Method for preparing exosome by simulating atherosclerosis microenvironment

Abstract

The invention discloses a method for preparing an exosome by simulating an atherosclerosis microenvironment, and relates to the technical field of biology, and the method comprises the following steps: (1) separation and culture of umbilical cord mesenchymal stem cells; (2) establishing a microenvironment for simulating the coronary atherosclerotic heart disease, and culturing in combination with a three-dimensional bracket; (3) extracting an exosome; the three-dimensional scaffold is obtained by taking an acellular matrix, a temperature-sensitive material and polypyrrole nanoparticles as printing materials through low-temperature deposition 3D printing; a composite three-dimensional stent containing an acellular matrix, a temperature-sensitive material Pluronic F127 and polypyrrole nanoparticles is constructed by adopting a low-temperature deposition 3D printing technology, the coronary atherosclerotic heart disease pathological microenvironment is simulated, and the coronary atherosclerotic heart disease is obtained by combining the technical means of dynamic mechanical regulation and electrical stimulation. According to the application, the umbilical cord mesenchymal stem cells are successfully induced to secrete the exosome of which the secretion amount is remarkably increased and the functionality is enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for preparing exosomes by simulating the atherosclerotic microenvironment. Background Art

[0002] Coronary atherosclerotic heart disease (CAD) is one of the main causes of death globally, and its pathological basis is the formation and development of coronary atherosclerotic plaques. In recent years, exosomes derived from mesenchymal stem cells (MSCs) have shown great potential in the treatment of CAD due to their functions such as immunomodulation, promoting angiogenesis, and inhibiting apoptosis. However, the therapeutic effect of MSCs-derived exosomes obtained under conventional culture conditions on CAD is limited.

[0003] Studies have shown that there are pathological microenvironments such as chronic inflammation, oxidative stress, and endothelial dysfunction in the bodies of CAD patients, and these factors can affect the functions of MSCs and the secretion of exosomes. Therefore, culturing MSCs by simulating the CAD microenvironment may induce the secretion of exosomes with stronger anti-CAD activity.

[0004] For example, in the Chinese patent with the application number CN202210959119.6, a method for preparing extracellular exosomes based on culturing on a decellularized matrix three-dimensional scaffold is disclosed. This invention belongs to the field of tissue engineering and regenerative medicine. The method for preparing exosomes provided by this invention includes the following steps: inoculating mesenchymal stem cells on a decellularized matrix three-dimensional scaffold, culturing, and then separating to obtain exosomes. This invention uses a decellularized matrix as a raw material to construct a biomimetic natural cell growth microenvironment from two aspects of material composition and spatial structure, which can further optimize the biological functions of exosomes and fully exert the therapeutic potential of exosomes.

[0005] The above solution optimizes the biological functions of exosomes by constructing a biomimetic natural cell growth microenvironment. However, there are still defects such as static mechanical environment and single functional regulation. Summary of the Invention

[0006] By providing a method for preparing exosomes by simulating the atherosclerotic microenvironment in the embodiments of the present application, the problems in the prior art that the exosomes obtained under conventional culture conditions have limited therapeutic effect on coronary atherosclerotic heart disease, and the mechanical environment is static and the functional regulation is single during the exosome culture process are solved. By using a low-temperature deposition 3D printing technology to construct a composite three-dimensional scaffold containing a decellularized matrix, a thermosensitive material Pluronic F127, and polypyrrole nanoparticles, simulating the CAD pathological microenvironment, and combining technical means of dynamic mechanical regulation and electrical stimulation, the present application successfully induces umbilical cord mesenchymal stem cells to secrete exosomes with a significantly increased secretion amount and enhanced functionality.

[0007] An embodiment of the present application provides a method for preparing exosomes by simulating an atherosclerotic microenvironment, comprising the following steps:

[0008] (1) Isolation and culture of umbilical cord mesenchymal stem cells;

[0009] (2) Establishment of a microenvironment simulating coronary atherosclerotic heart disease and combined three-dimensional scaffold culture;

[0010] (3) Extraction of exosomes;

[0011] The three-dimensional scaffold uses acellular matrix, thermosensitive material and polypyrrole nanoparticles as printing materials and is obtained by low-temperature deposition 3D printing.

[0012] Further, the thermosensitive material is Pluronic F127, and the particle size of the polypyrrole nanoparticles is 50 - 100 nm.

[0013] Further, the mass percentage concentration of the acellular matrix is 6%; the mass percentage concentration of the thermosensitive material is 20%; the polypyrrole nanoparticles are dispersed in PBS buffer to form a polypyrrole suspension, and the mass percentage concentration of the polypyrrole suspension is 0.5%.

[0014] Further, the volume ratio of the acellular matrix, Pluronic F127 solution and polypyrrole suspension is 3:1:0.05.

[0015] Further, the microenvironment simulating coronary atherosclerotic heart disease in step (2) includes a culture medium containing the following components:

[0016] (a) Inflammatory factor: tumor necrosis factor-α (TNF-α), concentration 10 ng / mL;

[0017] (b) Oxidative stress inducer: hydrogen peroxide (H2O2), concentration 100 μM;

[0018] (c) Endothelial dysfunction inducer: oxidized low-density lipoprotein (ox-LDL), concentration 50 μg / mL.

[0019] Further, the exosome extraction method in step (3) is ultracentrifugation or kit method.

