Method for inducing exosomes by starvation combined with gradient hypoxia and exosomes prepared and applications thereof

Abstract

The application discloses a method for inducing exosomes by adopting starvation combined gradient hypoxia and the exosomes prepared by the method and application, and belongs to the technical field of biological medicine. In view of the problems of low yield and insufficient activity of the existing exosome preparation method, a gradient hypoxia domestication culture strategy is provided. The method comprises sequentially performing stage hypoxia starvation culture on seed cells under a higher oxygen concentration and a lower oxygen concentration. Through the combined gradient induction mode, the yield of exosomes can be significantly improved, which is more than 2 times that of a normal oxygen group and more than 30% that of a single hypoxia group, and the repair promotion and anti-inflammatory activity thereof is enhanced. The exosomes prepared by the application are uniform in particle size, high in purity and activity, the process is stable and controllable, and are suitable for large-scale production, and are suitable for use in the fields of regenerative medicine, drug delivery and clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing exosomes from starved supernatant induced by hypoxia conditions, and more particularly to a method for preparing high-yield, highly active exosomes using a hypoxia gradient induction method combined with starvation culture, as well as the application of the exosomes prepared thereby in the fields of regenerative medicine, drug delivery, and clinical treatment. Background Technology

[0002] Exosomes are extracellular vesicles formed by cells through processes such as endocytosis, fusion, and efflux, typically ranging in diameter from 30 to 150 nm. They carry various bioactive molecules, including proteins, mRNA, miRNA, and lipids, and serve as important mediators for intercellular communication and substance exchange. In recent years, research has revealed significant roles for exosomes in tissue repair, anti-inflammation, and immune regulation, making them a research hotspot in regenerative medicine and drug delivery.

[0003] Currently, the laboratory preparation and large-scale production of exosomes mostly employ normoxic cell culture. However, the yield of exosomes secreted by cells under normoxic conditions is low, and the enrichment of functional bioactive factors in the obtained exosomes is insufficient, resulting in limited repair and anti-inflammatory activities, which is difficult to meet the needs of clinical translation and industrial applications.

[0004] Studies have shown that a hypoxic environment can mimic the physiological or pathological hypoxic microenvironment in vivo, activate the hypoxia-inducible factor (HIF-1α) pathway in cells, and thus promote paracrine function. This not only upregulates the total amount of exosome secretion but also optimizes the composition of exosome contents (such as pro-angiogenic factors and anti-inflammatory factors), thereby enhancing their therapeutic potential.

[0005] Most existing techniques for preparing hypoxia-induced exosomes employ a single, constant hypoxia concentration (e.g., 1% or 5% O2) for culture. For example, patent publication CN111000868A discloses a hypoxia-treated stem cell exosome method that uses a constant hypoxia concentration of 1%–5% to stimulate stem cells. Patent publication CN110115769A uses a constant hypoxia concentration of 0.5%–5% to target and modify stem cells. However, these methods have the following drawbacks: 1) The induction conditions are singular, lacking a gradient acclimatization process, making cells prone to stress responses in abruptly changed hypoxia environment, affecting cell activity and the stability of exosome secretion; 2) Although yields are improved, they are still insufficient to support large-scale applications; 3) There is a lack of standardized downstream purification processes that match the hypoxia process, making it difficult to guarantee the purity and activity of exosomes. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to provide a method for inducing exosomes through a combination of starvation and gradient hypoxia, as well as the resulting exosomes and their applications. By simulating the physiological process of cells gradually adapting to a hypoxic microenvironment, this method significantly improves the yield and bioactivity of exosomes, and the process is stable and controllable, providing a new solution for the large-scale production and clinical application of exosomes.

[0007] To address the aforementioned technical problems, this invention provides a method for inducing exosomes using a combination of starvation and gradient hypoxia, comprising the following steps: starving cells, then culturing the starved cells under gradient hypoxia, collecting the culture supernatant, and separating exosomes; wherein the gradient hypoxia culture involves placing the cells in a first hypoxia concentration for 20 days. After 28 hours, it was then incubated under a second low oxygen concentration for 20 hours. 28h; wherein the first hypoxia concentration is higher than the second hypoxia concentration, and both the first hypoxia concentration and the second hypoxia concentration are lower than the normoxic concentration.

[0008] Furthermore, the starvation culture refers to culturing cells to a confluence of 70%. After reaching 80%, the medium was replaced with phenol red-free basal medium and cultured under normoxic conditions for 20 days. 28 hours.

[0009] Furthermore, the first low oxygen concentration is 8% by volume of oxygen. 12%, the second low oxygen concentration is 4% by volume of oxygen. 6%.

[0010] Furthermore, the cells subjected to starvation culture were from the 3rd to the 6th generation.

