Method for promoting proliferation of immature bone marrow-derived macrophages and polarization of immature bone marrow-derived macrophages to M1 through stress application culture and application of method

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a method for promoting proliferation of immature bone marrow-derived macrophages and polarization of the immature bone marrow-derived macrophages to M1 through stress application culture and application of the method. The in-vitro stress application culture method of the macrophages from the immature bone marrow has the characteristics of high stability, high amplification efficiency, polarization controllability, high purity, flexibility in application, low production cost and the like; the problems that a traditional cell factor induction method is low in amplification efficiency, poor in polarization controllability, insufficient in purity and the like are solved, the purity of the obtained M1 type macrophage reaches 90% or above, stability is high, and cost is low. The method can be applied to preparation of macrophage drugs for immunoregulation, anti-fibrosis, metabolism regulation, anti-tumor CAR-M and the like, tissue regeneration biological materials, anti-inflammatory drugs, in-vitro diagnosis products and the like, dosage forms are diversified, and a standardized cell preparation scheme is provided for the related biological medicine field.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a method for promoting proliferation and M1 polarization of immature bone marrow-derived macrophages through force culture and application thereof. BACKGROUND

[0002] As a key effector cell of the innate immune system, the phenotype polarization (such as M1 / M2 type) of macrophages plays an important role in immune response, inflammation regulation and disease progression. M1 type macrophages exhibit important application value in tumor immunotherapy, infectious disease model construction and tissue repair due to their significant anti-tumor immune activity, pathogen clearance ability and pro-inflammatory characteristics. However, the main challenge in realizing the above applications at present is that the traditional in vitro culture of macrophages mainly relies on cytokine (such as LPS, IFN-γ, etc.) induced differentiation. This method has problems such as limited in vitro expansion efficiency, poor controllability of polarization, low purity, and possible functional heterogeneity of primary cell population, and leads to the difficulty in meeting the needs of large-scale production of cell preparations and precise immune regulation.

[0003] In recent years, with the in-depth study of cell biomechanics, the regulation mechanism of mechanical signals (such as stretching, compression, shear force, extracellular matrix stiffness, etc.) in the microenvironment on the phenotype of macrophages has been gradually elucidated. Studies have shown that macrophages highly express mechanical sensitive receptors (such as integrins and Piezo1 ion channels) on their surface, which can regulate cell proliferation, polarization, phagocytosis and other functions through mechanical force-chemical signal transduction pathways. It is worth noting that specific mechanical parameters can significantly enhance the mechanical sensitivity of macrophages, and thus optimize their immune regulation function. However, the existing mechanical culture technology still has systematic defects: 1) lack of macrophage proliferation and polarization characteristic optimization system, leading to cell phenotype deviation; 2) significant differences between in vitro culture system and in vivo mechanical microenvironment, which seriously affects its translational application in anti-tumor immunotherapy, inflammatory disease model construction and other fields; 3) simple mechanical stimulation is difficult to achieve the dual goals of stable expansion and high-purity polarization required for clinical cell products.

[0004] In view of this, the present application proposes an in vitro force culture method, which provides a macrophage preparation scheme with high expansion efficiency, high purity, high polarization stability and low production cost for applications such as tissue damage repair and tumor immunotherapy, and fundamentally breaks through the technical bottleneck of the prior art. SUMMARY

[0005] The application provides an in-vitro culture method based on mechanical-biochemical factor co-induction, aiming to realize efficient proliferation of immature bone marrow-derived macrophages and obtain high-purity M1 type macrophages, solve technical problems of low amplification efficiency, low controllability of polarization and insufficient purity in a traditional cytokine induction method, and provide a standardized cell preparation scheme for fields of tissue fibrosis, tumor immunotherapy, inflammatory disease model construction and the like.

[0006] The application is realized by the following technical scheme: A method for promoting proliferation of immature bone marrow-derived macrophages and polarization of the macrophages to M1 type, comprising: under force culture conditions, using an induction medium containing biochemical factors to perform force culture on the immature bone marrow-derived macrophages; the biochemical factor is macrophage colony-stimulating factor (M-CSF). Further, the induction medium is DMEM complete medium containing 10 ng / mL M-CSF.