[0020] Further, the three-dimensional scaffold is also subjected to secondary addition. Specifically, the secondary addition is carried out by in-situ injection of the sol at 48 h in the middle stage of cell proliferation.

[0021] Further, the sol added secondly is composed of the following components: Pluronic F127 solution, polypyrrole suspension, and acellular matrix;

[0022] Among them, the mass percentage concentration of the Pluronic F127 solution is 10%;

[0023] The mass percentage concentration of the polypyrrole suspension is 0.2%;

[0024] The mass percentage concentration of the acellular matrix is 2%;

[0025] The volume ratio of the Pluronic F127 solution, acellular matrix to polypyrrole suspension is 5:1:0.02.

[0026] Further, the three-dimensional scaffold is arranged on an elastic culture plate which can be stretched. When adding secondly, the culture plate is stretched along the uniaxial direction to form a stretching gap, and the width of the stretching gap is 100 - 150 μm.

[0027] Further, 75% - 85% of the volume of the sol added secondly is injected into the stretched gap, and the remaining part is injected in situ.

[0028] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0029] Firstly, by using the simulation of the CAD microenvironment combined with three-dimensional scaffold culture to prepare exosomes, the problems of low exosome secretion efficiency and single function in traditional two-dimensional culture are solved. By simulating the in-vivo environment, the exosome secretion amount and anti-CAD activity are improved;

[0030] Secondly, by introducing the thermosensitive material Pluronic F127 and combining with the temperature regulation technology, the problems of the limitation of the static mechanical environment of the traditional scaffold on cell function and exosome secretion efficiency are solved. By utilizing the temperature responsiveness of Pluronic F127, the porosity and stiffness of the scaffold are dynamically regulated to simulate the in-vivo tissue mechanical environment, promote cell growth and function exertion, and improve exosome secretion and functionality;

[0031] Thirdly, by introducing polypyrrole and cooperating with the thermosensitive material and acellular matrix, the problems of single function of the traditional scaffold, low exosome yield and poor distribution, etc. are solved. By means of the conductivity of polypyrrole, the dynamic mechanical regulation of the thermosensitive material and the biological activity of the acellular matrix, multiple signal pathways are activated, the exosome yield and functional activity are enhanced, its distribution is optimized, and exosome secretion and functionality are improved.

[0032] Fourthly, the secondary addition is based on the principle of dynamic changes in the spatial requirements during cell proliferation. The initially constructed three-dimensional structure has limited pores, which will limit the subsequent proliferation space of cells. Through secondary addition, new scaffold materials are introduced in the mid-stage of cell proliferation. Utilizing the characteristics of thermosensitive materials, the pores of the initial scaffold are expanded at low temperature, facilitating the penetration and filling of the gaps by the sol of the secondary scaffold. After gelation, a new structure is formed to meet the spatial requirements of continuous cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the culture plate of the fourth embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs; the terms used in the specification of this invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0035] Example 1: A method for preparing exosomes by simulating the atherosclerotic microenvironment, comprising the following steps:

[0036] (1) Isolation and culture of umbilical cord mesenchymal stem cells: Umbilical cord mesenchymal stem cells are isolated from Wharton's jelly of umbilical cord by tissue explant adherence method or enzymatic digestion method, and cultured using α-MEM medium containing 10% fetal bovine serum;

[0037] (2) Establishment of a microenvironment simulating coronary atherosclerotic heart disease (CAD): The umbilical cord mesenchymal stem cells are inoculated on a culture plate, and a decellularized matrix three-dimensional scaffold is set on the culture plate. When the cell confluence reaches 80%-90%, the following components are added to the α-MEM medium containing 10% fetal bovine serum:

[0038] (a) Inflammatory factor: Tumor necrosis factor-α (TNF-α), with a concentration of 10 ng / mL;

[0039] (b) Oxidative stress inducer: Hydrogen peroxide (H2O2), with a concentration of 100 μM;

[0040] (c) Endothelial dysfunction inducer: Oxidized low-density lipoprotein (ox-LDL), with a concentration of 50 μg / mL;

[0041] The decellularized matrix three-dimensional scaffold is obtained by low-temperature deposition 3D printing using the decellularized matrix as the printing material. The specific steps are as follows:

[0042] Place the acellular matrix in the cartridge of a 3D printer and perform extrusion printing by means of piston extrusion. The extruded acellular cartilage matrix will rapidly solidify and form a scaffold on a freezing platform;

[0043] After printing is completed, place the scaffold in a freeze dryer for sublimation drying;

[0044] Place the scaffold after sublimation drying in a crosslinking agent for crosslinking;

[0045] The crosslinking agent is a 95% ethanol solution by volume containing ethyl-dimethylamine-propyl carbodiimide and n-hydroxysuccinimide, wherein the concentration of ethyl-dimethylamine-propyl carbodiimide is 50 mmol / L; the concentration of n-hydroxysuccinimide is 20 mmol / L;

[0046] After crosslinking, soak in PBS buffer for 2 h to remove the excess crosslinking agent; after rinsing with triple-distilled water, perform freeze-drying sublimation treatment again to obtain the acellular matrix three-dimensional scaffold;

[0047] The acellular matrix is prepared by a method including the following steps:

[0048] Perform decellularization treatment on animal tissue to obtain an acellular matrix homogenate; the animal tissue is cartilage tissue;

