Purification and culture method of placenta macrophages

Abstract

The invention relates to the technical field of cell engineering, in particular to a placenta macrophage purification and culture method. Comprising the following steps: (1) cutting placenta tissues into pieces, adding an enzymatic hydrolysate for digestion, adding a red blood cell lysis buffer for ice bath, and adding a basic culture medium for resuspending cells to obtain a resuspended cell mixed solution; (2) adding the resuspended cell mixed solution to the upper layer of the lymphocyte separation solution, centrifuging, and sucking out macrophages to obtain purified macrophages; and (3) inoculating the purified macrophages into an amplification culture medium, and culturing to obtain the macrophages subjected to amplification culture. The invention provides an efficient and stable placenta macrophage purification and culture method, through optimized enzymolysis separation, gradient centrifugal purification and a specific amplification culture medium formula, the purity, the multiplication capacity and the functional stability of the macrophage are remarkably improved, and the placenta macrophage purification and culture method has a practical application value.

Description

Technical Field

[0001] The present invention relates to the field of cell engineering technology, in particular to a method for purifying and culturing placental macrophages. Background Art

[0002] Macrophages are core effector cells of the innate immune system, performing diverse functions such as phagocytosis of pathogens, antigen presentation, regulation of inflammatory responses, and promotion of tissue repair. The in vitro expansion and functional regulation of macrophages are of great significance in research on tumor immunity, infectious diseases, tissue regeneration, and autoimmune diseases.

[0003] Currently, macrophages are primarily obtained through differentiation of peripheral blood monocytes, culture of bone marrow-derived macrophages, and immortalized cell line culture. However, these methods all have significant limitations: peripheral blood-derived macrophages have limited yields and low purity; bone marrow-derived macrophages require lengthy culture cycles and exhibit significant individual variability; and immortalized cell lines struggle to accurately reflect the physiological properties of cells. In contrast, placental tissue, a postpartum waste product, is widely available and rich in macrophages, making it an ideal cell source.

[0004] In the existing technology, the isolation and culture of placental macrophages face many technical difficulties. First, the placental tissue structure is complex and contains rich extracellular matrix and vascular network. Traditional enzymatic digestion methods often result in low cell yield and poor activity. Secondly, the mixing of impurities such as red blood cells and fibroblasts during the separation process seriously affects the accuracy of subsequent experiments. Furthermore, the proliferation capacity of primary macrophages under in vitro culture conditions is limited, and conventional culture media are difficult to maintain their long-term survival and functional stability.

[0005] To address these technical bottlenecks, there is an urgent need to develop an efficient and stable method for the purification and culture of placental macrophages. This method can achieve highly active tissue dissociation, high-purity cell isolation, efficient in vitro expansion, and stable functional maintenance, providing reliable technical support for the research and application of placental macrophages. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for purifying and culturing placental macrophages.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for purifying and culturing placental macrophages, comprising the following steps:

[0009] (1) mincing the placental tissue, adding an enzymatic solution for digestion, and filtering to obtain a filtrate; centrifuging after terminating the digestion, discarding the supernatant, adding a red blood cell lysis solution, ice bathing, centrifuging, discarding the supernatant, and washing; finally, centrifuging, discarding the supernatant, and adding a basal culture medium to resuspend the cells to obtain a resuspended cell mixture;

[0010] (2) adding the resuspended cell mixture to the upper layer of the lymphocyte separation medium, centrifuging, aspirating the macrophages at the interface of the basal culture medium and the lymphocyte separation medium, then adding the basal culture medium to the macrophages, centrifuging and discarding the supernatant to obtain purified macrophages;

[0011] (3) inoculating the purified macrophages into an expansion medium for culture to obtain expanded macrophages;

[0012] The expansion culture medium includes the following components: 3-5% (v / v) serum replacement, 0.8-1.2% (v / v) Primocin, 2-4mM L-glutamine, 10-16ng / mL granulocyte-macrophage colony-stimulating factor, 8-12ng / ml transforming growth factor-β, 24-30ng / mL interleukin-4, 35-45ng / mL interleukin-3, 5-9μg / mL TNFSF15 protein, 7-11ng / ml bone morphogenetic protein 4, and the rest is basal culture medium.

