Umbilical cord-derived mesenchymal stem cell exosome preparation, preparation method and application in preparation of osteoarthritis drugs

Abstract

The present application relates to the technical field of biological medicine, in particular to a kind of umbilical cord-derived mesenchymal stem cell exosome preparation, preparation method and application in preparation of osteoarthritis drugs.The preparation is prepared by the method.The method comprises pretreating umbilical cord-derived mesenchymal stem cells with a pretreatment agent;discard the pretreatment agent, and gradient centrifugation treatment is carried out to the pretreated umbilical cord-derived mesenchymal stem cells, to obtain exosome precipitate;the exosome precipitate is resuspended in a buffer containing protease inhibitor to obtain umbilical cord-derived mesenchymal stem cell exosome preparation.The application is the application of the preparation in the preparation of osteoarthritis drugs.The present application solves the problems of low yield and functional heterogeneity of EXO obtained by conventional culture method.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an umbilical cord-derived mesenchymal stem cell exosome preparation, its preparation method, and its application in the preparation of osteoarthritis drugs. Background Technology

[0002] Osteoarthritis (OA), the most common degenerative joint disease worldwide, is characterized by progressive destruction of articular cartilage and chronic inflammation. Currently, there are no effective drugs to stop this degeneration. Mesenchymal stem cells (MSCs) have become a research hotspot due to their potential for immunomodulation and tissue repair; however, their clinical application is limited by problems such as low transplant survival rate, low differentiation efficiency, significant variability in allogeneic formulations, homing failure, poor efficacy persistence, difficulty in quality control, tumorigenic risk, and stringent transportation requirements.

[0003] Mesenchymal stem cell exosomes are vesicles secreted by mesenchymal stem cells, containing bioactive substances, thus avoiding the problems associated with directly using mesenchymal stem cells. Furthermore, exosomes (EXOs) serve as intercellular communication carriers, delivering active substances such as miRNAs and proteins, and possess biological characteristics similar to their source cells, demonstrating potential as an alternative to cell therapy. However, EXOs obtained through conventional culture methods suffer from low yields and functional heterogeneity, making the exploration of optimization strategies still urgently needed.

[0004] In summary, there is a need to provide a preparation of umbilical cord-derived mesenchymal stem cell exosomes, a preparation method thereof, and its application in the preparation of osteoarthritis drugs, in order to solve the problems of low yield and functional heterogeneity of EXO obtained by conventional culture methods. Summary of the Invention

[0005] The purpose of this invention is to provide an umbilical cord-derived mesenchymal stem cell exosome preparation, its preparation method, and its application in the preparation of osteoarthritis drugs. The specific technical solution is as follows:

[0006] In a first aspect, the present invention provides a method for preparing an umbilical cord-derived mesenchymal stem cell exosome preparation, comprising:

[0007] Step S1: Pre-treat umbilical cord-derived mesenchymal stem cells with a pre-treatment agent; the pre-treatment agent includes astragaloside A, tanshinone IIA and icariin, and the molar ratio of the three is 10-20:5-10:20-40;

[0008] Step S2: Discard the pretreatment agent and perform gradient centrifugation on the pretreated umbilical cord-derived mesenchymal stem cells to obtain exosome precipitate;

[0009] Step S3: Resuspend the exosome precipitate in a buffer solution containing a protease inhibitor to obtain an umbilical cord-derived mesenchymal stem cell exosome preparation.

[0010] Optionally, the pretreatment includes diluting the pretreatment agent with serum-free DMEM / F12 medium, diluting the molar concentrations of astragaloside A, tanshinone IIA, and icariin to 10–20 μM, 5–10 μM, and 20–40 μM, respectively; seeding 3rd–5th generation umbilical cord-derived mesenchymal stem cells in multi-well plates, and after cell adhesion, replacing the medium with serum-free DMEM / F12 medium for synchronization culture for 12–24 h, then adding the serum-free DMEM / F12 medium containing the pretreatment agent; and culturing in an incubator at 37°C and 5% CO2 for 24–48 h.

