A method for in vitro expansion of peripheral blood mononuclear cells into macrophages

Abstract

The application discloses a method for in-vitro expansion and induction of peripheral blood mononuclear cells into macrophages, and relates to the field of biomedical technology. The method comprises the following steps: separating PBMC from peripheral blood, and sequentially culturing the PBMC by using expansion medium, differentiation medium, maturation medium and support medium, so as to obtain the macrophages. The above-mentioned culture media are all based on RPMI-1640 medium, the expansion medium further comprises fetal bovine serum, insulin, IL-3 and IL-6; the differentiation medium further comprises 1,25-dihydroxyvitamin D3, insulin, GM-CSF and M-CSF; the maturation medium further comprises GM-CSF, M-CSF, insulin and transferrin; and the support medium further comprises insulin, transferrin and sodium selenite. The method has the advantages of efficient expansion of PBMC, accurate induction of PBMC into mature macrophages, and maintenance of the stability of the differentiation state of the macrophages.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and in particular to a method for in vitro expansion and induction of peripheral blood mononuclear cells into macrophages. BACKGROUND

[0002] Peripheral blood mononuclear cells (PBMCs) are an important immune cell population and are widely used in basic research and clinical research. The main source of PBMCs is peripheral blood, and the importance of patient-derived PBMCs in clinical experiments is increasing. However, the expansion of PBMCs still faces many challenges. Due to its high growth requirement, slow expansion speed, and susceptibility to culture conditions, traditional expansion methods often cannot meet the demand for large-scale and stable expansion, and easily lead to cell death. More complexly, due to the low proportion of macrophages in PBMCs, and the difficulty in isolating and culturing patient-derived macrophages, there is a lack of sufficient macrophage resources in basic research and clinical applications, which limits the development of related immunology research.

[0003] Macrophages play a central role in immune response, inflammatory response, tumor immunity and other biological processes, therefore, being able to effectively expand macrophages, especially from patient-derived PBMCs, is of great significance for basic immunology research, disease model establishment and immunotherapy. However, the induction of macrophages from PBMCs is a technical challenge. Traditional induction of PBMC differentiation into macrophages is carried out in serum-containing medium, and the success rate of induction and differentiation effect are greatly affected by the stability of serum, and the number of induced PBMC differentiated macrophages is small.

[0004] Currently, there is no standardized technology for the expansion and induction of PBMC-derived macrophages, especially under serum-free or low-serum conditions. Developing a simple, efficient and controllable method for in vitro induction of PBMC expansion into macrophages will provide strong cell resource support for research in immunology, oncology, infectious diseases and other fields. SUMMARY

[0005] The present application aims to at least solve one of the technical problems in the prior art, and provides a method for in vitro expansion and induction of peripheral blood mononuclear cells into macrophages, to solve the problems of difficulty in in vitro expansion of PBMCs and lack of macrophage resources, and to provide an innovative method for in vitro expansion and induction of PBMCs into macrophages. This method will effectively improve the culture efficiency and proliferation capacity of PBMCs, can effectively avoid the problem of low induction success rate caused by batch-to-batch differences of serum, can induce PBMCs to differentiate into macrophages and maintain stability. It solves the deficiencies of the prior art and provides more reliable cell sources for basic research and clinical research.

[0006] The technical solution of the present application is as follows:

[0007] The first aspect of the present application provides a method for in vitro expansion and induction of peripheral blood mononuclear cells into macrophages, comprising the following steps:

[0008] S1, peripheral blood mononuclear cells are separated from peripheral blood, and the peripheral blood mononuclear cells are expanded and cultured using an expansion culture medium to obtain mononuclear granulocytes;

[0009] S2, the mononuclear granulocytes are cultured using a differentiation culture medium to obtain initially induced and differentiated macrophages;

[0010] S3, the macrophages in step S2 are cultured using a maturation culture medium to obtain differentiated and mature macrophages;

