Stem cell culture medium, and preparation method and use for antioxidant stem cell exosome

Abstract

Provided are a stem cell culture medium, and a stem cell exosome and a preparation method therefor. The stem cell culture medium comprises baicalin, quercetin, liquiritin and catechin.

Description

Stem cell culture medium, preparation methods and applications of stem cell exosomes with antioxidant properties Technical Field

[0001] This invention relates to the field of cell-related technologies, and in particular to stem cell culture media, methods for preparing stem cell exosomes with antioxidant properties, and their applications. Background Technology

[0002] Currently, methods for combating oxidative damage mainly include the use of antioxidants and lifestyle interventions. Antioxidants can be divided into endogenous antioxidants (such as superoxide dismutase and glutathione peroxidase) and exogenous antioxidants (such as vitamin C, vitamin E, and carotenoids). Although these antioxidants can scavenge free radicals and reduce oxidative damage to some extent, they also have some limitations.

[0003] Stem cells have long been a focus of attention in regenerative medicine due to their self-renewal and multi-lineage differentiation capabilities, offering immense potential for treating a wide range of diseases. One of the key mechanisms by which stem cells exert their effects is through the secretion of exosomes. Stem cell exosomes are nanoscale vesicles with a diameter of approximately 30-150 nm, secreted by stem cells into the extracellular environment. Exosomes contain a variety of bioactive components, such as proteins, nucleic acids (including mRNA and miRNA), and lipids. These components endow exosomes with diverse biological functions, such as regulating intercellular communication, participating in immune regulation, and promoting tissue repair.

[0004] A growing body of research indicates that stem cell exosomes can migrate to damaged tissue sites and regulate the function of target cells by delivering their contained bioactive substances, providing new insights for disease treatment and anti-aging.

[0005] However, exosomes obtained under traditional culture methods lack targeting and their antioxidant effects are not significant, which seriously affects the clinical application and industrial development of exosomes. Summary of the Invention

[0006] This invention provides a stem cell culture medium, a method for preparing stem cell exosomes with antioxidant properties, and their applications, in order to overcome the shortcomings of the prior art.

[0007] As a first aspect of the present invention, the present invention provides a stem cell culture medium with antioxidant properties, the stem cell culture medium comprising baicalin, quercetin, glycyrrhizin and catechin.

[0008] In this invention, the antioxidant stem cell culture medium uses a mixture of baicalin, quercetin, glycyrrhizin and catechin as components of the culture medium to stimulate the stem cells, thereby changing the oxidative stress state of the stem cells and enabling them to secrete exosomes with antioxidant effects, resulting in exosomes with higher antioxidant capacity.

[0009] Baicalin is a flavonoid compound extracted from plants such as Scutellaria baicalensis. It possesses various biological activities, such as anti-inflammatory and antibacterial effects. In this invention, it serves as a component of the stem cell culture medium, providing a favorable microenvironment for stem cell growth and functional maintenance. Quercetin is a flavonol compound widely found in plants, possessing antioxidant and anti-apoptotic properties, which helps regulate the physiological state of stem cells. Glycyrrhizin, derived from licorice, has certain immunomodulatory and antioxidant capabilities. It works synergistically with other components to promote normal stem cell metabolism and maintain activity. Catechins are one of the main active ingredients in tea, possessing powerful antioxidant effects. They can effectively scavenge free radicals, reduce oxidative stress damage to stem cells, and may also play a role in cell signaling, collectively promoting the healthy growth and functional performance of stem cells in the culture medium.

[0010] Among them, the scutellarin, quercetin, glycyrrhizin, and catechin selected in this invention all possess flavonoid compounds. The hydroxyl and carbonyl functional groups in their structures can prevent the generation of free radicals and scavenge their antioxidant activity, thereby avoiding oxidative damage. They can act as free radical scavengers, effectively removing reactive oxygen species such as hydrogen peroxide and hydroxyl radicals in the body, and protecting cells from oxidative stress damage.

[0011] The selected mixture of scutellarin, quercetin, glycyrrhizin, and catechins, compared to a single ingredient or any combination of two, allows the combined action of this mixture to enable stem cells to further secrete exosomes with antioxidant properties.

[0012] The corresponding culture media may also include some basic culture media, such as DMEM / F12 medium, which is used to provide basic nutrition for stem cell culture.

