Preparation method of small extracellular vesicles of human umbilical cord mesenchymal stem cells and application thereof

Abstract

The present application belongs to the field of stem cell biology, and relates to a preparation method of human umbilical cord mesenchymal stem cell small extracellular vesicles and application thereof. The preparation method of human umbilical cord mesenchymal stem cell small extracellular vesicles comprises the following steps: S1: under normoxic conditions, human umbilical cord mesenchymal stem cells are induced and cultured by a culture medium containing cobalt chloride to obtain a cell culture solution. S2: the cell culture solution is subjected to small extracellular vesicle extraction and purification treatment to obtain human umbilical cord mesenchymal stem cell small extracellular vesicles. The preparation method significantly improves the yield of small extracellular vesicles, which is about 60% higher than that of normoxic culture, and does not require expensive hypoxic equipment, has low cost, simple operation and is easy to scale up.

Description

Technical Field

[0001] This invention relates to the field of stem cell biology, specifically to a method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells and their application. Background Technology

[0002] In recent years, extracellular vesicles (EVs) have attracted widespread attention due to their crucial role in intercellular communication. Among them, small extracellular vesicles (sEVs) are a class of natural nanoparticles with a diameter of 30-150 nm, actively secreted by cells, and encapsulating bioactive molecules such as proteins, nucleic acids, and lipids from the parent cell. Small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) possess advantages such as low immunogenicity, good biocompatibility, and the ability to cross biological barriers.

[0003] The applications of small extracellular vesicles are extremely broad, extending far beyond the treatment of single diseases. Studies have found that in the field of tissue regeneration and repair, small extracellular vesicles can effectively promote the proliferation and migration of fibroblasts and keratinocytes, accelerating wound healing. In the fields of anti-aging and skin care, small extracellular vesicles can regulate the aging process of skin cells, promote collagen synthesis, and improve skin elasticity. In the field of drug delivery, small extracellular vesicles, with their natural targeting and low immunogenicity, are considered ideal nanomedicine carriers for delivering anticancer drugs, nucleic acid drugs, and more. These diverse applications create an urgent need for the large-scale, low-cost production of small extracellular vesicles.

[0004] However, in traditional culture preparation, the yield of small extracellular vesicles is low, often failing to meet the dosage requirements for clinical treatment. Although the yield can be improved by using special culture devices, these methods are often accompanied by high equipment investment and complex process control requirements, which to some extent limits their application in large-scale production.

[0005] Therefore, there is a need for a preparation method that does not rely on expensive equipment and can significantly increase the yield of small extracellular vesicles to meet the needs of their large-scale application in fields such as regenerative medicine. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] To address the problems of low yield and high preparation cost of small extracellular vesicles in existing technologies, this invention provides a method for preparing small extracellular vesicles from human umbilical cord mesenchymal stem cells and its application.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0010] In a first aspect, the present invention provides a method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells, comprising the following steps:

[0011] S1: Under normoxic conditions, human umbilical cord mesenchymal stem cells were induced and cultured in a medium containing cobalt chloride to obtain cell culture medium;

[0012] S2: Extract and purify small extracellular vesicles from the cell culture medium to obtain small extracellular vesicles of human umbilical cord mesenchymal stem cells.

[0013] In the method for preparing human umbilical cord mesenchymal stem cell small extracellular vesicles as described above, preferably, in step S1, human umbilical cord mesenchymal stem cells are induced and cultured in a culture medium containing cobalt chloride for 4-8 hours to obtain a cell culture medium; the concentration of cobalt chloride in the culture medium is 80-120 μmol / L.

[0014] In the method for preparing human umbilical cord mesenchymal stem cell small extracellular vesicles as described above, preferably, in step S1, human umbilical cord mesenchymal stem cells are prepared as follows: umbilical cords delivered by cesarean section at 34-42 weeks of gestation are taken, the outer membrane and umbilical arteries and veins are removed, Wharton's jelly is retained, the tissue is cut into small pieces and mixed with culture medium, and then primary culture is performed using the tissue block adherence method to obtain human umbilical cord mesenchymal stem cells.