[0011] S4, the macrophages in step S3 are cultured using a support culture medium to obtain stable macrophages;

[0012] The components of the expansion culture medium include: RPMI-1640 culture medium, GM-CSF, M-CSF, fetal bovine serum, insulin, IL-3 and IL-6;

[0013] The components of the differentiation culture medium include: RPMI-1640 culture medium, 1,25-dihydroxyvitamin D3, insulin, GM-CSF and M-CSF;

[0014] The components of the maturation culture medium include: RPMI-1640 culture medium, GM-CSF, M-CSF, insulin and transferrin;

[0015] The components of the support culture medium include: RPMI-1640 culture medium, insulin, transferrin and sodium selenite.

[0016] In one specific embodiment of the present application, in S1, the addition amount of each component in the expansion culture medium is as follows:

[0017] The final concentration of GM-CSF is 19 ng / mL~21 ng / mL, the final concentration of M-CSF is 19 ng / mL~21 ng / mL, the addition volume of fetal bovine serum is 9%~11% of the volume of RPMI-1640 culture medium, the final concentration of insulin is 190 nM~210 nM, the final concentration of IL-3 is 9 ng / mL~11 ng / mL, and the final concentration of IL-6 is 19 ng / mL~21 ng / mL.

[0018] Preferably, the final concentration of GM-CSF is 19.5 ng / mL-20.5 ng / mL, the final concentration of M-CSF is 19.5 ng / mL-20.5 ng / mL, the volume of fetal bovine serum added is 9.5%-10.5% of the volume of RPMI-1640 medium, the final concentration of insulin is 195 nM-205 nM, the final concentration of IL-3 is 9.5 ng / mL-10.5 ng / mL, and the final concentration of IL-6 is 19.5 ng / mL-20.5 ng / mL.

[0019] More preferably, the final concentration of GM-CSF is 20 ng / mL, the final concentration of M-CSF is 20 ng / mL, the volume of fetal bovine serum added is 10% of the volume of RPMI-1640 medium, the final concentration of insulin is 200 nM, the final concentration of IL-3 is 10 ng / mL, and the final concentration of IL-6 is 20 ng / mL.

[0020] In one specific embodiment of the present application, in S2, the added amount of each component in the differentiation medium is as follows:

[0021] Preferably, the final concentration of 1,25-dihydroxyvitamin D3 is 9 nM-11 nM, the final concentration of insulin is 190 nM-210 nM, the final concentration of GM-CSF is 19 ng / mL-21 ng / mL, and the final concentration of M-CSF is 19 ng / mL-21 ng / mL.

[0022] Preferably, the final concentration of 1,25-dihydroxyvitamin D3 is 9.5 nM-10.5 nM, the final concentration of insulin is 195 nM-205 nM, the final concentration of GM-CSF is 19.5 ng / mL-20.5 ng / mL, and the final concentration of M-CSF is 19.5 ng / mL-20.5 ng / mL.

[0023] More preferably, the final concentration of 1,25-dihydroxyvitamin D3 is 10 nM, the final concentration of insulin is 200 nM, the final concentration of GM-CSF is 20 ng / mL, and the final concentration of M-CSF is 20 ng / mL.

[0024] In one specific embodiment of the present application, in S3, the added amount of each component in the mature medium is as follows:

[0025] The final concentration of GM-CSF is 19 ng / mL-21 ng / mL, the final concentration of M-CSF is 19 ng / mL-21 ng / mL, the final concentration of insulin is 190 nM-210 nM, and the final concentration of transferrin is 0.9 μg / mL-1.1 μg / mL based on RPMI-1640 medium.

[0026] Preferably, the final concentration of GM-CSF is 19.5 ng / mL-20.5 ng / mL, the final concentration of M-CSF is 19.5 ng / mL-20.5 ng / mL, the final concentration of insulin is 195 nM-205 nM, and the final concentration of transferrin is 0.95 μg / mL-1.05 μg / mL based on RPMI-1640 medium.