[0013] Preferably, the basal culture medium is one of DMEM, RPMI 1640 or IMDM.

[0014] Preferably, the enzymatic hydrolysis solution in step (1) is: TrypLUS digestion solution and type IV collagenase.

[0015] Preferably, the final concentration of the TrypLUS digestive solution added is 0.05-0.1% (v / v); the final concentration of the type IV collagenase added is 120-160 U / ml.

[0016] Preferably, the digestion time in step (1) is 8 to 12 minutes, and the number of digestions is 2 to 3 times.

[0017] Preferably, the conditions for the first centrifugation in step (1) are: 3-5°C, 1500-1800 r / min, 5-15 min;

[0018] The conditions for the second centrifugation are: 3-5°C, 800-1200 rpm, 4-8 min;

[0019] The conditions for the third centrifugation are 3-5°C, 900-1200 r / min, and 4-10 min.

[0020] Preferably, the ice bath time in step (1) is 26 to 32 minutes.

[0021] Preferably, the conditions for the first centrifugation in step (2) are: 3-5°C, 600-800 r / min, 15-25 min;

[0022] The conditions for the second centrifugation are: 3-5°C, 500-700 r / min, 10-16 min.

[0023] Preferably, the seeding density of the purified macrophages in step (3) is 2 to 5×10 5 pcs / ml.

[0024] Preferably, the culture temperature in step (3) is 36-38° C., and the carbon dioxide concentration is 4-6%.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This method uses TrypLUS digestion solution and type IV collagenase for synergistic digestion, combined with a three-step low-temperature centrifugation method for cell separation, to effectively reduce cell damage and improve the efficiency of placental tissue dissociation. Furthermore, gradient centrifugation using lymphocyte separation solution precisely enriches macrophages at the interface, significantly improving the purity of macrophage separation.

[0027] The proliferation culture medium provided by the present invention provides essential nutrients for macrophage growth, proliferation, and differentiation at all stages, effectively maintaining macrophage growth. The culture medium contains multiple cytokines, ultimately yielding large quantities of stable, high-purity, high-quality macrophages, which is of great significance for clinical treatment and research.

[0028] Among them, serum substitutes can replace traditional fetal bovine serum (FBS), providing essential growth factors, hormones, lipids and proteins to support cell adhesion and basal metabolism. Primocin is a broad-spectrum antibiotic that effectively prevents bacterial and fungal contamination. L-glutamine is a key substrate for cellular energy metabolism. Granulocyte-macrophage colony-stimulating factor can enhance the proliferation ability of macrophages and induce macrophage polarization to a pro-inflammatory phenotype (M1 type). Transforming growth factor-β inhibits excessive inflammatory response and promotes macrophage differentiation to an anti-inflammatory phenotype (M2 type). Interleukin 4 strongly induces M2 polarization, inhibits the secretion of M1-related inflammatory factors (such as TNF-α, IL-6), and enhances the phagocytic function of macrophages. Interleukin 3, as a hematopoietic growth factor, supports the survival and expansion of macrophage precursor cells. Enhance the ability of macrophages to respond to pathogens. TNFSF15 protein and bone morphogenetic protein 4 are used in combination to achieve the technical effect of improving cell expansion efficiency.