[0049] Add an acetic acid solution to the acellular matrix homogenate, stir, and then allow part of the acetic acid to volatilize to obtain the acellular matrix;

[0050] The acetic acid solution is continuously added dropwise to the acellular matrix homogenate until the acellular matrix homogenate changes from milky white to transparent;

[0051] The temperature of the stirring is 0-8 °C and the time is 12-72 h;

[0052] The method for acetic acid volatilization is to volatilize acetic acid by magnetic stirring under an exhaust fan;

[0053] The mass percentage concentration of the acellular matrix is 6%;

[0054] (3) Extraction of exosomes: Collect cell culture supernatant and extract exosomes by ultracentrifugation or kit method;

[0055] The specific implementation of the method for culturing umbilical cord mesenchymal stem cell exosomes in a simulated CAD microenvironment is as follows:

[0056] (I) Isolation and culture of umbilical cord mesenchymal stem cells

[0057] 1. Take the umbilical cord of a healthy full-term newborn and cut out Wharton's jelly tissue under sterile conditions;

[0058] 2. Cut the tissue blocks into pieces and isolate umbilical cord mesenchymal stem cells by tissue block adherence method or enzyme digestion method;

[0059] 3. Inoculate the isolated umbilical cord mesenchymal stem cells into a culture flask and culture them in α-MEM medium containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2;

[0060] 4. Change the medium every 2 - 3 days and passage when the cell confluence reaches 80% - 90%;

[0061] (II) Establishment of a simulated CAD microenvironment

[0062] 1. Inoculate the third-generation umbilical cord mesenchymal stem cells into a culture plate at a density of 1×10 5 cells / well, and set a decellularized matrix three-dimensional scaffold on the culture plate;

[0063] 2. When the cell confluence reaches 80% - 90%, add the following components to the α-MEM medium containing 10% fetal bovine serum:

[0064] (a) Inflammatory factor: Tumor necrosis factor-α (TNF-α), with a concentration of 10 ng / mL;

[0065] (b) Oxidative stress inducer: Hydrogen peroxide (H2O2), with a concentration of 100 μM;

[0066] (c) Endothelial dysfunction inducer: Oxidized low-density lipoprotein (ox-LDL), with a concentration of 50 μg / mL;

[0067] 3. Continue to culture for 24 - 72 hours;

[0068] (III) Extraction and identification of exosomes

[0069] 1. Collect the cell culture supernatant, centrifuge at 300 g for 10 minutes to remove cell debris;

[0070] 2. Transfer the supernatant to an ultracentrifuge tube and centrifuge at 100,000 g for 70 minutes, discard the supernatant;

[0071] 3. Resuspend the precipitate with PBS and centrifuge at 100,000 g for 70 minutes, discard the supernatant to obtain exosomes;

[0072] The control group is exosomes cultured under conventional culture conditions;

[0073] Performance detection: 1. Use a nanoparticle tracking analyzer to detect the exosome secretion amount; the results show that compared with the control group, the exosome secretion amount of umbilical cord mesenchymal stem cells in the simulated CAD microenvironment culture group is significantly increased; the detection results are shown in Table 1 below:

[0074] Group Exosome concentration (particles / mL) Control group (1.2±0.3)×1011 Example 1 (3.5±0.5)×1011

[0075] Table 1

[0076] 2. The expression levels of exosome marker proteins such as TSG101, CD63, and Alix in exosomes were detected by Western blot; the results showed that, compared with the control group, the expression levels of TSG101, CD63, and Alix proteins in exosomes of umbilical cord mesenchymal stem cells cultured in the simulated CAD microenvironment were significantly up-regulated; the detection results are shown in Table 2 below:

[0077] Protein Group Relative expression level (normalized) TSG101 Control group 1.0±0.1 Example 1 1.7±0.2 CD63 Control group 1.0±0.1 Example 1 2.0±0.3 Alix Control group 1.0±0.1 Example 1 1.8±0.2

[0078] Table 2

[0079] 3. The protective effect of exosomes on ox-LDL-induced vascular endothelial cell injury was detected by the CCK-8 method; the results showed that, compared with the control group, exosomes of umbilical cord mesenchymal stem cells cultured in the simulated CAD microenvironment could significantly increase the survival rate of ox-LDL-induced vascular endothelial cells and inhibit cell apoptosis;

[0080] 1) Human umbilical vein endothelial cells (HUVECs) were seeded in 96-well plates and cultured until confluence;

[0081] 2) 50 μg / mL ox-LDL was added to induce HUVECs injury;

[0082] 3) Exosomes of the control group and Example 1 group (10 8 particles / mL) were added respectively and co-cultured for 24 hours;

[0083] 4) The cell survival rate was detected by the CCK-8 method, and the cell apoptosis rate was detected by Annexin V-FITC / PI double staining; the detection results are shown in Table 3 below:

[0084] Group Cell survival rate (%) Cell apoptosis rate (%) Control group 100±5 5.0±1.0 ox-LDL group 62.5±6.8 32.5±3.5 ox-LDL + Control group exosomes 70.3±5.5 25.0±2.8 ox-LDL + Example 1 exosomes 85.2±4.2 12.8±1.5

[0085] Table 3

[0086] 4. Flow cytometry was used to detect the effect of exosomes of umbilical cord mesenchymal stem cells cultured in the simulated CAD microenvironment on ox-LDL-induced macrophage polarization. The results showed that, compared with the control group, exosomes of umbilical cord mesenchymal stem cells cultured in the simulated CAD microenvironment could significantly inhibit the polarization of macrophages induced by ox-LDL to the M1 type and promote their polarization to the M2 type.

