Culture method and application of macrophages

Abstract

The invention provides a macrophage culture method and application thereof, the culture method comprises the following steps: human PBMC is adopted, an induction medium is added for induced differentiation into macrophages, the induction medium comprises a basic medium, serum, M-CSF and 5-15 [mu] M norcantharidin, and liquid supplementation or liquid replacement is carried out every 2-3 days; on the fifth day, 100 U / mL of IFN-gamma is added into the induction culture medium to induce macrophages; and continuously culturing for 2 days to obtain corresponding macrophages, centrifuging to take cells and supernate, and detecting the cells and the supernate. By adopting the method, the M1 type polarization of the macrophages can be inhibited, and a new application prospect is provided for the anti-inflammation of the macrophages.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell culture, in particular to a macrophage culture medium and application thereof. BACKGROUND

[0002] As the core effector cell of the body's innate immune system, macrophages play a crucial role in immune defense, inflammation regulation, tissue repair and disease development. Macrophages can be divided into M1 phenotype with pro-inflammatory and anti-tumor effects and M2 phenotype with anti-inflammatory and pro-tumor progression effects. However, in actual disease treatment scenarios, over-activated M1-type macrophages often lead to uncontrolled inflammatory reactions, causing tissue damage and even exacerbating disease progression. For example, in rheumatoid arthritis, acute lung injury and other inflammatory diseases, the massive infiltration and over-activation of M1-type macrophages are important causes of disease deterioration. Therefore, if a drug or active ingredient that can effectively inhibit the activation of macrophage M1 phenotype can be found, it will provide a new treatment idea and intervention means for such inflammation-related diseases. SUMMARY

[0003] In view of this, the present application aims to solve the problem of macrophage culture being activated to M1 type in the prior art.

[0004] The present application provides a macrophage culture method, which uses human PBMC and is cultured under specific conditions to inhibit macrophage M1 polarization.

[0005] Specifically, the first aspect of the present application provides a macrophage culture method, comprising the following steps: using human PBMC, adding an induction medium to induce differentiation into macrophages, the induction medium comprising a basic medium, serum, M-CSF and 5-15 μM norcantharidin, wherein the liquid is supplemented or replaced every 2-3 days; On the 5th day, 100 U / mL of IFN-γ is added to the induction medium to induce macrophages; Continue to culture for 2 days to obtain corresponding macrophages, centrifuge to obtain cells and supernatant, and detect them.

[0006] In the present application, the corresponding human PBMC can be obtained after being collected and frozen and then resuscitated. Before adding the induction medium, resuspension inoculation and other operations are performed. Specifically, resuscitate the human PBMC cells, resuspend the PBMC in the basic medium, inoculate 0.5-0.75 x 10 7 6 / mL, 2 mL / well in a 6-well plate, and remove the non-adherent cells after adherent culture for 2 hours.

[0007] Specifically, in the present application, human PBMCs are used. Compared with animal-derived PBMCs, the gene expression, signaling pathways, surface markers, and cytokine profiles of the obtained macrophages are completely consistent with human diseases and physiological states, and 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 providing protection for subsequent clinical applications.

[0008] In the induction medium, the base medium and serum are used to provide basic nutrients, vitamins, amino acids, and inorganic salts necessary for cell growth, proliferation, and differentiation. Serum provides additional growth factors, hormones, and adhesion factors to create a stable microenvironment for cell survival and differentiation.

[0009] M-CSF is a macrophage colony-stimulating factor, which is a key cytokine for inducing monocytes to differentiate into macrophages. It activates downstream signaling pathways by binding to its receptor, drives monocytes to adhere, changes morphology, and expresses macrophage-specific markers, is the core inducer of macrophage differentiation, and is the basis for obtaining high-purity and high-yield macrophages.