[0015] The method for preparing human umbilical cord mesenchymal stem cell small extracellular vesicles as described above preferably involves cutting the tissue into small pieces and preparing a tissue homogenate, mixing it with culture medium, adding it to a culture flask, and performing primary culture at 37°C and 5% CO2 using the tissue block adhesion method. During the culture process, the medium is changed for the first time on day 5, the second time on day 10-12, and the third time on day 14, until the cell confluence reaches 80-90% on day 14. When the cell confluence reaches 80%-90%, the cells are digested with trypsin, then digestion is stopped, and the cells are pipetted to form a single-cell suspension for cell seeding and passage until the P3-P4 generation is reached.

[0016] In the method for preparing human umbilical cord mesenchymal stem cell small extracellular vesicles as described above, preferably, in step S1, under normoxic conditions, the obtained P3-P4 generation human umbilical cord mesenchymal stem cells with a viability ≥95% are processed at a rate of 8000-10000 cells / cm³. 2 The culture medium was inoculated at a density of 80-120 μmol / L in culture flasks. After 3 days of culture, the medium was replaced with one containing cobalt chloride to achieve a cobalt chloride concentration of 80-120 μmol / L. The culture was then induced at 37°C and 5% CO2.

[0017] The method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells as described above, preferably, in step S1, the preparation method of the culture medium containing cobalt chloride is as follows: add cobalt chloride solution to the serum-free complete culture medium for mesenchymal stem cells, filter it through a 0.22 μm filter membrane for sterilization, and obtain a culture medium with a cobalt chloride concentration of 80-120 μmol / L.

[0018] In the method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells as described above, preferably, step S2 includes the following steps:

[0019] S21: Centrifuge the cell culture medium at 4℃, centrifuge at 2500g-3500g for 25-35min, and obtain the supernatant after filtration.

[0020] S22: The supernatant was subjected to tangential flow filtration. The tangential flow filtration system was equipped with a filter membrane with a molecular weight cutoff of 0.45 μm. The peristaltic pump speed was adjusted to ≤100 rpm / min and the transmembrane pressure was controlled to ≤1.0 bar. The supernatant was concentrated to 10%±2% of the original volume. Then, physiological saline was added for replacement. The flow rate of the peristaltic pump was adjusted to ≤50 rpm / min to obtain the original solution of small extracellular vesicles.

[0021] S23: The collected small extracellular vesicle stock solution was sterilized by filtration using a 0.22μm vesicle filter with an initial pressure difference ≤0.05MPa. During the filtration process, the pressure difference was controlled between 0.1-0.2MPa to obtain small extracellular vesicles of human umbilical cord mesenchymal stem cells.

[0022] Secondly, the present invention provides an application of the small extracellular vesicles prepared by the above preparation method in the preparation of anti-aging skin care products.

[0023] Thirdly, the present invention provides an application of the small extracellular vesicles prepared by the above preparation method in the preparation of a drug for treating skin trauma.

[0024] Fourthly, the present invention provides an application of the small extracellular vesicles prepared by the above preparation method in the preparation of drug delivery carriers.

[0025] (III) Beneficial Effects

[0026] This invention utilizes cobalt chloride to induce the culture of human umbilical cord mesenchymal stem cells under normoxic conditions, effectively mimicking a hypoxic microenvironment and activating the intracellular hypoxia-inducible factor HIF-1α signaling pathway. mTOR and 4EBP-1 act as key nodes, regulating protein synthesis and cell proliferation, thereby significantly promoting the synthesis and secretion of small extracellular vesicles. Experimental verification shows that the protein concentration of small extracellular vesicles obtained by the method of this invention is increased by more than 60% compared to normoxic culture, and the overall yield is increased by approximately 60%, effectively solving the problem of insufficient small extracellular vesicle yield in traditional methods and providing a sufficient material basis for large-scale application.

[0027] Furthermore, the preparation method of this invention does not rely on expensive physical hypoxia culture equipment. Highly efficient induction culture can be achieved simply by adding cobalt chloride solution to a conventional culture medium, reducing equipment investment costs by more than 80%. The operation process is compatible with standard cell culture techniques, requiring no complex gas control or professional training, thus significantly simplifying the production process. This simple and economical preparation method is easily implemented in existing cell culture production lines, significantly improving the production efficiency and scalability of small extracellular vesicles, and meeting the urgent needs of regenerative medicine and other fields for high-yield, low-cost small extracellular vesicles. Attached Figure Description

[0028] Figure 1 This is a morphological diagram of human umbilical cord mesenchymal stem cells after culture.

[0029] Figure 2 These are TEM images of the small extracellular vesicles obtained in Example 1 under different scales.

