M2 type macrophage-derived cell vesicle simulant as well as preparation method and application thereof

Abstract

The invention provides a preparation method of an M2 type macrophage-derived cell vesicle simulant and an application of the simulant in relieving foreign body reaction after a biological 3D printed body is implanted. The M2 type macrophage-derived cell vesicle simulant is obtained by carrying out hypotonic lysis and mechanical extrusion on M2 type macrophages obtained by induced polarization. Researches find that the vesicle simulant has the typical characteristics of high yield, high uptake rate, capability of relieving foreign body reaction after the biological printing body is implanted, and the like. Therefore, the vesicle mimetics can be widely used as various bio-ink additives to alleviate foreign body reactions after implantation.

Description

Technical Field

[0001] The present invention belongs to the field of bioprinting, and more particularly, relates to an M2 macrophage-derived cell vesicle mimetic for alleviating foreign body reaction after implantation of a bio-3D printed body and a preparation method thereof. Background Art

[0002] A bio-3D printed body refers to medical devices, tissue engineering scaffolds, tissue organs and other products manufactured by a 3D bio-printer based on bio-inks. After implantation, a bio-3D printed body is often recognized as a foreign body by the host immune system, which then leads to a strong inflammatory response, and ultimately a fibrotic coating is formed around the implant, separating the implant from the host tissue and resulting in the failure of transplantation or even serious complications. Current methods for eliminating foreign body reactions of bio-3D printed bodies include improving the biocompatibility of materials and adding anti-inflammatory drugs. For example, the hydrogel optical fiber disclosed in Chinese Patent CN118181740A is composed of materials with good biocompatibility, is more compatible with biological tissues, and can reduce the rejection reaction of organisms to foreign substances, reducing potential immune reactions and foreign body reactions. However, this patent prepares an optical fiber by selecting a highly biocompatible material (Pluronic F-127) to prepare a bio-ink, emphasizing that it can reduce foreign body reactions, but there is no data in its experimental data to support that it can reduce foreign body reactions. In fact, most of the biological materials used in current bio-ininks are materials with high biocompatibility, but foreign body reactions still occur after implantation. This patent is only an optimization of a certain biological material, with limited scope of application and unclear in vivo effects. Another example is that the self-assembled 3D printed scaffold with anti-inflammatory and bone repair effects disclosed in Chinese Patent CN118252979A, after being implanted into the body, the encapsulant is destroyed after contacting with body fluid, and anti-inflammatory drugs such as puerarin can be released in time to inhibit inflammation and promote bone repair, and can better promote bone repair by eliminating foreign body reactions. However, this patent prepares a printed scaffold and then loads an anti-inflammatory drug such as puerarin to reduce foreign body reactions. On the one hand, the loading method of anti-inflammatory drugs is complex, requires the use of additional reagents, and has higher costs and complexity; on the other hand, the specific anti-foreign body reaction ability is limited by the types and efficacy of the selected anti-inflammatory drugs, and the effect of a single drug component is limited. In contrast, the material of the present patent is derived from anti-inflammatory cells, has high biocompatibility and cell affinity, and has multiple anti-inflammatory components, with stronger effects.

[0003] M2 macrophages refer to alternatively activated macrophages. The vesicles secreted by M2 macrophages have anti-inflammatory properties. CN 117721080A discloses an engineered nanovesicle derived from M2 macrophages based on Raw264.7 macrophages. This engineered nanovesicle derived from M2 macrophages has high preparation efficiency and exhibits excellent effects in promoting cell proliferation and migration, improving angiogenesis, regulating inflammatory responses, etc. However, the yield of existing cell vesicle mimics derived from M2 macrophages needs to be further improved, and their anti-inflammatory properties are much lower than those of parental cells. There is no report on cell vesicle mimics derived from M2 macrophages that can alleviate the foreign body reaction after the implantation of bio-3D printed bodies. Summary of the Invention

[0004] To solve the problems such as the strong foreign body reaction caused after the implantation of existing bio-printed bodies into the host and the low integration efficiency with the host, on the one hand, the present invention provides a method for preparing a cell vesicle mimic derived from M2 macrophages, including the following steps:

[0005] Lyse the M2 macrophages in a hypotonic lysis buffer to obtain an M2 macrophage lysate; the hypotonic lysis buffer is made from the following raw materials: Tris-HCl with a final concentration of 8 - 12 mM; sucrose with a final concentration of 20 - 30 mM; EGTA with a final concentration of 0.047 - 0.087 mM; D-mannitol with a final concentration of 60 - 90 mM and a protease / phosphatase inhibitor mixture to make up to 100 ml.

