Method for promoting exosome secretion through magnetic stimulation and application thereof

Abstract

The invention provides a method for promoting exosome secretion through magnetic stimulation and application of the method, and belongs to the technical field of biological medicine. The method comprises the following steps: S1, preparing magnetic hydrogel; s2, inoculating cells on the magnetic hydrogel; s3, dynamically loading a magnetic field to realize remote magnetic stimulation of the cells; s4, collecting a cell supernatant; and S5, extracting and purifying the exosome. Magnetic force stimulation is applied remotely through a magnetic field, the size of magnetic nanoparticles is extremely small, the generated movement and deformation can be positioned to the subcellular scale, mechanical stimulation can be uniformly and accurately applied to stem cells, the exosome secretion efficiency can be greatly improved, and the exosome secretion efficiency can be improved. Compared with cells cultured on a conventional cell culture plate, the cells on the magnetic hydrogel loaded with the dynamic magnetic field have the advantages that the quantity of generated exosomes is obviously increased and reaches 10 times or more. According to the method, mechanical stimulation on the cells is realized under the non-contact condition, and the method can be applied to various complex cell culture environments.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for promoting exosome secretion through magnetic stimulation and an application thereof. Background Art

[0002] Exosomes are extracellular vesicles that are currently considered to be potential carriers for detecting biomarkers and delivering physiological signals. Depending on the cell of origin, they can contain many components of the cell, including DNA, RNA, lipids, metabolites, and cytoplasmic and cell surface proteins. The proteins, metabolites, and nucleic acids they deliver to recipient cells can effectively change their biological responses. This intrinsic property of regulating cell life activities in complex pathways increases their potential utility in the treatment and control of many diseases. Currently, more than 100 clinical trials have verified the efficacy of exosomes secreted by human mesenchymal stem cells (MSCs) in the treatment of various diseases. In addition, they can promote collagen synthesis and delay skin aging; enhance fibroblast proliferation, migration, and angiogenic activity of endothelial cells, promote skin regeneration and repair; mediate intercellular communication, inhibit collagen deposition, and treat scar hyperplasia. The easy modification of exosomes also enhances their potential in targeted therapy and broadens their application scenarios. Although the therapeutic potential of MSCs-derived exosomes has been verified, the high cost of stem cell culture, the low efficiency of exosome production and isolation, and the difficulty of large-scale production all pose major challenges to their clinical application. Therefore, finding a suitable strategy to promote MSCs to secrete exosomes will help further advance their clinical application.

[0003] At present, a variety of methods have been explored to promote the secretion of exosomes by mesenchymal stem cells, mainly based on biological, chemical regulation or physical stimulation. However, biochemical regulation methods, such as growth factors, are usually expensive and are accompanied by unpredictable risks in use. Therefore, promoting the secretion of exosomes by stem cells through physical stimulation is a more promising direction. Existing research results have shown that directly applying external mechanical stimulation can enhance the number of exosomes secreted by stem cells. For example, under laminar flow, mechanical shear stress can promote the secretion of exosomes. Compared with the static control group, the direct application of cyclic stress loads will also increase the exosomes secreted by stem cells. These results together show that actively applying mechanical stimulation to cells is a feasible way to promote the secretion of exosomes by stem cells. However, these technologies usually require expensive and bulky equipment, which limits their application development.

[0004] Chinese patents CN110295142A and CN116024163A both disclose the direct mixing of magnetic nanoparticles into stem cell culture medium, using the physical properties of magnetic nanoparticles to improve exosome secretion. However, this method will cause stem cells to engulf a large number of magnetic particles, affecting cell viability and physiological state; at the same time, the extracted exosomes also contain magnetic nanoparticles, which require additional purification and separation, affecting the therapeutic effectiveness of the exosomes.

[0005] At present, there is no report on a system that uses magnetic nanoparticles to stimulate stem cells to promote exosome secretion. This magnetic stimulation method can provide new ideas for enhancing exosome secretion. Summary of the invention

[0006] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide a method and application of promoting exosome secretion by magnetic stimulation, which utilizes magnetic nanoparticles as a medium for remotely applying mechanical stimulation, applies magnetic stimulation to cultured stem cells by precisely controlling the movement of the external magnetic field, and significantly improves the exosome yield.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The first object of the present invention is to provide a method for promoting exosome secretion through magnetic stimulation, comprising the following steps:

[0009] S1. Preparation of magnetic hydrogel.