[0011] Furthermore, the specific steps include: (1) Cell expansion culture: Seed cells were inoculated into a medium containing 5% Cells were cultured in DMEM / F12 medium containing 15% fetal bovine serum at 37°C and 5% CO2 until 70% confluence was achieved. 80%; (2) Starvation treatment: Discard the original culture medium and replace it with phenol red-free DMEM / F12 medium, and continue to culture at 37℃ and 5% CO2 for 20 days. 28 hours; (3) Gradient hypoxia induction: The cells treated in step (2) were transferred to a three-gas incubator, and first incubated at an oxygen volume fraction of 8%. Cultured at 12% concentration, 5% carbon dioxide concentration, and 37°C for 20 days 28 hours; then adjust the oxygen concentration to 4%. 6%, other conditions unchanged, continue training 20 Collect and culture supernatant over 28 hours; (4) Centrifuge the collected culture supernatant to remove cell debris and impurities and obtain hypoxia-induced exosomes.

[0012] Furthermore, the centrifugation in step (4) is performed at 4°C with 3000g for 10 minutes.

[0013] The exosomes prepared by the above method.

[0014] Furthermore, the exosomes have a particle size of 30 mm. The exosomes were 150 nm in size and expressed positively CD9, CD63, and TSG101, and negatively expressed Calnexin. The yield of the exosomes was more than 2 times higher than that of exosomes prepared by normoxic culture, more than 30% higher than that of exosomes prepared by single hypoxic culture, and more than 3 times higher than that of exosomes prepared by hyperoxic culture.

[0015] An exosome preparation comprising the above-described exosomes and a pharmaceutically acceptable carrier or excipient.

[0016] The use of the above-mentioned exosomes or exosome preparations in the preparation of drugs for tissue repair, anti-inflammation or drug delivery.

[0017] Compared with the prior art, the present invention has the following significant advantages: 1. Significantly Increased Yield: Through a unique gradient hypoxia acclimatization strategy of "10% O2 pretreatment + 5% O2 induction," cells can better adapt to the hypoxic environment, activating highly efficient exosome synthesis and secretion pathways. Experimental results show that compared with the normoxic culture group, the exosome yield prepared by the method of this invention is increased by more than 2 times; compared with the single 5% hypoxia culture group, the yield is increased by more than 30%.

[0018] 2. Enhanced biological activity: Gradient hypoxia induction significantly enriches exosomes with tissue repair-promoting and anti-inflammatory active factors (such as specific miRNAs and growth factors). In vitro cell experiments demonstrated that the exosomes of this invention promoted fibroblast migration and proliferation by more than 50% compared to the normoxic group, and reduced the expression level of the inflammatory factor TNF-α by more than 40% in the LPS-induced inflammation model.

[0019] 3. Stable and controllable process: The gradient induction method avoids stress-induced apoptosis caused by sudden environmental changes, resulting in high cell survival rate, stable culture process, and small batch-to-batch differences, making it very suitable for large-scale industrial production.

[0020] 4. High purity and uniform quality: Combining optimized differential centrifugation with size exclusion chromatography or ultracentrifugation purification process, the obtained exosomes have a particle size mainly concentrated between 30-150nm, positively expressing CD9, CD63, and TSG101 markers, and negatively expressing the endoplasmic reticulum protein Calnexin, which meets the standards of the International Society for Extracellular Vesicles (ISEV). Attached Figure Description

[0021] Figure 1 This is a particle size distribution diagram (NTA detection) of the exosomes obtained in Example 1 of the present invention.

[0022] Figure 2 Western blotting diagram for biomarker identification of starved cultured exosomes obtained in Example 1 of this invention (CD9, CD63, TSG101, Calnexin).

[0023] Figure 3 Electron micrographs of starved cultured exosomes obtained in Group A of Example 1, Group D of Example 2, and Group G of Example 3 of this invention.

[0024] Figure 4 This is a graph showing the healing rate in a scratch test.

[0025] Figure 5 The graph shows the quantitative results of HSF cell Transwell assay.

[0026] Figure 6 This is a graph showing the levels of the inflammatory factor TNF-α. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, it should be understood that the specific embodiments of this invention are only for explaining the invention and are not intended to limit the scope of protection of this invention. All equivalent substitutions or modifications made based on the technical solutions of this invention fall within the scope of protection of this invention.

[0028] The key to the method of inducing exosomes by starvation combined with gradient hypoxia in this invention is that it includes the steps of starvation culture and gradient hypoxia acclimatization culture of seed cells.

[0029] The starvation culture is as follows: after the cells are cultured to a confluence of 70-80%, the culture medium is replaced with phenol red-free DMEM / F12 medium (Baidi / L1041-500) and starved for 20-28 hours.