[0027] More preferably, the final concentration of GM-CSF is 20 ng / mL, the final concentration of M-CSF is 20 ng / mL, the final concentration of insulin is 200 nM, and the final concentration of transferrin is 1 μg / mL based on RPMI-1640 medium.

[0028] In one specific embodiment of the present application, in S4, the adding amount of each component in the support medium is as follows:

[0029] The final concentration of insulin is 190 nM-210 nM, the final concentration of transferrin is 0.9 μg / mL-1.1 μg / mL, and the final concentration of sodium selenite is 9 nM-11 nM based on RPMI-1640 medium.

[0030] Preferably, the final concentration of insulin is 195 nM-205 nM, the final concentration of transferrin is 0.95 μg / mL-1.05 μg / mL, and the final concentration of sodium selenite is 9.5 nM-10.5 nM based on RPMI-1640 medium.

[0031] More preferably, the final concentration of insulin is 200 nM, the final concentration of transferrin is 1 μg / mL, and the final concentration of sodium selenite is 10 nM based on RPMI-1640 medium.

[0032] In one specific embodiment of the present application, the culture method in S1 comprises:

[0033] The peripheral blood mononuclear cells are suspended in the expansion medium and placed in a 37℃, 5% CO2 incubator for culture; the cell proliferation is observed every 2 days, and the expansion medium is supplemented according to the cell density to maintain the culture environment; during the expansion process, the state, number and activity of the cells are monitored;

[0034] Culture to the 3rd day~15th day, collect the cell culture fluid, centrifuged at 25℃, obtain mononuclear granulocytes.

[0035] In one embodiment of the present application, in S2, the amplified mononuclear granulocytes are inoculated into the differentiation culture medium and cultured at 37℃, 5% CO2 for 2-4 days to complete the initial differentiation induction.

[0036] In one embodiment of the present application, in S3, the macrophages that have completed the initial induction differentiation are added to the mature culture medium and cultured at 37℃, 5% CO2 for 1-3 days to promote the maturation of macrophages, thereby obtaining differentiated mature macrophages.

[0037] In one embodiment of the present application, in S4, the differentiated mature macrophages are added to the support culture medium and cultured at 37℃, 5% CO2 for 4-6 days, thereby obtaining stable macrophages.

[0038] The second aspect of the present application provides a macrophage obtained by in vitro expansion and induction using the above method.

[0039] The macrophage obtained above can be used for cell experiments in medical research

[0040] The present application has at least one of the following beneficial effects:

[0041] 1. The present application has the advantage of efficiently expanding PBMC. In the traditional PBMC culture method, the cell expansion effect is poor due to low cell density, incomplete differentiation or cell stress, and even cell death. However, by changing the culture medium formula, the present application effectively completes the expansion of PBMC in the culture condition of 10% fetal bovine serum by adding appropriate types and concentrations of additives GM-CSF, M-CSF, insulin, IL-3 and IL-6, which ensures the proliferation efficiency while maintaining the stability and usability of PBMC.

[0042] 2. This invention can efficiently induce PBMC differentiation in a serum-free environment, prevent abnormal differentiation, and overcome serum dependence. Under serum-free conditions, this invention uses a stepwise culture method, first employing differentiation medium and then maturation medium, to more precisely regulate the differentiation and maturation process of macrophages. On one hand, this invention induces PBMC differentiation into macrophages by adding 1,25-dihydroxyvitamin D3, GM-CSF, and M-CSF; on the other hand, it promotes macrophage maturation by using GM-CSF, M-CSF, insulin, and transferrin. Through these two aspects, it precisely induces PBMC differentiation into mature macrophages and maintains a stable macrophage differentiation state. By precisely controlling the above additives under serum-free conditions, the influence of batch factors and external interference in serum on cell differentiation is avoided, enabling more precise regulation of the macrophage differentiation and maturation process, making its phenotype and function comparable to macrophages under traditional serum conditions.

