Preparation process of three-dimensional umbilical cord mesenchymal stem cell exosome and application of three-dimensional umbilical cord mesenchymal stem cell exosome in medicines and medical beauty

Abstract

The invention discloses a preparation method of a three-dimensional umbilical cord mesenchymal stem cell exosome and application of the three-dimensional umbilical cord mesenchymal stem cell exosome in the fields of medicines and medical beauty. The preparation method comprises the step of amplifying umbilical cord mesenchymal stem cells (UC-MSCs) by adopting a three-dimensional culture system. Experiments show that IGF-1, EGF, VEGF, TGF-beta, PDGF, FGF and the like in the exosome subjected to three-dimensional culture are beneficial to skin repair and oxidation resistance, and the content of cell active factors for promoting collagen secretion is remarkably increased. The umbilical cord mesenchymal stem cell exosome significantly improves the fibroblast proliferation activity, and UV-induced skin fibroblast damage model construction and function verification experiments also prove that the exosome can promote the HA level recovery of damaged skin fibroblasts. The invention provides an efficient and safe biological material for tissue repair.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a preparation process of three-dimensional umbilical cord mesenchymal stem cell exosomes and its application in medicine and medical aesthetics. Background Art

[0002] Mesenchymal stem cell exosomes (MSC-exosomes) are attracting significant attention in regenerative medicine and cosmetics as cell-free therapeutics due to their immunomodulatory, angiogenic, and anti-inflammatory properties. Umbilical cord mesenchymal stem cells (UC-MSCs) are a preferred cell source for exosome production due to their abundant availability and strong proliferation capacity. However, existing technologies face the following bottlenecks: 1. Low culture efficiency: UC-MSCs cultured in traditional two-dimensional planar culture are prone to dedifferentiation, resulting in insufficient exosome secretion and unstable expression of functional proteins.

[0003] 2. Insufficient functional regulation: Existing culture media lack directional induction components, resulting in low expression of key repair factors (such as TGF-β1 and FGF-2) in exosomes, limiting their application in complex injury repair.

[0004] To address these issues, some studies have attempted to improve culture conditions. For example, some existing technologies have increased VEGF expression by 2.1-fold by adding dexamethasone and ascorbic acid to induce UC-MSCs. Others have used human umbilical cord matrigel culture media, increasing exosome yield by 30%. However, these methods still suffer from issues such as the lack of a three-dimensional culture system and insufficient exosome purity. Furthermore, while industry standards cover exosome quality testing (such as CD63 / CD81 / TSG101 marker detection), they do not address standardized evaluation of functional activity.

[0005] Therefore, there is an urgent need to develop an efficient three-dimensional culture-exosome purification combined process and establish its application system in drug delivery and skin repair. Summary of the Invention

[0006] The present invention first provides a method for preparing three-dimensional umbilical cord mesenchymal stem cell exosomes, which comprises the following steps: (1) separation and extraction of umbilical cord mesenchymal stem cells; (2) culturing umbilical cord mesenchymal stem cells; and (3) exosome extraction.

[0007] In certain embodiments, the (2) umbilical cord mesenchymal stem cell culture is performed using three-dimensional culture.

[0008] In certain embodiments, the three-dimensional culture utilizes a three-dimensional scaffold.

[0009] In certain embodiments, the three-dimensional scaffold is a porous chitosan scaffold with a porosity of >90% and a pore size of 100-200 μm.

[0010] In certain embodiments, the culture medium used for the three-dimensional culture is DMEM / F12 basal culture medium + basic fibroblast growth factor bFGF, epidermal growth factor EGF and berberine.

[0011] In certain embodiments, the culture medium used for the three-dimensional culture is DMEM / F12 basal medium + 5-50 ng / mL basic fibroblast growth factor bFGF, 5-50 ng / mL epidermal growth factor EGF, and 5-50 ng / mL berberine.

[0012] In certain embodiments, the culture medium used for the three-dimensional culture is DMEM / F12 basal medium + 10 ng / mL basic fibroblast growth factor bFGF, 10 ng / mL epidermal growth factor EGF, and 10 ng / mL berberine.

[0013] The present invention also provides a three-dimensional umbilical cord mesenchymal stem cell exosome, which is prepared by the above method.

[0014] The present invention also provides an application of the three-dimensional umbilical cord mesenchymal stem cell exosomes as described above, wherein the application is for preparing medicines or medical aesthetic materials.