[0087] 1) THP-1 cells were induced to differentiate into macrophages;

[0088] 2) 50 μg / mL ox-LDL was added to induce macrophage polarization;

[0089] 3) Add exosomes of umbilical cord mesenchymal stem cells (10 8 particles / mL) to the control group and the group cultured in a simulated CAD microenvironment respectively, and co-culture for 24 hours;

[0090] 4) Use flow cytometry to detect the expression levels of macrophage surface markers CD86 (M1 type) and CD206 (M2 type); the detection results are shown in Table 4 below:

[0091]

[0092] Table 4

[0093] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0094] Exosomes are prepared by culturing in a simulated CAD microenvironment combined with a three-dimensional scaffold, which solves the problems of low exosome secretion efficiency and single function in traditional two-dimensional culture. By simulating the in-vivo environment, the exosome secretion amount and anti-CAD activity are improved;

[0095] The three-dimensional scaffold is constructed based on decellularized matrix and forms a biomimetic structure through low-temperature deposition 3D printing technology. The decellularized matrix retains the biochemical components and physical structure of the natural extracellular matrix, can simulate the three-dimensional microenvironment of cells in vivo. The pore structure and mechanical properties of the three-dimensional scaffold can promote cell-matrix interaction and activate the mechanical signal pathway, thereby regulating cell proliferation, differentiation and paracrine function; the three-dimensional scaffold simulates the in-vivo microenvironment to break through the limitations of traditional two-dimensional culture, enhances the interaction between cells through the three-dimensional spatial structure, and promotes the physiological activity of umbilical cord mesenchymal stem cells; the mechanical stimulation and biochemical signals provided by the three-dimensional scaffold can enhance the exosome secretion efficiency and content loading; the natural extracellular matrix components in the decellularized matrix endow exosomes with specific therapeutic functions by regulating cell signal pathways;

[0096] The pathological microenvironment of CAD is characterized by chronic inflammation (TNF-α), oxidative stress (H2O2) and endothelial dysfunction (ox-LDL). By adding these three factors to act on exosomes, TNF-α activates the NF-κB pathway and induces umbilical cord mesenchymal stem cells to secrete anti-inflammatory factors; H2O2 triggers an oxidative stress response and up-regulates the expression of antioxidant enzymes and stress-related proteins; ox-LDL simulates lipid deposition in atherosclerosis and promotes umbilical cord mesenchymal stem cells to secrete angiogenic factors; by simulating the pathological signals of CAD, umbilical cord mesenchymal stem cells are induced to secrete targeted repair exosomes; anti-inflammatory, antioxidant and angiogenic factors are enriched in exosomes, enhancing their regulatory ability on endothelial injury and macrophage polarization;

[0097] The mechanical support of the three-dimensional scaffold promotes the adhesion and spreading of umbilical cord mesenchymal stem cells, enhances their sensitivity to microenvironmental stimuli. At the same time, CAD-related factors regulate the biosynthesis and release of exosomes synergistically with the mechanical signaling pathway by activating cell surface receptors; the ECM components of the three-dimensional scaffold promote the enrichment of miRNAs in exosomes through integrin signaling, while CAD microenvironmental stimuli regulate post-translational protein modification through epigenetic modification, jointly optimizing the functional molecular profile of exosomes;

[0098] In this example, umbilical cord mesenchymal stem cells were cultured by simulating the CAD microenvironment, and exosomes with stronger anti-CAD activity were successfully induced to secrete. Compared with the umbilical cord mesenchymal stem cell exosomes obtained under conventional culture conditions, the exosomes obtained in the present invention have the following advantages: increased secretion: culturing by simulating the CAD microenvironment can significantly increase the secretion of umbilical cord mesenchymal stem cell exosomes; protein expression change: culturing by simulating the CAD microenvironment can up-regulate the expression levels of proteins such as TSG101, CD63, and Alix in umbilical cord mesenchymal stem cell exosomes; enhanced anti-CAD activity: the exosomes of umbilical cord mesenchymal stem cells in the group cultured by simulating the CAD microenvironment have a stronger protective effect on ox-LDL-induced vascular endothelial cell injury, and can inhibit the polarization of macrophages into the M1 type and promote their polarization into the M2 type;

[0099] This example first proposed a method for culturing umbilical cord mesenchymal stem cell exosomes by simulating the CAD microenvironment, providing a new idea for obtaining exosomes with stronger anti-CAD activity. Through experiments, it was verified that culturing by simulating the CAD microenvironment can significantly increase the secretion of umbilical cord mesenchymal stem cell exosomes and up-regulate the expression levels of proteins such as TSG101, CD63, and Alix. It was confirmed that the exosomes of umbilical cord mesenchymal stem cells in the group cultured by simulating the CAD microenvironment have a stronger protective effect on ox-LDL-induced vascular endothelial cell injury, and can inhibit the polarization of macrophages into the M1 type and promote their polarization into the M2 type; the method of the present invention is simple to operate, easy to implement, and has good application prospects.