[0030] The gradient hypoxia acclimatization culture is as follows: cells that have been starved for 20-28 hours are placed in a first hypoxia concentration (8%-12%) for a first set culture time, and then placed in a second hypoxia concentration (4%-6%) for a second set culture time; wherein, the first hypoxia concentration is higher than the second hypoxia concentration.

[0031] As a preferred embodiment of the present invention, the starvation gradient hypoxia acclimatization culture specifically involves: first, culturing the cells under conditions of 8%-12% oxygen volume fraction for 20-28 hours, and then culturing the cells under conditions of 4%-6% oxygen volume fraction for another 20-28 hours.

[0032] As a preferred embodiment of the present invention, the method specifically includes the following steps: (1) Cell expansion culture: Seed cells were passaged and seeded in DMEM / F12 medium (Gibco / C11330500BT) containing 5-15% fetal bovine serum (EXCELL / FSP50) and cultured in a normal oxygen incubator at 37℃ and 5% CO2 until the cell confluence reached 70-80%.

[0033] (2) Starvation treatment: Discard the culture medium, wash with 0.9% sodium chloride injection, and replace with phenol red-free DMEM / F12 basal medium (Baidi / L1041-500). Continue to culture in a normal oxygen incubator at 37℃ and 5% CO2 for 20-28 hours.

[0034] (3) Gradient hypoxia induction: Transfer the cells treated in step (2) to a three-gas incubator and first culture them at an oxygen concentration of 8%-12%, a carbon dioxide concentration of 5%, and a temperature of 37°C for 20-28 hours; then adjust the oxygen concentration to 4%-6%, a carbon dioxide concentration of 5%, and a temperature of 37°C, and continue to culture for 20-28 hours, and collect the culture supernatant.

[0035] (4) Centrifuge the recovered culture supernatant to remove cell debris and impurities in the supernatant, and recover the supernatant to obtain hypoxia-induced exosomes.

[0036] As a preferred embodiment of the present invention, the basic culture medium in step (1) is DMEM / F12 culture medium (Gibco / C11330500BT), and the volume fraction of the fetal bovine serum (EXCELL / FSP500) is 5%-15%.

[0037] As a preferred embodiment of the present invention, the basic culture medium in step (1) is DMEM / F12 culture medium (Gibco / C11330500BT), which needs to be warmed to 37°C before use.

[0038] As a preferred embodiment of the present invention, the phenol red-free basal culture medium in step (2) is phenol red-free DMEM / F12 culture medium (Baidi / L1041-500).

[0039] As a preferred embodiment of the present invention, the phenol red-free basal culture medium in step (2) is phenol red-free DMEM / F12 culture medium (Baidi / L1041-500), which needs to be warmed to 37°C before use.

[0040] As a preferred embodiment of the present invention, the centrifugation parameters in step (4) are: centrifugation at 3000g for 10min and centrifugation temperature of 4℃.

[0041] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0042] Example 1 1. Isolation and culture of umbilical cord mesenchymal stem cells: Primary umbilical cord mesenchymal stem cells (UC-MSCs) were obtained by cutting up umbilical cords from healthy newborns and using the tissue block adherence method. They were cultured and passaged in DMEM / F12 complete medium (Gibco / C11330500BT) containing 5% fetal bovine serum (FBS) (EXCELL / FSP500) at 37°C and 5% CO2.

[0043] 2. Experimental grouping and treatment: UC-MSCs in good growth condition from generation P5 were selected and treated with 2×10⁻⁶ cells / mL. 4 The cells were seeded at a density of 100 cells / cm² into 6 T-175 culture flasks. When the cell confluence reached approximately 70%, the following treatment was performed: (1) Divide the 6 T-175 culture flasks into three groups: A, B and C, with 2 T-175 culture flasks in each group.

[0044] (2) Starvation culture: Discard the old culture medium, wash with 0.9% sodium chloride injection, and replace with phenol red-free DMEM / F12 culture medium (Baidi / L1041-500) for 24h.

[0045] (3) Gradient low oxygen group (Group A): Two bottles of starved cultured cells from Group A were placed in a three-gas incubator and the parameters were set as follows: 37℃, 5% CO2, 10% O2, and cultured for 20h; then the oxygen concentration was adjusted to 5%, and other conditions remained unchanged, and cultured for another 20h.

[0046] (4) Single hypoxia group (Group B): Two bottles of starved cultured cells from Group B were placed in a three-gas incubator at 37°C, 5% CO2, and 5% O2 and cultured continuously for 48 hours.

[0047] (5) Normoa group (C group): Two bottles of starved cells from group C were placed in a regular incubator at 37°C and 5% CO2 (approximately 21% O2) and cultured continuously for 48 hours.

[0048] 3. Collection of starvation culture supernatant: Collect the starvation culture supernatant from groups A, B, and C respectively, and label them A, B, and C. Centrifuge the starvation culture supernatant from groups A, B, and C at 4℃: 3000g for 10 min, and collect the supernatant for later use.