[0010] The magnetic hydrogel is synthesized by using magnetic nanoparticles and acrylamide. After preparing a hydrogel prepolymer solution, a magnetic nanoparticle aqueous solution is added, mixed evenly and poured into a mold to form a gel insulated from air. The gelled gel sheet is washed with deionized water to remove excess magnetic nanoparticles, thus obtaining a magnetic hydrogel.

[0011] S2. Cell seeding on magnetic hydrogels.

[0012] Coating type I collagen on the magnetic hydrogel to obtain a collagen-modified magnetic hydrogel, and then resuspending the cells and seeding them on the collagen-modified magnetic hydrogel;

[0013] S3, dynamically load magnetic field to achieve remote magnetic stimulation of cells,

[0014] After inoculating the cells, they were cultured in complete medium and then switched to serum-free medium. During serum-free culture, the magnetic field was applied for 1 hour every 12 hours. After 3 to 4 cycles, the magnetic hydrogel and cells were transferred to the living cell workstation, and the magnetic field was applied statically and dynamically. The strength of the applied magnetic field was controlled by controlling the distance between the living cell workstation and the magnet.

[0015] S4. Collect cell supernatant.

[0016] collecting the supernatant of the cells after magnetic stimulation in step S3;

[0017] S5. Extraction and purification of exosomes

[0018] The cell supernatant obtained in step S4 is subjected to ultracentrifugation to remove dead cells and membrane debris, and then extracted and purified to obtain exosomes.

[0019] Furthermore, the cells include human mesenchymal stem cells.

[0020] Furthermore, the magnetic nanoparticles are prepared by a solvent thermal method, and the mass fraction of the magnetic nanoparticles in the hydrogel prepolymer solution is 0.3% to 2%.

[0021] Furthermore, the particle size of the magnetic nanoparticles is 50 to 200 nm.

[0022] Furthermore, the type I collagen is mouse type I collagen, and the collagen and the magnetic hydrogel are bonded using a heterobifunctional protein cross-linking agent, sulfosuccinimide 6-(4'-azido-2'-nitrophenylamino)hexanoate.

[0023] Furthermore, in step S2, before inoculating cells, the collagen-modified magnetic hydrogel is rinsed with PBS and then sterilized by external light; the cells are washed with PBS, digested with a 0.25% by volume trypsin solution for 3 to 5 minutes, and then the digested cells are centrifuged at 300g to 350g for 3 to 5 minutes. Finally, the cells are resuspended and inoculated on the collagen-modified magnetic hydrogel.

[0024] Furthermore, in step S3, the dynamic loading of the magnetic field is achieved by moving the permanent magnet.

[0025] Furthermore, the distance between the living cell workstation and the magnet is 5 to 15 mm, the magnetic induction intensity corresponds to 80 to 250 mT, and the period of loading the magnetic field is 1 s to 6 s.

[0026] Furthermore, step S4 is specifically as follows: centrifuging the supernatant obtained in step S3 at 4°C, 300g for 10min to 15min to remove cells; then centrifuging at 2000g for 10min to 15min to remove dead cells; then centrifuging at 10000g for 10min to 15min to remove membrane fragments, collecting the supernatant, and finally ultracentrifuging at 100000g for 60min to 70min to obtain the precipitate as exosomes.

[0027] The second objective of the present invention is to provide a substance prepared by the above-mentioned method of promoting exosome secretion by magnetic stimulation, and its application in the preparation of collagen and immunotherapy drugs.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention proposes a method for promoting exosome secretion by magnetic stimulation. By applying magnetic stimulation over a long distance through a magnetic field, mechanical stimulation of cells can be achieved without contact, and can be applied to a variety of complex cell culture environments. On the other hand, the magnetic nanoparticles themselves are extremely small in size, and the movement and deformation they produce can be localized to the subcellular scale, and mechanical stimulation can be applied to stem cells uniformly and accurately. The efficiency of exosome secretion can be greatly improved. Compared with cells cultured on conventional cell culture plates, cells on magnetic hydrogels loaded with dynamic magnetic fields produce significantly more exosomes, reaching more than 10 times.

[0030] (2) The method provided by the present invention was used to prepare exosomes, and the number of exosomes was significantly increased. At the single cell level, human mesenchymal stem cells on magnetic hydrogels loaded with dynamic magnetic fields under the same conditions produced more than 10 times the number of exosomes produced by cells cultured on ordinary culture plates, reaching about 4×10 per cell. 5 indivual.