[0006] Subject the M2 macrophage lysate to mechanical extrusion and centrifugal resuspension to obtain the cell vesicle mimic derived from M2 macrophages.

[0007] On the second aspect, the present invention provides a cell vesicle mimic derived from M2 macrophages.

[0008] On the third aspect, the present invention provides an application of a cell vesicle mimic derived from M2 macrophages in the preparation of a raw material for reducing the foreign body reaction after the implantation of a bio-3D printed body.

[0009] On the fourth aspect, the present invention provides a modified bioink. The raw materials for preparing the modified bioink include the aforementioned cell vesicle mimic derived from M2 macrophages and bioink.

[0010] On the fifth aspect, the present invention provides a bio-3D printed body. The raw materials for preparing the bio-3D printed body include the aforementioned cell vesicle mimic derived from M2 macrophages, bioink, and a crosslinking agent.

[0011] With the above technical solutions, the present invention has at least the following advantages and beneficial effects: The M2 macrophage vesicle mimetic provided by the present invention has a high yield, and has the characteristics of good biocompatibility, high cell uptake rate, and can effectively inhibit the foreign body reaction. It can be added to a variety of bioinks without affecting the basic properties and printing effect of the bioinks, and has a wide application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Electron microscopy detection imaging diagram;

[0013] Figure 2 NTA detection result diagram;

[0014] Figure 3 Cell uptake experiment result diagram;

[0015] Figure 4 Anti-foreign body reaction ability fiber capsule thickness result diagram;

[0016] Figure 5 Anti-foreign body reaction ability collagen density result diagram. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] Bio 3D printing is an emerging product that integrates life science, materials science, and manufacturing science. It provides new ideas and methods for medical fields such as organ transplantation and is of great significance for the development of regenerative medicine. However, after the bio 3D printed body is implanted, it is often recognized as a foreign body by the host immune system, which in turn leads to a strong inflammatory reaction, and finally a fibrotic coating is formed around the implant, separating the implant from the host tissue and resulting in the failure of transplantation or even serious complications. Developing a safe and effective method to reduce the foreign body reaction is of great significance for ensuring the transplantation effect of bio-printed bodies.

[0018] Research shows that macrophages play an indispensable role in the immune response caused by grafts. They are involved in antigen presentation, providing co-stimulatory signals, producing cytokines, communicating with other immune cells, and immune regulation. In addition, it has also been confirmed that macrophages can directly mediate the foreign body reaction and form antigen-specific and memory immune responses. M2 macrophages can control the foreign body reaction through signal pathways such as PI3K, and at the same time can clear the residual substances of damaged cells and tissues, and promote angiogenesis to accelerate the repair process. Therefore, M2 macrophages are expected to be the key to alleviating the foreign body reaction after the implantation of bio 3D printed bodies.

[0019] However, the direct application of macrophages has limitations in cell sources, unstable phenotypes in vivo, and potential ethical issues. Non-cellular derivatives of macrophages, especially cell vesicles, can circumvent these risks. Traditional cell vesicles are usually obtained by ultracentrifugation purification using cell culture media, with low yields, difficult preparation, and high costs. In recent years, cell vesicle mimics obtained by mechanical extrusion have gained popularity. Although the yield has increased, their anti-inflammatory properties are far lower than those of parental cells. Against this background, multiple embodiments of the present invention design a method for preparing M2 macrophage vesicle mimics and add them to bioink for application in alleviating the foreign body reaction after the implantation of bio-3D printed bodies.

[0020] Some embodiments of the present invention provide a method for preparing M2 macrophage-derived cell vesicle mimics and their application in reducing the foreign body reaction after the implantation of bio-3D printed bodies, solving problems such as strong foreign body reactions caused by existing bio-printed bodies after implantation into the host and low integration efficiency with the host.