[0029] The present invention provides an efficient and stable method for purifying and culturing placental macrophages. Through optimized enzymatic separation, gradient centrifugation purification and specific expansion culture medium formulation, the purity, proliferation ability and functional stability of macrophages are significantly improved, and the method has practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0031] Figure 1 This is a graph showing the proliferation activity of macrophages in Experimental Example 2. DETAILED DESCRIPTION

[0032] The present invention provides a method for purifying and culturing placental macrophages, comprising the following steps:

[0033] (1) mincing the placental tissue, adding an enzymatic solution for digestion, and filtering to obtain a filtrate; centrifuging after terminating the digestion, discarding the supernatant, adding a red blood cell lysis solution, ice bathing, centrifuging, discarding the supernatant, and washing; finally, centrifuging, discarding the supernatant, and adding a basal culture medium to resuspend the cells to obtain a resuspended cell mixture;

[0034] (2) adding the resuspended cell mixture to the upper layer of the lymphocyte separation medium, centrifuging, aspirating the macrophages at the interface of the basal culture medium and the lymphocyte separation medium, then adding the basal culture medium to the macrophages, centrifuging and discarding the supernatant to obtain purified macrophages;

[0035] (3) The purified macrophages are inoculated into an expansion medium for culture to obtain expanded macrophages.

[0036] In the present invention, the expansion medium comprises the following components: 3-5% (v / v) serum replacement, 0.8-1.2% (v / v) Primocin, 2-4 mM L-glutamine, 10-16 ng / mL granulocyte-macrophage colony-stimulating factor, 8-12 ng / ml transforming growth factor-β, 24-30 ng / mL interleukin-4, 35-45 ng / mL interleukin-3, 5-9 μg / mL TNFSF15 protein, 7-11 ng / ml bone morphogenetic protein 4, and the remainder is basal medium;

[0037] Preferably, the medium comprises 4% (v / v) serum replacement, 0.9-1.1% (v / v) Primocin, 3mM L-glutamine, 11-15ng / mL granulocyte-macrophage colony-stimulating factor, 9-11ng / ml transforming growth factor-β, 25-29ng / mL interleukin-4, 37-43ng / mL interleukin-3, 6-8μg / mL TNFSF15 protein, 8-10ng / ml bone morphogenetic protein 4, and the remainder is basal culture medium;

[0038] Further preferably, 4% (v / v) serum replacement, 1% (v / v) Primocin, 3mM L-glutamine, 12-14ng / mL granulocyte-macrophage colony-stimulating factor, 10ng / ml transforming growth factor-β, 26-28ng / mL interleukin-4, 39-41ng / mL interleukin-3, 7μg / mL TNFSF15 protein, 9ng / ml bone morphogenetic protein 4, and the remainder are basal culture medium; more preferably, 4% (v / v) serum replacement, 1% (v / v) Primocin, 3mM L-glutamine, 13ng / mL granulocyte-macrophage colony-stimulating factor, 10ng / ml transforming growth factor-β, 27ng / mL interleukin-4, 40ng / mL interleukin-3, 7μg / mL TNFSF15 protein, 9ng / ml bone morphogenetic protein 4, and the remainder are basal culture medium.

[0039] In the present invention, the basal culture medium is: one of DMEM, RPMI 1640 or IMDM; preferably DMEM.

[0040] In the present invention, the enzymatic hydrolysis solution in step (1) is: TrypLUS digestion solution and type IV collagenase.

[0041] In the present invention, the final concentration of the TrypLUS digestive solution added is 0.05-0.1% (v / v); preferably 0.06-0.09% (v / v); more preferably 0.07-0.08% (v / v); and more preferably 0.08% (v / v).

[0042] In the present invention, the final concentration of the type IV collagenase added is 120-160 U / ml, preferably 130-150 U / ml, and more preferably 140 U / ml.

[0043] In the present invention, the digestion time in step (1) is 8 to 12 minutes, and the number of digestions is 2 to 3 times; preferably, the digestion time is 9 to 11 minutes, and the number of digestions is 3 times; further preferably, the digestion time is 10 minutes, and the number of digestions is 3 times.

[0044] In the present invention, the conditions for the first centrifugation in step (1) are: 3-5°C, 1500-1800 r / min, 5-15 min; preferably 4°C, 1600-1700 r / min, 7-13 min; more preferably 4°C, 1700 r / min, 9-11 min; more preferably 4°C, 1700 r / min, 10 min.

