norcantharidin is used as an early inhibitor of M1 polarization in this system. Using this method, macrophage M1 polarization can be inhibited, providing a new application prospect for macrophage anti-inflammatory treatment. Attached Figure Description

[0035] Figure 1 shows the flow cytometry detection of CCR7 molecules in macrophages from each experimental group of this invention.

[0036] Figure 2 is a bar chart showing the flow cytometry results of CCR7 molecules in macrophages from each experimental group in this invention;

[0037] Figure 3 shows the flow cytometry detection of CD64 molecules in macrophages from each experimental group of this invention;

[0038] Figure 4 is a bar chart showing the flow cytometry results of CD64 molecules in macrophages from each experimental group in this invention.

[0039] Figure 5 shows the flow cytometry detection of CD40 molecules in macrophages in each experimental group of this invention;

[0040] Figure 6 is a bar chart showing the flow cytometry results of CD40 molecules in macrophages from each experimental group in this invention.

[0041] Figure 7 shows the JC-1 flow cytometry results of mitochondrial membrane potential in macrophages in each experimental group of this invention;

[0042] Figure 8 is a bar chart of the JC-1 flow cytometry detection results of mitochondrial membrane potential of macrophages in each experimental group of the present invention;

[0043] Figure 9 shows the flow cytometry detection of macrophages phagocytizing FITC-labeled dextran in each experimental group of the present invention.

[0044] Figure 10 is a bar chart showing the detection results of macrophages phagocytizing FITC-labeled dextran in each experimental group of the present invention.

[0045] In the bar chart, ns indicates no statistically significant difference, and **** indicates a highly statistically significant difference. Detailed Implementation

[0046] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0047] The inventors of this invention demonstrated through experiments that adding norepinephrine to the induction culture medium at a corresponding concentration range during the early stage of macrophage culture inhibited M1 type polarization of macrophages.

[0048] In the initial stage of culture, the corresponding induction medium includes basal medium, serum, M-CSF, and 5-15 μM norcantharidin. The corresponding basal medium can be AIM-V or KBM581, with AIM-V medium being preferred; the corresponding serum is autologous plasma, serum substitutes, or fetal bovine serum, with autologous plasma being preferred.

[0049] During the first 0-5 days of culture, the cells were cultured using the aforementioned induction medium. On the 5th day, additional IFN-γ was added for polarization, and macrophages were obtained by continuing the culture.

[0050] Specifically, the present invention is verified using the following experimental groups and testing methods.

[0051] Experimental group 1:

[0052] In this experimental group, the corresponding induction medium included the following components:

[0053] AIM-V culture medium + 10% autologous plasma + 100 ng / mL M-CSF + 5 μM norcantharidin;

[0054] AIM-V medium is the basal medium, and the following steps are used to culture the corresponding macrophages:

[0055] S1: Resuscitate human PBMCs and resuspend them in basal medium at 0.5~0.75×10⁻⁶. 7 Cells were seeded at 1 / mL and 2mL / well in 6-well plates. After adhering and culturing for 2 hours, non-adhering cells were removed.

[0056] S2: Replace with the culture medium described above in experimental group 1 and continue culturing for 5 days, replenishing with fresh culture medium every 2 days;

[0057] S3: On day 5 of culture, add an additional 100 U / m of IFN-γ to the existing culture medium and continue culture for 2 days;

[0058] S4: After culturing for 7 days, macrophages were scraped off using a cell scraper, and the cells and supernatant were collected by centrifugation.

[0059] Experimental group 2:

[0060] In this experimental group, the corresponding induction medium included the following components:

[0061] AIM-V culture medium + 10% autologous plasma + 100 ng / mL M-CSF + 10 μM norcantharidin;

[0062] The cultivation method for experimental group 2 was the same as that for experimental group 1.