[0043] 3. This invention reduces cell variability and improves safety. The invention employs a serum-free induction differentiation method, avoiding the variability that may arise from serum use in traditional methods, such as batch differences and contamination. This helps improve the consistency of cell culture and the reproducibility of results. For applications requiring precise cell function assessment or clinical treatment, this method provides a more reliable cell source.

[0044] 4. This invention can maintain the survival of macrophages. The supporting culture medium of this invention does not contain serum, thus avoiding the influence of serum on differentiated and mature macrophages and preventing them from being further induced into functionally exhausted macrophages. Furthermore, the supporting culture medium in this invention not only provides nutrients for the growth of differentiated and mature macrophages, but also the sodium selenite in it prevents stress damage to macrophage cells caused by the in vitro culture environment. Attached Figure Description

[0045] Figure 1 The following is a diagram showing the results of culture in Example 1. In the diagram, A shows the change in cell number over time after culture in the amplification medium; B shows the cell number on day 1 and day 5 of culture in the amplification medium; and C shows peripheral blood mononuclear cells (PBMCs) before induction and macrophages after induction.

[0046] Figure 2 The figures show the results of culture and differentiation of Example 1 and Comparative Examples 1-11. In the figures, A represents macrophages after expansion and stepwise induction in Example 1; B-J represent the results of culture / differentiation of Comparative Examples 1-11. Detailed Implementation

[0047] In order to make the technical problems, technical solutions and beneficial effects solved by the present application more clear and explicit, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0048] Example 1

[0049] The present embodiment provides a method for in vitro expansion and induction of peripheral blood mononuclear cells into macrophages, which comprises two parts of cell culture expansion of PBMC and serum-free induction of PBMC differentiation into macrophages, and the specific method is as follows:

[0050] 1. Cell separation

[0051] Single nuclear cells (PBMC) are separated from peripheral blood, and PBMC are separated by conventional density gradient centrifugation and placed in RPMI-1640 medium. In the present embodiment, lymphocyte separation medium produced by Beijing Solabio Technology Co., Ltd. (lymphocyte separation medium produced by other companies can also be used by those skilled in the art) is used to separate PBMC according to the instructions. Anti-CD14 antibody produced by Dako Biotechnology Co., Ltd. is used for incubation, and CD14+ mononuclear cells are sorted by flow sorting.

[0052] 2. Expansion culture

[0053] (1) Preparation of culture medium and additives:

[0054] RPMI-1640 is used as the basic culture medium, and the following substances are supplemented to prepare the expansion culture medium, and the specific method is as follows:

[0055] The expansion culture medium is prepared by adding GM-CSF, M-CSF, fetal bovine serum, insulin, IL-3 and IL-6 to RPMI-1640 medium, wherein the final concentration of GM-CSF is 20 ng / mL, the final concentration of M-CSF is 20 ng / mL, the volume of fetal bovine serum added is 10% of the volume of RPMI-1640 medium, the final concentration of insulin is 200 nM (200 nmol / L), the final concentration of IL-3 is 10 ng / mL, and the final concentration of IL-6 is 20 ng.

[0056] (2) Culture method:

[0057] PBMC is suspended in the expansion culture medium and placed in a 37℃, 5% CO2 incubator for culture. The cell proliferation is observed every 2 days, and the expansion culture medium is appropriately supplemented to maintain the culture environment according to the cell density.

[0058] During the expansion process, monitor the status, number, and viability of the cells. Ensure that the culture environment is stable, avoid contamination, and collect and count the expanded PBMC cells at the end of the culture.

[0059] On the 11th day of culture, collect the cell culture fluid, centrifuge at 300 g for 10 minutes at 25°C, and store the obtained peripheral blood mononuclear cells in liquid nitrogen using serum-free cell freezing solution.