[0021] Reagents and instruments:

[0022] J774A.1 (Wuhan Punosai Life Science Co., Ltd., mouse BALB / c mononuclear macrophages); fetal bovine serum (FBS); interleukin 4 (IL-4); DMEM (dulbecco's modified eagle medium) high-glucose medium; Aminomethane Hydrochlorid or Tris (Hydroxymethyl), Tris-HCl; EGTA (ethylene glycol bis(α-aminoethyl ether) tetraacetic acid); protease / phosphatase inhibitor mixture (cocktail, purchased from Thermo Fisher); D-Mannitol; PBS (phosphate buffered saline); polycarbonate membrane (Moger Machinery (Shanghai) Co., Ltd., LiposoEasy LE-1); microextruder (manufacturer, model); ultrasonic crusher (Newzhik SCIENTZ-IID); C57BL / 6 mice (Beijing Weishang Lide Biotechnology Co., Ltd.)

[0023] In some embodiments, a method for preparing M2 macrophage-derived cell vesicle mimics is provided. The M2 macrophage-derived cell vesicle mimics are obtained by hypotonic lysis and mechanical extrusion of induced polarized M2 macrophages, and specifically include the following steps:

[0024] Lyse the M2 macrophages in a hypotonic lysis buffer to obtain an M2 macrophage lysate. The hypotonic lysis buffer is prepared from the following raw materials: Tris-HCl with a final concentration of 8-12 mM, further preferably 8, 9, 10, 11, or 12 mM; sucrose with a final concentration of 20-30 mM, further preferably 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mM; EGTA with a final concentration of 0.047-0.087 mM, further preferably 0.047, 0.057, 0.067, 0.077, or 0.087 mM; D-mannitol with a final concentration of 60-90 mM, further preferably 60, 65, 70, 75, 80, 85, or 90 mM; and a protease / phosphatase inhibitor mixture to make up to 100 ml. In a further preferred embodiment, 1 dish of cells (about 10^6 cells) is lysed with 1-2 ml of the lysate, but the present invention does not make specific limitations.

[0025] Subject the M2 macrophage lysate to mechanical extrusion and centrifugal resuspension to obtain the M2 macrophage-derived cell vesicle mimetic.

[0026] Multiple embodiments of the present invention show that each cell produces approximately 1-5×10^4 cell vesicles, which is much higher than the yield of natural cell vesicles. Multiple embodiments of the present invention show that the present invention first removes nuclear material and then mainly obtains vesicles through the cell membrane, with a stronger anti-foreign body reaction ability; while the prior art generally directly extrudes cells without removing the nucleus and cytoplasm through a hypotonic solution. Since the nucleus and cytoplasm contain nuclear materials such as DNA / RNA, there is a greater possibility of causing an immune response and weakening the anti-inflammatory characteristics of the vesicles themselves.

[0027] In some further embodiments, a method for preparing an M2 macrophage-derived cell vesicle mimetic is provided, and the lysis includes the following steps:

[0028] Add the M2 macrophages to the hypotonic lysis buffer and then perform ultrasonic treatment to obtain an ultrasonic mixture. In a further embodiment, the ultrasonic treatment method is to ultrasonically treat the sample for 1-10 min at 2-6°C with an ultrasonic crusher, and further preferably, the ultrasonic treatment method is to ultrasonically treat the sample for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 min at 2, 3, 4, 5, or 6°C with an ultrasonic crusher.

[0029] Centrifuge the ultrasonic mixture to obtain a first supernatant, which is the M2 macrophage lysate. In a further embodiment, the centrifugation method is to centrifuge the ultrasonic mixture at 10,000 - 14,000×g, 2 - 6°C for 10 - 20 min to remove unbroken cell nuclei and cells. A further centrifugation method is to centrifuge the ultrasonic mixture at 10,000, 11,000, 12,000, 13,000 or 14,000×g, 2, 3, 4, 5 or 6°C for 10, 15 or 20 min.