[0011] Optionally, the pretreatment agent may be an organic solvent, including DMSO or ethanol.

[0012] Optionally, the culture method for the 3rd to 5th generation umbilical cord-derived mesenchymal stem cells includes:

[0013] First, the umbilical cord sample was washed multiple times with saline solution and cut into 2-3 cm segments. After removing blood vessels, it was longitudinally dissected, Wharton's jelly tissue was separated, and then cut into 1-2 mm pieces. 3 Tissue block;

[0014] Next, the tissue blocks were evenly spread in the culture flask, DMEM / F12 medium containing serum was added, and the culture was carried out in an incubator with a culture temperature of 37°C and a volume percentage of 5% CO2.

[0015] Then, after culturing for 24–48 hours, the serum-containing DMEM / F12 medium was replaced to remove non-adherent cells, thus culturing the first generation of umbilical cord-derived mesenchymal stem cells.

[0016] Finally, the serum-containing DMEM / F12 culture medium is replaced every 2 to 3 days until the 3rd to 5th generation umbilical cord-derived mesenchymal stem cells are cultured.

[0017] Optionally, the gradient centrifugation process includes a first centrifugation process, a second centrifugation process, and a third centrifugation process performed sequentially, with the centrifugal force and centrifugation time increasing sequentially.

[0018] Optionally, the centrifugal force used in the first centrifugation process is 300-500×g, and the centrifugation time is 10-20min.

[0019] Optionally, the centrifugal force used in the second centrifugation process is 2000–5000 × g, and the centrifugation time is 20–30 min.

[0020] Optionally, the third centrifugation process uses a centrifugal force of 100,000 × g and a centrifugation time of 50–70 min.

[0021] In a second aspect, the present invention provides an umbilical cord-derived mesenchymal stem cell exosome preparation, which is prepared using the aforementioned method for preparing umbilical cord-derived mesenchymal stem cell exosome preparations.

[0022] In a third aspect, the present invention provides the application of the aforementioned umbilical cord-derived mesenchymal stem cell exosome preparation in the preparation of osteoarthritis drugs.

[0023] The application of the technical solution of the present invention has at least the following beneficial effects:

[0024] This invention provides a method for preparing an umbilical cord-derived mesenchymal stem cell exosome preparation, which can produce a more targeted, safer, and easily standardized umbilical cord-derived mesenchymal stem cell exosome preparation, solving the problems of low yield and functional heterogeneity of EXO obtained by conventional culture methods. Specifically, in terms of targeting, the umbilical cord-derived mesenchymal stem cell exosome preparation significantly enhances chondrocyte viability and reduces synovial inflammation and cartilage defects, effectively alleviating the progression of osteoarthritis without abnormal calcification or immune rejection, providing a cell-free therapy strategy with significant effects for targeted treatment of osteoarthritis. In terms of safety, the umbilical cord-derived mesenchymal stem cell exosome preparation exhibits higher safety because it does not require cell transplantation, reducing the risk of tumorigenesis. In terms of standardized production, the preparation method used in this invention has the advantages of simple and easily controllable parameters, facilitating standardized production, increasing yield, and ensuring functional homogeneity.

[0025] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the figures. Attached Figure Description

[0026] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0027] Figure 1 This is a graph showing the results of the analysis of the particle size and concentration of the umbilical cord-derived mesenchymal stem cell exosome preparation using the nanoparticle tracking analysis method (NTA) in Example 1.

[0028] Figure 2 This is a graph showing the results of Western blot analysis of the proteins TSG101, CD9, and CD63 in the umbilical cord-derived mesenchymal stem cell exosome preparation in Example 1.

[0029] Figure 3 This is a diagram showing the results of identifying the shape of the umbilical cord-derived mesenchymal stem cell exosome preparation using transmission electron microscopy with negative staining in Example 1.