[0013] In an optional embodiment of the first aspect of the present invention, the antioxidant stem cell culture medium comprises 1-20 μg / mL baicalin, 1-10 μM quercetin, 1-20 μg / mL glycyrrhizin, and 0.1-2 μg / mL catechin.

[0014] Specifically, the concentration of baicalin added is 1-20 μg / mL, which means that the content of baicalin in each milliliter of culture medium is between 1 μg and 20 μg. Specifically, the concentration of baicalin can be 1 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, or 20 μg / mL.

[0015] The concentration range of quercetin is 1 to 10 μM, which specifically means that the molar concentration of quercetin in each liter of culture medium is between 1 μmol and 10 μmol. Specifically, the concentration of quercetin in the culture medium can be 1 μM, 3 μM, 5 μM, 8 μM, or 10 μM.

[0016] The concentration of glycyrrhizin added is 1 to 20 μg / mL, which specifically refers to the content of glycyrrhizin in each milliliter of culture medium being between 1 μg and 20 μg. Specifically, the concentration of glycyrrhizin can be 1 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, or 20 μg / mL.

[0017] The addition concentration of catechin is 0.1 to 2 μg / mL, which specifically refers to the content of catechin in each milliliter of culture medium being between 0.1 μg and 2 μg. The specific concentration of catechin can be 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, or 2 μg / mL.

[0018] Within this concentration range, the effectiveness and stability of each component in the culture medium can be ensured, while avoiding toxic effects on stem cells.

[0019] In an optional embodiment of the first aspect of the present invention, the stem cell culture medium comprises 20 μg / mL baicalin, 2.5 μM quercetin, 20 μg / mL glycyrrhizin, and 0.5 μg / mL catechin.

[0020] Even better, stem cell culture medium at this concentration can further enhance the antioxidant properties of the corresponding exosomes while avoiding toxic effects on stem cells.

[0021] As a second aspect of the present invention, a method for preparing stem cell exosomes with antioxidant properties is provided, comprising: culturing stem cells in any of the culture media described in the first aspect of the present invention, and collecting the exosomes produced by the stem cells.

[0022] In this invention, the antioxidant stem cell exosomes are obtained from stem cells cultured using the culture medium of the first invention. In the corresponding mixture culture system, the stem cells are stimulated to separate antioxidant exosomes.

[0023] In an optional embodiment of the second aspect of the present invention, the stem cells are cultured for 3-4 days. Specifically, the corresponding culture time can be determined based on cell growth and exosome production.

[0024] In an optional embodiment of the second aspect of the present invention, the extraction of exosomes specifically includes the purification of the exosomes.

[0025] The purpose of exosome purification is to remove impurities and cell debris from the culture medium to obtain high-purity exosomes. The purification of exosomes can be achieved by methods such as differential centrifugation, ultrafiltration, or immunomagnetic bead collection.

[0026] Ultracentrifugation is a classic method that separates substances of different sizes and densities by gradually increasing centrifugal force, thus obtaining exosome precipitates. Ultrafiltration uses membranes with different pore sizes to separate exosomes based on their size. Immunomagnetic bead separation is based on specific markers on the exosome surface; it utilizes the binding properties of magnetic beads to antibodies to specifically capture exosomes. Purification can improve the stability and efficacy of exosomes in subsequent applications, making their effects in skincare products or pharmaceuticals more precise and reliable.

[0027] In one specific embodiment of the present invention, the purification method for exosomes is a combination of centrifugation and ultrafiltration. Specifically, it can be as follows: collect the supernatant of exosomes after culturing for 3-4 days; centrifuge the collected supernatant, filter the supernatant, and collect the filtered supernatant to obtain antioxidant-enhancing exosomes.

[0028] In an optional embodiment of the second aspect of the present invention, the stem cells are selected from hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, neural stem cells, and induced pluripotent stem cells.

[0029] In an optional embodiment of the second aspect of the present invention, the stem cells are mesenchymal stem cells.

[0030] Mesenchymal stem cells are widely available, such as from bone marrow and adipose tissue, and have advantages such as easy acquisition, strong expansion capacity, and low immunogenicity.