[0030] Figure 3 TEM images of small extracellular vesicles cultured using existing techniques at different scales. Detailed Implementation

[0031] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] This invention provides a method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells, comprising the following steps:

[0033] S1: Under normoxic conditions, human umbilical cord mesenchymal stem cells were induced and cultured in a medium containing cobalt chloride to obtain cell culture medium.

[0034] S2: Extract and purify small extracellular vesicles from the cell culture medium to obtain small extracellular vesicles of human umbilical cord mesenchymal stem cells.

[0035] This invention utilizes cobalt chloride to induce the culture of human umbilical cord mesenchymal stem cells under normoxic conditions, effectively mimicking a hypoxic microenvironment and activating the intracellular hypoxia-inducible factor HIF-1α signaling pathway. mTOR and 4EBP-1 act as key nodes, regulating protein synthesis and cell proliferation, thereby significantly promoting the synthesis and secretion of small extracellular vesicles. Experimental verification shows that the protein concentration of small extracellular vesicles obtained by the method of this invention is more than 80% higher than that obtained by normoxic culture, and the overall yield is increased by approximately 60%, effectively solving the problem of insufficient small extracellular vesicle yield in traditional methods and providing a sufficient material basis for large-scale application.

[0036] Furthermore, the preparation method of this invention does not rely on expensive physical hypoxia culture equipment. Highly efficient induction culture can be achieved simply by adding cobalt chloride solution to a conventional culture medium, reducing equipment investment costs by more than 80%. The operation process is compatible with standard cell culture techniques, requiring no complex gas control or professional training, thus significantly simplifying the production process. This simple and economical preparation method is easily implemented in existing cell culture production lines, significantly improving the production efficiency and scalability of small extracellular vesicles, and meeting the urgent needs of regenerative medicine and other fields for high-yield, low-cost small extracellular vesicles.

[0037] Preferably, in step S1, human umbilical cord mesenchymal stem cells are induced and cultured for 4-8 hours in a culture medium containing cobalt chloride to obtain a cell culture medium, wherein the concentration of cobalt chloride in the culture medium is 80-120 μmol / L.

[0038] Preferably, in step S1 above, human umbilical cord mesenchymal stem cells are obtained as follows: a healthy full-term cesarean section umbilical cord at 34-42 weeks of gestation is taken, rinsed repeatedly three times with sterile saline to remove surface blood and connective tissue and avoid contamination by impurities, then the outer membrane and umbilical arteries and veins are removed, Wharton's jelly is retained, the tissue is cut into small pieces and mixed with culture medium, and then primary culture is performed using the tissue block adherence method to obtain human umbilical cord mesenchymal stem cells.

[0039] Specifically, after the tissue was minced and prepared into a tissue homogenate, it was mixed with a culture medium containing 1% penicillin and antibiotics, and then added to a culture flask. Primary culture was carried out at 37°C and 5% CO2 using the tissue block adhesion method. The medium was changed for the first time on day 5, the second time on day 10-12, and the third time on day 14, until the cell confluence reached 80-90% on day 14. The culture medium in the culture flask was a serum-free complete culture medium for mesenchymal stem cells containing 1% penicillin and antibiotics.

[0040] After 14 days of culture, when the cell confluence reaches 80-90%, digest with 0.25% trypsin for 2-3 minutes, then add culture medium to stop the digestion, and pipette to prepare a single-cell suspension. Then, distribute the suspension at a density of 1 cm³. 2The cells were passaged at a rate of 5000-8000 cells per cell in the culture area to obtain P1 generation human umbilical cord mesenchymal stem cells, which were then cultured to P3-P4 generation.

[0041] Preferably, in step S1 above, under normoxic conditions, human umbilical cord mesenchymal stem cells of passages P4-P5 with a viability ≥95% are introduced at a rate of 8000-10000 cells / cm³. 2 The culture medium was inoculated at a density in T75 culture flasks. After culturing for 3 days, the medium was replaced with one containing cobalt chloride to achieve a cobalt chloride concentration of 80-120 μmol / L. The culture was then induced at 37°C and 5% CO2.

[0042] The preparation method of the culture medium containing cobalt chloride is as follows: add cobalt chloride solution to the serum-free complete culture medium for mesenchymal stem cells, filter it through a 0.22 μm filter membrane to remove bacteria, and obtain a culture medium with a cobalt chloride concentration of 80-120 μmol / L. Store at -20℃ and the shelf life is 3 months.