[0060] 3. Serum-free induction of PBMC differentiation into macrophages

[0061] (1) Preparation of culture medium:

[0062] Prepare differentiation medium, maturation medium, and support medium using RPMI-1640 as the base medium, according to the following methods:

[0063] Differentiation medium: Add 1,25-dihydroxyvitamin D3, insulin, GM-CSF, and M-CSF to RPMI-1640 medium as the base medium; the final concentration of 1,25-dihydroxyvitamin D3 is 10 nM, the final concentration of insulin is 200 nM, the final concentration of GM-CSF is 20 ng / mL, and the final concentration of M-CSF is 20 ng / mL.

[0064] Maturation medium: Add GM-CSF, M-CSF, insulin, and transferrin to RPMI-1640 medium as the base medium; the final concentration of GM-CSF is 20 ng / mL, the final concentration of M-CSF is 20 ng / mL, the final concentration of insulin is 200 nM, and the final concentration of transferrin is 1 μg / mL.

[0065] Support medium: Add insulin, transferrin, and sodium selenite to RPMI-1640 medium as the base medium; the final concentration of insulin is 200 nM, the final concentration of transferrin is 1 μg / mL, and the final concentration of sodium selenite is 10 nM.

[0066] (2) Culture method:

[0067] Seed the expanded PBMC cells into the differentiation medium and incubate at 37°C, 5% CO2 for 3 days to complete the initial differentiation induction.

[0068] Discard the initial differentiation medium in the culture dish, add the cells that have completed the initial induction differentiation into the maturation medium, and incubate at 37°C, 5% CO2 for 2 days to promote the maturation of macrophages.

[0069] The cell culture medium in the culture dish is replaced with the support culture medium, and the stable macrophages are obtained by culturing at 37°C under 5% CO2 for 5 days. The stable macrophages obtained in the support culture medium can be used for cell experiments in medical research.

[0070] The results are shown in Figure 1 and Figure 2 , as shown in Figure 1 , the number of cells cultured using the amplification culture medium of the present embodiment gradually increases with the culture time, as shown in Figure 1 , the number of cells cultured using the amplification culture medium of the present embodiment gradually increases with the culture time, as shown in Figure 1 , the number of cells cultured using the amplification culture medium of the present embodiment gradually increases with the culture time, as shown in Figure 2 , the number of cells cultured using the amplification culture medium of the present embodiment gradually increases with the culture time, as shown in

[0071] Comparative Example 1

[0072] The difference from Example 1 is that "Step 3, serum-free induction of PBMC differentiation into macrophages" is changed to "simultaneous differentiation, maturation and support culture, instead of step-by-step", and the others are the same as Example 1.

[0073] The specific method is as follows:

[0074] 1. Cell separation: same as Example 1.

[0075] 2. Amplification culture: same as Example 1.

[0076] 3. Serum-free induction of PBMC differentiation into macrophages:

[0077] (1) Preparation of culture medium:

[0078] The additives in the differentiation culture medium, the maturation culture medium and the support culture medium are added together in RPMI-1640 to form a new culture medium, and the concentration of each additive is the sum of the concentrations of each additive in Example 1. The specific method is as follows:

[0079] RPMI-1640 was used as the basal medium, to which 1,25-dihydroxyvitamin D3, insulin, GM-CSF, M-CSF, transferrin, and sodium selenite were added. The final concentrations of 1,25-dihydroxyvitamin D3, insulin, GM-CSF, M-CSF, transferrin, and sodium selenite were 10 nM.

[0080] (2) Cultivation method:

[0081] The expanded PBMC cells were seeded into the new culture medium and cultured at 37°C and 5% CO2 for 7 days.

[0082] Culture results as follows Figure 2 As shown in B in the figure, it can be seen from the figure that compared to Figure 2 In Example A, compared to Example 1, the number of cells undergoing differentiation, maturation, and support culture simultaneously was small, and the cells varied in morphology and size, exhibiting an increase in elongated spindle-shaped, irregular, and slightly blurred-edge cells; the cytoplasm was loose and contained vacuoles, resulting in weak refraction; and the pseudopodia were slender and disordered. This suggests that the cells exhibit multi-level differentiation and cannot be directly used for cell experiments in medical research.