[0031] In a second aspect of this invention, the stem cell exosomes prepared by the above-described method exhibit significant antioxidant properties due to the culture of stem cells in a specific culture medium. Because of these significant antioxidant properties, these exosomes have potential applications in intercellular tissue repair and the treatment or prevention of antioxidant-related diseases. For example, in skin tissue, their antioxidant properties can neutralize free radicals generated by external factors such as ultraviolet radiation, reducing oxidative damage to skin cells and delaying skin aging; in vivo, they can alleviate oxidative stress damage to organs and tissues, promoting the repair and regeneration of damaged tissues.

[0032] As a third aspect of the invention, protection is claimed for a stem cell exosome prepared by any of the methods described in the second aspect of the invention.

[0033] The stem cell exosomes of the present invention are isolated after being stimulated and cultured using the culture system of the first aspect of the present invention. Compared with stem cell exosomes cultured in normal culture medium, the stem cell exosomes secreted by the exosomes have higher antioxidant capacity.

[0034] This invention utilizes stem cell exosomes generated after stimulation, which exhibit superior antioxidant and anti-aging properties, demonstrating better performance compared to other methods in anti-cellular oxidative death assays, apoptosis assays, and anti-aging assays. The stem cell exosomes of this invention have broad applications in cellular antioxidant damage prevention, anti-aging, and related cellular antioxidant and anti-aging processes.

[0035] As a fourth aspect of the invention, protection is claimed for a composition comprising stem cell exosomes prepared by any of the methods described in any of the second aspects of the invention and excipients for use in skin care products or pharmaceuticals.

[0036] In a fourth aspect of the invention, the composition includes stem cell exosomes prepared by the method and excipients that can be used in the fields of skincare products or pharmaceuticals.

[0037] The corresponding excipients in the skincare product field refer to excipients that can be added to skincare products, such as water, surfactants, and moisturizers, which can better disperse stem cell exosomes in the product, maintaining product stability and user comfort. Specific details are not limited in this solution. After adding the corresponding stem cell exosomes and skincare excipients, the composition possesses antioxidant and anti-aging effects, and can function as an antioxidant and anti-aging product. The composition can be used in skincare products with antioxidant and / or anti-aging properties. These skincare products can be aqueous, cream, powder, or oil-based, such as toners, lotions, and face creams.

[0038] In the pharmaceutical field, excipients that can be used include pharmaceutically acceptable carriers, excipients, and diluents that are compatible with the active pharmaceutical ingredient. These excipients help to formulate stem cell exosomes into suitable dosage forms, such as tablets, capsules, and injections, to facilitate drug storage, transportation, and use, enabling exosomes to be accurately delivered to the target site to exert their antioxidant and therapeutic effects.

[0039] The stem cell culture medium proposed in this invention uses a mixture of baicalin, quercetin, glycyrrhizin, and catechins as its components to stimulate the stem cells, altering their oxidative stress state and enabling them to secrete exosomes with antioxidant properties. These exosomes exhibit superior antioxidant capacity. The corresponding stem cell exosomes demonstrate excellent antioxidant performance. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0041] Figure 1 shows the toxic effects of different concentrations of baicalin on umbilical cord mesenchymal stem cells;

[0042] Figure 2 shows the toxic effects of different concentrations of quercetin on umbilical cord mesenchymal stem cells;

[0043] Figure 3 shows the toxic effects of different concentrations of glycyrrhizin on umbilical cord mesenchymal stem cells;

[0044] Figure 4 shows the toxic effects of different concentrations of catechins on umbilical cord mesenchymal stem cells;

[0045] Figure 5 shows examples of exosome particle size (NTA) in different experimental groups;

[0046] Figure 6 shows examples of transmission electron microscopy (TEM) images of exosomes from different experimental groups;

[0047] Figure 7 shows flow cytometry examples of apoptosis detection in different experimental groups;

[0048] Figure 8 shows examples of β-galactosidase staining in different experimental groups. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0050] This invention provides stem cell exosomes with antioxidant properties, produced from stem cells cultured in a specific medium containing a mixture of baicalin, quercetin, glycyrrhizin, and catechins. Compared to existing dedicated stem cell culture media, these exosomes exhibit superior antioxidant and anti-aging properties.

[0051] Specifically, in the embodiments of the present invention, mesenchymal stem cells are used as cultured cells for verification, specifically umbilical cord mesenchymal stem cells.

[0052] Umbilical cord mesenchymal stem cell exosomes in this embodiment of the invention are prepared using the following steps:

[0053] S1: Preparation of culture medium

[0054] Prepare stem cell culture media containing the corresponding components according to the specified ratio, wherein the corresponding stem cell culture media contains the basal culture medium DMEM / F12 medium.