[0031] (3) The method provided by the present invention for promoting exosome secretion by magnetic stimulation does not require biological or chemical additives, and avoids changing the physiological functions of stem cells and exosomes, compared with the traditional biochemical method for improving exosome secretion. This method drives the movement of magnetic nanoparticles through the magnetic field and then drags the gel to deform, which can achieve more precise physical stimulation, increase the number of exosomes produced by each cell, and has the advantages of low cost, simple process, remote control, and extremely high biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A schematic diagram of a method for promoting exosome secretion by magnetic stimulation provided by the present invention;

[0033] Figure 2 Schematic diagram of the magnetic stimulation process caused by dynamic loading magnetic field;

[0034] Figure 3 This is a schematic diagram of the magnetic induction intensity corresponding to the magnet distance;

[0035] Figure 4 This is a real photo of the prepared magnetic hydrogel;

[0036] Figure 5 The figure shows the test results of storage modulus and loss modulus of magnetic hydrogel;

[0037] Figure 6 This is the modulus change diagram of magnetic hydrogel when loaded with magnetic field;

[0038] Figure 7This is the SEM image of the magnetic hydrogel before and after magnetic field loading. The pore size becomes smaller after magnetic field loading.

[0039] Figure 8 The fluorescence comparison of stem cells cultured on magnetic hydrogel before and after magnetic field loading;

[0040] Fig. 9 This is a graph showing the change in the proportion of live cells of stem cells cultured on magnetic hydrogels before and after magnetic field loading;

[0041] Fig.10 This is a graph showing the changes in stem cell proliferation after magnetic hydrogels with different mass fraction gradients were loaded with magnetic fields;

[0042] Fig.11 This is a typical TEM image of exosomes prepared by the method provided by the present invention;

[0043] Fig.12 is the particle size distribution diagram of exosomes;

[0044] Fig.13 A comparison of the number of exosomes secreted by a single cell on different substrates;

[0045] Fig.14 Comparison of the particle size distribution of exosomes secreted by single cells on different substrates;

[0046] Fig.15 The graphs show the results of calcium ion staining before and after magnetic field loading at different magnetic particle mass fractions;

[0047] Fig.16 This is a quantitative comparison chart of calcium ion fluorescence intensity in stem cells;

[0048] Fig.17 This is the result graph showing the effect of the mass fraction of 50 nm magnetic particles on the exosome yield;

[0049] Fig.18 This is the result graph showing the effect of the mass fraction of 100 nm magnetic particles on the exosome yield;

[0050] Fig.19 This is the result graph showing the effect of the mass fraction of 200 nm magnetic particles on the exosome yield;

[0051] Fig. 20 This is a diagram showing the therapeutic effect of magnetic stimulation of exosomes on damaged endometrial stromal cells;

[0052] Fig.21 This is a graph showing the effect of magnetic stimulation on the total protein content of exosomes. DETAILED DESCRIPTION

[0053] In order to explain the technical solution and beneficial effects of the present invention more clearly, the present invention is described in detail below in conjunction with the accompanying drawings and embodiments. It should be clear that the reference drawings described are only some embodiments of the present invention and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0055] Example 1

[0056] This embodiment provides a method for enhancing the secretion efficiency of stem cell exosomes through magnetic stimulation based on a magnetic hydrogel platform with dynamic loading magnetic field. The principle is as shown in the attached figure. Figure 1 As shown, the specific steps are as follows:

[0057] (1) Preparation of magnetic nanoparticles

[0058] Magnetic nanoparticles (MNPs) with particle sizes of 50nm, 100nm and 200nm were prepared by solvothermal method. Diethylene glycol (DEG) and ethylene glycol (EG) were mixed together with a total volume of 80mL. The respective ratios were adjusted according to the different particle sizes prepared. 50nm corresponds to DEG:EG=64:16, 100nm corresponds to DEG:EG=60:20, and 200nm corresponds to DEG:EG=50:30. 2.16gFeCl3·6H2O, 8g anhydrous sodium acetate, and 0.2g polyacrylic acid were dissolved and mixed in the freshly prepared solution. The entire dissolution process was carried out under ultrasonic stirring. First, FeCl3·6H2O was dissolved and stirred until the solution was no longer obviously turbid, and then anhydrous sodium acetate and polyacrylic acid were added in sequence to gradually dissolve and stir. The yellow solution obtained above was added to a high-temperature reactor and heated continuously at 200℃ for 12 hours. After completion, it was naturally cooled to room temperature. At this time, the upper layer of the reactant obtained is a basically colorless and transparent solution, and the lower layer of precipitation is mainly uniformly dispersed magnetic nanoparticles. Remove the upper clear liquid, and wash the lower clear liquid alternately with water and ethanol. The whole process is repeated for at least ten times. In the last step, add an appropriate amount of deionized water to prepare an aqueous solution for use.