[0049] 4. Exosome analysis: (1) Particle size analysis: Exosome samples were analyzed using a nanoparticle tracking analyzer. The results showed that the particle size distribution of exosomes in the gradient hypoxia group of this invention ranged from 50 to 180 nm (see...). Figure 1 The average particle size is approximately 66 nm (see Table 1).

[0050] (2) Biomarker detection: Western blotting was used to detect biomarkers on the surface of exosomes. The results showed that the exosomes in the gradient hypoxia group of this invention positively expressed CD9, CD63 and ALIX (see...). Figure 2 It meets the standards for exosome identification.

[0051] (3) Yield Comparison: Exosome protein concentration was determined using a BCA protein quantification kit, and the total protein content reflected the relative exosome yield. The exosome yield (based on total protein) in the gradient hypoxia group of this invention was 2.52 times that of the normoxic group and 1.4 times that of the single hypoxia group (i.e., a 40% increase in yield). Specific data are shown in Table 2. The absorbance of the mesenchymal stem cell sample purified by ultracentrifugation was measured at a specific wavelength of 562 nm, and the sample absorbance was 0.193 (R0). 2 The value was >0.99, and the calculated sample concentration was 38.046 μg / mL.

[0052] (4) Observe and photograph under a transmission electron microscope, as shown in the image. Figure 3 .

[0053] Example 2 1. Isolation and culture of umbilical cord mesenchymal stem cells: Primary umbilical cord mesenchymal stem cells (UC-MSCs) were obtained by cutting up umbilical cords from healthy newborns and using the tissue block adherence method. They were cultured and passaged in DMEM / F12 complete medium (Gibco / C11330500BT) containing 10% fetal bovine serum (FBS) (EXCELL / FSP500) at 37°C and 5% CO2.

[0054] 2. Experimental grouping and treatment: UC-MSCs in good growth condition from generation P5 were selected and treated with 2×10⁻⁶ cells / mL. 4The cells were seeded at a density of 100 cells / cm² into 6 T-175 culture flasks. When the cell confluence reached approximately 75%, the following treatment was performed: (1) Divide the 6 T-175 culture flasks into three groups: D, E and F, with 2 T-175 culture flasks in each group.

[0055] (2) Starvation culture: Discard the old culture medium, wash with 0.9% sodium chloride injection, and replace with phenol red-free DMEM / F12 culture medium (Baidi / L1041-500) for 24h.

[0056] (3) Gradient low oxygen group (Group D): Two bottles of starved cultured cells from Group D were placed in a three-gas incubator and the parameters were set as follows: 37℃, 5% CO2, 10% O2, and cultured for 24h; then the oxygen concentration was adjusted to 5%, and other conditions remained unchanged, and cultured for another 24h.

[0057] (4) Single hypoxia group (Group E): Two bottles of starved cultured cells from Group E were placed in a three-gas incubator at 37°C, 5% CO2, and 5% O2 and cultured continuously for 48 hours.

[0058] (5) Normoa group (F group): Two bottles of starved cells from group F were placed in a regular incubator at 37°C and 5% CO2 (about 21% O2) and cultured continuously for 48 hours.

[0059] 3. Collection of starvation culture supernatant: Collect the starvation culture supernatant from groups D, E, and F respectively, and label them D, E, and F. Centrifuge the starvation culture supernatant from groups D, E, and F at 4℃: 3000g for 10 minutes, and collect the supernatant for later use.

[0060] 4. Exosome analysis: (1) Particle size analysis: Exosome samples were analyzed using a nanoparticle tracking analyzer. The results showed that the particle size distribution of exosomes in the gradient hypoxia group of this invention ranged from 50 to 180 nm (see...). Figure 1 The average particle size is approximately 89 nm (see Table 1).

[0061] (2) Yield Comparison: Exosome protein concentration was determined using a BCA protein quantification kit, and the relative exosome yield was reflected by the total protein content. The exosome yield (based on total protein) in the gradient hypoxia group of this invention was 2.49 times that of the normoxic group and 1.28 times that of the single hypoxia group. Specific data are shown in Table 2. The absorbance of the mesenchymal stem cell sample purified by ultracentrifugation at a specific wavelength of 562 nm was 0.201 (R0). 2 The value was >0.99, and the calculated sample concentration was 47.105 μg / mL.

[0062] Example 3 1. Isolation and culture of umbilical cord mesenchymal stem cells: Primary umbilical cord mesenchymal stem cells (UC-MSCs) were obtained by cutting up umbilical cords from healthy newborns and using the tissue block adherence method. They were cultured and passaged in DMEM / F12 complete medium (Gibco / C11330500BT) containing 15% fetal bovine serum (FBS) (EXCELL / FSP500) at 37°C and 5% CO2.