[0015] In certain embodiments, the application is the preparation of a medicine or medical cosmetic material that promotes skin healing.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects This invention utilizes a three-dimensional culture system to expand umbilical cord mesenchymal stem cells (UC-MSCs). Experiments have shown that the exosomes in this three-dimensional culture significantly increase the levels of cellular active factors, such as IGF-1, EGF, VEGF, TGF-β, PDGF, and FGF, which contribute to skin repair and anti-oxidation, and promote collagen secretion. Umbilical cord mesenchymal stem cell exosomes significantly enhance fibroblast proliferation. A UV-induced skin fibroblast injury model and functional validation experiments also demonstrate that the exosomes of this invention can promote the restoration of HA levels in damaged skin fibroblasts. This invention provides a highly efficient and safe biomaterial for tissue repair. DETAILED DESCRIPTION

[0017] The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0018] The present invention first provides a method for preparing three-dimensional umbilical cord mesenchymal stem cell exosomes, which comprises the following steps: (1) separation and extraction of umbilical cord mesenchymal stem cells; (2) culturing umbilical cord mesenchymal stem cells; and (3) exosome extraction.

[0019] In certain embodiments, the (2) umbilical cord mesenchymal stem cell culture is performed using three-dimensional culture.

[0020] In certain embodiments, the three-dimensional culture utilizes a three-dimensional scaffold.

[0021] In certain embodiments, the three-dimensional scaffold is a porous chitosan scaffold with a porosity of >90% and a pore size of 100-200 μm.

[0022] In certain embodiments, the culture medium used for the three-dimensional culture is DMEM / F12 basal culture medium + basic fibroblast growth factor bFGF, epidermal growth factor EGF and berberine.

[0023] In certain embodiments, the culture medium used for the three-dimensional culture is DMEM / F12 basal medium + 5-50 ng / mL basic fibroblast growth factor bFGF, 5-50 ng / mL epidermal growth factor EGF, and 5-50 ng / mL berberine.

[0024] In certain embodiments, the culture medium used for the three-dimensional culture is DMEM / F12 basal medium + 10 ng / mL basic fibroblast growth factor bFGF, 10 ng / mL epidermal growth factor EGF, and 10 ng / mL berberine.

[0025] The present invention also provides a three-dimensional umbilical cord mesenchymal stem cell exosome, which is prepared by the above method.

[0026] The present invention also provides an application of the three-dimensional umbilical cord mesenchymal stem cell exosomes as described above, wherein the application is for preparing medicines or medical aesthetic materials.

[0027] In certain embodiments, the application is the preparation of a medicine or medical cosmetic material that promotes skin healing or anti-aging.

[0028] The specific embodiments apply the following.

[0029] Example 1 Culture medium Medium 1: 10% FBS (fetal bovine serum) in low-glucose DMEM; 10 ng / mL basic fibroblast growth factor (bFGF), 10 ng / mL epidermal growth factor (EGF), and 10 ng / mL berberine (CAS No. 2086-83-1).

[0030] Culture medium 2 (Comparative Example 1): low-glucose DMEM containing 10% FBS (fetal bovine serum); 15 ng / mL basic fibroblast growth factor (bFGF); and 15 ng / mL epidermal growth factor (EGF).

[0031] Culture medium 3 (Comparative Example 2): low-glucose DMEM containing 10% FBS (fetal bovine serum); 15 ng / mL basic fibroblast growth factor bFGF and 15 ng / mL berberine.

[0032] Culture medium 4 (Comparative Example 3): low-glucose DMEM containing 10% FBS (fetal bovine serum); 15 ng / mL epidermal growth factor (EGF) and 15 ng / mL berberine.

[0033] Culture medium 5 (Comparative Example 4): low-glucose DMEM containing 10% FBS (fetal bovine serum); 30 ng / mL basic fibroblast growth factor bFGF.

[0034] Culture medium 6 (Comparative Example 5): low-glucose DMEM containing 10% FBS (fetal bovine serum); 30 ng / mL epidermal growth factor (EGF).

[0035] Culture medium 7 (Comparative Example 6): 10% FBS (fetal bovine serum) low-glucose DMEM; 30 ng / mL berberine.

[0036] Culture medium 8 (Comparative Example 7): low-glucose DMEM containing 10% FBS (fetal bovine serum).