[0030] Figure 4 These are the chondrocyte viability test results from Example 2;

[0031] Figure 5 This is a diagram showing the results of hematoxylin-eosin (H&E) staining evaluation of a mouse knee arthritis model in Example 3;

[0032] Figure 6 This is a diagram showing the quantitative results of safranin O-fast green staining in a mouse knee arthritis model in Example 3;

[0033] Figure 7 This is a graph showing the results of hematoxylin-eosin (H&E) staining evaluation of the sham-operated group, DMM model group, and treatment group in Example 3;

[0034] Figure 8 This is a graph showing the quantitative results of safranin O-fast green staining in Example 3 for the sham surgery group, DMM model group, and treatment group;

[0035] Among them, Figure 4 "CellViability-CCK8" indicates CCK-8 cell viability assay; in Figure 7 and Figure 8 The “Sham” in the text refers to the sham surgery group. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0037] Example 1:

[0038] A method for preparing an umbilical cord-derived mesenchymal stem cell exosome preparation includes:

[0039] Step S1: Pre-treat umbilical cord-derived mesenchymal stem cells using a pre-treatment agent; the pre-treatment agent includes astragaloside A (AS-IV, purity ≥98%), tanshinone IIA (Tan IIA, purity ≥98%), and icariin (ICA, purity ≥95%), and the molar ratio of the three is 10:5:20.

[0040] Step S2: Discard the pretreatment agent and perform gradient centrifugation on the pretreated umbilical cord-derived mesenchymal stem cells to obtain exosome precipitate;

[0041] Step S3: Resuspend the exosome precipitate in PBS buffer containing protease inhibitors to obtain an umbilical cord-derived mesenchymal stem cell exosome preparation.

[0042] The pretreatment agent uses an organic solvent, including DMSO or ethanol; specifically, DMSO is used to dissolve astragaloside A, tanshinone IIA and icariin to form a mother liquor; in the mother liquor, the molar concentrations of astragaloside A, tanshinone IIA and icariin are 10 mM, 5 mM and 20 mM respectively.

[0043] The pretreatment includes diluting the pretreatment agent (i.e., the stock solution) with serum-free DMEM / F12 medium (i.e., DMEM / F12 medium without FBS fetal bovine serum), diluting the molar concentrations of the astragaloside A, tanshinone IIA, and icariin to 10 μM, 5 μM, and 20 μM, respectively, with the volume percentage of the organic solvent being less than or equal to 0.1%; and seeding 3rd to 5th generation umbilical cord-derived mesenchymal stem cells into 6-well plates (seedling density of 5 × 10⁶ cells / well). 4 Cells were placed in wells and, after adhesion, were replaced with serum-free DMEM / F12 medium and cultured synchronously for 12 hours to eliminate interference from basal exosome secretion. Then, serum-free DMEM / F12 medium containing the pretreatment agent was added. The cells were cultured in an incubator at 37°C and 5% CO2 for 48 hours.

[0044] The culture methods for the 3rd to 5th generation umbilical cord-derived mesenchymal stem cells include:

[0045] First, the umbilical cord sample was washed multiple times with saline solution and cut into 2-3 cm segments. After removing blood vessels, it was longitudinally dissected, Wharton's jelly tissue was separated, and then cut into 1-2 mm pieces. 3 Tissue block;

[0046] Next, the tissue blocks were evenly spread in a T75 culture flask, and DMEM / F12 medium containing serum was added. The flask was then cultured in an incubator at 37°C with 5% CO2. The DMEM / F12 medium contained 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (in a 1:1 ratio). DMEM / F12 cell culture medium is a medium that can be used for umbilical cord mesenchymal stem cell culture and can be purchased directly.

[0047] Then, after culturing for 48 hours, the serum-containing DMEM / F12 medium was replaced to remove non-adherent cells, thus culturing the first generation of umbilical cord-derived mesenchymal stem cells.

[0048] Finally, the serum-containing DMEM / F12 culture medium was changed every 3 days until the 3rd to 5th generation umbilical cord-derived mesenchymal stem cells were cultured.