[0055] S2: Culture of umbilical cord mesenchymal stem cells

[0056] Third-generation umbilical cord mesenchymal stem cells were taken, seeded into culture flasks, and cultured for 3 days with the corresponding culture medium.

[0057] In this step, third-generation umbilical cord mesenchymal stem cells were used for culture. The corresponding culture environment was an incubator at 37°C and 5% CO2. During the culture process, the mixture of baicalin, quercetin, glycyrrhizin, and catechins gradually affected the stem cell's metabolism and secretion functions, thereby giving the secreted exosomes superior antioxidant properties.

[0058] The isolation and passage culture method for umbilical cord mesenchymal stem cells is as follows: umbilical cord samples are collected under sterile conditions; then they are rinsed with PBS buffer containing double antibody solution and minced.

[0059] The shredded umbilical cord tissue pieces were evenly inoculated at the bottom of the culture flask. DMEM was used as the basal medium, with 10% FBS and 1% antibiotic solution added. The inoculated culture flask was then placed in an incubator at 37°C and 5% CO2.

[0060] When the cell fusion rate of mesenchymal stem cells reaches about 70%-80%, passage should be performed.

[0061] S3: Isolation and purification of exosomes

[0062] After 3 days of culture, the corresponding exosome supernatant is collected for separation and purification. In this embodiment of the invention, the separation and purification method is as follows:

[0063] The collected exosome supernatant was centrifuged to remove microbubbles or protein aggregates. The supernatant was then collected and filtered using an exosome separation and enrichment nanochip. The filtered solution was then used to obtain the stem cell exosome solution.

[0064] Specifically, it includes the following sub-steps:

[0065] S31: Centrifuge the collected exosome supernatant at 4℃ and 12000g for 30min to remove microvesicles or protein aggregates, and take the supernatant.

[0066] S32: Dilute the supernatant with PBS solution.

[0067] S33: Filter the diluted supernatant using a 0.45μm filter, and collect the second supernatant after filtration.

[0068] S34: Filter the supernatant twice using a 0.22μm filter to remove apoptotic bodies and microvesicles, and collect the filtered supernatant.

[0069] S35: Exosome separation and enrichment nanochip filtration is used, and the filtered solution is the stem cell exosome solution.

[0070] The following experiments are provided simultaneously in the specific embodiments of the present invention for verification.

[0071] 1. Toxicity tests of different concentrations of components on umbilical cord mesenchymal stem cells

[0072] The first aspect of this invention is used to verify the toxicity of different concentrations of components to umbilical cord mesenchymal stem cells. Specifically, in this invention, baicalin, quercetin, glycyrrhizin, and catechin are all flavonoid compounds. To determine their toxic effects on mesenchymal stem cells, in the first aspect of this invention, P3 generation umbilical cord mesenchymal stem cells were cultured for 3 days using different concentrations of baicalin, quercetin, glycyrrhizin, and catechin, and then the cell viability at each concentration was detected using a CCK-8 assay kit.

[0073] 1.1 Toxicity test of different concentrations of baicalin on umbilical cord mesenchymal stem cells

[0074] P3 generation umbilical cord mesenchymal stem cells were cultured for 3 days with baicalin at concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL. Cell viability at each concentration was then detected using a CCK-8 assay kit. The results are shown in Table 1 and Figure 1.

[0075] Table 1. Effects of different concentrations of baicalin on the toxicity of umbilical cord mesenchymal stem cells. Note: There were no significant differences between the different concentrations of baicalin groups compared with the blank control group (p > 0.5).

[0076] As can be seen from Table 1 and Figure 1, the addition of 1-20 μg / mL baicalin did not have a significant effect on umbilical cord mesenchymal stem cells. However, 20 μg / mL baicalin had a certain effect on improving the survival rate of umbilical cord mesenchymal stem cells. Therefore, the concentration range of baicalin in this invention is 1-20 μg / mL, preferably 20 μg / mL.

[0077] 1.2 Toxicity test of different concentrations of quercetin on umbilical cord mesenchymal stem cells

[0078] P3 generation umbilical cord mesenchymal stem cells were cultured for 3 days with quercetin at concentrations of 1 μM, 2.5 μM, 5 μM, and 10 μM. Cell viability at each concentration was then detected using a CCK-8 assay kit. The results are shown in Table 2 and Figure 2.