[0043] Preferably, step S2 includes the following steps:

[0044] S21: Centrifuge the cell culture medium at 4℃ for 25-35 minutes at a centrifugal force of 2500g-3500g, and obtain the supernatant after filtration.

[0045] S22: The supernatant was subjected to tangential flow filtration. The tangential flow filtration system was equipped with a filter membrane with a molecular weight cutoff of 0.45 μm. The peristaltic pump speed was adjusted to ≤100 rpm / min and the transmembrane pressure was controlled to ≤1.0 bar. The supernatant was concentrated to 10%±2% of its original volume. Then, physiological saline was added for replacement. The flow rate of the peristaltic pump was adjusted to ≤50 rpm / min to obtain the original solution of small extracellular vesicles.

[0046] S23: The collected human umbilical cord mesenchymal stem cell small extracellular vesicle stock solution was sterilized by filtration using a 0.22μm vesicle filter with an initial pressure difference ≤0.05MPa. During the filtration process, the pressure difference should be controlled between 0.1MPa and 0.2MPa to obtain human umbilical cord mesenchymal stem cell small extracellular vesicles.

[0047] The small extracellular vesicles prepared by the method of the present invention can be used to prepare anti-aging skin care products, drugs for treating skin trauma, and drug delivery carriers.

[0048] To further clarify the present invention and its technological advancements, the following description is provided in conjunction with specific embodiments and technical effects.

[0049] Example 1

[0050] This embodiment provides a method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells, comprising the following steps:

[0051] S1: Harvest the umbilical cord from a healthy full-term cesarean section at 38 weeks of gestation. Remove the outer membrane and umbilical arteries and veins, retain Wharton's jelly, mince the tissue, prepare a tissue homogenate, mix it with culture medium, and then add it to a culture flask. Primary culture is performed at 37°C and 5% CO2 using the tissue block adhesion method. The first medium change is performed on day 5, the second on day 11, and the third on day 14. On day 14, the cell confluence reaches 86.7%. Then, digest with 0.25% trypsin for 2 minutes, add culture medium to stop digestion, and pipette to prepare a single-cell suspension. Cells are then cultured at a density of 1 cm³. 2 Cells were passaged at a rate of 8000 cells per culture flask to obtain P1 generation human umbilical cord mesenchymal stem cells, which were then cultured to P3-P4 generations. The culture medium in the culture flasks was serum-free complete mesenchymal stem cell culture medium containing 1% penicillin-dextrose antibody.

[0052] Under normoxic conditions, human umbilical cord mesenchymal stem cells of passages P4-P5 with a viability ≥95% were cultured at 8000 cells / cm³. 2 The cells were inoculated into culture flasks at a density of [insert density here], and after 3 days of culture, the medium was replaced with one containing cobalt chloride to achieve a cobalt chloride concentration of 100 μmol / L. The cells were then induced to culture at 37°C and 5% CO2 for 6 hours. The preparation method of the cobalt chloride-containing medium in this step is as follows: Cobalt chloride solution was added to serum-free complete medium for mesenchymal stem cells, and the solution was filtered through a 0.22 μm filter membrane to obtain a 100 μmol / L cobalt chloride stock solution, which was then stored at -20°C.

[0053] S2: The cell culture medium was centrifuged at 3000g for 30 min at 4℃, and the supernatant was obtained after filtration. The supernatant was then subjected to tangential flow filtration using a membrane with a molecular weight cutoff of 0.45 μm. The peristaltic pump speed was adjusted to 50 rpm / min, and the transmembrane pressure was controlled at 1.0 bar. The supernatant was concentrated to 10% of its original volume, and then physiological saline was added for replacement. The peristaltic pump flow rate was adjusted to 30 rpm / min to obtain the small extracellular vesicle stock solution. The collected small extracellular vesicle stock solution was sterilized using a 0.22 μm vesicle filter with an initial pressure difference of 0.04 MPa. The pressure difference was controlled at 0.1 MPa during filtration to obtain small extracellular vesicles of human umbilical cord mesenchymal stem cells.