[0030] In some further embodiments, a method for preparing an M2 macrophage-derived cell vesicle mimetic is provided, and the mechanical extrusion includes the following steps:

[0031] Extrude the M2 macrophage lysate through a polycarbonate membrane to obtain a second supernatant; store the second supernatant at -80°C. In a further embodiment, the mechanical extrusion method is: extrude the M2 macrophage lysate (the supernatant after centrifugation) through a 100 - 300 nm polycarbonate membrane 1 - 15 times using a microextruder, and store the extruded second supernatant at -60 - -100°C. Extrude the M2 macrophage lysate (the supernatant after centrifugation) through a 100, 200 or 300 nm polycarbonate membrane 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 times using a microextruder, and store the extruded second supernatant at -60, -70, -80, -90 or -100°C.

[0032] In some further embodiments, a method for preparing an M2 macrophage-derived cell vesicle mimetic is provided, and the mechanical extrusion further includes the following steps:

[0033] Take the second supernatant and perform centrifugation to remove larger vesicles to obtain a third supernatant; take the third supernatant and obtain a microporous membrane filtrate through a microporous membrane. In a further embodiment, the centrifugation conditions for removing larger vesicles are as follows: Medium-speed thaw the sample (the second supernatant stored frozen) at 35-40 °C, transfer the sample to a new centrifuge tube, centrifuge at 1000-3000 × g, 2-6 °C for 10-50 min. Transfer the supernatant to a new centrifuge tube and centrifuge again at 8000-12000 × g, 2-6 °C for 30-60 min to remove larger vesicles. Further centrifugation conditions for removing larger vesicles are as follows: Medium-speed thaw the sample at 35 °C, 36 °C, 37 °C, 38 °C, 39 °C or 40 °C, transfer the sample to a new centrifuge tube, centrifuge at 1000, 2000 or 3000 × g, 2, 3, 4, 5 or 6 °C for 10, 20, 30, 40 or 50 min. Transfer the supernatant to a new centrifuge tube and centrifuge again at 8000, 9000, 10000, 11000 or 12000 × g, 2, 3, 4, 5 or 6 °C for 30, 40, 50 or 60 min to remove larger vesicles. In a further embodiment, the method for microporous membrane treatment is as follows: Take the supernatant (the third supernatant), filter it through a 0.40-0.60 μm filter membrane, and collect the filtrate (the microporous membrane filtrate). Further method for microporous membrane treatment is as follows: Take the supernatant (the third supernatant), filter it through a 0.40, 0.45, 0.50, 0.55 or 0.60 μm filter membrane, and collect the filtrate (the microporous membrane filtrate).

[0034] In some further embodiments, a method for preparing an M2 macrophage-derived cell vesicle mimetic is provided, and the centrifugation and resuspension include the following steps:

[0035] The filtrate is subjected to ultracentrifugation to obtain a centrifugate; the centrifugate is resuspended to obtain the M2 macrophage-derived cell vesicle mimetic. In a further embodiment, the method of centrifugation and resuspension is as follows: Transfer the filtrate to a new centrifuge tube, select an ultracentrifuge rotor, at 2-6 °C, 8000-12000×g, ultracentrifuge for 40-100 min to obtain a centrifugate. Remove the supernatant from the centrifugate, resuspend it with 5-15 mL of pre-cooled 1×PBS, and store it at -60 to -100 °C. Further ultracentrifugation conditions are as follows: Transfer the filtrate to a new centrifuge tube, select an ultracentrifuge rotor, at 2, 3, 4, 5 or 6 °C, 8000, 9000, 10000, 11000 or 12000×g, ultracentrifuge for 40, 50, 60, 70, 80, 90 or 100 min to obtain a centrifugate. Remove the supernatant from the centrifugate, resuspend it with 5, 10 or 15 mL of pre-cooled 1×PBS, and store it at -60, -70, -80, -90 or -100 °C. In a further embodiment, the centrifugation and resuspension are carried out twice. When resuspending for the second time, take 50-150 μL of pre-cooled 1×PBS, and further take 50, 100 or 150 μL of pre-cooled 1×PBS.