[0079] Table 2. Effects of different concentrations of quercetin on the toxicity of umbilical cord mesenchymal stem cells Note: Compared with the blank control group, there were significant differences in each concentration of quercetin group (p < 0.5).

[0080] As shown in Table 2 and Figure 2, the effects of adding 1-10 μM quercetin on cell viability varied, with significant differences compared to the blank control group observed in concentrations ranging from 2.5 μM to 10 μM. Specifically, quercetin at concentrations within the range of 2.5-5 μM significantly improved sample viability, while excessively high concentrations, such as 10 μM, significantly reduced sample viability. Therefore, in this invention, the preferred amount of quercetin added is 2.5-5 μM, with 2.5 μM being the most preferred.

[0081] 1.3 Toxicity test of different concentrations of glycyrrhizin on umbilical cord mesenchymal stem cells

[0082] P3 generation umbilical cord mesenchymal stem cells were cultured for 3 days with glycyrrhizin at concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL. Cell viability at each concentration was then detected using a CCK-8 assay kit. The results are shown in Table 3 and Figure 3.

[0083] Table 3. Effects of different concentrations of glycyrrhizin on the toxicity of umbilical cord mesenchymal stem cells Note: Compared with the blank control group, there were significant differences in each concentration of glycyrrhizin group (p < 0.5).

[0084] As can be seen from Table 3 and Figure 3, the addition of 1-20 μg / mL glycyrrhizin has a promoting effect on the survival rate of umbilical cord mesenchymal stem cells, and it is determined that the promoting effect on the survival of umbilical cord mesenchymal stem cells increases with the increase of the added amount. Therefore, the concentration range of glycyrrhizin in this invention is 1-20 μg / mL, preferably 20 μg / mL.

[0085] 1.4 Toxicity test of different concentrations of catechins on umbilical cord mesenchymal stem cells

[0086] P3 generation umbilical cord mesenchymal stem cells were cultured for 3 days with catechins at concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL. Cell viability at each concentration was then detected using a CCK-8 assay kit. The results are shown in Table 4 and Figure 4.

[0087] Table 4. Effects of different concentrations of catechins on the toxicity of umbilical cord mesenchymal stem cells Note: Compared with the blank control group, there were significant differences in each concentration of catechin group (p < 0.5).

[0088] As can be seen from Table 4 and Figure 4, the addition of 0.1-2 μg / mL catechin has a certain effect on the survival rate of umbilical cord mesenchymal stem cells. Among them, 0.5 μg / mL catechin has a promoting effect on the survival of umbilical cord mesenchymal stem cells. Therefore, the preferred amount of catechin is 0.5 μg / mL catechin.

[0089] Based on the above tests, the first aspect of the specific embodiments of the present invention determines the optimal concentrations for culture of each compound: 20 μg / mL baicalin, 2.5 μM quercetin, 20 μg / mL glycyrrhizin, and 0.5 μg / mL catechin.

[0090] 2. Detection of exosome shape and particle size

[0091] As a second aspect of the specific embodiments of the present invention, the following experimental groups are provided for control testing to verify whether the exosomes extracted from the corresponding culture medium have any impact on the shape and particle size.

[0092] Experimental group:

[0093] Experimental group A: Blank control culture medium for umbilical cord mesenchymal stem cell exosomes;

[0094] Experimental Group B: Umbilical cord mesenchymal stem cell exosomes containing a mixed culture medium containing baicalin, quercetin, glycyrrhizin, and catechin;

[0095] Experimental group C: Umbilical cord mesenchymal stem cell exosomes in a mixed culture medium of baicalin and glycyrrhizin;

[0096] Experimental group D: umbilical cord mesenchymal stem cell exosomes in a mixed culture medium containing quercetin and catechin;

[0097] In each experimental group, the concentrations of baicalin, quercetin, glycyrrhizin, and catechin were 20 μg / mL, 2.5 μM, 20 μg / mL, and 0.5 μg / mL, respectively.

[0098] Specifically, in this embodiment, umbilical cord mesenchymal stem cells were cultured in a stem cell culture medium containing a mixture of 20 μg / mL baicalin, 2.5 μM quercetin, 20 μg / mL glycyrrhizin, and 0.5 μg / mL catechin, with a normal stem cell-specific culture medium serving as a blank control. After 3 days of culture, exosomes were extracted from the supernatant, and their particle size, distribution, and morphology were analyzed using NTA and TEM. The extraction method is the same as in the first aspect of this embodiment and will not be repeated here.