[0100] Example 2: In the above Example 1, a three-dimensional scaffold combined with a simulated CAD microenvironment was used to culture and prepare exosomes, which solved the problems of low exosome secretion efficiency and single function in traditional two-dimensional culture. By simulating the in vivo environment, the exosome secretion amount and anti-CAD activity were improved. On the basis of Example 1, further improvements were made to further increase the exosome secretion.

[0101] The printing material includes a temperature-sensitive material, and the temperature-sensitive material is Pluronic F127; the mass percentage concentration of the decellularized matrix is 6%, the mass percentage concentration of Pluronic F127 is 20%, and the volume ratio of the decellularized matrix to the Pluronic F127 solution is 3:1;

[0102] Mix the decellularized matrix with Pluronic F127 solution, stir well until homogeneous to form a composite matrix material as the printing material;

[0103] Use the composite matrix material as the printing material and obtain a composite three-dimensional scaffold through low-temperature deposition 3D printing;

[0104] In step (ii), seed the third-generation umbilical cord mesenchymal stem cells at a density of 1×10 5 cells / well on the composite three-dimensional scaffold; maintain at 4°C in the initial stage (0 - 2 hours);

[0105] Cell seeding period (2 - 24 hours): Set the temperature to increase gradually from 4°C to 37°C at a heating rate of 0.5°C / min to promote uniform cell distribution;

[0106] Cell proliferation period (24 - 72 hours): Maintain at 37°C and add CAD microenvironment stimulating factors (TNF-α 10 ng / mL, H2O2 100 μM, ox-LDL 50 μg / mL);

[0107] Based on the experiment in Example 1, the difference between this experiment and the experiment in Example 1 is that the printing material also includes Pluronic F127; in this experiment, the mass percentage concentration of the decellularized matrix is 6%, the mass percentage concentration of Pluronic F127 is 20%, and the volume ratio of the decellularized matrix to the Pluronic F127 solution is 3:1.

[0108] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0109] By introducing the thermosensitive material Pluronic F127 and combining temperature regulation technology, the problem that the static mechanical environment of traditional scaffolds restricts cell function and exosome secretion efficiency is solved. Utilizing the temperature responsiveness of Pluronic F127, dynamically regulate the porosity and stiffness of the scaffold, simulate the in vivo tissue mechanical environment, promote cell growth and function, and improve exosome secretion and functionality;

[0110] As a triblock copolymer (PEO-PPO-PEO), the temperature responsiveness of Pluronic F127 is based on the micellization process driven by hydrophobic interactions. At low temperatures, i.e., below the critical micelle temperature, the material is in a liquid sol state, with molecular chains freely dispersed, forming a highly fluid solution with high porosity and large pore sizes. The high-porosity state is conducive to the uniform distribution of cells within the scaffold and mass exchange. When the temperature rises to the critical micelle temperature and above, the hydrophobic PPO chain segments aggregate to form the micelle core, and the hydrophilic PEO chain segments wrap around the outer layer. The micelles form a three-dimensional crosslinked network through physical entanglement, and the material transforms into a gel state, with reduced porosity and a significantly increased storage modulus. The change in the material's stiffness can simulate the mechanical environment of natural tissues, providing cells with a growth condition closer to that in vivo.

[0111] The thermosensitive property of Pluronic F127 combines with bioactive components in the decellularized matrix, such as collagen and glycoproteins, to form a dynamic composite three-dimensional scaffold. At the low-temperature sol state, the high porosity facilitates the uniform distribution of cells. At the high-temperature gel state, the scaffold stiffness is enhanced, simulating the mechanical environment of natural tissues and promoting cell growth and function. The extracellular matrix components in the decellularized matrix bind to the Pluronic F127 molecular chains through hydrogen bonds and hydrophobic interactions. This binding improves the stability of the scaffold and also retains the bioactive signals in the decellularized matrix, providing necessary biochemical stimuli for cells.

[0112] By regulating the temperature at different stages, the dynamic changes in the physical properties of the scaffold are achieved, providing a suitable growth environment for cells. The low-temperature sol state can promote cell seeding and initial distribution, while the high-temperature gel state simulates the tissue stiffness in vivo, enhancing the interaction between cells and the matrix, promoting cell growth and function, and improving the secretion efficiency of exosomes. The scaffold states at different stages can affect the loading of exosome contents, making it spatiotemporally specific and enhancing the functional activity of exosomes. The early high porosity promotes the enrichment of pro-angiogenic factors, and the late high stiffness induces the upregulation of anti-inflammatory factor expression, enabling exosomes to perform different functions at different stages, such as promoting angiogenesis in the early stage and anti-inflammatory and immunomodulatory effects in the late stage.

[0113] The thermosensitive material regulates the physical properties of the scaffold, namely porosity and stiffness, through phase change, adapting to the cell growth requirements in stages, breaking through the limitations of static scaffolds, and providing a more suitable growth environment for cells. The decellularized matrix provides bioactive signals, and Pluronic F127 provides dynamic mechanical support. The two work together to activate the mechanical-biochemical dual signaling pathway, optimizing the secretion and function of exosomes, and significantly improving both the yield and functional activity of exosomes.