[0036] In some further embodiments, a method for preparing an M2 macrophage-derived cell vesicle mimetic is provided, which includes the step of directing the polarization of murine macrophages J774A.1 into M2 macrophages. The method for inducing the polarization of the M2 macrophages specifically includes the following steps:

[0037] Culture murine macrophages in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. When the cell confluence reaches 70-80%, add 20 ng / ml IL-4 to the DMEM high-glucose medium, and culture it in an incubator at 37 °C and 5% CO2 for 24 h to obtain the M2 macrophages. Preferably, the murine macrophages include murine Raw264.7 macrophages or murine J774A.1 macrophages; more preferably, the murine macrophages are murine J774A.1 macrophages.

[0038] In some other embodiments, an M2 macrophage-derived cell vesicle mimetic is provided, which is prepared by any of the foregoing method embodiments.

[0039] In some other embodiments, the application of the M2 macrophage-derived cell vesicle mimetic in the preparation of a raw material for reducing the foreign body reaction after implantation of a bio-3D printed body is provided.

[0040] In some other embodiments, a modified bioink is involved. The raw materials for preparing the modified bioink include the aforementioned M2 macrophage-derived cell vesicle mimics and bioink. Preferably, the raw materials for preparing the bioink include gelatin and sodium alginate. Further preferably, the raw materials for preparing the bioink further include 5 parts by weight of gelatin, 3 parts by weight of sodium alginate, and 9 parts by weight of PBS buffer solution. Even further preferably, the raw materials for preparing the modified bioink further include PBS buffer solution for dissolving the M2 macrophage-derived cell vesicle mimics, and 1 ml of PBS buffer solution contains 10^ 10 M2 macrophage vesicle mimics; even further preferably, the volume ratio of the PBS buffer solution containing 10^ 10 M2 macrophage vesicle mimics to the bioink is 1:1.

[0041] In some other embodiments, a biological 3D printed body is involved. The raw materials for preparing the biological 3D printed body include the aforementioned M2 macrophage-derived cell vesicle mimics, bioink, and cross-linking agent. Preferably, the cross-linking agent is calcium chloride.

[0042] The present invention will be further described below in conjunction with specific embodiments.

[0043] Example 1 A method for preparing M2 macrophage-derived cell vesicle mimics

[0044] 1. Maintain mouse J774A.1 macrophages in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. When the cell confluence reaches 70-80%, add IL-4 with a final concentration of 20 ng / ml to the medium, and place it in a 37°C, 5% CO2 incubator for 24 h to obtain polarized macrophages (M2 macrophages).

[0045] 2. Use a cell scraper to collect the polarized macrophages and lyse them in a hypotonic lysis buffer containing 10 mM Tris-HCl, 25 mM sucrose, 0.067 mM EGTA, 75 mM D-mannitol, and a protease / phosphatase inhibitor mixture.

[0046] 3. Ultrasonically treat the sample (i.e., the sample obtained by adding M2 macrophages to the hypotonic lysis buffer) at 4°C for 5 min using an ultrasonic crusher.

[0047] 4. Centrifuge the mixture (ultrasonic mixture) at 12000×g, 4°C for 15 min to remove unbroken cell nuclei and cells.

[0048] 5. Extrude the M2 macrophage lysate (the supernatant after centrifugation) through a 200 nm polycarbonate membrane 8 times using a microextruder, and store the extruded supernatant (the second supernatant) at -80 °C.

[0049] 6. Thaw the sample at medium speed at 37 °C, transfer the sample to a new centrifuge tube, and centrifuge at 2000×g at 4 °C for 30 min.

[0050] 7. Transfer the supernatant to a new centrifuge tube and centrifuge again at 10000×g at 4 °C for 45 min to remove larger vesicles.

[0051] 8. Take the supernatant, filter it through a 0.45 μm filter membrane, and collect the filtrate.

[0052] 9. Transfer the filtrate to a new centrifuge tube, select an ultracentrifuge rotor, and ultracentrifuge at 100000×g at 4 °C for 70 min.

[0053] 10. Remove the supernatant, resuspend it with 10 mL of pre-cooled 1×PBS, select an ultracentrifuge rotor, and ultracentrifuge again at 100000×g at 4 °C for 70 min.

[0054] 11. Remove the supernatant, resuspend it with 100 μL of pre-cooled 1×PBS, and store it at -80 °C.