[0099] In the experiments of this invention, the normal stem cell culture medium corresponding to the blank control group was a commercially available culture medium, the corresponding brand was Meallcell, and the product specification was MAO 108 500mL.

[0100] The particle size distribution of exosomes was detected using nanoparticle tracking analysis (NTA), and the morphology of exosomes was observed using transmission electron microscopy (TEM). Specific detection results are shown in Table 5 and Figures 5 and 6 below.

[0101] Table 5. Exosome particle size

[0102] As can be seen from Table 5 and Figures 5 and 6, the particle size distribution of umbilical cord mesenchymal stem cell exosomes differs under different culture medium conditions, and specific culture medium components can increase the number of exosomes in a specific particle size range.

[0103] The embodiments of the present invention demonstrate that different culture media have a certain influence on the secreted exosomes, specifically in terms of different particle sizes.

[0104] 3. Exosome function testing

[0105] As a third aspect of the specific embodiments of the present invention, the present invention provides corresponding experiments and tests for verifying exosomes with antioxidant properties.

[0106] 3.1 Anti-oxidative death experiment

[0107] Experimental groups: blank control group, H2O2 model group, H2O2+A group, H2O2+B group, H2O2+C group, H2O2+D group

[0108] This experiment uses an H2O2-induced damage model. H2O2 has a significant damaging effect on cells and significantly reduces cell survival rate. By verifying the cell survival rate in this model, we can verify whether different exosome groups can improve cell damage caused by H2O2, thereby verifying their antioxidant capacity.

[0109] Immortalized human keratinocytes (HaCat cells) in the logarithmic growth phase were seeded in 96-well plates, 5 × 10⁶ cells / well. 3 Exosomes were cultured in adherent culture for 24 hours, and divided into blank control group, H2O2 model group, and H2O2+A (5×10⁶ exosomes) group. 7 Particles / mL group, H2O2+B (exosomes 5×10) 7 Particles / mL group, H2O2+C (exosomes 5×10) 7 Particles / mL group, H2O2+D group (exosomes 5×10) 7 Cells were divided into groups (particles / mL) and treated separately. After 24 hours, the cell viability of each group was detected using a CCK-8 assay kit. The results are as follows:

[0110] Table 6. Relative cell survival rate (%) in each group Note: a indicates a significant difference compared with the model group (P < 0.05); b indicates a significant difference compared with the blank control umbilical cord mesenchymal stem cell exosome group (Group A) (P < 0.05); c indicates a significant difference compared with the umbilical cord mesenchymal stem cell exosome group (Group B) in a mixed culture medium containing baicalin, quercetin, glycyrrhizin, and catechins (P < 0.05).

[0111] Table 6 shows that the cell survival rate in group B reached over 79%, which was significantly higher than that in other groups. This indicates that group B significantly improved the cell survival rate after H2O2 damage at this concentration and had significant antioxidant properties.

[0112] In conjunction with the second aspect of the embodiments of the present invention, it is shown that different culture medium components not only affect particle size but also their antioxidant properties. Compared with the four experimental groups, experimental group B of the present invention, namely the culture medium containing a mixture of baicalin, quercetin, glycyrrhizin and catechin in the embodiments of the present invention, is more effective in stimulating the corresponding stem cells to secrete exosomes with antioxidant properties.

[0113] 3.2 Apoptosis Experiment

[0114] This experiment aimed to investigate the effects of different treatments on the survival rate of immortalized human keratinocytes (HaCat cells). By setting up a blank control group, an H2O2 model group, and treatment groups with different combinations of additives (A and B) and H2O2, the effects of these treatments on cell apoptosis were observed, thereby evaluating the role of different treatments in cell survival.

[0115] Immortalized human keratinocytes (HaCat cells) in the logarithmic growth phase were seeded in 6-well plates, 5 × 10⁶ cells / well. 5 Cells / well, adherent cultured for 24 h, and divided into blank control group, H2O2 model group, and H2O2+A (2×10⁻⁶ cells / well). 8 Particles / mL) group, H2O2+B (5×10) 7 Particles / mL) group, H2O2+B (2×10) 8 The particles were grouped into groups (particles / mL), and after 24 hours, they were stained using the Annexin V / PI apoptosis detection kit and then detected by flow cytometry. The results are shown in Figure 7 and Table 7.