[0054] Example 2

[0055] This embodiment provides a method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells, comprising the following steps:

[0056] S1: Harvest the umbilical cord from a healthy full-term cesarean section at 34 weeks of gestation. Remove the outer membrane and umbilical arteries and veins, retain Wharton's jelly, mince the tissue, prepare a tissue homogenate, mix it with culture medium, and then add it to a culture flask. Primary culture is performed at 37°C and 5% CO2 using the tissue block adhesion method. The first medium change is performed on day 5, the second on day 11, and the third on day 14. On day 14, cell confluence reaches 89%. Then, digest with 0.25% trypsin for 3 minutes, add culture medium to stop digestion, and pipette to prepare a single-cell suspension. Cells are then cultured at a density of 1 cm³. 2 Cells were passaged at a rate of 8000 cells per culture flask to obtain P1 generation human umbilical cord mesenchymal stem cells, which were then cultured to P3-P4 generations. The culture medium in the culture flasks was serum-free complete mesenchymal stem cell culture medium containing 1% penicillin-dextrose antibody.

[0057] Under normoxic conditions, human umbilical cord mesenchymal stem cells of passages P4-P5 with a viability ≥95% were cultured at a rate of 9000 cells / cm². 2 The cells were inoculated into the culture medium at a density of [insert density here], and after 3 days of culture, the medium was replaced with one containing cobalt chloride to achieve a cobalt chloride concentration of 80 μmol / L. The cells were then induced to culture at 37°C and 5% CO2 for 8 hours. The preparation method of the cobalt chloride-containing culture medium in this step is as follows: Cobalt chloride solution was added to serum-free complete culture medium for mesenchymal stem cells, and the solution was filtered through a 0.22 μm filter membrane to obtain a cobalt chloride stock solution with a concentration of 80 μmol / L, which was then stored at -20°C.

[0058] S2: The cell culture medium was centrifuged at 3000g for 25 min at 4℃, and the supernatant was obtained after filtration. The supernatant was then subjected to tangential flow filtration using a membrane with a molecular weight cutoff of 0.45 μm. The peristaltic pump speed was adjusted to 40 rpm / min, and the transmembrane pressure was controlled at 1.0 bar. The supernatant was concentrated to 12% of its original volume, and then physiological saline was added for replacement. The peristaltic pump flow rate was adjusted to 30 rpm / min to obtain the small extracellular vesicle stock solution. The collected small extracellular vesicle stock solution was sterilized using a 0.22 μm vesicle filter with an initial pressure difference of 0.05 MPa. The pressure difference was controlled at 0.2 MPa during filtration to obtain human umbilical cord mesenchymal stem cell small extracellular vesicles.

[0059] Example 3

[0060] This embodiment provides a method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells, comprising the following steps:

[0061] S1: Harvest the umbilical cord from a healthy full-term cesarean section at 42 weeks of gestation. Remove the outer membrane and umbilical arteries and veins, retain Wharton's jelly, mince the tissue, prepare a tissue homogenate, mix it with culture medium, and then add it to a culture flask. Primary culture is performed at 37°C and 5% CO2 using the tissue block adhesion method. The first medium change is performed on day 5, the second on day 11, and the third on day 14. On day 14, cell confluence reaches 84%. Then, digest with 0.25% trypsin for 2 minutes, add culture medium to stop digestion, and pipette to prepare a single-cell suspension. Cells are then cultured at a density of 1 cm³. 2 Cells were passaged at a rate of 9000 cells per culture flask to obtain P1 generation human umbilical cord mesenchymal stem cells, which were then cultured to P3-P4 generations. The culture medium in the culture flasks was serum-free complete mesenchymal stem cell culture medium containing 1% penicillin-dextrose antibody.

[0062] Under normoxic conditions, human umbilical cord mesenchymal stem cells of passages P4-P5 with a viability ≥95% were cultured at a rate of 10,000 cells / cm³. 2 The cells were inoculated into culture flasks at a density of [insert density here], and after 3 days of culture, the medium containing cobalt chloride was replaced with a cobalt chloride stock solution to achieve a cobalt chloride concentration of 120 μmol / L. The culture was then induced at 37°C and 5% CO2 for 4 hours. The preparation method of the cobalt chloride-containing medium in this step is as follows: cobalt chloride solution was added to serum-free complete mesenchymal stem cell culture medium, and the solution was filtered through a 0.22 μm filter membrane for sterilization to obtain a cobalt chloride stock solution with a concentration of 120 μmol / L, which was then stored at -20°C.