[0059] (2) Preparation of magnetic nanoparticle composite hydrogel

[0060] Magnetic hydrogels were synthesized using magnetic nanoparticles (MNPs) and acrylamide (AM), with a total monomer content of 5% w / v. N,N'-methylenebisacrylamide (BIS) was used as a crosslinker. The crosslinking density of all samples was 0.03% of the total weight of the monomers. After preparing the hydrogel prepolymer, different volumes of 3% w / v aqueous solutions of magnetic nanoparticles were added thereto and mixed evenly by oscillation. The solution was poured into a flat mold with a thickness of about 1 mm and covered with a plastic sheet to isolate the air for gelation. After about 1 hour of gelation, the gel sheet was rinsed with deionized water to remove excess magnetic nanoparticles.

[0061] (3) Cell seeding on magnetic hydrogels

[0062] Type I collagen (0.6% w / v in 6 mM acetic acid) was coated on the hydrogel to promote cell adhesion on the hydrogel, and the protein was bonded to the hydrogel using a heterobifunctional protein crosslinker sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (Sulfo-SANPAH). Afterwards, the collagen-modified magnetic hydrogel was stored in PBS at 4°C until further cell culture. Human mesenchymal stem cells within ten passages were cultured in a humidified atmosphere of 5% CO2 at 37°C using complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. Before inoculating cells, the prepared collagen-modified magnetic hydrogel was rinsed with PBS, and then the hydrogel was sterilized with 254 nm ultraviolet light for at least 1 hour. The cells were washed with PBS and digested from the culture flask with 0.25% (v / v) trypsin-EDTA solution for 3 to 5 minutes. The digested cells were then centrifuged at 300g for 3 to 5 minutes. Finally, the cells were resuspended and seeded on the prepared collagen-modified magnetic hydrogels.

[0063] (4) Dynamic loading of magnetic field to achieve remote magnetic stimulation

[0064] After inoculating the cells, use complete medium to culture for 24 hours and then switch to serum-free medium for 48 hours. During serum-free culture, a magnetic field is loaded for 1 hour every 12 hours, at which time the magnetic hydrogel and cells are transferred to the living cell workstation. The dynamic loading of the magnetic field is achieved by moving a permanent magnet. The crank slider structure can quickly realize the cyclic reciprocating motion of the target object in mechanical engineering. A permanent magnet with a size of 20mm cube is fixed at the front end of the slider. The frequency of the reciprocating loading of the magnetic field is controlled by changing the slider movement speed. The strength of the applied magnetic field is controlled by controlling the distance between the living cell workstation and the magnet. The magnetic field induction intensity is measured using a Tesla meter. The distance between the living cell workstation and the magnet is 5 to 15mm. The magnetic induction intensity is measured by a Tesla meter to be 80 to 250mT. The period of loading the magnetic field is 1s to 6s. The more preferred result is a loading distance of 10mm and a loading period of 6s. The loading process is as follows Figure 2As shown, when the static magnetic field is applied, the distance between the magnet and the gel is kept constant, and the stem cell supernatant is obtained after culturing under the above conditions. The corresponding magnetic induction intensity on the magnetic gel is as follows Figure 3 As shown, at this time, the magnetic induction intensity corresponding to the 10mm loading distance is 120mT.

[0065] (5) Extraction of exosomes

[0066] The cell culture supernatant was centrifuged at 4°C, 300g for 10 min, 2000g for 10 min, and 10000g for 10 min to remove cells, dead cells, and membrane fragments, and the supernatant was collected. Finally, the exosome particles were collected by ultracentrifugation at 100000g for 70 min, and the precipitate was resuspended in PBS and stored at -80°C for later use.

[0067] Example 2

[0068] This embodiment characterizes the changes in the mechanical properties of the gel caused by the magnetic field.