[0007] Further, the preparation of the immature bone marrow-derived macrophages comprises the following steps: (1) A mouse is sacrificed by cervical dislocation, and then the mouse is soaked in 75% ethanol for disinfection, transferred to a clean bench, and then femur and tibia of the mouse are taken and the muscle and connective tissue above the femur and tibia are removed, and then the femur and tibia are washed with PBS containing 3% penicillin-streptomycin mixed solution. (2) The two ends of the femur and tibia are cut off, and then a 1 mL syringe containing DMEM medium (DMEM+10% serum+2% double antibody) is used to repeatedly aspirate and blow the bone marrow cavity, until the bone marrow cavity is washed to be nearly transparent, and then a cell suspension is collected, the cell suspension is filtered through a 70 μm filter screen, and the cell suspension is centrifuged at 1700 rpm for 5 min. (3) The supernatant is discarded, the cells are resuspended with 2 mL PBS, 14 mL red blood cell lysis solution (product composition: NH4Cl, NaHCO3, EDTA-Na2 and deionized water; storage condition: 2-8°C; working solution preparation: accurately take 1 mL 10x red blood cell lysis solution and add 9 mL deionized water to prepare 1x working solution) is added, and then the mixture is uniformly blown and mixed, lysis for one minute, PBS is added to 50 mL to terminate lysis, and then the mixture is centrifuged at 1700 rpm for 5 min. (4) The supernatant is discarded, the cells are resuspended with 15 mL DMEM medium, and then the cells are washed twice, the supernatant is discarded, the cells are resuspended with freshly prepared DMEM complete medium (DMEM+12% serum+1% double antibody), and then the cells are counted by using a full-automatic cell counter. (5) 10 ng / mL M-CSF is added to the DMEM complete medium, and then the cells are plated into a T25 cell culture bottle (1x10 6The 5 mL culture solution is transferred to a cell culture box at 37 DEG C and 5% CO2 saturated humidity, and cultured; After 48 h, the cells are half-replaced with fresh culture medium and cytokines, and after 72 h, full replacement is performed, and fresh prepared induction culture medium is added to the T25 cell culture bottle, and the cells are continuously cultured in the cell culture box.

[0008] Further, the force culture condition is that the cells are placed on a shaker in a cell incubator at 37 DEG C and 5% CO2 saturated humidity, and cultured at a speed of 50 rpm for 12 h, and high-efficiency proliferation and M1-type polarization of the cells are realized through mechanical-biochemical factor co-induction.

[0009] Further, the specific operation of the force culture includes that after the immature bone marrow-derived macrophages normally cultured for 3, 4 or 5 days are replaced with fresh induction culture medium, the cells are placed on the shaker for force culture for 12 h; after culture, the cell purity and function are verified by SDS-PAGE, flow cytometry or activity detection, and the cells are stored in liquid nitrogen for standby after sub-packaging.

[0010] An application of the immature bone marrow-derived macrophages obtained by the method, for preparation of any one of the following products: (1) an anti-tumor CAR-M cell therapeutic drug or product; (2) a biological material product for promoting tissue regeneration; (3) a cell drug or product for regulating inflammation; (4) an anti-fibrosis cell drug or exosome product; (5) a cell drug or exosome product for regulating fat metabolism; (6) a product for in vitro diagnosis and screening of tumor prognosis, tissue regeneration function, organ fibrosis and fat metabolism.

[0011] Further, the dosage form of the product or drug includes cryopreserved living cells, exosome freeze-dried powder, a temperature-sensitive in-situ gel preparation, a biodegradable scaffold composite preparation, microspheres, a pre-filled injection / spray, a microencapsulated sustained-release preparation or a microfluidic chip integrated kit.

[0012] Further, the double antibody is a penicillin-streptomycin mixed solution.

[0013] Compared with the prior art, the present application has the following advantages: 1. The immature bone marrow-derived macrophage in-vitro forced culture method adopted by the application has the characteristics of strong stability (good cell morphological characteristics, low cell mortality), high expansion efficiency (high cell proliferation activity), controllable polarization, high purity (M1 type macrophage purity up to > 90%), flexible application (diversified dosage forms), and low production cost (simplified process), and provides a standardized cell preparation scheme for the fields of tumor immunity, inflammatory disease treatment, tissue engineering, and other biological and pharmaceutical fields, and is suitable for popularization and application.