[0055] Example 2 A method for preparing a cell vesicle mimetic derived from M2 macrophages

[0056] Induction of M2 macrophage polarization: Maintain mouse J774A.1 macrophages in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. When the cell confluence reaches 70-80%, add 20 ng / ml IL-4 to the medium and culture it in an incubator at 37 °C and 5% CO2 for 24 h. Verify the macrophage polarization effect by PCR and immunofluorescence.

[0057] Preparation of cell vesicle mimetic derived from M2 macrophages: Use a cell scraper to collect the polarized macrophages and lyse them in a hypotonic lysis buffer containing 10 mM Tris-HCl, 25 mM sucrose, 0.067 mM EGTA, 75 mM D-mannitol, and a protease / phosphatase inhibitor mixture. Sonicate the sample at 4 °C for 5 min using a sonicator. Centrifuge the mixture at 12000×g (where "g" represents the acceleration due to gravity on Earth, approximately 9.8 m / s 2。×g centrifugation is mainly used to describe the multiple relationship between the centrifugal force generated by a centrifuge during operation and the earth's gravity. ), 4°C, 15 min to remove unbroken cell nuclei and cells. The supernatant was extruded through a 200 nm polycarbonate membrane 8 times using a microextruder, and the extruded supernatant was stored at -80°C. The sample was thawed at medium speed at 37°C, transferred to a new centrifuge tube, and centrifuged at 2000×g, 4°C, for 30 min. The supernatant was transferred to a new centrifuge tube and centrifuged again at 10000×g, 4°C, for 45 min to remove larger vesicles. The supernatant was taken, filtered through a 0.45 μm filter membrane, and the filtrate was collected. The filtrate was transferred to a new centrifuge tube, an ultracentrifugation rotor was selected, and ultracentrifugation was performed at 100000×g, 4°C, for 70 min. The supernatant was removed, resuspended with 10 mL of pre-cooled 1×PBS, an ultracentrifugation rotor was selected, and ultracentrifugation was performed again at 100000×g, 4°C, for 70 min. The supernatant was removed, resuspended with 100 μL of pre-cooled 1×PBS, and stored at -80°C to obtain M2 macrophage-derived cell vesicle mimics.

[0058] Comparative Example 1:

[0059] CN117721080A An engineered nanovesicle derived from M2 macrophages, the preparation process is as follows: I. Preparation of M2 macrophages The Raw264.7 macrophages ( RAW 264.7 (mouse monocyte macrophage leukemia cells) (STR identification correct) https: / / www.procell.com.cn / view / 1380.html) After resuscitation, after 3 passages, continue to culture in 10% FBS high-glucose DMEM medium. When the fusion rate of Raw264.7 macrophages reaches 50% - 60%, add IL-4 with a final concentration of 10 ng / mL, and after culturing for 24 h, induce to obtain M2 macrophages. II. Preparation of engineered nanovesicles derived from M2 macrophages (1) Collect the above-mentioned induced M2 macrophages with good growth status and resuspend them with phosphate buffer (PBS) to form a single-cell suspension; (2) Pass the single-cell suspension through microporous filter membranes with pore sizes of 1.2 μm, 0.6 μm, and 0.22 μm in sequence; (3) Repeat the operation in step (2) three times to obtain extruded vesicles; (4) Centrifuge the extruded vesicles at 100000g for 70 min, collect precipitate a, add PBS to resuspend precipitate a and then centrifuge at 100000g for 70 min to collect precipitate b to obtain engineered nanovesicles derived from M2 macrophages.

[0060] Test Example 1

[0061] The morphology, particle size, and uptake effect of the M2 macrophage-derived cell vesicle mimetic of Example 2 were evaluated by transmission electron microscopy, NTA nanoparticle tracking analysis technology (Nanoparticle Tracking Analysis), and cell uptake experiments.