[0045] In the present invention, the conditions for the second centrifugation in step (1) are: 3-5°C, 800-1200 r / min, 4-8 min; preferably 4°C, 900-1100 r / min, 5-7 min; more preferably 4°C, 1000 r / min, 6 min.

[0046] In the present invention, the conditions for the third centrifugation in step (1) are 3-5°C, 900-1200 r / min, 4-10 min; preferably 4°C, 1000-1100 r / min, 5-9 min; more preferably 4°C, 1100 r / min, 8 min.

[0047] In the present invention, the ice bath time in step (1) is 26 to 32 minutes, preferably 27 to 31 minutes, more preferably 28 to 30 minutes, and even more preferably 30 minutes.

[0048] In the present invention, the conditions for the first centrifugation in step (2) are: 3-5°C, 600-800 r / min, 15-25 min; preferably 4°C, 700 r / min, 17-23 min; more preferably 4°C, 700 r / min, 20 min.

[0049] In the present invention, the conditions for the second centrifugation in step (2) are: 3-5°C, 500-700 r / min, 10-16 min; preferably 4°C, 600 r / min, 11-15 min; more preferably 4°C, 600 r / min, 15 min.

[0050] In the present invention, the seeding density of the purified macrophages in step (3) is 2 to 5×10 5 / ml; preferably 5×10 5 pcs / ml.

[0051] In the present invention, the culture temperature in step (3) is 36-38° C., and the carbon dioxide concentration is 4-6%; preferably, the culture temperature is 37° C., and the carbon dioxide concentration is 5%.

[0052] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] A method for purifying and culturing placental macrophages, comprising the following steps:

[0055] (1) Cut the placenta tissue into pieces with a size of 2 ± 0.5 mm 3 , adding an enzymatic solution for digestion, and filtering to obtain a filtrate; the enzymatic solution comprises: TrypLUS digestion solution (added at a final concentration of 0.05% (v / v)) and type IV collagenase (added at a final concentration of 120 U / ml); the digestion time is 8 minutes, and the number of digestions is 2; after each digestion, the digestion solution is filtered through a 120-mesh cell strainer, and the filtrate is collected;

[0056] Helios serum substitute was added to terminate the digestion, and the cells were centrifuged (3°C, 1500 rpm, 5 min). The supernatant was discarded, and red blood cell lysis buffer was added for an ice bath (26 min). The cells were centrifuged (3°C, 800 rpm, 4 min), and the supernatant was discarded. The cells were washed three times with PBS, and finally centrifuged (3°C, 900 rpm, 4 min). The supernatant was discarded, and the cells were resuspended in basal medium to obtain a resuspended cell mixture.

[0057] (2) Add 5 ml of Ficoll-Gastrografin lymphocyte separation medium (density 1.007 g / L) to a 15 ml centrifuge tube, slowly add the resuspended cell mixture to the upper layer of Ficoll-Gastrografin lymphocyte separation medium, centrifuge (3°C, 600 r / min, 15 min), aspirate the macrophages at the interface of the basal culture medium and lymphocyte separation medium, then add basal culture medium to the macrophages, centrifuge (3°C, 500 r / min, 10 min), and discard the supernatant to obtain purified macrophages;

[0058] (3) The purified macrophages were inoculated (inoculation density was 3×10 5 cells / ml) in an expansion medium (temperature of 36° C., carbon dioxide concentration of 4%) to obtain expanded macrophages;

[0059] The expansion medium is composed of the following components: 3% (v / v) serum replacement, 0.8% (v / v) Primocin, 2mM L-glutamine, 10ng / ml granulocyte-macrophage colony-stimulating factor, 8ng / ml transforming growth factor-β, 24ng / mL interleukin-4, 35ng / mL interleukin-3, 5μg / mL TNFSF15 protein, 7ng / ml bone morphogenetic protein 4, and the rest is basal medium; the basal medium is: IMDM.