[0014] 2. The method of the application solves the problems of low expansion efficiency, poor controllable polarization, and insufficient purity of traditional cytokine induction method, and the obtained M1 type macrophage has a purity of more than 90%, strong stability, and low cost. The method can be applied to prepare macrophage drugs for immune regulation, anti-fibrosis, metabolic regulation, anti-tumor CAR-M, tissue regeneration biomaterials, anti-inflammatory drugs, and in-vitro diagnostic products, and has diversified dosage forms, thereby providing a standardized cell preparation scheme for related biological and pharmaceutical fields. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Principal component analysis of gene expression of immature BMDM in the control group and the treatment group; Figure 2 Expression profile analysis of immature BMDM in the control group and the treatment group; Figure 3 Effect of forced culture on cell proliferation process in the control group and the treatment group; Figure 4 Effect of forced culture on cell polarization process in the control group and the treatment group; Figure 5 Protective effect of bone marrow-derived macrophages after forced culture on acute lung injury; Figure 6 BMDM after forced culture significantly improves the survival rate of mice with LPS-induced acute lung injury; Figure 7 Effect of BMDM after forced culture on the body weight of obese mice. DETAILED DESCRIPTION

[0016] In order to further explain the application, the following specific examples are combined for illustration.

[0017] Example 1: Promoting effect of forced culture on the proliferation of immature BMDM Bone marrow-derived macrophages were prepared by the following method: cervical dislocation of mice, immersion in 75% ethanol for disinfection, transfer to a clean bench, take the femur and tibia of mice, and remove the muscle and connective tissue on top, wash with PBS containing 3% double antibody. The two ends of the femur and tibia were cut off, and the bone marrow cavity was repeatedly aspirated and blown with a 1 mL syringe containing DMEM medium (DMEM + 10% serum + 2% double antibody), and the bone marrow cavity was washed until it was nearly transparent. The cell suspension was filtered with a 70 μm filter, and the cell suspension was centrifuged at 1700 rpm for 5 min. The supernatant was discarded, resuspended with 2 mL PBS, added with 14 mL red blood cell lysis solution and mixed well, lysed for one minute, added with PBS to 50 mL to terminate lysis. Centrifuged at 1700 rpm for 5 min. Discard the supernatant, resuspend the cells with 15 mL of DMEM medium, and wash twice. Discard the supernatant, resuspend with freshly prepared DMEM complete medium (DMEM + 12% serum + 1% double antibody), and count with a full-automatic cell counter. Add 10 ng / mL M-CSF to the DMEM complete medium, and plate the cells to a T25 cell culture bottle (1 x 10 6 The cells were cultured in a cell incubator at 37°C, 5% CO2 saturated humidity. After 48 h, the cells were half-replaced with fresh medium and cytokines. After 72 h, the cells were fully replaced with fresh induction medium, and the T25 cell culture bottle was placed in the cell incubator for continuous culture.

[0018] The in vitro forced culture of immature bone marrow-derived macrophages was constructed by the following method: normal culture to the fourth day, discard the original culture medium, replace the culture medium in the cell culture bottle with fresh induction medium, and place the macrophages in a shaking bed of a cell incubator at 37°C, 5% CO2 saturated humidity. Experimental groups: control group, n = 5, normal culture in the cell incubator for 6 h, 12 h, 18 h; treatment group, n = 5, forced culture in the cell incubator (shaking speed adjusted to 50 rpm) for 6 h, 12 h, 18 h. Both groups were induced to obtain immature bone marrow-derived macrophages; the activity was detected, and the purity and function were verified.

[0019] The principal component analysis of the conventional transcriptome sequencing of immature BMDM is shown in Figure 1 The results show that there is a significant difference in gene expression between the control group and the treatment group, and this difference is particularly significant after 12 h of forced culture, indicating that forced culture has an important impact on the gene expression pattern of immature BMDM.

[0020] The expression profile analysis of the conventional transcriptome sequencing of immature BMDM is shown in Figure 2As shown in the figure. The results showed that the main changes at the whole transcriptome level occurred at 12 h of intensified culture, and at this time point, the expression levels of genes related to cell proliferation in the treatment group were significantly higher than those in the control group, indicating that 12 h of intensified culture affected the proliferation process of immature BMDM.

[0021] Figure 2 In the image: A) Heatmap of RNA-seq analysis results for the control and treatment groups; B) Expression profiles of genes related to cell proliferation for the control and treatment groups.

[0022] After normal culture for three days, the original culture medium was discarded, and the culture medium in the cell culture flask was replaced with fresh induction medium. The macrophages were then placed in a shaker in a 37℃, 5% CO2 saturated humidity cell incubator. Experimental groups: Control group (n=5), cultured normally in a cell incubator; Treatment group (n=5), cultured with increased pressure in a cell incubator (shaker speed adjusted to 50 rpm). Both groups were cultured for 12 h on days three, four, and five. BMDM proliferation was detected by RT-qPCR and EdU proliferation assay.