[0114] Example 3: In Example 2 above, by introducing the thermosensitive material Pluronic F127 and combining temperature regulation technology, the problem that the static mechanical environment of traditional scaffolds restricts cell function and exosome secretion efficiency is solved. Utilizing the temperature responsiveness of Pluronic F127, the porosity and stiffness of the scaffold are dynamically regulated to simulate the in vivo tissue mechanical environment, promoting cell growth and function, and improving exosome secretion and functionality. To further improve exosome secretion, it is further improved on the basis of Example 2.

[0115] The printing material further includes polypyrrole nanoparticles, and the particle size of the polypyrrole nanoparticles is 50 - 100 nm;

[0116] Disperse the polypyrrole nanoparticles in PBS buffer to form a polypyrrole suspension, and the mass percentage concentration of the polypyrrole suspension is 0.5%;

[0117] The volume ratio of the decellularized matrix, Pluronic F127 solution, and polypyrrole suspension is 3:1:0.05;

[0118] The preparation of the printing material is specifically as follows:

[0119] Mix the decellularized matrix with the Pluronic F127 solution and stir well until homogeneous;

[0120] Slowly add the polypyrrole suspension, continue stirring, and then perform ultrasonic treatment to disperse the nanoparticles to form a composite conductive matrix material as the printing material;

[0121] Use the composite conductive matrix material as the printing material and obtain a composite conductive three-dimensional scaffold by low-temperature deposition 3D printing;

[0122] After inoculating cells on the composite conductive three-dimensional scaffold in step (2), gradually heat up to 37°C. During the cell proliferation period, apply a direct current pulsed electric field with an electric field strength of 50 mV / mm and a frequency of 1 Hz for 1 h per day.

[0123] Based on the experiment in Example 2, the difference between this experiment and the experiment in Example 2 is that the printing material further includes polypyrrole nanoparticles; in this experiment, the mass concentration of the polypyrrole suspension is 0.5%, and the volume ratio of the decellularized matrix, Pluronic F127 solution, and polypyrrole suspension is 3:1:0.05;

[0124] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0125] By introducing polypyrrole and synergizing with temperature-sensitive materials and decellularized matrix, the problems of single function, low exosome yield and poor distribution of traditional scaffolds are solved. By leveraging the conductivity of polypyrrole, the dynamic mechanical regulation of temperature-sensitive materials and the biological activity of decellularized matrix, multiple signaling pathways are activated, the exosome yield and functional activity are enhanced, its distribution is optimized, and the exosome secretion and functionality are improved.

[0126] Polypyrrole is a conductive polymer material whose conductivity can simulate the in vivo electrical microenvironment. When an electrical stimulus is applied, polypyrrole can activate cell membrane ion channels, such as calcium ion channels, thereby promoting ion influx and then regulating intracellular signaling pathways. Polypyrrole can enhance cell-cell communication, promote cell metabolic activity and paracrine function through electrical signal transmission. At the same time, it can regulate mitochondrial function, induce exosomes to enrich energy metabolism-related proteins such as ATP synthase, and enhance the energy support ability of exosomes for damaged tissues. Based on these characteristics, it is introduced into the temperature-sensitive dynamic scaffold system to achieve the regulation of cell behavior and exosome function.

[0127] The conductivity of polypyrrole can activate specific signaling pathways, upregulate the expression of related proteins, promote the fusion of exosome vesicles with the plasma membrane, increase exosome yield, and at the same time increase the expression of exosome marker proteins, enhance vesicle integrity, and improve exosome secretion efficiency. Electrical stimulation induces exosomes to enrich energy metabolism-related proteins, increases ATP content, and enhances the energy supply to ischemic tissues. It can also promote the expression of angiogenesis-promoting factors and anti-inflammatory factors in exosomes, comprehensively enhancing the functional activity of exosomes.

[0128] At 4 °C in the sol state, the printing material has high porosity and low conductivity, and polypyrrole nanoparticles are evenly dispersed in it. At this time, the high porosity promotes the uniform distribution of cells. At 37 °C in the gel state, the high stiffness of the printing material is combined with the high conductivity of polypyrrole to enhance the efficiency of electrical signal transmission and activate the mechanosensitive pathway. The decellularized matrix in the printing material provides biochemical signals, and the extracellular matrix components it contains can promote cell proliferation. The temperature-sensitive material provides mechanical signals by regulating the mechanical properties of the scaffold to promote cell adhesion. Polypyrrole provides electrical signals to activate intracellular signaling pathways and enhance exosome secretion. The three form a triple-signal synergistic effect. Through the triple-signal synergistic effect, multiple pathways are activated, the exosome secretion and functional molecule loading are optimized, and the exosome yield and functional activity are comprehensively improved.

[0129] The high porosity of the scaffold synergizes with the conductive network formed by polypyrrole. The high porosity ensures the uniform distribution of cells and exosomes, and the conductive network guides the directional migration of exosomes to the damaged area through electrical stimulation. The conductivity of polypyrrole enhances the penetration ability of exosomes into tissues, making it easier to penetrate dense tissues and improving the targeted delivery efficiency. The enriched energy metabolism proteins extend the functional activity time of exosomes in the ischemic environment, enabling them to play a more persistent role at the damaged site.

[0130] Example 4: In Example 3 above, by introducing polypyrrole and synergizing with the temperature-sensitive material and decellularized matrix, the problems of single function of traditional scaffolds, low exosome production and poor distribution are solved. By leveraging the conductivity of polypyrrole, the dynamic mechanical regulation of the temperature-sensitive material and the biological activity of the decellularized matrix, multiple signaling pathways are activated, the exosome production and functional activity are enhanced, its distribution is optimized, and the exosome secretion and functionality are improved. To further improve the exosome secretion, it is further improved on the basis of Example 3.