[0116] Table 7. Cell viability (%) in each group Note: a indicates a significant difference compared to the model group, P < 0.05; b indicates a significant difference compared to group A, P < 0.05.

[0117] As shown in Table 7 and Figure 7, the concentration of exosome A used in the experiment did not effectively improve H2O2-induced cell damage, while the addition of exosome B effectively improved the cell survival rate after H2O2 damage, and the protective effect increased with increasing concentration, indicating that exosome B has good application potential in improving H2O2-induced cell damage.

[0118] 3.3 Anti-aging experiment

[0119] This experiment aimed to investigate the effects of different treatments on the senescence of immortalized human keratinocytes (HaCat cells). A blank control group, an H2O2 model group, and treatment groups with different combinations of exosomes (A and B) and H2O2 were set up. β-galactosidase staining was used to detect cell senescence, and the degree of cell senescence was assessed by the percentage of positive cells. The specific experimental methods are as follows:

[0120] Immortalized human keratinocytes (HaCat cells) in the logarithmic growth phase were seeded in 24-well plates, 2 × 10⁶ cells / well. 4 Exosomes were cultured in adherent culture for 24 hours, and divided into blank control group, H2O2 model group, and H2O2+A (5×10⁶ exosomes) group. 7 Particles / mL group, H2O2+B (exosomes 5×10)7 Particles / mL) group, H2O2+B (2×10) 8 The cells were divided into groups (particles / mL) and stained with a β-galactosidase staining kit after 24 hours. The cells were photographed under a microscope and the percentage of positive cells was analyzed using ImageJ software. The results are shown in Figure 8 and Table 8.

[0121] Table 8. Percentage of cells positive for β-galactosidase staining in each group Note: a indicates a significant difference compared to the model group, P < 0.05; b indicates a significant difference compared to group A, P < 0.05.

[0122] Figure 8 and Table 8 show that exosome A can alleviate H2O2-induced cell senescence to some extent, but the effect is not as good as that of exosome B. Exosome B can effectively reduce the degree of cell senescence, and the anti-senescence effect is enhanced with increasing concentration, showing potential application value in combating H2O2-induced cell senescence.

[0123] In summary, the stem cell exosomes of this invention exhibit improved antioxidant and anti-aging properties, demonstrating potential in relevant antioxidant and anti-aging applications. One such application is the incorporation of these stem cell exosomes into skincare products or pharmaceuticals, which then possess antioxidant and anti-aging effects.

[0124] 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A stem cell culture medium, characterized by, The stem cell culture medium includes baicalin, quercetin, glycyrrhizin, and catechin.

2. The stem cell culture medium of claim 1, wherein, The stem cell culture medium comprises 1–20 μg / mL baicalin, 1–10 μM quercetin, 1–20 μg / mL glycyrrhizin, and 0.1–2 μg / mL catechin.

3. The stem cell culture medium of claim 1, wherein, The stem cell culture medium includes 20 μg / mL baicalin, 2.5 μM quercetin, 20 μg / mL glycyrrhizin, and 0.5 μg / mL catechin.

4. A method of preparing stem cell exosomes having antioxidant properties, characterized by, This includes culturing stem cells in the culture medium described in any one of claims 1-3 and collecting the exosomes produced by the stem cells.

5. The method of claim 4, wherein the antioxidant is selected from the group consisting of ascorbic acid, tocopherol, and a mixture thereof. The stem cells were cultured for 3-4 days.

6. The method of claim 4, wherein the antioxidant is selected from the group consisting of ascorbic acid, tocopherol, and a mixture thereof. It also includes the purification of exosomes.

7. The method of claim 4, wherein the antioxidant is selected from the group consisting of ascorbic acid, tocopherol, and a mixture thereof. The stem cells are selected from one of the following: hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, neural stem cells, and induced pluripotent stem cells. 8.The method of claim 4, wherein the antioxidant is selected from the group consisting of ascorbic acid, tocopherol, and a mixture thereof. The stem cells mentioned are mesenchymal stem cells.

9. A stem cell exosome prepared by the method according to any one of claims 4-8.

10. A composition comprising stem cell exosomes prepared by the method of any one of claims 4-8 and excipients acceptable in the fields of skincare or pharmaceutical products.

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