[0023] The effects of enhanced culture on cell proliferation in the control and treatment groups, such as Figure 3 As shown in the figure. The results showed that intensive culture for 12 h on the third and fourth days promoted the expression of cell proliferation-related genes and enhanced EdU proliferation activity in the treatment group, indicating that intensive culture for 12 h on the fourth day had a certain promoting effect on the proliferation process of immature BMDM.

[0024] Figure 3 In the table: A) RT-qPCR results of cell proliferation-related genes in the control and treatment groups on days 3, 4, and 5; B) EdU staining results in the control and treatment groups on days 3 and 4; C) Statistical results of EdU staining data in the control and treatment groups on days 3 and 4 (n = 10). *For comparison with the control group, *P < 0.05, **P < 0.01.

[0025] Table 1 Summary of RT-qPCR Primer Sequences

[0026] Example 2: The promoting effect of enhanced culture on the M1-type polarization of immature BMDM After normal culture for three days, the original culture medium was discarded, and the culture medium in the cell culture flask was replaced with fresh induction medium. The macrophages were then placed in a shaker in a 37℃, 5% CO2 saturated humidity cell incubator. Experimental groups: Control group (n=5), cultured normally in a cell incubator; Treatment group (n=5), cultured with enhanced shaking (shaker speed adjusted to 50 rpm) in a cell incubator. Both groups were cultured for 12 h on days three, four, and five, and the purity and function of M1 macrophages were assessed.

[0027] The effect of force loading culture on the polarization process of control and treatment groups of cells is shown in Figure 4 The results show that force loading culture for 12 h on the third and fourth days promotes immature BMDMs to secrete pro-inflammatory cytokines in the treatment group, indicating that force loading culture for 12 h on the fourth day promotes immature BMDMs to polarize into Ml type macrophages.

[0028] Example 3: Protective effect of BMDMs on LPS-induced acute lung injury mice SPF level male C57BL / 6 mice, 4 months old, weighing 24-30 g, were provided by Guangzhou Laboratory Animal Experiment Center (Ethical Number: GZLAB-AUCP-2023-01-A04). All mice were adaptively fed in a SPF level barrier environment for 1 week before the implementation of the study, and the temperature and humidity were suitable, with a 12 / 12 hour light / dark cycle. The mice were fed with standard mouse feed during feeding, and free water. Experimental grouping: negative control group (NC group), n = 5, given the same volume of sterile normal saline by intranasal instillation; acute lung injury group (LPS group), n = 5, intranasal instillation of LPS, the dose of drug administration is 30 mg / kg; macrophage treatment group (Therapy group), n = 5, 2 h after intranasal instillation of LPS (30 mg / kg), intraperitoneal injection of 1 million bone marrow-derived macrophages (normal culture in vitro to the sixth day).

[0029] The tissue specimens were immediately fixed with 4% paraformaldehyde (pH 7.4) for 24 h to maintain morphological integrity. After fixation, the samples were sequentially dehydrated (70%~100% gradient ethanol), transparentized (xylene), and paraffin-impregnated (60°C). The embedded tissues were cut into 5 μm thick sections using a rotary microtome (Leica), and the sections after HE staining and Masson staining were imaged under a full-automatic slide scanning system (Olympus).

[0030] The results of lung tissue pathological staining are shown in Figure 5 The results show that BMDM treatment significantly reduces LPS-induced lung tissue damage, as evidenced by reduced inflammatory cell infiltration, improved alveolar structure, and reduced collagen fiber deposition area, indicating that BMDMs have a certain improvement effect on lung injury.

[0031] BMDMs significantly improve the survival rate of acute lung injury mice Figure 6 .

[0032] Example 4: Effect of BMDMs on the body weight of obese mice SPF level male C57BL / 6 mice, 4 months old, weighing 24-30 g, were provided by Guangzhou Laboratory Animal Experiment Center (Ethical Number: GZLAB-AUCP-2023-01-A04). All mice were acclimated for 1 week in a SPF barrier environment before the study was implemented, with suitable temperature and humidity, and a 12 / 12 hour light / dark cycle. Mice in the blank control group were fed standard mouse feed during the feeding period, with free access to water; mice in the model control group and macrophage treatment group were fed high-fat feed (casein (20%), L-cystine (0.39%), corn starch (21.2%), maltodextrin (7.1%), sucrose (11.3%), soybean oil (2.5%), lard (15.5%), cellulose (5%), mixed minerals (S10026), mixed vitamins (V10001), choline hydrochloride tartrate (0.2%)), n = 5 for each group of mice; two months later, the blank control group and the model group were given the same volume of sterile normal saline intraperitoneally; the macrophage treatment group (Therapy group), n = 5, was given 200 uL of 1 million bone marrow-derived macrophages (normally cultured in vitro for 5 days) intraperitoneally, once every other week, for a total of 4 weeks of administration.