[0037] Example 2 Preparation of three-dimensional umbilical cord mesenchymal stem cell exosomes The specific preparation method of three-dimensional umbilical cord mesenchymal stem cell exosomes includes the following steps: 1. Tissue Pretreatment and Enzymatic Hydrolysis 1. Raw material acquisition and cleaning: In a sterile operating environment, obtain healthy sheep umbilical cord tissue, rinse with physiological saline to remove residual blood, and then remove blood vessels and membranous tissue to obtain purified umbilical cord matrix segments.

[0038] 2. Mechanical homogenization: Cut the umbilical cord tissue into 1-3 mm³ pieces and physically crush them using a tissue grinder (2000 rpm for 5 min) to ensure that the particle size is ≤1 mm. Aliquot the pieces into 50 mL centrifuge tubes for later use.

[0039] 3. Enzymatic digestion: Add an equal volume of pre-cooled type I collagenase solution (concentration 0.25%) to the tissue homogenate and digest with shaking at 37°C for 300 minutes. Then, add phosphate buffer containing 0.25% EDTA to terminate the reaction and obtain the enzymatic digestion mixture.

[0040] 2. Cell Isolation and Culture 4. Cell pellet preparation: Centrifuge the enzymatic hydrolysis mixture at 1500 rpm for 5 minutes (4°C), discard the supernatant and retain the bottom cell pellet. Resuspend the cell pellet in 10 mL of PBS buffer and purify the single-cell suspension by passing it through a 70 μm cell sieve and centrifuging it at 2000 rpm for 5 minutes.

[0041] 5. Primary Culture: Inoculate the cell suspension into a T75 culture flask containing DMEM / F12 complete medium (with 10% fetal bovine serum) and culture in a 37°C, 5% CO2 incubator. Change the medium by half every 3 days. Subculture cells after reaching 80% confluency (approximately 7-10 days).

[0042] 3. Cell Expansion and Identification 6. Subculture: When cells reach 80% confluency, discard the old culture medium and digest with 0.25% trypsin-EDTA solution for 3-5 minutes. Observe cell detachment under a microscope and add serum-supplemented culture medium to terminate digestion. Collect the cell suspension by pipetting, centrifuge at 400 × g for 3 minutes, resuspend, and subculture at a 1:3 ratio to the third passage.

[0043] 7. Phenotypic Verification: Flow cytometry was performed on cells at passage 3 to confirm that the CD73 / CD90 / CD105 positivity rate was >98% and that CD14 / CD34 / CD45 was negative, consistent with the characteristics of umbilical cord mesenchymal stem cells (UC-MSCs).

[0044] 4. Three-dimensional Culture and Exosome Harvesting 8. Culture umbilical cord mesenchymal stem cells to the third generation, discard the supernatant, add 10 mL of new culture medium 1, and use three-dimensional culture (initial density 2×10 5 cells / mL), three-dimensional scaffold: chitosan porous scaffold (porosity>90%, pore size 100-200μm).

[0045] The chitosan porous scaffold was prepared as follows: 2.0 g of chitosan powder was weighed and added to 100 mL of a 1% (volume) acetic acid solution. The solution was then placed in a 37°C water bath with continuous magnetic stirring until completely dissolved, resulting in a 2% chitosan acetic acid solution. 1.0 mL of this solution was then evenly added dropwise to each well of a 24-well culture plate. The plate was pre-chilled at 4°C for 6 hours, then transferred to a -25°C freezer overnight, and finally freeze-dried in a freeze dryer at -75°C for 24 hours. After initial drying, 1.0 mL of 0.10 mol / L sodium hydroxide solution was slowly added to each well, and the plate was allowed to hydrate for 20 minutes. The plate was then rinsed thoroughly with phosphate-buffered saline (PBS) for 2 minutes and this rinse cycle was repeated three times. Finally, the sample was dried in a freeze dryer again until a white, cylindrical solid scaffold was formed, completing the preparation of the chitosan porous scaffold.

[0046] Culture conditions: Continue culturing at 37°C, 5% CO2, and 2% O2. When cell confluency reaches 80%, collect the cell supernatant. Filter the harvested supernatant through a 0.22μm filter. Centrifuge the filtered cell culture medium at 12,000g for 20 minutes, resuspend the pellet in sterile PBS, and store it at 4°C for a short period of time. This is the umbilical cord mesenchymal stem cell exosomes.

[0047] Comparative Example 1 Except that medium 2 was used to replace medium 1, other conditions were the same as those in Example 2.

[0048] Comparative Example 2 Except that medium 3 was used to replace medium 1, other conditions were the same as those in Example 2.