[0060] Example 2

[0061] A method for purifying and culturing placental macrophages, comprising the following steps:

[0062] (1) Cut the placenta tissue into pieces with a size of 2 ± 0.5 mm 3 , adding an enzymatic solution for digestion, and filtering to obtain a filtrate; the enzymatic solution comprises: TrypLUS digestion solution (added at a final concentration of 0.1% (v / v)) and type IV collagenase (added at a final concentration of 160 U / ml); the digestion time is 12 minutes, and the number of digestions is 3; after each digestion, the digestion solution is filtered through a 120-mesh cell strainer, and the filtrate is collected;

[0063] Helios serum substitute was added to terminate the digestion, and the mixture was centrifuged (5°C, 1800 rpm, 15 min). The supernatant was discarded, and red blood cell lysis buffer was added for an ice bath (32 min). The mixture was centrifuged (5°C, 1200 rpm, 8 min), and the supernatant was discarded. The cells were washed three times with PBS, and finally centrifuged (5°C, 1200 rpm, 10 min). The supernatant was discarded, and the cells were resuspended in basal medium to obtain a resuspended cell mixture.

[0064] (2) Add 5 ml of Ficoll-glucamine lymphocyte separation medium (density 1.007 g / L) to a 15 mL centrifuge tube, slowly add the resuspended cell mixture to the upper layer of Ficoll-glucamine lymphocyte separation medium, centrifuge (5°C, 800 r / min, 25 min), aspirate the macrophages at the interface of the basal culture medium and lymphocyte separation medium, then add basal culture medium to the macrophages, centrifuge (5°C, 700 r / min, 16 min), and discard the supernatant to obtain purified macrophages;

[0065] (3) The purified macrophages were inoculated (inoculation density was 5×10 5 cells / ml) in an expansion medium (temperature of 38° C., carbon dioxide concentration of 6%) to obtain expanded macrophages;

[0066] The expansion medium is composed of the following components: 5% (v / v) serum replacement, 1.2% (v / v) Primocin, 4mM L-glutamine, 16ng / mL granulocyte-macrophage colony-stimulating factor, 12ng / ml transforming growth factor-β, 30ng / mL interleukin 4, 45ng / mL interleukin 3, 9μg / mL TNFSF15 protein, 11ng / ml bone morphogenetic protein 4, and the rest is basal medium; the basal medium is: RPMI 1640.

[0067] Example 3

[0068] A method for purifying and culturing placental macrophages, comprising the following steps:

[0069] (1) Cut the placenta tissue into pieces with a size of 2 ± 0.5 mm 3 , adding an enzymatic solution for digestion, and filtering to obtain a filtrate; the enzymatic solution comprises: TrypLUS digestion solution (added at a final concentration of 0.09% (v / v)) and type IV collagenase (added at a final concentration of 140 U / ml); the digestion time is 10 minutes, and the number of digestions is 3; after each digestion, the digestion solution is filtered through a 120-mesh cell strainer, and the filtrate is collected;

[0070] Helios serum substitute was added to terminate the digestion, and the cells were centrifuged (4°C, 1700 rpm, 10 min). The supernatant was discarded, and red blood cell lysis buffer was added for an ice bath (30 min). The cells were centrifuged (4°C, 1000 rpm, 6 min), and the supernatant was discarded. The cells were washed three times with PBS, and finally centrifuged (4°C, 1100 rpm, 8 min). The supernatant was discarded, and the cells were resuspended in basal medium to obtain a resuspended cell mixture.