[0049] In step S2, the serum-free DMEM / F12 medium containing the pretreatment agent is discarded, and the culture is continued for 48 hours in serum-free DMEM / F12 medium. Subsequently, the serum-free DMEM / F12 medium after culture is collected, and the pretreated umbilical cord-derived mesenchymal stem cells are subjected to gradient centrifugation to obtain exosome precipitate.

[0050] The gradient centrifugation process includes a first centrifugation process, a second centrifugation process, and a third centrifugation process performed sequentially, with the centrifugal force and centrifugation time increasing sequentially.

[0051] The first centrifugation process uses a centrifugal force of 300×g and a centrifugation time of 10min to remove suspended cells.

[0052] The second centrifugation process uses a centrifugal force of 2000×g and a centrifugation time of 20min to remove large particle fragments. Subsequently, the centrifuged liquid is filtered through a 0.22μm filter membrane to remove impurities.

[0053] The third centrifugation process uses a centrifugal force of 100,000 × g, a centrifugation time of 70 min, and a centrifugation temperature of 4 °C to enrich exosomes. After discarding the supernatant, the exosome precipitate is gently resuspended in PBS buffer pre-cooled to 4 °C, and ultracentrifugation is repeated once to improve purity.

[0054] The umbilical cord-derived mesenchymal stem cell exosome preparations prepared in Example 1 were subjected to the following identifications:

[0055] (1) The particle size and concentration of the umbilical cord-derived mesenchymal stem cell exosome preparation were analyzed using the nanoparticle tracking analysis (NTA) method. The results are as follows: Figure 1 As shown. The instrument used for nanoparticle tracking analysis (NTA) was Zetaview (Particle Metrix); the experimental conditions were: laser wavelength 488 nm, detection temperature 25 °C, each exosome preparation sample was measured three times, each measurement lasted 60 seconds, the sample dilution solution was sterile PBS (pH 7.4), and the injection volume was 0.5 mL.

[0056] Depend on Figure 1 It is known that the particle detection concentration of the exosome preparation sample is 9.2E+7 (i.e., 9.2 × 10⁻⁷). 7 Particles / mL (dilution factor 2000), the calculated original concentration is 1.8E+11 (i.e. 1.8 × 10⁻¹¹). 11The particle size distribution was 138.2 nm, which is within the typical particle size range of exosomes (30–200 nm). The particle size distribution of the exosome preparation samples showed a unimodal characteristic, with no obvious impurity peaks or large particles (>300 nm) interfering, indicating that the exosome preparation samples had high purity and that no significant contamination or aggregation was introduced during the preparation process.

[0057] (2) The proteins TSG101, CD9, and CD63 of the umbilical cord-derived mesenchymal stem cell exosome preparation were identified by Western blot. The results are as follows: Figure 2 As shown (in) Figure 2 EXO1 and EXO2 represent two umbilical cord-derived mesenchymal stem cell exosome preparation samples taken from Example 1; MSC represents umbilical cord-derived mesenchymal stem cells without pretreatment; CALNEXIN represents a type I integrated endoplasmic reticulum membrane protein, typically located between the endoplasmic reticulum and cytosol. A negative CALNEXIN result is considered one of the conditions for obtaining relatively pure exosomes. Western blot was used to extract proteins from the exosome preparation samples using RIPA lysis buffer (containing protease inhibitors); subsequently, SDS-PAGE electrophoresis was performed to separate the proteins, which were then transferred to PVDF or NC membranes and blocked with 5% skim milk at room temperature for 1 hour; then, primary antibody (incubated overnight at 4°C, the primary antibody being antibodies against CD9, CD63, TSG101, and CALNEXIN) and secondary antibody (incubated at room temperature for 1 hour, the secondary antibody being an HRP-labeled antibody) were added sequentially. After each antibody incubation, the samples were washed three times with TBST (5 minutes each time); finally, the expression of the target proteins was analyzed by ECL chemiluminescence imaging.