[0063] S2: The cell culture medium was centrifuged at 2500g for 30 min at 4℃, and the supernatant was obtained after filtration. The supernatant was then subjected to tangential flow filtration using a membrane with a molecular weight cutoff of 0.45 μm. The peristaltic pump speed was adjusted to 60 rpm / min, and the transmembrane pressure was controlled at 1.0 bar. The supernatant was concentrated to 8% of its original volume, and then physiological saline was added for replacement. The peristaltic pump flow rate was adjusted to 50 rpm / min to obtain the small extracellular vesicle stock solution. The collected small extracellular vesicle stock solution was sterilized using a 0.22 μm vesicle filter with an initial pressure difference of 0.03 MPa. The pressure difference was controlled at 0.1 MPa during filtration to obtain small extracellular vesicles of human umbilical cord mesenchymal stem cells.

[0064] Comparative Example 1

[0065] This comparative example provides a method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells. The difference from Example 1 is that cobalt chloride is not used, and the induction culture time in step S1 is 5 days.

[0066] Comparative Example 2

[0067] This comparative example provides a method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells. The difference from Example 1 is that after replacing the culture medium containing cobalt chloride, the concentration of cobalt chloride in the culture medium is 40 μmol / L.

[0068] Comparative Example 3

[0069] This comparative example provides a method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells. The difference from Example 1 is that in step S2, after replacing the culture medium containing cobalt chloride, the concentration of cobalt chloride in the culture medium is 300 μmol / L.

[0070] The small extracellular vesicles prepared in Examples 1-3 and Comparative Examples 1-3 were analyzed and detected:

[0071] Figure 1 This is a morphological image of human umbilical cord mesenchymal stem cells after culture, as shown in Example 1. Figure 1 It is known that the human umbilical cord mesenchymal stem cells cultured in this invention have a long spindle-shaped adherent cell morphology. Furthermore, long spindle-shaped human umbilical cord mesenchymal stem cells were also cultured in Examples 2-3.

[0072] Figure 2 These are TEM images of the small extracellular vesicles obtained in Example 1 under different scales. Figure 3 TEM morphology image of small extracellular vesicles cultured using existing techniques.

[0073] pass Figure 2 as well as Figure 3 It can be seen that the morphology of the extracellular vesicles of human umbilical cord mesenchymal stem cells induced by cobalt chloride in Example 1 is the same as that of the extracellular vesicles of human umbilical cord mesenchymal stem cells induced by non-cobalt chloride culture, both exhibiting a cup-shaped structure. Therefore, the use of cobalt chloride does not affect the morphology of the extracellular vesicles. In addition, the morphology of the extracellular vesicles prepared in Examples 2-3 is the same as that in Example 1, all exhibiting a cup-shaped structure.

[0074] Furthermore, the Coulter method analysis showed that the particle size of the small extracellular vesicles prepared in Examples 1-3 was concentrated in the range of 30-150 nm, which meets the standard for small extracellular vesicles. Western blotting analysis showed that the small extracellular vesicles prepared in Examples 1-3 were positive for CD63, CD9, and CD81, confirming the purity and authenticity of the products.

[0075] To comprehensively evaluate the superiority of the preparation method of the present invention, a systematic evaluation of the yield (expressed as protein concentration) and key biological functions (immunomodulatory activity) of the small extracellular vesicles obtained in each embodiment and comparative example was conducted. Specific test results are as follows:

[0076] 1. Analysis of extracellular vesicle production (represented by BCA protein concentration):

[0077] The total protein concentration in small extracellular vesicle suspensions of Examples 1-3 and Comparative Examples 1-3 at the same concentration was determined using the BCA protein quantification method, and this concentration was used as the core indicator for measuring sEV yield. A saline group was also included as a negative control group to eliminate interference from the solvent itself. The specific BCA content is shown in Table 1.

[0078] Table 1. Statistical table of BCA content in small extracellular vesicles prepared in Examples 1-3 and Comparative Examples 1-3.

[0079]

[0080] As shown in Table 1, the average sEV protein concentration in Comparative Example 1, which was not induced by cobalt chloride, was 2.095 mg / mL. This data represents the basal secretion level of human umbilical cord mesenchymal stem cells under conventional normoxic culture conditions.