[0049] Comparative Example 3 Except that medium 4 was used instead of medium 1, other conditions were the same as those in Example 2.

[0050] Comparative Example 4 Except that medium 5 was used instead of medium 1, other conditions were the same as those in Example 2.

[0051] Comparative Example 5 Except that medium 6 was used to replace medium 1, other conditions were the same as those in Example 2.

[0052] Comparative Example 6 Except that medium 7 was used instead of medium 1, other conditions were the same as those in Example 2.

[0053] Comparative Example 7 Except that medium 8 was used to replace medium 1, other conditions were the same as those in Example 2.

[0054] Example 3 Analysis of 3D Umbilical Cord Mesenchymal Stem Cell Exosomes The active factors in the three-dimensional umbilical cord mesenchymal stem cell exosomes of Example 2 and Comparative Examples 1 to Comparative Examples 7 were detected using ELISA to detect IGF-1, EGF, VEGF, TGF-β, PDGF, and FGF, which are cellular active factors that contribute to skin repair and anti-oxidation and promote collagen secretion. The relative expression levels of each group were calculated based on the expression levels of Comparative Example 7. The results are shown in Table 1. The relative expression levels of the active factors in Example 2 were significantly improved, indicating that the culture medium has a significant effect and that the components have achieved a synergistic effect.

[0055] Table 1. Relative expression levels of each active factor Experimental group IGF-1 EGF VEGF TGF-β PDGF FGF Example 2 7.2±0.6 9.2±0.8 6.1±0.7 9.1±0.2 4.5±0.6 5.1±0.5 Comparative Example 1 4.1±0.1 5.1±1.1 4.1±0.4 6.1±0.3 3.0±0.4 4.4±0.8 Comparative Example 2 4.3±0.3 5.9±0.8 4.9±0.5 6.2±0.9 3.5±0.2 4.2±1.1 Comparative Example 3 4.2±0.5 4.8±0.8 4.6±0.6 6.7±0.7 2.6±0.5 3.1±0.7 Comparative Example 4 2.6±0.2 3.8±1.3 2.1±0.3 4.9±0.8 1.5±0.4 2.8±0.3 Comparative Example 5 2.5±0.3 3.9±0.7 2.2±0.2 4.3±0.7 1.3±0.2 1.4±0.4 Comparative Example 6 2.9±0.4 2.7±0.4 2.5±0.3 3.1±1.1 1.4±0.1 1.1±0.2

[0056] Example 4: Effect of umbilical cord mesenchymal stem cell exosomes on fibroblast proliferation Experimental materials and groups NIH / 3T3 mouse embryonic fibroblasts in the logarithmic proliferation phase were selected and cultured at 5×10 5 Cells were seeded into 96-well plates at a density of cells / mL, with five parallel wells per group. The experiment was divided into a control group (complete DMEM medium) and experimental groups (containing culture media with different formulations of umbilical cord mesenchymal stem cell exosomes prepared in Example 2 and Comparative Examples 1-7). The final exosome concentration in all treatment groups was uniformly 40 μg / mL.

[0057] Operation process 1. Cell attachment treatment: After the cells have attached (approximately 24 hours), remove the original culture medium and add complete culture medium containing CCK-8 reagent (10 μL / well) and incubate at 37°C for 2 hours.

[0058] 2. Initial detection: Use a microplate reader to measure the absorbance (OD) at 450 nm and record the initial proliferation data.

[0059] 3. Exosome Intervention: Discard the liquid in the wells and gently rinse twice with PBS buffer. The experimental group received a culture medium containing a specific formula (final exosome concentration of 40 μg / mL), while the control group maintained the basal culture medium.

[0060] 4. Long-term culture and terminal assay: Continue culturing for 96 hours and repeat the OD measurement (450 nm).

[0061] Table 2. Effects of umbilical cord mesenchymal stem cell exosomes on fibroblast proliferation (96h) Experimental group 450 nm Example 2 8.7±0.3 Comparative Example 1 3.6±0.2 Comparative Example 2 3.5±0.2 Comparative Example 3 4.3±0.4 Comparative Example 4 3.3±0.1 Comparative Example 5 2.7±0.2 Comparative Example 6 2.8±0.1 Comparative Example 7 1.9±0.3 control group 1.5±0.2 Result Analysis Experimental data showed (Table 2) that the exosomes from umbilical cord mesenchymal stem cells derived from Example 2 significantly enhanced the proliferation activity of fibroblasts (P<0.01, relative to the control group), and its effect was better than that of other formula groups, indicating that the exosomes have the best pro-proliferation effect.