[0071] (2) Add 5 ml of Ficoll-glucamine lymphocyte separation medium (density 1.007 g / L) to a 15 mL centrifuge tube, slowly add the resuspended cell mixture to the upper layer of Ficoll-glucamine lymphocyte separation medium, centrifuge (4°C, 700 r / min, 20 min), aspirate the macrophages at the interface of the basal culture medium and lymphocyte separation medium, then add basal culture medium to the macrophages, centrifuge (4°C, 600 r / min, 13 min), and discard the supernatant to obtain purified macrophages;

[0072] (3) The purified macrophages were inoculated (inoculation density was 2×10 5 cells / ml) in an expansion medium (temperature of 37° C., carbon dioxide concentration of 5%) to obtain expanded macrophages;

[0073] The expansion medium is composed of the following components: 4% (v / v) serum replacement, 1% (v / v) Primocin, 3mM L-glutamine, 13ng / mL granulocyte-macrophage colony-stimulating factor, 10ng / ml transforming growth factor-β, 26ng / mL interleukin-4, 40ng / mL interleukin-3, 7μg / mL TNFSF15 protein, 9ng / ml bone morphogenetic protein 4, and the rest is basal medium; the basal medium is: DMEM.

[0074] Comparative Example 1

[0075] The other methods were the same as those in Example 3, except that no TNFSF15 protein was added.

[0076] Comparative Example 2

[0077] The other methods were the same as those in Example 3, except that TNFSF15 protein and bone morphogenetic protein 4 were not added.

[0078] Comparative Example 3

[0079] The other methods were the same as those in Example 3, except that the amount of bone morphogenetic protein 4 added was increased to 30 ng / ml.

[0080] Comparative Example 4

[0081] The other methods were the same as those in Example 3, except that the TrypLUS digestion solution was replaced with a neutral protease with a final concentration of 0.1 g / mL.

[0082] Experimental Example 1

[0083] Before inoculation of the purified macrophages in step (3) of Example 3 and Comparative Example 4, 1×10 6 Cells were centrifuged with 1 ml of culture medium at 500 g for 10 min at 4°C, the supernatant discarded, and 1 ml of pre-chilled methanol was added. The cells were incubated at -20°C for 20 min. The cells were centrifuged at 500 g for 10 min at 4°C, the supernatant discarded, and the cells were washed twice with PBS. The cells were centrifuged at 500 g for 10 min at 4°C, the supernatant discarded, and nonspecific antigens were inactivated with PBS containing 50:1 fetal bovine serum. The cells were incubated at room temperature for 30 min. The cells were centrifuged at 500 g for 10 min at 4°C, the supernatant discarded, and the cells were washed once with PBS. The cells were stained with 200 μl of 1× PBS-BSA-anti-macrophage antibody (FITC-labeled) in a centrifuge tube. As a control, 200 μl of 1× PBS-BSA was added to another centrifuge tube and incubated at room temperature in the dark for 45 min. The cells were centrifuged at 500 g for 10 min at 4°C, the supernatant discarded, and the cells were washed twice with PBS. The cells were centrifuged at 500 g for 10 min at 4°C, the supernatant discarded. After adding 500 μl of PES to resuspend the cells, the cells were transferred to a flow cytometer tube for flow cytometric analysis. The results are shown in Table 1.

[0084] Table 1 Cell positive rate

[0085] Group 1 2 3 average Example 3 98.1％ 97.5％ 98.4％ 98.0％ Comparative Example 4 92.3％ 89.8％ 90.7％ 90.9％ control group 0.21％ 0.18％ 0.18％ 0.19％

[0086] As shown in Table 1, the average positive rate of cells in Example 3 was 98.0%, while that in the control group was 0.19%, indicating that the purity of the macrophages in Example 3 was high and met the requirements of later experiments. However, the purity of the macrophages in Comparative Example 4 was significantly lower than that in Example 3, indicating that different enzymatic hydrolysis protocols can have a significant impact on the later purification process.

[0087] Experimental Example 2

[0088] CCK8 proliferation assay, primary macrophages isolated from placenta were cultured at 2×10 2 Cells were seeded / well in a 96-well cell culture plate. After adding the expansion medium prepared in Example 3, the plates were incubated at 37°C, 5% CO2 for 24, 48, 72, 96, 120, and 144 hours. 10 μl of CCK-8 solution was added to each well, shaken to mix, and incubated for another 2 hours. The culture was terminated, and the absorbance of each well was measured using an enzyme-linked immunosorbent assay (ELISA) at a wavelength of 450 nm to characterize cell proliferation activity. The results are shown in Table 2.