[0063] Experimental group 3:

[0064] In this experimental group, the corresponding culture medium includes the following components:

[0065] AIM-V culture medium + 10% autologous plasma + 100 ng / mL M-CSF + 15 μM norcantharidin;

[0066] The cultivation method for experimental group 3 was the same as that for experimental group 1.

[0067] Control group:

[0068] In this experimental group, the corresponding induction medium included the following components:

[0069] AIM-V culture medium + 10% autologous plasma + 100 ng / mL M-CSF;

[0070] The culture method for the control group was the same as that for experimental group 1.

[0071] Related tests

[0072] The expression of M1-type markers CD40, CD64, and CCR7, mitochondrial membrane potential (JC-1 staining), and phagocytic function were detected in centrifuged cells from experimental groups 1-3 and the control group.

[0073] Test 1: Effects of different concentrations of norcantharidin on the M1 macrophage phenotype

[0074] CCR7, CD64, and CD40 are all internationally recognized characteristic surface markers of M1 macrophages. Their expression levels directly reflect the degree of M1 polarization, as specifically demonstrated below:

[0075] CCR7: A key molecule for the maturation and migration of M1 macrophages. High expression indicates a stronger ability to recruit to inflammatory sites and participate in immune responses, and is a core marker of M1 polarization.

[0076] CD64: A specific marker of phagocytic function of M1 macrophages, it is highly expressed only on the surface of activated M1 macrophages and is directly associated with the core immune effect of clearing pathogens and foreign substances, which is the opposite of the low expression characteristic of M2 macrophages;

[0077] CD40: A key molecule for immune activation and antigen presentation of M1 macrophages. High expression of CD40 can promote the interaction between macrophages and T cells and initiate adaptive immunity. It is an important functional marker of M1 polarization.

[0078] In this test, cells from experimental groups 1-3 and the control group were collected for flow cytometry antibody staining. The antibodies used were APC anti-human CCR7, PerCP / Cyanine5.5 anti-human CD64, and PE anti-human CD40. The test results are shown in Table 1 and Figures 1-6 below.

[0079] Table 1. Effects of different concentrations of norcantharidin on the M1 macrophage phenotype.

[0080]

[0081] Note: 'a' indicates a highly significant difference compared to the control group, P < 0.001.

[0082] The positive rates of CCR7, CD64, and CD40 can directly reflect the activation status and cell proportion of M1 macrophages, and different concentrations of norcantharidin exhibit a concentration-dependent regulatory effect on them.

[0083] As can be clearly seen from the table and Figures 1-6 above, in the control group, the M1 polarization was strongest in the group without the addition of norcanthalassemia. The positive rates of the three M1 markers were all at the high level of the group, and the polarization characteristics were the strongest. That is, in the absence of norcanthalassemia, macrophages are more likely to maintain the M1 polarization state.

[0084] Compared with the control group without added cantharidin, the regulation of 5 μM cantharidin (experimental group 1) was different, significantly reducing the positive rates of CCR7 and CD64 while increasing the positive rate of CD40. This concentration of cantharidin selectively regulated M1 macrophages: it inhibited the activation of M1 macrophages related to migration and antigen binding, but upregulated the expression of CD40, which is related to the initiation of immune response. In contrast, 10 μM and 15 μM cantharidin (experimental groups 2 and 3) both showed inhibitory effects on the expression of M1 macrophage markers. The decrease in the positive rates of CCR7 and CD64 was much greater than that in experimental group 1, and there was no significant difference in the regulatory effects of these two concentrations. The positive rate of CD40 was also significantly lower than that of the control group. Only the CD40 positive rate in experimental group 3 slightly increased but did not return to the level of the control group.

[0085] This shows that norcantharidin does not simply promote or inhibit the polarization of M1 macrophages. At low concentrations, it exhibits selective functional regulation, while at higher concentrations, it mainly inhibits the activation of M1 macrophages.