[0063] 2. Experimental grouping and treatment: UC-MSCs in good growth condition from generation P5 were selected and treated with 2×10⁻⁶ cells / mL. 4 The cells were seeded at a density of 1 cell / cm² in 6 T-175 culture flasks. When the cell confluence reached approximately 80%, the following treatment was performed: (1) Divide the 6 T-175 culture flasks into three groups: G, H and I, with 2 T-175 culture flasks in each group.

[0064] (2) Starvation culture: Discard the old culture medium, wash with 0.9% sodium chloride injection, and replace with phenol red-free DMEM / F12 culture medium (Baidi / L1041-500) for 24h.

[0065] (3) Gradient hypoxia group (G group): Two bottles of starved cultured cells from group G were placed in a three-gas incubator and the parameters were set as follows: 37℃, 5% CO2, 10% O2, and cultured for 28h; then the oxygen concentration was adjusted to 5%, and other conditions remained unchanged, and cultured for another 28h.

[0066] (4) Single hypoxia group (H group): Two bottles of starved cultured cells from group H were placed in a three-gas incubator at 37°C, 5% CO2, and 5% O2 and cultured continuously for 48 hours.

[0067] (5) Normoa group (Group I): Two bottles of starved cells from Group I were placed in a regular incubator at 37°C and 5% CO2 (approximately 21% O2) and cultured continuously for 48 hours.

[0068] 3. Collection of starvation culture supernatant: Collect the starvation culture supernatant of the three groups of cells (G, H, and I) separately, and label them as G, H, and I. Centrifuge the starvation culture supernatant of the three groups of cells at 4°C: 3000g for 10 minutes, and collect the supernatant for later use.

[0069] 4. Exosome analysis: (1) Particle size analysis: Exosome samples were analyzed using a nanoparticle tracking analyzer. The results showed that the particle size distribution of exosomes in the gradient hypoxia-induced group of this invention ranged from 50 to 180 nm (see...). Figure 1 The average particle size is approximately 106 nm (see Table 1).

[0070] (2) Yield Comparison: Exosome protein concentration was determined using a BCA protein quantification kit, and the relative exosome yield was reflected by the total protein content. The exosome yield (based on total protein) in the gradient hypoxia group of this invention was 2.63 times that of the normoxic group and 1.52 times that of the single hypoxia group. Specific data are shown in Table 2. The absorbance of the mesenchymal stem cell sample purified by ultracentrifugation at a specific wavelength of 562 nm was 0.212 (R0). 2 The value was >0.99, and the calculated sample concentration was 59.317 μg / mL.

[0071] Table 1. Average particle size data of starved cultured exosomes obtained in Examples 1, 2, and 3 of this invention, as detected by a nanoparticle tracking analyzer.

[0072] Table 2 shows the comparison data of exosome production under different culture conditions in Examples 1, 2, and 3 of this invention.

[0073] Example 4 1. To systematically compare the effects of different high oxygen concentrations on exosome production, this embodiment sets up a high oxygen culture group with an oxygen concentration of 30%.

[0074] 2. Cell culture and starvation treatment: P5 generation UC-MSCs were cultured to a confluence of about 75%, and then starved for 24 hours using phenol red-free DMEM / F12, the same method as in Example 1.

[0075] 3. Hyperoxygen-induced culture (30% O2): After starvation treatment, the cells were transferred into a three-gas incubator, and the oxygen concentration was set to 30%, the CO2 concentration to 5%, and the temperature to 37°C. The cells were cultured continuously for 48 hours.

[0076] 4. Exosome collection and quantification: Collect culture supernatant, centrifuge to remove cell debris, and determine the total protein content of exosomes using the BCA method.

[0077] 5. Results: The exosome production in the 30% hyperoxia group was 18.7 μg / 10. 6 Cells, compared to the normoxia group (23.6 μg / 10) 6 Cellular count decreased by 20.8%, compared to the gradient hypoxia group (58.9 μg / 10 cells). 6 Cell viability decreased by 68.3%. Cell viability assays showed that after 48 hours of culture in 30% hyperoxia, the cell viability was 89.2% (compared to 95.5% in the gradient hypoxia group). Exosome migration-promoting activity (scratch wound healing rate at 24 hours) was only 32%, significantly lower than that in the gradient hypoxia group (75%). This indicates that a 30% oxygen concentration significantly induced oxidative stress in the cells, inhibiting exosome secretion.

[0078] Example 5 1. Cell culture and starvation treatment: Same as in Example 2.

[0079] Hyperoxia-induced culture (50% O2): After starvation treatment, cells were transferred into a three-gas incubator, with oxygen concentration of 50%, CO2 concentration of 5%, and temperature of 37°C, and cultured continuously for 48 hours.