[0131] The three-dimensional scaffold is subjected to a secondary addition. Specifically, the secondary addition is carried out by in-situ injecting the sol at the mid-cell proliferation stage (48 hours).

[0132] The sol for secondary addition consists of the following components: Pluronic F127 solution, polypyrrole suspension, and decellularized matrix.

[0133] Among them, the mass percentage concentration of the Pluronic F127 solution is 10%.

[0134] The mass percentage concentration of the polypyrrole suspension is 0.2%.

[0135] The mass percentage concentration of the decellularized matrix is 2%.

[0136] The volume ratio of the Pluronic F127 solution, the decellularized matrix to the polypyrrole suspension is 5:1:0.02.

[0137] The steps of the secondary addition are specifically as follows:

[0138] A1. Cool down to 4°C at the mid-cell proliferation stage (48 hours) to make the primary scaffold return to the sol state and expand the pores.

[0139] A2. In-situ inject the secondary scaffold sol (10 μL / well), and the sol penetrates into the gaps of the primary scaffold at low temperature.

[0140] A3. Heat up to 37°C to trigger the gelation of the secondary sol, form a hierarchical pore structure, maintain at 37°C, and continue the culture.

[0141] An experiment is carried out on the basis of Example 3. As Experiment 1 of Example 4, the difference between this experiment and the experiment of Example 3 is that this example conducts a secondary addition.

[0142] As Figure 1 shown, the culture plate is an elastic culture plate that can be stretched. When performing the secondary addition, the culture plate is stretched along the uniaxial direction to form a stretching gap, and the width of the stretching gap is 100 - 150 μm.

[0143] 75%-85% of the volume of the second addition of the sol is injected into the stretched gap, and the remaining part is injected in situ;

[0144] Based on Experiment 1, Experiment 2 of Example 4 was carried out. The difference between this experiment and Experiment 1 is that 80% of the volume of the second addition of the sol is injected into the stretched gap, and the remaining part is injected in situ.

[0145] Performance detection was carried out to detect the experimental results of Examples 2 to 4. The experimental results are shown in Table 5 below:

[0146] Group Cell proliferation rate (OD value) Exosome concentration Example 1 1.0 <![CDATA[3.5×10 11 > Example 2 1.4 <![CDATA[6.8×10 11 > Example 3 1.6 <![CDATA[9.5×10 11 > Example 4 (Experiment 1) 2.0 <![CDATA[1.2×10 12 > Example 4 (Experiment 2) 2.5 <![CDATA[1.5×10 12 >

[0147] Table 5

[0148] The technical solutions in the embodiments of the present application described above have at least the following technical effects or advantages:

[0149] Based on the principle of the dynamic change of the spatial requirements during cell proliferation, the pores of the initially constructed three-dimensional structure are limited, which will limit the subsequent proliferation space of cells. Through the second addition, new scaffold materials are introduced in the middle stage of cell proliferation. Using the characteristics of the thermosensitive material, the pores of the initial scaffold are expanded at low temperature, facilitating the penetration and filling of the gap by the second scaffold sol. After gelation, a new structure is formed to meet the spatial requirements of continuous cell proliferation;

[0150] The flexible and highly porous scaffold formed by the second addition expands the cell growth space, optimizes the cell proliferation space, avoids overcrowding of cells, and is conducive to the continuous proliferation of cells; the hierarchical pore structure promotes the interaction between cells and the matrix, thereby enhancing the secretion of exosomes and improving the functional activity of exosomes at the same time;

[0151] The high stiffness of the initial scaffold provides mechanical support to guide cell adhesion and initial proliferation; the flexible and highly porous second scaffold expands the growth space. The two form a dynamic pore hierarchy coordination, adapting to the mechanical and spatial requirements of different stages of cell proliferation; the conductive polymer polypyrrole in the second scaffold and the polypyrrole in the initial scaffold form a continuous conductive network, ensuring the stability of the electric stimulation transmission efficiency, enabling the seamless integration of conductivity and biological activity during the second addition, and continuously playing the role of promoting cell metabolic activity and paracrine function; the second sol has high fluidity at low temperature and can seamlessly fill the gaps in the initial scaffold; under the gel state, the second scaffold and the original structure are physically cross-linked through the thermosensitive material to form an overall mechanical adaptability, ensuring the stability and functionality of the scaffold structure; the concentration of the thermosensitive material added in the second addition is relatively low, forming a gradient mechanics with the high concentration added in the first addition to avoid interfacial stress concentration;

[0152] The hierarchical pore structure provides a more suitable growth environment for cells, promotes the interaction between cells and the matrix, and thus enhances the secretion of exosomes; exosomes are generated in a more optimized environment, and their functional activity is improved. By optimizing the cell growth space and maintaining the conductive network, while increasing the yield of exosomes, their quality is also guaranteed;