[0062] Transmission electron microscopy: High-resolution transmission electron microscope (HR-TEM, HT-7700, Hitachi, Japan); 10 μL of the vesicles was taken out and dropped onto a copper grid for precipitation for 1 min, and the floating liquid was blotted off with filter paper. 10 μL of uranyl acetate was dropped onto the copper grid for precipitation for 1 min, and the floating liquid was blotted off with filter paper. It was dried at room temperature for several minutes. Electron microscopy detection and imaging were performed at 580 kv, and the results are shown in Figure 1 。

[0063] NTA: ZetaView, PARTICLE METRIX, Germany. The frozen sample was taken out, thawed in a 25°C water bath, and placed on ice. The sample was diluted with 1×PBS and directly used for NTA detection. The results are shown in Figure 2 。

[0064] Cell uptake: M2-EVM was labeled with PKH26 (Solarbio, China), and the concentration was diluted to 9×10 10 cells / ml with 1×PBS solution. For fluorescence analysis, three types of subcutaneous implanted functional cells were selected for uptake experiments, L929 cells (fibroblasts, 5×10 4 cells per well), C166 cells (vascular endothelial cells, 2.5×10 4 cells per well), and J774A.1 cells (macrophages, 2.5×10 4 cells per well) were seeded in a 48-well plate with a 9-mm round cell slide and cultured for 24 hours. Then, 10 μL of the PKH26-labeled M2-EVMS suspension was added to each well. After incubation for 6 hours, the cells were stained with DAPI and phalloidin. Images were collected by a confocal microscope (SP8, Leica, Germany). The results are shown in Figure 3 。

[0065] Test Example 2

[0066] Verification of the ability of the M2 macrophage-derived cell vesicle mimetic to reduce the foreign body reaction after implantation of a bio-3D printed body: Taking the gelatin-alginate bioink as an example, the anti-foreign body reaction ability of the vesicle mimetic was verified.

[0067] Preparation of the biological 3D printing body: Weigh 0.5 g of gelatin and 0.3 g of sodium alginate and place them in a small beaker. Add 9 ml of PBS buffer solution, seal the beaker, dissolve it in a water bath at 70 °C, and gently stir with a glass rod until the solution is uniform without solute particles. After sterilization using the pasteurization method, seal it and store it at 4 °C to obtain the gelatin-sodium alginate bioink; Dissolve 2.5 g of calcium chloride solid powder in 100 ml of deionized water, stir magnetically until fully dissolved, sterilize it by high-pressure steam, seal it and store it at 4 °C to obtain the crosslinking agent. Mix 9 ml of sterile gelatin-sodium alginate hydrogel and 1 ml of PBS buffer solution containing 10^ 10 (10 to the 10th power) M2 macrophage vesicle mimics, and then load them into a pre-filled syringe. Use a sterile three-way device to mix them thoroughly to obtain the bioink containing M2 macrophage vesicle mimics; Load this bioink into the printing cylinder and install it in a 3D printer. Adjust the printing nozzle temperature to 4 °C, the platform temperature to 0 °C, the air pressure to 0.15 MPa, and the nozzle moving speed to 8 cm / s, and start printing. After printing, crosslink it with the crosslinking agent at 0 °C for 10 min. After crosslinking, remove the crosslinking agent to obtain the biological 3D printing body containing M2 macrophage vesicle mimics.

[0068] Verification of the anti-foreign body reaction ability of the vesicle mimics: Use 8-week-old female C57BL / 6 mice as the subcutaneous implantation subjects. After anesthesia with isoflurane gas, make a 1.5-cm incision under the skin on the back of the mice, bluntly separate to form a subcutaneous cavity, and implant the biological 3D printing body containing M2 macrophage vesicle mimics, with three parallel samples in each group. At 7 days or 14 days after implantation, sacrifice the mice, harvest the skin-containing printing body for further evaluation. After fixing with 4% paraformaldehyde for 6 h, dehydrate and embed it in paraffin sections, and perform hematoxylin and eosin (H&E) staining and Masson staining.

[0069] Compared with the control group (gelatin-sodium alginate bioink without adding M2 macrophage-derived cell vesicle mimics), after subcutaneous implantation of the biological 3D printing body added with M2 macrophage-derived cell vesicle mimics, the thickness of the fibrous capsule formed around the implant at 7 days and 14 days decreased significantly, and there was less infiltration of inflammatory cells, indicating a milder foreign body reaction. The results are shown in Figure 4 .