[0083] Comparative Example 2

[0084] The difference from Example 1 is that the amplification culture and induction culture are performed simultaneously instead of in separate steps; otherwise, they are the same as in Example 1.

[0085] The specific method is as follows:

[0086] 1. Cell isolation: Same as in Example 1.

[0087] 2. Simultaneous amplification culture and induction culture:

[0088] (1) Preparation of culture medium:

[0089] Add the additives from the amplification medium, differentiation medium, maturation medium, and support medium together to RPMI-1640 to form a new medium. The concentration of each additive is the sum of the concentrations of each additive in Example 1. The specific method is as follows:

[0090] RPMI-1640 was used as the basal medium, to which fetal bovine serum, IL-3, IL-6, 1,25-dihydroxyvitamin D3, insulin, GM-CSF, M-CSF, transferrin, and sodium selenite were added. The volume of fetal bovine serum added was 10% of the volume of RPMI-1640 medium. The final concentrations of IL-3, IL-6, 1,25-dihydroxyvitamin D3, insulin, GM-CSF, and M-CSF were 10 ng / mL, 20 ng, 10 nM, 600 nM, 60 ng / mL, 2 μg / mL, and 10 nM, respectively.

[0091] (2) Cultivation method:

[0092] The isolated PBMC cells were seeded into the above culture medium and cultured at 37°C and 5% CO2 for 18 days.

[0093] Culture results as follows Figure 2 As shown in C in the figure, it can be seen from the figure that compared to Figure 2 In Example A, compared to Example 2, cell expansion was poor, with a significant decrease in number and inconsistent cell morphology and size. The cells were characterized by an increase in spindle-shaped cells with slightly blurred edges; the cytoplasm was loose and contained vacuoles, resulting in weak refraction; and the pseudopodia were long, thin, and disordered. This suggests poor cell expansion and multi-level differentiation, making them unsuitable for direct use in cell experiments for medical research.

[0094] Comparative Example 3

[0095] The difference from Example 1 is that the amplification culture medium does not contain "GM-CSF, M-CSF", but the rest is the same as Example 1.

[0096] Culture results as follows Figure 2 As shown in D in the figure, it can be seen from the figure that compared to Figure 2 In Example A, compared to Example 3, cell expansion was poor, with a significant decrease in cell number, inconsistent cell morphology and size, and the presence of dead cells. These cells exhibited an increase in elongated spindle-shaped, irregular, and slightly blurred-edge cells; increased cytoplasmic granules containing vacuoles resulted in weak refraction; and most cells appeared dull and lacked luster. This indicates poor cell expansion and condition, making them unsuitable for direct use in cell experiments for medical research.

[0097] Comparative Example 4

[0098] The difference from Example 1 is that the amplification culture medium does not contain "IL-3, IL-6", but the rest is the same as Example 1.

[0099] Culture results as follows Figure 2 As shown in E in the figure, it can be seen from the figure that compared to Figure 2In Example A, compared to Example 4, cell expansion was poor. The number of cells was low, and many were dark in color and in poor condition, exhibiting an increase in spindle-shaped, star-shaped, irregularly shaped, and slightly blurred-edge cells. Cytoplasmic granules were increased, containing vacuoles and showing weak refraction. Some cells showed elongated pseudopodia; most cells were generally dull and lacked luster. This suggests poor cell expansion, poor cell condition, and multi-level differentiation, making them unsuitable for direct use in cell experiments for medical research.

[0100] Comparative Example 5

[0101] The difference from Example 1 is that the differentiation medium does not contain "GM-CSF, M-CSF", but the rest is the same as in Example 1.