[0080] 2. Exosome collection and quantification: Same as in Example 2.

[0081] 3. Results: The exosome production in the 50% hyperoxia group was only 10.3 μg / 10. 6 Cell counts decreased by 56.4% compared to the normoxic group and by 82.5% compared to the gradient hypoxia group. Cell viability plummeted to 58.4%, and numerous rounded and detached cells were observed under a microscope. The collected exosomes showed significantly increased levels of pro-inflammatory factors (such as IL-6) and extremely low levels of pro-repair growth factors (VEGF, HGF). This confirms that the extreme hyperoxia environment severely disrupts cellular homeostasis and is completely unsuitable for exosome production.

[0082] Example 6 1. Isolation and culture of umbilical cord mesenchymal stem cells: Primary umbilical cord mesenchymal stem cells (UC-MSCs) were obtained by cutting up umbilical cords from healthy newborns and using the tissue block adherence method. They were cultured and passaged in DMEM / F12 complete medium (Gibco / C11330500BT) containing 15% fetal bovine serum (FBS) (EXCELL / FSP500) at 37°C and 5% CO2.

[0083] 2. Experimental grouping and treatment: UC-MSCs in good growth condition from generation P5 were selected and treated with 2×10⁻⁶ cells / mL. 4 The cells were seeded at a density of 1 cell / cm² into 6 T-175 culture flasks. When the cell confluence reached approximately 80%, the following treatment was performed. (1) Divide the 6 T-175 culture flasks into three groups: J, K and L, with 2 T-175 culture flasks in each group.

[0084] (2) Starvation culture: Discard the old culture medium, wash with 0.9% sodium chloride injection, and replace with phenol red-free DMEM / F12 culture medium (Baidi / L1041-500) for 24h. (3) Gradient hypoxia group (J group): Place two bottles of starved cells from group J into a three-gas incubator and set the parameters as follows: 37℃, 5% CO2, 10% O2, and culture for 28h; then adjust the oxygen concentration to 5%, keep other conditions unchanged, and continue to culture for 28h.

[0085] (4) Reverse gradient group (K group): The two bottles of starved cultured cells in the K group were first placed in a three-gas incubator of 37℃, 5% CO2, and 5% O2 and cultured continuously for 24h. Then they were transferred to a three-gas incubator of 37℃, 5% CO2, and 10% O2 and cultured for another 24h.

[0086] (5) Single hypoxia group (L group): Two bottles of starved cultured cells from the L group were placed in an incubator at 37°C, 5% CO2, and 5% O2 and cultured continuously for 48 hours.

[0087] (6) Collection of starvation culture supernatant: Collect the starvation culture supernatant of the three groups of cells J, K and L respectively, and label them J, K and L.

[0088] (7) Centrifuge the supernatant of the starvation culture of groups J, K and L at 4℃: centrifuge at 3000g for 10 minutes and take the supernatant for later use.

[0089] Example 7 1. Isolation and culture of umbilical cord mesenchymal stem cells: Primary umbilical cord mesenchymal stem cells (UC-MSCs) were obtained by cutting up umbilical cords from healthy newborns and using the tissue block adherence method. They were cultured and passaged in DMEM / F12 complete medium (Gibco / C11330500BT) containing 15% fetal bovine serum (FBS) (EXCELL / FSP500) at 37°C and 5% CO2.

[0090] 2. Experimental grouping and treatment: UC-MSCs in good growth condition from generation P5 were selected and treated with 2×10⁻⁶ cells / mL. 4 The cells were seeded at a density of 1 cell / cm² in 6 T-175 culture flasks. When the cell confluence reached approximately 80%, the following treatment was performed: (1) Divide the 6 T-175 culture flasks into three groups: M, N and O, with 2 T-175 culture flasks in each group.

[0091] (2) Starvation culture: Discard the old culture medium, wash with 0.9% sodium chloride injection, and replace with phenol red-free DMEM / F12 culture medium (Baidi / L1041-500) for 24h.

[0092] (3) The present invention group (Group M): Two bottles of starved cultured cells from Group M were placed in a three-gas incubator and the parameters were set as follows: 37°C, 5% CO2, 10% O2, and cultured for 28 hours; then the oxygen concentration was adjusted to 5%, and other conditions remained unchanged, and cultured for another 28 hours.

[0093] (4) Large span group (N group): The two bottles of starved cultured cells in group N were first placed in a three-gas incubator of 37℃, 5% CO2, and 21% O2 and cultured continuously for 24h. Then they were transferred to a three-gas incubator of 37℃, 5% CO2, and 5% O2 and cultured for another 24h.