[0086] Overall, the results showed that with the addition of norcanthalassemia and an increase in concentration from 5 μM to 15 μM, the positive rate of M1 markers showed a significant decreasing trend, and the degree of M1 polarization was gradually inhibited. The corresponding CD64 marker was the most sensitive, dropping sharply from 87.77% to 28.13%, a decrease of 68%, indicating that norcanthalassemia in this range had the most significant inhibitory effect on the core phagocytic function of M1 macrophages, and the higher the concentration, the stronger the inhibition. CCR7 steadily decreased from 88.77% to 53.90%, a decrease of 39%, indicating that although the concentration of norcanthalassemia in this range increased, it continuously weakened the maturation and migration ability of M1 macrophages. Based on the above data, it can be clearly concluded that the culture method using norcanthalassemia in this concentration range has the effect of inhibiting the polarization of M1 macrophages.

[0087] 2. Effects of different concentrations of norcantharidin on macrophage metabolism

[0088] Macrophages from experimental groups 1-3 and the control group were collected and mitochondrial membrane potential was detected by flow cytometry using the JC-1 kit (Beyotime C2006) according to its instructions. At higher mitochondrial membrane potentials, JC-1 aggregates in the mitochondrial matrix, forming J-aggregates and producing red fluorescence. At lower mitochondrial membrane potentials, JC-1 does not aggregate in the mitochondrial matrix and remains a monomer, producing green fluorescence. The relative ratio of red to green fluorescence is commonly used to measure the proportion of mitochondrial depolarization. Furthermore, the transition of JC-1 from red to green fluorescence can also serve as an early indicator of apoptosis. The detection results are shown in Figures 7-8 and Table 2.

[0089] Table 2. Effects of different concentrations of norcantharidin on mitochondrial membrane potential in macrophages

[0090]

[0091] The Aggregates / Monomers ratio directly reflects the functional conformational balance of M1 polarization-related molecules. The higher the ratio, the higher the proportion of functional aggregates, which is more conducive to M1 polarization signal transduction; the lower the ratio, the lower the proportion of aggregates, and the more M1 polarization is suppressed.

[0092] Data showed that the target component exhibited a concentration-dependent biphasic effect in regulating the ratio and macrophage M1 polarization: the ratio in the control group without the component was 4.05±0.04, while in experimental group 1, the ratio decreased significantly by about 30%, indicating that it inhibited M1 polarization and that energy metabolism might shift towards the low-energy-consuming, high-FAO (mitochondrial fatty acid oxidation) type of M2. In experimental group 2, with the addition of 10 μM, the ratio rebounded to 3.99±0.08 (no significant difference from the control group), and the inhibitory effect on M1 polarization was alleviated, suggesting that the component's interference with conformational homeostasis was weakened at this concentration. In experimental group 3 with a high concentration of the component, the ratio further increased significantly to 5.76±0.08 (a), which was significantly higher than that of the control group. Although high membrane potential is usually associated with M1 polarization, this value was too high and accompanied by early apoptosis characteristics, indicating that this concentration might induce cytotoxicity or excessive mitochondrial hyperpolarization, rather than simple M1 inhibition.

[0093] 3. Effects of different concentrations of norcantharidin on macrophage phagocytic function

[0094] Based on the above groupings, after cell collection, the phagocytic capacity of macrophages was detected using FITC-labeled Dextran (MW4,000) reagent (procedure according to product instructions, Beyotime ST2930-50mg). Flow cytometry was then used for analysis. The phagocytic function of macrophages in each group was assessed; a higher percentage of FITC-labeled Dextran-positive cells indicated stronger phagocytic function of M2 macrophages.

[0095] Among them, the phagocytic capacity of M1 macrophages can be detected by CD64 positivity rate as mentioned above. The core function of M1 macrophages is to participate in innate immune response and clear invading pathogens and abnormally proliferating tumor cells. They have higher phagocytic activity against such foreign substances. On the other hand, the core function of M2 macrophages is to participate in tissue repair and maintain immune tolerance. They have a more prominent phagocytic capacity against apoptotic / necrotic cells and metabolic debris of their own origin.