[0069] In order to verify the mechanical properties of magnetic hydrogels, the changes in modulus and pore size of hydrogels under external magnetic field excitation were measured. A rheometer was used to test the dynamic mechanical properties of magnetic hydrogels. The surface water of the magnetic hydrogels was dried, and after prestressing the gel, the gel shear modulus was measured using an oscillating shear mode. The surface pore size of the gel was observed using a scanning electron microscope. Before observation under a scanning electron microscope, the gel sample was first frozen in liquid nitrogen and then freeze-dried under vacuum. After confirming that the water was fully drained, a small sample was fixed on a metal bracket and the image was recorded at an accelerating voltage of 15kV.

[0070] refer to Figure 4 , is the prepared magnetic hydrogel. The whole is black, and the magnetic nanoparticles are evenly distributed in the gel.

[0071] refer to Figure 5 , are the storage modulus (black dots) and loss modulus (red dots) of the magnetic gel. The storage modulus is much larger than the loss modulus, and the gel can be approximately regarded as a linear elastic material.

[0072] refer to Figure 6 , is the change in modulus of magnetic hydrogel when magnetic field is loaded. It can be found that the modulus of gel gradually increases and finally stabilizes, which takes about 20-30 minutes. This shows that magnetic field can enhance the stiffness of magnetic hydrogel, and there is a saturation upper limit.

[0073] refer to Figure 7 It can be seen that the stiffness of the magnetic hydrogel increases and the pore size decreases when the magnetic field is loaded. The magnetic field drives the movement of magnetic nanoparticles and causes the mechanical properties of the magnetic hydrogel to change.

[0074] Example 3

[0075] This study investigated the effects of magnetic stimulation on cell proliferation and apoptosis.

[0076] The ratio of live cells to dead cells was observed using the Calcein-AM / PI live / dead cell double staining kit. 4 / cm 2 The cells were inoculated on the gel substrate at a density of 100 μM. After complete attachment, the cells were magnetically stimulated for 48 h under the above conditions, and the culture medium was removed. Calcein-AM solution and PI solution were added to 1×Assay Buffer and mixed thoroughly. The final concentration of Calcein-AM in the working solution was 2 μM, and the final concentration of PI was 4.5 μM. The cells were incubated at 37°C for 20 minutes. At this time, live cells were excited with green fluorescence, and dead cells were excited with red fluorescence.

[0077] The CCK8 method was used to detect the active substances of the cells in the control group and after magnetic stimulation. 4 / cm 2 The cells were inoculated on the gel substrate at a density of 100 μg / ml. After complete attachment, the cells were magnetically stimulated for 48 h under the above conditions. Fresh culture medium containing CCK8 reagent (10% v / v) was replaced for each group of conditions. The cells were incubated at 37°C for 2 h, and DMSO was added at room temperature for 15 min to completely dissolve the active substances. The dissolved solutions of each group were collected in a 96-well plate, and the absorbance of each group was calibrated using an ELISA reader.

[0078] refer to Figure 8 and Fig. 9 The results showed that the proportion of cell apoptosis did not change significantly after magnetic stimulation, indicating that magnetic loading would not induce cell apoptosis.

[0079] refer to Fig.10 The CCK8 results showed that compared with the control group, magnetic hydrogels with different mass fraction gradients did not produce obvious toxic effects on MSCs after loading the magnetic field, and there was no significant difference in cell proliferation.

[0080] Example 4

[0081] This example characterizes the effect of magnetic stimulation on the concentration and size distribution of exosomes, and compares the effects of various culture conditions (1) culture flask (2) culture flask plus magnetic field (3) polyacrylamide hydrogel (PA gel) (4) magnetic nanoparticle composite hydrogel (5) magnetic nanoparticle composite hydrogel loaded with static magnetic field (6) magnetic nanoparticle composite hydrogel loaded with dynamic magnetic field on the efficiency of stem cell secretion of exosomes. Compared with magnetic gel, PA gel only replaces the magnetic particles with deionized water, and the other ratios are the same.

[0082] Transmission electron microscopy (TEM) was used to observe the morphology and size of exosomes, and nanoparticle size analyzer (NTA) was used to obtain concentration and size distribution information.

[0083] refer to Fig.11 and Fig.12 , indicating that the exosomes exhibited a distinct cup-shaped structure and the particle size was between 100nm and 200nm.