[0010] Crucially, the inventors of the present application found that the addition of norcantharidin before the addition of IFN-γ can inhibit subsequent polarization to M1 type. Norcantharidin is used as an early inhibitor of M1 polarization in this system, and its core role is to intervene and inhibit the M1 polarization pathway at the early stage of differentiation, thereby affecting the M1 polarization of macrophages. Specifically, norcantharidin and M-CSF are added to the culture of human PBMCs in the early stage of differentiation.

[0011] In the initial induction medium culture, liquid replenishment or replacement is performed every 2-3 days to maintain a stable norcantharidin inhibition concentration, ensuring the persistence and stability of the inhibition effect during the 7-day culture period, and also providing a stable culture environment for the differentiation process lasting for several days, ensuring the healthy growth of cells.

[0012] IFN-γ 100 U / mL is added on the 5th day as a polarization induction pressure.

[0013] In an alternative embodiment of the first aspect of the present application, the concentration of M-CSF is 50-200 ng / mL.

[0014] In the present embodiment, the concentration of M-CSF is in the range of 50-200 ng / mL, which ensures that it can provide a strong enough signal stimulus to drive most monocytes to start the differentiation program, while ensuring maximum differentiation efficiency, avoiding cost waste caused by excessive addition of cytokines, and preventing unintended cell reactions or functional deviations that may be caused by excessive signals.

[0015] In an alternative embodiment of the first aspect of the application, the norcantharidin is added in an amount of 5-10 μM.

[0016] In an alternative embodiment of the first aspect of the application, the norcantharidin is added in an amount of 5 μM.

[0017] In an alternative embodiment of the first aspect of the application, the norcantharidin is added in an amount of 10 μM.

[0018] In a preferred experimental protocol of the application, the norcantharidin is added in an amount of 5-10 μM, which has been proven to effectively interfere with the IFN-γ mediated M1 polarization signaling pathway. Among them, 5 μM is determined as the most preferred implementation concentration. Under the condition of the most preferred concentration, the macrophages obtained by the method of the application, compared with the control group without adding norcantharidin, using only M-CSF and IFN-γ induction, the characteristic performance of M1 depolarization is significantly, and at 10 μM, the data of each group shows inhibition of M1 polarization, but its inhibition effect is relatively alleviated.

[0019] In an alternative embodiment of the first aspect of the application, the serum is selected from one of autologous plasma, serum substitute, fetal bovine serum.

[0020] Among them, the serum is preferably autologous plasma, which is derived from the human PBMC donor itself, which can completely eliminate heterologous immune response and batch difference, and provide the closest factor composition to the in vivo environment of the donor.

[0021] In an alternative embodiment of the first aspect of the application, the basal medium is AIM-V or KBM581. Preferably, the culture medium is AIM-V medium.

[0022] In an alternative embodiment of the first aspect of the application, the detection includes one or more of the detection of M1 type indicators CD40, CD64, CCR7 expression, mitochondrial membrane potential JC-1 staining and phagocytic function.

[0023] Specifically, the application is also directed to the cultured macrophages, and the polarity of the macrophages is detected to determine whether the macrophages exist to inhibit M1 polarization.

[0024] 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.

[0025] 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.

[0026] 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).

[0027] 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.

[0028] 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: 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.

[0029] Beneficial effects: 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

[0030] Figure 1 This is a flow cytometry image of CCR7 molecules in macrophages from various experimental groups in this invention. Figure 2 This is a bar chart showing the flow cytometry results of CCR7 molecules in macrophages from each experimental group in this invention. Figure 3 This is a flow cytometry image of CD64 molecules in macrophages from each experimental group of this invention. Figure 4 This is a bar chart showing the flow cytometry results of CD64 molecules in macrophages from each experimental group in this invention. Figure 5 This is a flow cytometry image of CD40 molecules in macrophages from each experimental group of this invention. Figure 6 This is a bar chart showing the flow cytometry results of CD40 molecules in macrophages from each experimental group in this invention. Figure 7 This is a flow cytometry result of the mitochondrial membrane potential of macrophages in each experimental group of this invention. Figure 8 This is a bar chart showing the JC-1 flow cytometry results of mitochondrial membrane potential of macrophages in each experimental group of this invention. Figure 9 The image shows flow cytometry data of macrophages phagocytosing FITC-labeled dextran in each experimental group of this invention. Figure 10 This is a bar chart showing the detection results of macrophages phagocytosing FITC-labeled dextran in each experimental group of this invention.