[0081] The average sEV protein concentrations in Examples 1-3 were 3.945 mg / mL, 3.74 mg / mL, and 3.905 mg / mL, respectively. Compared to Comparative Example 1, the yield of Example 1 increased by approximately 88.3%, Example 2 by approximately 78.5%, and Example 3 by approximately 86.2%. The average increase for all three exceeded 80%. Furthermore, the yields of Comparative Examples 2 and 3 were significantly lower than those of Examples 1-3, indicating that the concentration of cobalt chloride directly affects the yield. This significant difference demonstrates that the present invention, through induction with a specific concentration of cobalt chloride under normoxic conditions, can effectively activate the HIF-1α signaling pathway in hUC-MSCs. mTOR and 4EBP-1, as key nodes, regulate protein synthesis and cell proliferation, thereby greatly promoting the synthesis and secretion of small extracellular vesicles and significantly increasing their yield. This directly solves the problem of low yield of small extracellular vesicles in the prior art.

[0082] 2. Analysis of the immunomodulatory function of small extracellular vesicles (represented by the inhibition of lymphocyte proliferation):

[0083] To evaluate the biological activity of the small extracellular vesicles obtained in each example and comparative example, an in vitro lymphocyte proliferation inhibition assay was performed. This assay is a key indicator for evaluating the immunomodulatory capacity of mesenchymal stem cell-derived sEVs. A negative control group and a positive control group were set up to ensure the validity and reliability of the experimental system; specific inhibition rate data are shown in Table 2. In the negative control group, phosphate-buffered saline (PBS) was used instead of the sEV sample. This group was designed to simulate a blank condition without any intervention to determine the basal proliferation level of lymphocytes under standard stimulation, thereby eliminating the influence of solvents or the operation itself on the experimental results. The positive control group used cyclosporine A, a known potent immunosuppressant, as a reference standard to verify the maximum response of the experimental system.

[0084] Table 2. Statistical table of the inhibition rate of lymphocyte proliferation by small extracellular vesicles prepared in Examples 1-3 and Comparative Examples 1-3.

[0085]

[0086] As shown in Table 2, the control group (Example 1) without cobalt chloride induction culture exhibited an average inhibition rate of 25.55% on lymphocyte proliferation by sEVs. This data represents the baseline immunomodulatory level of sEVs secreted by human umbilical cord mesenchymal stem cells under conventional normoxic culture conditions.

[0087] The average inhibition rates of sEVs in Examples 1-3 were 32.05%, 42.95%, and 35.30%, respectively. Compared with Comparative Example 1, the function of Example 1 was enhanced by approximately 25.4%, Example 2 by approximately 68.1%, and Example 3 by approximately 38.1%. Furthermore, the inhibition rates of Comparative Examples 2 and 3 were significantly lower than those of Examples 1-3, indicating that the concentration of cobalt chloride directly affects the functional properties of sEVs. These significant differences demonstrate that the present invention, through induction with a specific concentration of cobalt chloride under normoxic conditions, significantly increases sEV yield while effectively enhancing or at least fully preserving its key immunomodulatory functions, rather than producing a large number of functionally deficient products. This indicates that the sEVs prepared by the method of the present invention are not only abundant but also of high quality, fundamentally solving the problem of high production costs caused by insufficient yield of small extracellular vesicles in existing technologies, and providing a high-quality material basis for large-scale applications.

[0088] In summary, this invention, through systematic experimental verification, demonstrates that the method of cobalt chloride-induced culture under normoxic conditions can efficiently prepare small extracellular vesicles of human umbilical cord mesenchymal stem cells with both high yield and strong function. This product not only solves the bottleneck problem of high production costs due to low yield in existing technologies, but more importantly, its superior immunomodulatory activity has been confirmed.

[0089] Based on the above-mentioned superior technical effects, the hUC-MSC-sEV prepared by this invention is particularly suitable for the following application areas:

[0090] ① Application in the preparation of anti-aging skincare products: Skin aging is closely related to persistent low-grade chronic inflammation. The powerful immunomodulatory and anti-inflammatory functions of the sEV in this invention can effectively intervene in this process, reduce inflammatory damage to skin tissue, thereby delaying signs of aging and improving skin texture. Combined with its yield increase of over 80%, it provides ample material support for the development of high-performance, cost-effective, and mass-producible high-end skincare products.

[0091] ② Application in the preparation of drugs for treating skin trauma: Wound healing is a complex and dynamic process in which precise regulation of the inflammatory phase is crucial. Excessive or persistent inflammatory responses can hinder tissue regeneration and lead to scar formation. The significant immunomodulatory capabilities exhibited by the sEV of this invention enable it to effectively balance the level of inflammation at the wound site, creating a more favorable microenvironment for tissue repair, thereby accelerating wound healing and reducing scarring. Its high-yield characteristics also meet the high-dose requirements of biological agents in wound treatment.