[0102] Culture results as follows Figure 2 As shown in F in the figure, it can be seen from the figure that compared to Figure 2 In example A, compared to example 5, the number of cells increased was small, and some cells grew in clusters, making them unsuitable for direct use in cell experiments in medical research.

[0103] Comparative Example 6

[0104] The difference from Example 1 is that the differentiation medium does not contain "1,25-dihydroxyvitamin D3", but the rest is the same as in Example 1.

[0105] Culture results as follows Figure 2 As shown in G in the figure, it can be seen from the figure that compared to Figure 2 In the A group, the cell expansion effect of Comparative Example 6 was poor, with cell overlap, local accumulation, and most cells exhibiting elongated, disordered pseudopodia. The differentiation direction was uncontrollable, making it unsuitable for direct use in cell experiments in medical research.

[0106] Comparative Example 7

[0107] The difference from Example 1 is that: instead of maturation culture, the differentiated cells are directly inoculated into the support medium for culture, otherwise the same as Example 1.

[0108] Culture results as follows Figure 2 As shown in H in the figure, it can be seen from the figure that compared to Figure 2 In Example A, compared to Example 7, cell expansion was poor, with a significant decrease in cell number, an increase in irregularly shaped cells with slightly blurred edges, increased cytoplasmic granules containing vacuoles, and weak refraction; some cells showed elongated and disordered pseudopodia. This indicates poor cell expansion and poor cell condition, making them unsuitable for direct use in cell experiments for medical research.

[0109] Comparative Example 8

[0110] The difference from Example 1 is that the mature culture medium does not contain "transferrin", but the rest is the same as Example 1.

[0111] Culture results as followsFigure 2 As shown in Figure I, it can be seen from the figure that compared to Figure 2 In sample A, the cell expansion effect was poor compared to ratio 8. The number of cells was low, and the number of dead cells was significantly increased. Approximately half of the cells appeared dull and lackluster, with reduced transparency, blurred, rough, or ruptured borders. Increased granules or vacuolation were observed in the cytoplasm. Some cells did not adhere well to the culture medium. Therefore, it cannot be directly used for cell experiments in medical research.

[0112] Comparative Example 9

[0113] The difference from Example 1 is that no supporting culture is performed, and the mature cells are directly used for cell experiments in medical research. Otherwise, it is the same as Example 1.

[0114] Culture results as follows Figure 2 As shown in J in the figure, it can be seen from the figure that compared to Figure 2 In the A sample, the culture effect of Comparative Example 9 was poor, with a small number of cells, varying cell sizes, and vacuoles in the cytoplasm, indicating poor cell expansion and slightly poor cell condition, making it unsuitable for direct use in cell experiments in medical research.

[0115] Comparative Example 10

[0116] The difference from Example 1 is that the support culture medium does not contain "transferrin", but the rest is the same as Example 1.

[0117] Culture results as follows Figure 2 As shown in K in the figure, it can be seen from the figure that compared to Figure 2 In sample A, the culture results in control ratio 10 were poor, with a low cell count and poor cell condition. Half of the cells appeared as a dull gray color with reduced transparency, and increased granules or vacuolation were visible in the cytoplasm. Therefore, it cannot be directly used for cell experiments in medical research.

[0118] Comparative Example 11

[0119] The difference from Example 1 is that the support culture medium does not contain sodium selenite; otherwise, the contents are the same as in Example 1.

[0120] Culture results as follows Figure 2 As shown in the figure, L represents the difference between the two. Figure 2 In sample A, the culture results of Comparative Example 11 were poor, with a low cell count and poor cell condition. Half of the cells appeared dull and lacked luster, with reduced transparency and increased granules or vacuolation in the cytoplasm. Therefore, it cannot be directly used for cell experiments in medical research.