[0070] Compared with the control group, after subcutaneous implantation of the biological 3D printing body added with M2 macrophage-derived cell vesicle mimics, the level of collagen deposition in the fibrous capsule formed around the implant at 7 days and 14 days decreased significantly, indicating that the macrophage-derived vesicles successfully reduced the degree of fibrosis caused by the foreign body reaction of the implant. The results are shown in Figure 5 .

[0071] The above-described embodiments are only described as the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of M2 macrophage-derived cell vesicle mimics, characterized in that, It includes the following steps: Lyse the M2 macrophages in a hypotonic lysis buffer to obtain an M2 macrophage lysate; the hypotonic lysis buffer is made from the following raw materials: Tris-HCl with a final concentration of 8 - 12 mM; sucrose with a final concentration of 20 - 30 mM; EGTA with a final concentration of 0.047 - 0.087 mM; D-mannitol with a final concentration of 60 - 90 mM and a protease / phosphatase inhibitor mixture to make up to 100 ml; Subject the M2 macrophage lysate to mechanical extrusion and centrifugal resuspension to obtain the M2 macrophage-derived cell vesicle mimetic.

2. The preparation method according to claim 1, characterized in that, The lysis includes the following steps: Add the M2 macrophages to the hypotonic lysis buffer and then perform ultrasonic treatment to obtain an ultrasonic mixture; Centrifuge the ultrasonic mixture to obtain a first supernatant, which is the M2 macrophage lysate.

3. The preparation method according to claim 2, characterized in that, The mechanical extrusion includes the following steps: Extrude the M2 macrophage lysate through a polycarbonate membrane to obtain a second supernatant; Store the second supernatant at -80 °C.

4. The preparation method according to claim 3, characterized in that, The mechanical extrusion further includes the following steps: Take the second supernatant and perform centrifugation to remove larger vesicles to obtain a third supernatant; Take the third supernatant and pass it through a microporous filter membrane to obtain a microporous filter membrane filtrate.

5. The preparation method according to claim 4, characterized in that, The centrifugal resuspension includes the following steps: Ultracentrifuge the filtrate to obtain a centrifugate; Resuspend the centrifugate to obtain the M2 macrophage-derived cell vesicle mimetic.

6. The preparation method according to any one of claims 1 to 5, characterized in that, The method for inducing polarization of the M2 macrophages includes the following steps: Culture mouse macrophages in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. When the cell confluence reaches 70 - 80%, add 20 ng / ml IL-4 to the DMEM high-glucose medium and culture it in an incubator at 37 °C and 5% CO2 for 24 h to obtain the M2 macrophages; preferably, the mouse macrophages include mouse Raw264.7 macrophages or mouse J774A.1 macrophages; more preferably, the mouse macrophages are mouse J774A.1 macrophages.

7. A cell-derived vesicle mimetic of M2 macrophages, characterized in that, The M2 macrophage-derived cell vesicle mimetic is prepared by the method of any one of claims 1 - 8.

8. Use of an M2 macrophage-derived cell vesicle mimetic as claimed in claim 7 in the preparation of a raw material for reducing the foreign body reaction after implantation of a bio-3D printed body.

9. A modified bioink, characterized in that, The raw materials for preparing the modified bioink include the M2 macrophage-derived cell vesicle mimetic as claimed in claim 7 and the bioink; preferably, the raw materials for preparing the bioink include gelatin and sodium alginate; more preferably, the raw materials for preparing the bioink further include 5 parts by weight of gelatin, 3 parts by weight of sodium alginate, and 9 parts by weight of PBS buffer solution; still more preferably, the raw materials for preparing the modified bioink further include the PBS buffer solution for dissolving the M2 macrophage-derived cell vesicle mimetic, and 1 ml of the PBS buffer solution contains 10^ 10 M2 macrophage vesicle mimetics; still more preferably, the volume ratio of the PBS buffer solution containing 10^ 10 M2 macrophage vesicle mimetics to the bioink is 1:

1.

10. A biological 3D printed body, characterized in that, The raw materials for preparing the bio-3D printed body include the M2 macrophage-derived cell vesicle mimetic as claimed in claim 7, a bioink, and a crosslinking agent; preferably, the crosslinking agent is calcium chloride.

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