[0094] (5) Small span group (O group): Two bottles of starved cultured cells from group O were placed in an incubator at 37℃, 5% CO2, and 8% O2 and cultured continuously for 24 hours; then transferred to a three-gas incubator at 37℃, 5% CO2, and 6% O2 and cultured for another 24 hours.

[0095] (6) Collection of starvation culture supernatant: Collect the starvation culture supernatant of the three groups of cells (M, N, and O) and label them as M, N, and O respectively.

[0096] (7) Centrifuge the supernatant of the three groups of starvation culture at 4°C: centrifuge at 3000g for 10 minutes and take the supernatant for later use.

[0097] Example 8: Validation of the synergistic effect of starvation and gradient hypoxia (factorial design) A 2×2 factorial design was used: Group 1: No starvation + normoxic culture (baseline) Group 2: Starvation (24h) + normoxic culture Group 3: No starvation + gradient hypoxia (10%→5%) Group 4 (This invention): Includes starvation + gradient hypoxia Results: The yield of group 1 was 23.6 μg / 10 6 Cells: Group 2: 28.3 μg (1.20-fold); Group 3: 42.5 μg (1.80-fold); Group 4: 58.9 μg (2.50-fold). If a simple additive approach were used, the expected yield would be 28.3 + 42.5 - 23.6 = 47.2 μg (2.00-fold), but the actual yield was 2.50-fold. The synergistic coefficient CI = 2.50 / 2.00 = 1.25 > 1, demonstrating a significant synergistic effect between starvation and gradient hypoxia.

[0098] Example 9: Detection of exosomes' ability to promote cell migration (cell scratch assay) To quantitatively assess the potential of exosomes obtained under different culture conditions to promote tissue repair, this embodiment uses the classic Wound Healing Assay to detect the promoting effect of exosomes on the migration ability of human skin fibroblasts (HSF).

[0099] 1. Experimental Methods (1) Exosome samples: The culture supernatants of normoxic group (21% O2), single hypoxic group (5% O2), gradient hypoxic group of the present invention (10%→5% O2) and hyperoxic group (40% O2) were collected respectively. The exosomes were purified by ultracentrifugation, quantified by BCA method and resuspended in PBS for later use.

[0100] (2) Scratch test procedure: HSF cells were loaded at 5 × 10 5 Inoculate one well per well into a 6-well plate and culture according to standard methods until the fusion rate reaches over 90%.

[0101] Using a 200 μL sterile pipette tip, draw a vertical line down the center of each well. Gently wash twice with PBS to remove detached cells. Replace with serum-free DMEM medium and add exosomes from different sources to a final concentration of 20 μg / mL. Perform three replicates per group.

[0102] At 0 hours (immediately after scratching), 12 hours, and 24 hours, the scratch area was measured using ImageJ software, and the scratch healing rate at each time point was calculated: Healing rate (%) = (Scratch area at 0h - Scratch area at observation) / Scratch area at 0h × 100%. Cell migration rate was calculated: Migration rate (μm / h) = (Scratch width at 0h - Scratch width at observation) / 2 / Culture time. The scratch width was obtained by dividing the area by the scratch length (a fixed value).

[0103] 2. Experimental Results The scratch healing rate and migration rate of fibroblasts in each experimental component at different time points are as follows: Figure 4 , Figure 5 As shown in Table 3.

[0104] Table 3 Comparison of scratch healing rate and migration rate of fibroblasts treated with different exosomes (Mean±SD, n=3)

[0105] The above results fully demonstrate that the exosomes prepared by the "starvation combined with gradient hypoxia induction" method of the present invention have the strongest cell migration-promoting activity, and the effect is significantly better than the existing single hypoxia induction method and hyperxia culture method, providing solid in vitro pharmacodynamic evidence for its application in the field of tissue repair.

[0106] Example 10: Detection of exosome inflammatory factor activity (LPS-induced macrophage inflammation model) 1. Experimental Methods: RAW264.7 mouse macrophages were cultured at a concentration of 1×10⁻⁶ cells / mL. 5 Each exosome was seeded per well in a 24-well plate, stimulated with 1 μg / mL LPS, and simultaneously added with exosomes from different sources (20 μg / mL). After 24 hours of culture, the supernatant was collected, and the levels of TNF-α, IL-6, and IL-1β were detected by ELISA.

[0107] 2. Results: The TNF-α level in the normoxia group was 850±45 pg / mL, in the hypoxia-only group it was 520±32 pg / mL, and in the present invention group it was 320±28 pg / mL (a decrease of 62.4% compared to normoxia and 38.5% compared to hypoxia-only, p<0.01). The hyperoxia group (40% O2) had a TNF-α level of 890±50 pg / mL, with no anti-inflammatory effect. The trends of IL-6 and IL-1β detection results were consistent. Figure 6 .