[0121] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical solution and inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A method for in vitro expansion of peripheral blood mononuclear cells into macrophages, characterized by, The method comprises the following steps: S1, peripheral blood mononuclear cells are separated from peripheral blood, and the peripheral blood mononuclear cells are subjected to expansion culture using an expansion culture medium to obtain monocyte granulocytes; S2, the monocyte granulocytes are subjected to culture using a differentiation culture medium to obtain initially induced and differentiated macrophages; S3, the macrophages in step S2 are subjected to culture using a maturation culture medium to obtain differentiated and mature macrophages; S4, the macrophages in step S3 are subjected to culture using a support culture medium to obtain stable macrophages; The components of the expansion culture medium are RPMI-1640 culture medium, GM-CSF, M-CSF, fetal bovine serum, insulin, IL-3 and IL-6; The components of the differentiation culture medium are RPMI-1640 culture medium, 1,25-dihydroxyvitamin D3, insulin, GM-CSF and M-CSF; The components of the maturation culture medium are RPMI-1640 culture medium, GM-CSF, M-CSF, insulin and transferrin; The components of the support culture medium are RPMI-1640 culture medium, insulin, transferrin and sodium selenite; The addition amounts of the components in the expansion culture medium are as follows: taking RPMI-1640 culture medium as a basic culture medium, the final concentration of GM-CSF is 19 ng / mL-21 ng / mL, the final concentration of M-CSF is 19 ng / mL-21 ng / mL, the addition volume of fetal bovine serum is 9%-11% of the volume of the RPMI-1640 culture medium, the final concentration of insulin is 190 nM-210 nM, the final concentration of IL-3 is 9 ng / mL-11 ng / mL, and the final concentration of IL-6 is 19 ng / mL-21 ng / mL; The addition amounts of the components in the differentiation culture medium are as follows: taking RPMI-1640 culture medium as a basic culture medium, the final concentration of 1,25-dihydroxyvitamin D3 is 9 nM-11 nM, the final concentration of insulin is 190 nM-210 nM, the final concentration of GM-CSF is 19 ng / mL-21 ng / mL, and the final concentration of M-CSF is 19 ng / mL-21 ng / mL; The addition amounts of the components in the maturation culture medium are as follows: taking RPMI-1640 culture medium as a basic culture medium, the final concentration of GM-CSF is 19 ng / mL-21 ng / mL, the final concentration of M-CSF is 19 ng / mL-21 ng / mL, the final concentration of insulin is 190 nM-210 nM, and the final concentration of transferrin is 0.9 μg / mL-1.1 μg / mL; The addition amounts of the components in the support culture medium are as follows: taking RPMI-1640 culture medium as a basic culture medium, the final concentration of insulin is 190 nM-210 nM, the final concentration of transferrin is 0.9 μg / mL-1.1 μg / mL, and the final concentration of sodium selenite is 9 nM-11 nM.

2. The method of claim 1, wherein, The culture method in S1 comprises the following steps: The peripheral blood mononuclear cells are suspended in an expansion medium and placed in a 37 DEG C incubator with 5% CO2 for culture; the cell proliferation is observed every 2 days, and the expansion medium is supplemented according to the cell density to maintain the culture environment; during the expansion, the state, quantity and viability of the cells are monitored; The cell culture fluid is collected and centrifuged at 25 DEG C to obtain mononuclear granulocytes.

3. The method of claim 1, wherein, The culture method in S2 comprises: The expanded mononuclear granulocytes are inoculated into a differentiation medium and placed in a 37 DEG C incubator with 5% CO2 for 2-4 days to complete initial differentiation induction.

4. The method of claim 1, wherein, The culture method in S3 comprises: The macrophages completing initial induction differentiation are added into a maturation medium and placed in a 37 DEG C incubator with 5% CO2 for 1-3 days to promote maturation of the macrophages, thereby obtaining differentiated mature macrophages.

5. The method of claim 1, wherein, The culture method in S4 comprises: The differentiated mature macrophages are added into a support medium and placed in a 37 DEG C incubator with 5% CO2 for 4-6 days to obtain stable macrophages.

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