[0062] Example 5: Medical beauty application Construction and functional verification of UV-induced skin fibroblast damage model 1. Cell Model Construction 1. Cell Expansion and Plating Human foreskin fibroblasts (HFF-1) in the logarithmic growth phase were cultured in DMEM high-glucose medium (Gibco) containing 10% fetal bovine serum and expanded to the logarithmic growth phase at 37°C, 5% CO2, and saturated humidity. The cell density was adjusted to 1×10 5cells / well, seeded in a 96-well plate (Corning), and cultured for 12 h until the cell attachment rate reached 80%.

[0063] 2. Photodamage Model Establishment The original culture medium was discarded, and cells were irradiated with a 311 nm UVB light source (CL-1000M ultraviolet therapy device) at a gradient dose of 50 mJ / cm² (the irradiation time was verified by preliminary experiments) to establish a UV-induced skin photoaging model. Cells in the model group were rinsed three times with PBS buffer before proceeding to the functional verification phase.

[0064] 2. Extracellular Matrix Metabolic Function Detection 3. Experimental Grouping and Intervention The model cells were divided into 10 groups: Blank control group (Group C): HFF-1 cells not irradiated with UVB; Model control group (M group): 100 μL PBS was given after UVB irradiation; Experimental groups (E1-E8 groups): After UVB irradiation, 100 μL of intervention solution containing exosomes from Example 2 to Comparative Example 7 (final concentration 10 mg / mL) was administered respectively.

[0065] 4. Functional index testing Cells in each group were incubated at 37°C, 5% CO₂, for 24 hours, and the supernatants were collected. Hyaluronic acid (HA) secretion was quantified using an ELISA assay (R&D Systems). The data (Table 3) showed that UVB irradiation decreased HA secretion in HFF-1 cells (P < 0.001, relative to the blank control group). However, the exosome-treated group (Example 2) showed the best HA recovery, significantly superior to the other treatment groups (P < 0.01, relative to the PBS group).

[0066] Table 3. HA content in UV-damaged HFF-1 cell model Experimental group HA (μg / L) Blank control group 80.2±6.1 PBS group 38.5±3.6 Example 2 77.1±4.5 Comparative Example 1 64.1±4.6 Comparative Example 2 67.3±3.9 Comparative Example 3 65.2±5.7 Comparative Example 4 63.5±4.2 Comparative Example 5 59.4±5.1 Comparative Example 6 56.1±4.1 Comparative Example 7 50.1±7.4 The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing three-dimensional umbilical cord mesenchymal stem cell exosomes, characterized by: The preparation method comprises the following steps: (1) separation and extraction of umbilical cord mesenchymal stem cells; (2) culturing umbilical cord mesenchymal stem cells; and (3) extraction of exosomes.

2. The method according to claim 1, characterized in that The (2) umbilical cord mesenchymal stem cell culture is carried out using three-dimensional culture.

3. The method according to claim 2, characterized in that The three-dimensional culture uses a three-dimensional scaffold.

4. The method according to claim 3, characterized in that The three-dimensional scaffold is a chitosan porous scaffold with a porosity of >90% and a pore size of 100-200 μm.

5. The method according to claim 1, characterized in that The culture medium used for the three-dimensional culture is DMEM / F12 basic culture medium plus basic fibroblast growth factor bFGF, epidermal growth factor EGF and berberine.

6. The method according to claim 3, characterized in that The culture medium used for the three-dimensional culture is DMEM / F12 basic culture medium + 5-50 ng / mL basic fibroblast growth factor bFGF, 5-50 ng / mL epidermal growth factor EGF and 5-50 ng / mL berberine.

7. The method according to claim 5, characterized in that The culture medium used for the three-dimensional culture is DMEM / F12 basic culture medium + 10 ng / mL basic fibroblast growth factor bFGF, 10 ng / mL epidermal growth factor EGF and 10 ng / mL berberine.

8. A three-dimensional umbilical cord mesenchymal stem cell exosome, characterized in that: The exosomes are prepared by the method according to any one of claims 1 to 7.

9. A use of the three-dimensional umbilical cord mesenchymal stem cell exosomes according to claim 8, characterized in that: The application is the preparation of medicines or medical aesthetic materials.

10. The use according to claim 9, characterized in that The medicine or medical cosmetic material is a medicine or material that promotes skin healing.