[0031] In the bar chart, ns indicates no statistically significant difference, and **** indicates a highly statistically significant difference. Detailed Implementation

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Specifically, the present invention is verified using the following experimental groups and testing methods.

[0037] Experimental group 1: In this experimental group, the corresponding induction medium included the following components: AIM-V culture medium + 10% autologous plasma + 100 ng / mL M-CSF + 5 μM norcantharidin; AIM-V medium is the basal medium, and the following steps are used to culture the corresponding macrophages: 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. 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; 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; S4: After culturing for 7 days, macrophages were scraped off using a cell scraper, and the cells and supernatant were collected by centrifugation.

[0038] Experimental group 2: In this experimental group, the corresponding induction medium included the following components: AIM-V culture medium + 10% autologous plasma + 100 ng / mL M-CSF + 10 μM norcantharidin; The cultivation method for experimental group 2 was the same as that for experimental group 1.

[0039] Experimental group 3: In this experimental group, the corresponding culture medium includes the following components: AIM-V culture medium + 10% autologous plasma + 100 ng / mL M-CSF + 15 μM norcantharidin; The cultivation method for experimental group 3 was the same as that for experimental group 1.

[0040] Control group: In this experimental group, the corresponding induction medium included the following components: AIM-V culture medium + 10% autologous plasma + 100 ng / mL M-CSF; The culture method for the control group was the same as that for experimental group 1.

[0041] Related tests 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.

[0042] Test 1: Effects of different concentrations of norcantharidin on the M1 macrophage phenotype 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: 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. 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; 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.

[0043] 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 / Cyanine 5.5 anti-human CD64, and PE anti-human CD40. The test results are shown in Table 1 below. Figures 1-6 As shown.

[0044] Table 1. Effects of different concentrations of norcantharidin on the M1 macrophage phenotype.

[0045] Note: 'a' indicates a highly significant difference compared to the control group, P < 0.001.

[0046] 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.

[0047] From the above table and Figures 1-6 It is evident that in the control group, where no norcanthone was added, the M1 polarization was the strongest, and the positive rates of the three M1 markers were all at a high level, with the strongest polarization characteristics. In other words, without norcanthone, macrophages are more likely to maintain the M1 polarization state.

[0048] 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 between the two concentrations. The positive rate of CD40 was also significantly lower than that in 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.

[0049] 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.

[0050] 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.

[0051] 2. Effects of different concentrations of norcantharidin on macrophage metabolism 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 as follows: Figures 7-8 As shown in Table 2.

[0052] Table 2. Effects of different concentrations of norcantharidin on mitochondrial membrane potential in macrophages

[0053] 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.

[0054] 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.

[0055] 3. Effects of different concentrations of norcantharidin on macrophage phagocytic function 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.

[0056] 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.

[0057] Table 3. Effects of different concentrations of norcantharidin on macrophage phagocytic function.

[0058] 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).

[0059] Combined with Table 3 and Figures 9-10In the control group, no norcantharidin was added, and its phagocytic function positivity rate was 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.

[0060] 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.

[0061] 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 and induced to differentiate into macrophages by adding induction medium, which included basal medium, serum, M-CSF and 5-15 μM norepinephrine, with replenishment or replacement of medium every 2-3 days. On day 5, 100 U / mL of IFN-γ was added to the induction medium to induce macrophages; Continue culturing for 2 days to obtain the corresponding macrophages. Centrifuge to collect the cells and supernatant for analysis.

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 5-10 μM.

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

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

7. 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.

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

9. 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.

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

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