[0092] ③ Application in the preparation of drug delivery carriers: As a natural nanoscale biological carrier, sEVs have attracted much attention due to their low immunogenicity and good biocompatibility. This invention enables the large-scale, low-cost production of sEVs. Furthermore, while delivering anticancer drugs, the sEVs of this invention also possess anti-inflammatory properties that help improve the immunosuppressed tumor microenvironment, achieving a synergistic therapeutic effect.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells, characterized in that, Includes the following steps: S1: Under normoxic conditions, human umbilical cord mesenchymal stem cells were induced and cultured in a medium containing cobalt chloride to obtain cell culture medium; S2: Extract and purify small extracellular vesicles from the cell culture medium to obtain small extracellular vesicles of human umbilical cord mesenchymal stem cells.

2. The method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step S1, human umbilical cord mesenchymal stem cells are induced and cultured for 4-8 hours in a culture medium containing cobalt chloride to obtain cell culture medium; the concentration of cobalt chloride in the culture medium is 80-120 μmol / L.

3. The method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step S1, human umbilical cord mesenchymal stem cells are prepared as follows: umbilical cords delivered by cesarean section at 34-42 weeks of gestation are taken, the outer membrane and umbilical arteries and veins are removed, Wharton's jelly is retained, the tissue is minced and mixed with culture medium, and then primary culture is performed using the tissue block adherence method to obtain human umbilical cord mesenchymal stem cells.

4. The method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells according to claim 3, characterized in that, After the tissue was minced and homogenized, it was mixed with the culture medium and then added to a culture flask. Primary culture was carried out at 37°C and 5% CO2 using the tissue block adhesion method. The medium was changed for the first time on day 5, the second time on day 10-12, and the third time on day 14, until the cell confluence reached 80-90% on day 14. When the cell confluence reached 80%-90%, the cells were digested with trypsin, and then digestion was stopped. The cells were pipetted to form a single-cell suspension and then seeded and passaged until the P3-P4 generation.

5. The method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step S1, under normoxic conditions, the obtained P3-P4 generation human umbilical cord mesenchymal stem cells with a viability ≥95% are processed at a rate of 8000-10000 cells / cm³. 2 The culture medium was inoculated at a density of 80-120 μmol / L in culture flasks. After 3 days of culture, the medium was replaced with one containing cobalt chloride to achieve a cobalt chloride concentration of 80-120 μmol / L. The culture was then induced at 37°C and 5% CO2.

6. The method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells according to claim 5, characterized in that, In step S1, the preparation method of the culture medium containing cobalt chloride is as follows: Cobalt chloride solution is added to the serum-free complete culture medium for mesenchymal stem cells, and after filtration through a 0.22 μm filter membrane for sterilization, a culture medium with a cobalt chloride concentration of 80-120 μmol / L is obtained.

7. The method for preparing small extracellular vesicles of human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, Step S2 includes the following steps: S21: Centrifuge the cell culture medium at 4℃, centrifuge at 2500g-3500g for 25-35min, and obtain the supernatant after filtration. S22: The supernatant was subjected to tangential flow filtration. The tangential flow filtration system was equipped with a filter membrane with a molecular weight cutoff of 0.45 μm. The peristaltic pump speed was adjusted to ≤100 rpm / min and the transmembrane pressure was controlled to ≤1.0 bar. The supernatant was concentrated to 10%±2% of the original volume. Then, physiological saline was added for replacement. The flow rate of the peristaltic pump was adjusted to ≤50 rpm / min to obtain the original solution of small extracellular vesicles. S23: The collected small extracellular vesicle stock solution was sterilized by filtration using a 0.22μm vesicle filter with an initial pressure difference ≤0.05MPa. During the filtration process, the pressure difference was controlled between 0.1-0.2MPa to obtain small extracellular vesicles of human umbilical cord mesenchymal stem cells.

8. The application of the small extracellular vesicles prepared by the preparation method according to any one of claims 1-7 in the preparation of anti-aging skin care products.

9. The use of a small extracellular vesicle prepared by the preparation method according to any one of claims 1-7 in the preparation of a medicament for treating skin trauma.

10. The application of small extracellular vesicles prepared by the preparation method according to any one of claims 1-7 in the preparation of drug delivery carriers.

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