[0084] refer to Fig.13 , it can be seen that the exosome secretion efficiency in the culture flask is 5.7×10 4 The magnetic field of the culture bottle is 5.2×10 4 pcs, PA glue is 2.5×10 4 The magnetic gel is 2.6×10 4 The dynamic magnetic field of magnetic gel loading is 4.4×10 4 The dynamic magnetic field of magnetic gel loading is 6.5×10 4 This shows that the effect of static magnetic field on cells themselves does not significantly affect exosome secretion; magnetic hydrogel does not significantly affect exosome secretion compared with PA gel, while magnetic gel loaded with static magnetic field will promote the enhancement of exosome secretion, and exosome secretion is significantly increased to 2.6 times after loading with dynamic magnetic field.

[0085] refer to Fig.14 It can be seen that magnetic field stimulation, magnetic hydrogel and magnetic force stimulation in this article will not cause significant changes in the particle size of exosomes, indicating that the physical characteristics of exosomes remain basically unchanged.

[0086] Example 5

[0087] This study characterized the effect of magnetic stimulation on the calcium ion concentration of stem cells and compared the changes in intracellular calcium ion concentration of PA gel, 0.3% mass fraction magnetic gel, and 2% mass fraction magnetic gel before and after dynamic loading of the magnetic field.

[0088] This experiment used live cell Ca 2+ The cells were stained with a dye based on Fluo-4 AM dye, which is a live cell dye. After preparing the cell sample, the cell culture medium was first removed, and the Fluo-4 AM dye was diluted in PBS and incubated with the cell sample in a 37°C incubator for 30 minutes, with a working concentration of 1μM. After the staining was completed, the dye was removed, washed with PBS, and then the cell culture medium was added and incubated in a 37°C incubator for another 30 minutes. A dynamic magnetic field was applied for 1 hour during the staining process, and then a confocal microscope was used to quickly capture fluorescence images and quantify the fluorescence intensity in each cell.

[0089] refer to Fig.15 and Fig.16 , it can be seen that when there is no magnetic field, the intracellular Ca 2+The concentration is generally low. When the dynamic magnetic field is applied, the Ca 2+ The concentration was slightly increased, and the Ca in the cells of the magnetic hydrogel-corresponding experimental group 2+ The concentration was significantly increased, and the magnetic hydrogel with a larger mass fraction promoted the cell Ca 2+ The concentration increased more significantly.

[0090] Example 6

[0091] This study compared the different enhancing effects of magnetic stimulation on exosome secretion when the mass fraction of magnetic nanoparticles was changed.

[0092] By changing the mass fraction of magnetic nanoparticles in the hydrogel prepolymer solution, the stiffness and magnetic stimulation performance of the hydrogel can be regulated. In this embodiment, the enhancement effect of magnetic hydrogels with particle sizes of 50nm, 100nm, and 200nm and mass fractions of 0.3%, 1.2%, and 2% was studied, and a dynamic magnetic field was loaded at this time.

[0093] refer to Fig.17 and Fig.18 and Fig.19 By comparison, it can be seen that when the magnetic nanoparticle size is small, the magnetic stimulation effect is poor. When the magnetic nanoparticle size and mass fraction increase, the response of stem cells to the same magnetic field stimulation increases accordingly. However, when the concentration of magnetic particles is further increased, the secretion of exosomes decreases, which indicates that there is an optimal concentration. When the other experimental conditions are kept unchanged, the best magnetic nanoparticle-hydrogel ratio can be optimized. When the magnetic nanoparticle size is 100nm, the highest exosome secretion efficiency is achieved at a mass fraction of 1.2%.

[0094] Example 7

[0095] This example verifies and compares the therapeutic effects of magnetically stimulated exosomes and conventionally extracted exosomes on damaged endometrial stromal cells.

[0096] The extracted exosomes were used to treat damaged human endometrial stromal cells (provided by Anhui Medical University). The in vitro damage conditions were 60μmol / L mifepristone for 48 hours and exosome treatment for 18 hours. The number of stem cells corresponding to the commonly extracted exosomes and the magnetically stimulated exosomes was equivalent, and the control group was added with an equal amount of PBS.

[0097] refer to Fig. 20 The results of the scratch experiment showed that the magnetically stimulated cell exosomes had a significantly improved therapeutic effect on damaged endometrial stromal cells. Compared with the exosomes extracted from ordinary cells, the migration ability of damaged cells was further enhanced, promoting faster wound healing.

[0098] Example 8

[0099] This study explored the effect of magnetic stimulation on the protein content of exosomes produced by stem cells.