[0096] Table 3. Effects of different concentrations of norcantharidin on macrophage phagocytic function.

[0097]

[0098] Note: a indicates a highly significant difference compared to the control group (P < 0.001); b indicates a significant difference compared to the control group (P < 0.05).

[0099] Referring to Table 3 and Figures 9-10, the control group, without the addition of norcantharidin, had a phagocytic function positivity rate of 25.80±2.60%, representing the baseline phagocytic level. In experimental group 1, the positivity rate significantly increased to 54.57±0.70%, a significant difference from the control group, indicating that low concentrations of norcantharidin can significantly enhance the phagocytic function of M2 macrophages. For experimental group 2, the positivity rate decreased to 32.00±0.66%, lower than experimental group 1, but still higher than the control group, indicating that the enhancing effect of phagocytic function at medium concentrations was somewhat weakened, but not fully recovered. For experimental group 3, the positivity rate was close to the control group level, indicating that high concentrations of norcantharidin can reverse the enhancing effect of low concentrations, restoring phagocytic function to its baseline state.

[0100] Based on all experimental data (M1 characteristic marker expression, aggregate / monomer ratio, phagocytic function), experimental group 1 (containing 5 μM low-concentration norcantharidin) showed a significant difference in mitochondrial membrane potential compared to the control group, significantly reducing the aggregate / monomer ratio of M1 polarization-related molecules to 2.80±0.08. This group exhibited the most significant inhibitory effect on macrophage M1 polarization, indicating that it directly disrupted the signal transduction basis of M1 polarization. This was accompanied by a significant decrease in the positive rates of core M1 markers such as CCR7 and CD64. Although low concentrations showed an enhancing effect on CD40 positivity, considering the maturation migration, immune activation, and conformational balance of key molecules in M1 polarization, experimental group 1 was the group that achieved significant inhibition of the expression of core M1 polarization characteristic markers and molecular functional conformation. In experimental group 2, all data showed inhibition of M1 polarization, but the inhibitory effect was relatively mild. The high concentration in experimental group 3 suggests that it may induce cytotoxicity or excessive mitochondrial hyperpolarization.

[0101] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for culturing macrophages, characterized in that, Includes the following steps: Human PBMCs were used to induce differentiation into macrophages by adding induction medium, which included basal medium, serum, M-CSF and 5-10 μM norcantharidin. The medium was replenished or changed every 2-3 days. On the 5th day, 100 U / mL IFN-γ was added to the induction medium to induce macrophages. After culturing for another 2 days, the corresponding macrophages were obtained. The cells and supernatant were collected by centrifugation and then analyzed.

2. The method for culturing macrophages according to claim 1, characterized in that, Before adding the induction medium for differentiation induction, the following steps are included: resuspending human PBMCs in basal medium at a concentration of 0.5~0.75×10⁻⁶. 7 Cells were seeded at 1 / mL and 2mL / well in 6-well plates. After adhering and culturing for 2 hours, non-adhering cells were removed.

3. The method for culturing macrophages according to claim 1, characterized in that, The concentration of M-CSF in the induction medium is 50-200 ng / mL.

4. The method for culturing macrophages according to claim 1, characterized in that, The amount of norcantharidin added to the induction medium is 5 μM.

5. The method for culturing macrophages according to claim 1, characterized in that, The amount of norcantharidin added to the induction medium is 10 μM.

6. The method for culturing macrophages according to claim 1, characterized in that, The serum is selected from autologous plasma, serum substitutes, or fetal bovine serum.

7. The method for culturing macrophages according to claim 1, characterized in that, The basal culture medium is AIM-V or KBM581.

8. The method for culturing macrophages according to claim 1, characterized in that, The detection includes one or more of the following: detection of M1 type markers CD40, CD64, and CCR7 expression; mitochondrial membrane potential JC-1 staining; and phagocytic function.

9. The application of a macrophage culture method as described in any one of claims 1-8 in macrophage culture.

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