[0108] Example 11: Analysis of exosome contents (miRNA and growth factors) qPCR analysis showed that the expression levels of miR-21, miR-126, and miR-146a in the exosomes of this invention were 2.1±0.2 times, 1.8±0.2 times, and 2.3±0.3 times that of the hypoxia-only group, respectively (p<0.05). ELISA analysis of growth factor content in exosomes showed that the VEGF content in the present invention group was 1.9±0.2 times that of the hypoxia-only group, and the HGF content was 2.2±0.2 times. The mechanism of enhanced exosome activity in this invention was explained at the molecular level of inclusions.

[0109] Example 12: Process stability verification (three batches replicated) Three consecutive batches of gradient hypoxia exosomes were independently prepared, and the yield, particle size, and cell viability were measured. The results are shown in Table 4. The yield CV was 1.9%, the particle size CV was 2.2%, and the viability CV was 0.37%, demonstrating that the method is stable and controllable and suitable for large-scale production.

[0110] Table 4. Repeatability verification data for three batches

[0111] Example 13: Mechanism Validation – HIF-1α Pathway Activation Detection Western blotting analysis of HIF-1α protein levels at the end of cell culture revealed that the HIF-1α expression level in the experimental group was 2.3 times that of the hypoxia-only group and 5.1 times that of the normoxic group. qPCR analysis of downstream genes VEGF and GLUT1 mRNA levels showed that the expression levels in the experimental group were upregulated by 2.0-fold and 1.8-fold, respectively, compared to the hypoxia-only group. After treatment with the HIF-1α inhibitor YC-1 (10 μM), exosome production in the experimental group decreased to levels close to those in the normoxic group, demonstrating that gradient hypoxia mediates exosome production enhancement through the HIF-1α pathway.

[0112] In summary, this invention significantly improves the yield and biological activity of umbilical cord mesenchymal stem cell exosomes by using a gradient hypoxia-induced starvation culture supernatant preparation strategy of "10% O2-5% O2", providing a new technical platform for exosomes in regenerative medicine and clinical treatment.

[0113] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for inducing exosomes using a combination of starvation and gradient hypoxia, characterized in that, Includes the following steps: Cells were subjected to starvation culture, followed by gradient hypoxia culture. The culture supernatant was collected, and exosomes were isolated. The gradient hypoxia culture consisted of culturing cells at a first hypoxia concentration for 20-28 hours, followed by culturing them at a second hypoxia concentration for 20-28 hours. The first hypoxia concentration was higher than the second hypoxia concentration, and both the first and second hypoxia concentrations were lower than normoxic concentrations. The starvation culture consisted of culturing cells to a confluence of 70%-80%, followed by replacing the medium with phenol red-free basal medium and starving them under normoxic conditions for 20-28 hours. The first hypoxia concentration was 8%-12% oxygen by volume, and the second hypoxia concentration was 4%-6% oxygen by volume.

2. The method according to claim 1, characterized in that, Cells subjected to starvation culture are at passages 3-6.

3. The method according to claim 1, characterized in that, Specifically, the following steps are included: (1) Cell expansion culture: Seed cells were inoculated in DMEM / F12 medium containing 5%-15% fetal bovine serum and cultured at 37℃ and 5% CO2 until the cell confluence was 70%-80%; (2) Starvation treatment: Discard the original culture medium and replace it with phenol red-free DMEM / F12 medium. Continue to culture at 37℃ and 5% CO2 for 20-28 hours. (3) Gradient hypoxia induction: Transfer the cells treated in step (2) to a three-gas incubator and culture them for 20-28 hours at 8%-12% oxygen volume fraction, 5% carbon dioxide concentration, and 37°C. Then adjust the oxygen concentration to 4%-6% while keeping other conditions unchanged and continue culturing for 20-28 hours. Collect the culture supernatant. (4) Centrifuge the collected culture supernatant to remove cell debris and impurities and obtain hypoxia-induced exosomes.

4. The method according to claim 3, characterized in that, The centrifugation in step (4) is centrifugation at 3000g for 10 minutes at 4℃.

5. Exosomes prepared by the method according to any one of claims 1-4.

6. The exosome according to claim 5, characterized in that, The exosomes have a particle size of 30-150 nm, positively express CD9, CD63 and TSG101, and negatively express Calnexin. The yield of the exosomes is more than 2 times higher than that of exosomes prepared by normoxic culture, more than 30% higher than that of exosomes prepared by single hypoxic culture, and more than 3 times higher than that of exosomes prepared by hyperoxic culture.

7. An exosome preparation, characterized in that, It comprises the exosomes as described in claim 5 or 6, and a pharmaceutically acceptable carrier or excipient.

8. The use of the exosomes of claim 5 or 6 or the exosome formulation of claim 7 in the preparation of medicaments for tissue repair, anti-inflammation or drug delivery.

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