[0153] During the proliferation process, cells require more space. The initially constructed three-dimensional scaffold has limited pores and cannot meet the needs of continuous cell proliferation. By stretching the elastic culture plate, additional space can be created without destroying the original scaffold structure, providing more growth areas for cells; the secondarily added sol is a thermosensitive-conductive composite sol, which has good fluidity at low temperatures and can penetrate into the gaps formed by stretching. Using this property, most of the sol is injected into the stretching gaps, enabling the sol to better fill the newly formed space, and at the same time in-situ supplementing the remaining sol to ensure good integration of the new and old scaffold interfaces; through stretching and secondarily adding sol, a hierarchical pore structure is formed. Areas with different porosities can adapt to the needs of different stages of cell proliferation. At the same time, the continuity of the conductive network and bioactivity ensures the stable transmission of electrical and biological signals, promoting the normal metabolism and function of cells;

[0154] Stretching the culture plate forms stretching gaps. After adding sol in stages, a hierarchical pore structure is formed. The porosity of the stretching area increases, providing a broader growth space for cells; the non-stretching area maintains a relatively dense structure, providing certain mechanical support, making the entire scaffold structure more reasonable and better adapting to the mechanical and space requirements during cell proliferation. The optimized scaffold structure provides a more suitable growth environment for cells, promotes cell proliferation and migration. At the same time, the continuity of the conductive network ensures the effective transmission of electrical stimulation, activates the metabolic activity and paracrine function of cells, and promotes the secretion and functional improvement of exosomes; by adding sol in stages, the interface between the new and old scaffolds can be better fused, and the interface bonding strength is further enhanced, ensuring the stability and integrity of the scaffold structure;

[0155] By stretching the elastic culture plate, the position and amount of the secondarily added sol can be more accurately controlled, enabling the sol to better fill the areas that need to be optimized, further optimizing the porosity and mechanical properties of the three-dimensional structure, and providing a more suitable growth environment for cells; the optimized three-dimensional structure can more effectively expand the cell growth space, avoid overcrowding of cells, and thus improve the cell proliferation efficiency; the more optimized cell growth environment can increase the secretion amount and functional activity of exosomes, and is applicable to scenarios requiring large-volume tissue regeneration.

[0156] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing exosomes by simulating the atherosclerotic microenvironment, characterized in that, It includes the following steps: (1) Isolation and culture of umbilical cord mesenchymal stem cells; (2) Establishment of a microenvironment simulating coronary atherosclerotic heart disease and combined three-dimensional scaffold culture; (3) Extraction of exosomes; The three-dimensional scaffold uses decellularized matrix, thermosensitive material and polypyrrole nanoparticles as printing materials and is obtained by low-temperature deposition 3D printing.

2. The method for preparing exosomes by simulating an atherosclerotic microenvironment according to claim 1, wherein The thermosensitive material is Pluronic F127, and the particle size of the polypyrrole nanoparticles is 50-100 nm.

3. The method for preparing exosomes by simulating an atherosclerotic microenvironment according to claim 1, wherein, The mass percentage concentration of the decellularized matrix is 6%; the mass percentage concentration of the thermosensitive material is 20%; the polypyrrole nanoparticles are dispersed in PBS buffer to form a polypyrrole suspension, and the mass percentage concentration of the polypyrrole suspension is 0.5%.

4. The method for preparing exosomes by simulating an atherosclerotic microenvironment according to claim 3, wherein, The volume ratio of the decellularized matrix, Pluronic F127 solution and polypyrrole suspension is 3:1:0.

05.

5. The method for preparing exosomes by simulating an atherosclerotic microenvironment according to claim 1, wherein The microenvironment simulating coronary atherosclerotic heart disease described in step (2) includes a culture medium containing the following components: (a) Inflammatory factor: Tumor necrosis factor-α (TNF-α), concentration 10 ng / mL; (b) Oxidative stress inducer: Hydrogen peroxide (H2O2), concentration 100 μM; (c) Endothelial dysfunction inducer: Oxidized low-density lipoprotein (ox-LDL), concentration 50 μg / mL.

6. The method for preparing exosomes that mimic the atherosclerotic microenvironment according to claim 1, wherein The exosome extraction method described in step (3) is the ultracentrifugation method or the kit method.

7. The method for preparing exosomes by simulating an atherosclerotic microenvironment according to claim 1, wherein, The three-dimensional scaffold also undergoes a secondary addition. Specifically, at 48 h in the middle stage of cell proliferation, the secondary addition is carried out by in-situ injection of the sol.

8. The method for preparing exosomes by simulating an atherosclerotic microenvironment according to claim 7, wherein The sol for secondary addition is composed of the following components: Pluronic F127 solution, polypyrrole suspension, decellularized matrix; Among them, the mass percentage concentration of the Pluronic F127 solution is 10%; The mass percentage concentration of the polypyrrole suspension is 0.2%; The mass percentage concentration of the decellularized matrix is 2%; The volume ratio of the Pluronic F127 solution, decellularized matrix and polypyrrole suspension is 5:1:0.

02.

9. The method for preparing exosomes by simulating an atherosclerotic microenvironment according to claim 7, wherein, The three-dimensional scaffold is set on an elastic culture plate. The elastic culture plate can be stretched. During the secondary addition, the culture plate is stretched along the uniaxial direction to form a stretching gap, and the width of the stretching gap is 100-150 μm.

10. The method for preparing exosomes by simulating an atherosclerotic microenvironment according to claim 9, wherein 75%-85% of the volume of the sol for secondary addition is injected into the stretched gap, and the remaining part is injected in-situ.

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