[0089] Table 2 Proliferation activity

[0090]

[0091]

[0092] As can be seen from the contents recorded in Table 2, the proliferation ability of macrophages in Example 3 is strong, and the cell proliferation activity gradually increases during the culture period, which is significantly better than the data of other comparative examples. From the data of Comparative Examples 1 to 3, it can be seen that only by adopting the complete culture medium formula of the present invention can the best experimental effect be achieved. If some of the components are discarded or the amount of the components added is inappropriate, it will have a significant impact on the cell proliferation activity. From the data of Comparative Example 4, it can be seen that the settings of different enzymatic hydrolysis methods also have a significant effect on the cell proliferation activity.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for purifying and culturing placental macrophages, characterized in that: The steps include: (1) mincing the placental tissue, adding an enzymatic solution for digestion, and filtering to obtain a filtrate; centrifuging after terminating the digestion, discarding the supernatant, adding a red blood cell lysis solution, ice bathing, centrifuging, discarding the supernatant, and washing; finally, centrifuging, discarding the supernatant, and adding a basal culture medium to resuspend the cells to obtain a resuspended cell mixture; (2) adding the resuspended cell mixture to the upper layer of the lymphocyte separation medium, centrifuging, aspirating the macrophages at the interface of the basal culture medium and the lymphocyte separation medium, then adding the basal culture medium to the macrophages, centrifuging and discarding the supernatant to obtain purified macrophages; (3) inoculating the purified macrophages into an expansion medium for culture to obtain expanded macrophages; The expansion culture medium includes the following components: 3-5% (v / v) serum replacement, 0.8-1.2% (v / v) Primocin, 2-4 mM L-glutamine, 10-16 ng / ml granulocyte-macrophage colony-stimulating factor, 8-12 ng / ml transforming growth factor-β, 24-30 ng / ml interleukin-4, 35-45 ng / ml interleukin-3, 5-9 μg / ml TNFSF15 protein, 7-11 ng / ml bone morphogenetic protein 4, and the rest is basal culture medium.

2. The method for purifying and culturing placental macrophages according to claim 1, wherein: The basal culture medium is one of DMEM, RPMI 1640 or IMDM.

3. The method for purifying and culturing placental macrophages according to claim 1, wherein: The enzymatic hydrolysis solution in step (1) is: TrypLUS digestion solution and type IV collagenase.

4. The method for purifying and culturing placental macrophages according to claim 3, wherein: The final concentration of the TrypLUS digestive solution added is 0.05-0.1% (v / v); the final concentration of the type IV collagenase added is 120-160 U / ml.

5. The method for purifying and culturing placental macrophages according to claim 1, wherein: The digestion time in step (1) is 8 to 12 minutes, and the number of digestions is 2 to 3 times.

6. The method for purifying and culturing placental macrophages according to claim 1, wherein: The conditions for the first centrifugation in step (1) are: 3-5° C., 1500-1800 rpm, 5-15 min; The conditions for the second centrifugation are: 3-5°C, 800-1200 rpm, 4-8 min; The conditions for the third centrifugation are: 3-5°C, 900-1200 r / min, 4-10 min.

7. The method for purifying and culturing placental macrophages according to claim 1, wherein: The ice bath time in step (1) is 26 to 32 minutes.

8. The method for purifying and culturing placental macrophages according to claim 1, wherein: The conditions for the first centrifugation in step (2) are: 3-5° C., 600-800 rpm, 15-25 min; The conditions for the second centrifugation are: 3-5°C, 500-700 r / min, 10-16 min.

9. The method for purifying and culturing placental macrophages according to claim 1, wherein: The seeding density of the purified macrophages in step (3) is 2 to 5×10 5 pcs / ml.

10. The method for purifying and culturing placental macrophages according to claim 1, wherein: The culture temperature in step (3) is 36-38° C., and the carbon dioxide concentration is 4-6%.

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