[0058] Depend on Figure 2 It is known that exosome preparation samples EXO1 and EXO2 highly express proteins TSG101, CD9, and CD63, but do not express the protein Calnexin; the corresponding MSC protein expression is the opposite, indicating that exosome preparation samples EXO1 and EXO2 are relatively pure exosome samples.

[0059] (3) The shape of the umbilical cord-derived mesenchymal stem cell exosome preparation was identified using transmission electron microscopy with negative staining. The results are as follows: Figure 3 As shown. The transmission electron microscope (TEM) negative staining method was performed using a transmission electron microscope, model HT7800 / HT7700; the experimental conditions were as follows:

[0060] 1. Use a pipette to draw 20 μl of the exosome preparation sample and drop it onto a carbon membrane copper grid. Let it stand for 3-5 minutes, and then use filter paper to absorb the excess liquid.

[0061] 2. Drop 2% phosphotungstic acid onto a carbon film copper mesh, let it stand for 1-2 minutes, absorb excess liquid with filter paper, and air dry at 25°C.

[0062] 3. Observe under a transmission electron microscope and collect and analyze images.

[0063] Depend on Figure 3 It is known that the exosome preparation sample exhibits a saucer-like structure with a distinct membrane structure, clear three-dimensional structure, and a size of approximately 30-200 nm. This indicates that the exosome preparation sample has a typical exosome structure.

[0064] Example 2:

[0065] This study investigated the effect of the umbilical cord-derived mesenchymal stem cell exosome preparation prepared in Example 1 on the viability of mouse chondrocytes.

[0066] Experimental methods: Primary chondrocytes from 10-11 week old C57BL / 6J wild-type male mice (weighing 18-20g) were cultured at 1×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 1 / well in 96-well plates and pre-cultured for 24 hours in a serum-containing DMEM / F12 culture medium at 37°C with 5% CO2 until cell adhesion was achieved. The old culture medium was discarded, and the experimental group was treated with the umbilical cord-derived mesenchymal stem cell exosome preparation prepared in Example 1 (concentration 1×10⁻⁶). 10 The concentration of particles / mL was obtained by dilution with serum-free DMEM / F12 medium. The control group received equal volumes of conventional exosomes (NC-EXO) and blank medium (Control). Each group had 5 replicates. After culturing for 24 hours, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated in the dark for 2 hours. The absorbance (OD value) at 450 nm was immediately measured using a microplate reader. Cell viability was calculated using GraphPad Prism 9.0 ((experimental group OD value / control group OD value) × 100%). One-way ANOVA was used to compare differences between groups (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Results are shown below. Figure 4 .

[0067] Depend on Figure 4 It is known that, compared with the control group containing an equal amount of conventional exosomes (NC-EXO) and the control group containing blank culture medium (Control), the umbilical cord-derived mesenchymal stem cell exosome preparation prepared in Example 1 increased chondrocyte viability by about 30%, indicating that it can significantly enhance the cartilage repair potential.

[0068] Example 3:

[0069] This study investigated the therapeutic effect of an umbilical cord-derived mesenchymal stem cell exosome preparation prepared in Example 1 on knee osteoarthritis in mice.

[0070] Establishment of a mouse model of knee osteoarthritis:

[0071] Ten- to eleven-week-old wild-type male C57BL / 6J mice (weighing 18–20 g) were selected to induce a knee osteoarthritis model through medial meniscus instability (DMM) surgery. The specific procedure was as follows: After anesthesia with isoflurane inhalation, the medial aspect of the right hind limb knee joint was prepared and disinfected. A longitudinal incision (approximately 5 mm) was made along the medial aspect of the patellar ligament. The joint cavity was exposed by blunt dissection, and the medial meniscus-tibial ligament (MMTL) was microsurgically severed to disrupt meniscus stability. In the sham surgery group, only the ligament was exposed but not severed. The joint capsule and skin were sutured layer by layer. Postoperatively, meloxicam (5 mg / kg) was administered subcutaneously for analgesia for three consecutive days, and penicillin (50,000 U / kg) was administered to prevent infection. Postoperatively, gait abnormalities (limping) and joint swelling were observed in mice. Four weeks after modeling, mice were sacrificed, and knee joint samples were collected and fixed in 4% (meaning 40g paraformaldehyde per L solution) paraformaldehyde for 48 hours, followed by decalcification in 10% (meaning 100g EDTA per L solution) EDTA for 4 weeks. After paraffin embedding, 5μm sagittal serial sections were prepared. Hematoxylin-eosin (H&E) staining was used to assess synovitis and cartilage structure destruction, and Safranin O-Fixed Green staining was used to quantify chondroitin proteoglycan loss. For detailed results, see [link to relevant documentation]. Figure 5 and Figure 6 .Depend on Figure 5 and Figure 6 Histological examination showed that the mouse model of knee arthritis was successfully established.

[0072] Group experiment:

[0073] Mice were divided into three groups: a sham-operated group (n=24) of successfully established knee osteoarthritis models (C57BL / 6J mice) were randomly divided into three groups (n=8 / group): a sham-operated group (ligaments exposed but not severed), a DMM model group (untreated), and a treatment group (injected with an umbilical cord-derived mesenchymal stem cell exosome preparation prepared in Example 1, abbreviated as EXO). Starting from day 3 after modeling, the treatment group received EXO injections into the knee joint (dose: 100 μL / time) once a week for 4 weeks; the DMM model group and the sham-operated group received an equal volume of PBS. After the intervention, knee joint samples were collected from each mouse. The mouse knee joint samples were fixed in 4% paraformaldehyde for 48 hours, decalcified in 10% EDTA for 4 weeks, embedded in paraffin, and prepared into sagittal sections. Hematoxylin-eosin (H&E) staining was used to assess synovitis and cartilage structure destruction, and Safranin O-Fixed Green staining was used to quantify chondroitin proteoglycan loss. Specific results are detailed in [link to relevant documentation]. Figure 7 and Figure 8 .

[0074] Depend on Figure 7It is known that, as shown by hematoxylin-eosin (H&E) staining, compared with the untreated DMM model group, the synovial inflammation of the knee joint of mice treated with the umbilical cord-derived mesenchymal stem cell exosome preparation prepared in Example 1 was significantly reduced (inflammatory cell infiltration was reduced), the fibrosis and fissure formation of the cartilage surface were significantly improved, the chondrocytes were arranged in an orderly manner, the tidal line structure was intact, and the cartilage thickness was restored to a level close to that of the sham-operated group.

[0075] Depend on Figure 8 It was found that, through Safranin O-Fixed Green staining analysis, the cartilage of mice in the DMM model group was severely depleted of proteoglycans (GAGs), while the GAG ​​content in the treatment group was significantly restored. Furthermore, the Fast Green staining showed that the collagen fibers were tightly arranged and regularly oriented, which was in stark contrast to the extensive collagen network disintegration in the DMM model group.

[0076] comprehensive Figure 7 and Figure 8 The results show that the umbilical cord-derived mesenchymal stem cell exosome preparation prepared in Example 1 can effectively provide a cell-free treatment strategy for targeted therapy of knee osteoarthritis in mice.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method of preparing a preparation of umbilical cord-derived mesenchymal stem cell exosomes, characterized by, The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. First, the umbilical cord sample was washed several times with normal saline and cut into small pieces of 2-3 cm, after removing the blood vessels, it was cut longitudinally, the Wharton's jelly tissue was separated, and it was cut into 1-2 mm 3 tissue pieces; The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes.

2. The method of claim 1, wherein the umbilical cord-derived mesenchymal stem cell exosome preparation is prepared by the following steps of: The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application relates to a preparation method of umbilical cord-derived mesenchymal stem cell exosomes. The application

Images