[0100] The total protein content of exosomes in serum-free culture medium of human mesenchymal stem cells under the conditions of plastic substrate, polyacrylamide hydrogel, magnetic gel without magnetic field, magnetic gel loaded with static magnetic field, and magnetic gel loaded with dynamic magnetic field was counted respectively.

[0101] refer to Fig.21 ,The results showed that the total protein content of exosomes secreted by unit stem cells after magnetic stimulation was higher than that of other groups.

[0102] For matters not mentioned above, the prior art applies.

[0103] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention belongs may make modifications or supplements to the described specific embodiments or replace them in a similar manner, but they will not deviate from the direction of the present invention or exceed the scope defined by the attached claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above implementations based on the technical essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for promoting exosome secretion by magnetic stimulation, characterized in that: The following steps are involved: S1. Preparation of magnetic hydrogel. The magnetic hydrogel is synthesized by using magnetic nanoparticles and acrylamide. After preparing a hydrogel prepolymer solution, a magnetic nanoparticle aqueous solution is added, mixed evenly and poured into a mold to form a gel insulated from air. The gelled gel sheet is washed with deionized water to remove excess magnetic nanoparticles, thus obtaining a magnetic hydrogel. S2. Cell seeding on magnetic hydrogels. Coating type I collagen on the magnetic hydrogel to obtain a collagen-modified magnetic hydrogel, and then resuspending the cells and seeding them on the collagen-modified magnetic hydrogel; S3, dynamically load magnetic field to achieve remote magnetic stimulation of cells, After inoculating the cells, they were cultured in complete medium and then switched to serum-free medium. During serum-free culture, the magnetic field was applied for 1 hour every 12 hours. After 3 to 4 cycles, the magnetic hydrogel and cells were transferred to the living cell workstation and dynamically loaded with the magnetic field. The strength of the applied magnetic field was controlled by controlling the distance between the living cell workstation and the magnet. S4. Collect cell supernatant. collecting the supernatant of the cells after magnetic stimulation in step S3; S5. Extraction and purification of exosomes The cell supernatant obtained in step S4 is subjected to ultracentrifugation to remove dead cells and membrane debris, and then extracted and purified to obtain exosomes.

2. The method according to claim 1, characterized in that The cells include human mesenchymal stem cells.

3. The method according to claim 2, characterized in that The magnetic nanoparticles are prepared by a solvent thermal method, and the mass fraction of the magnetic nanoparticles in the hydrogel prepolymer solution is 0.3% to 2%.

4. The method according to claim 3, characterized in that The particle size of the magnetic nanoparticles is 50 to 200 nm.

5. The method according to claim 2, characterized in that The type I collagen is mouse type I collagen, and the collagen and the magnetic hydrogel are bonded by using a heterobifunctional protein cross-linking agent, sulfosuccinimide 6-(4'-azido-2'-nitrophenylamino)hexanoate.

6. The method according to claim 2, characterized in that In step S2, before inoculating cells, the collagen-modified magnetic hydrogel is rinsed with PBS and then sterilized by external light; the cells are washed with PBS, digested with a 0.25% by volume trypsin solution for 3 to 5 minutes, and then the digested cells are centrifuged at 300g to 350g for 3 to 5 minutes. Finally, the cells are resuspended and inoculated on the collagen-modified magnetic hydrogel.

7. The method according to claim 2, characterized in that In step S3, the dynamic loading of the magnetic field is achieved by moving the permanent magnet.

8. The method according to claim 7, characterized in that When loading the magnetic field, the distance between the living cell workstation and the magnet is 5 to 15 mm, and the magnetic induction intensity corresponds to 80 to 250 mT. A fixed distance is maintained when statically loading the magnetic field, and the period of dynamically loading the magnetic field is 1s to 6s.

9. The method according to claim 2, characterized in that Step S4 specifically comprises centrifuging the supernatant obtained in step S3 at 4°C, 300g for 10min-15min to remove cells; then centrifuging at 2000g for 10min-15min to remove dead cells; then centrifuging at 10000g for 10min-15min to remove membrane fragments, collecting the supernatant, and finally ultracentrifuging at 100000g for 60min-70min to obtain the precipitate as exosomes.

10. Use of a substance prepared by the method for promoting exosome secretion through magnetic stimulation according to any one of claims 2 to 9 in the preparation of collagen and immunotherapy drugs.

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