[0033] From Figure 7 As can be seen, the body weight of the control group mice remained at a low level, and the body weight of the high-fat model group mice increased significantly, with a very significant difference compared with the control group (P < 0.01), indicating that the high-fat feed successfully induced obesity in mice. p <0.05), indicating that bone marrow-derived macrophages (BMDM) can effectively reduce the body weight of high-fat-induced obese mice. p <0.05), and there was no significant difference (ns) compared with the control group, indicating that bone marrow-derived macrophages (BMDM) can effectively reduce the body weight of high-fat-induced obese mice.

[0034] The specific embodiments described above further illustrate the objectives, technical solutions, and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the protection scope of the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for promoting the proliferation and M1 polarization of immature bone marrow-derived macrophages through enhanced culture, characterized in that, include: Immature bone marrow-derived macrophages were subjected to enhanced culture under enhanced culture conditions using an induction medium containing biochemical factors. The biochemical factor is macrophage colony-stimulating factor M-CSF.

2. The method according to claim 1, characterized in that, The induction medium is DMEM complete medium containing 10 ng / mL M-CSF.

3. The method according to claim 1, characterized in that, The preparation of the immature bone marrow-derived macrophages includes the following steps: (1) Take the femur and tibia of mice, remove the muscle and connective tissue, and wash with PBS containing 3% penicillin-streptomycin mixed solution and double antibiotics; (2) Cut off both ends of the bone and rinse the bone marrow cavity with a syringe containing DMEM medium containing 10% serum + 2% penicillin and antibiotics. Collect the cell suspension, filter it through a filter screen and centrifuge it. (3) Discard the supernatant, resuspend the cells in PBS, add red blood cell lysis buffer to lyse for 1 min, add PBS to stop lysis and then centrifuge; (4) Wash the cells twice with DMEM medium and then resuspend them in DMEM complete medium containing 12% serum + 1% penicillin antibiotics; (5) Add 10 ng / mL M-CSF to the DMEM complete medium and inoculate the cells at a concentration of 1×10⁻⁶. 6 Inoculate at a density of 10 cells / mL into T25 culture flasks, with 5 mL of culture medium per flask, and incubate at 37°C in a 5% CO2 incubator. (6) After culturing for 48 h, half of the medium was replaced, and after 72 h, the medium was completely replaced and fresh induction medium was added. The culture was continued until immature bone marrow-derived macrophages were obtained.

4. The method according to claim 1, characterized in that, The enhanced culture conditions are as follows: the cells are placed on a shaker in a cell incubator at 37°C and 5% CO2 saturated humidity, and cultured at 50 rpm for 12 h. The cells are then induced to proliferate efficiently and polarize to the M1 type through the combined action of mechanical and biochemical factors.

5. The method according to claim 4, characterized in that, The specific operation of the enhanced culture includes: replacing the immature bone marrow-derived macrophages that have been cultured normally for three, four, or five days with fresh induction medium, and then placing them on the shaker for enhanced culture for 12 hours; after culture, the purity and function of the cells are verified by SDS-PAGE, flow cytometry, or activity detection, and the cells are aliquoted and stored in liquid nitrogen for later use.

6. The application of immature bone marrow-derived macrophages obtained by the method according to any one of claims 1 to 5, characterized in that, Used in the preparation of any of the following products: (1) Anti-tumor CAR-M cell therapy drugs or products; (2) Biomaterial products that promote tissue regeneration; (3) Cellular drugs or products that regulate inflammation; (4) Antifibrotic cell drugs or exosome products; (5) Cellular drugs or exosome products that regulate lipid metabolism (6) Products for in vitro diagnosis and screening of tumor prognosis, tissue regeneration function, organ fibrosis and lipid metabolism.

7. The application according to claim 6, characterized in that, The dosage forms of the products or drugs include cryopreserved live cells, exosome lyophilized powders, thermosensitive in-situ gel formulations, biodegradable scaffold composite formulations, microspheres, pre-filled injections / sprays, microencapsulated sustained-release formulations, or microfluidic chip integrated kits.

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