A culture method and application of stem cell exosomes for anti-aging

Abstract

The present invention belongs to the field of biomedicine or health care products, and specifically discloses a method for culturing stem cell exosomes for anti-aging and its application. The method significantly increases the exosome yield to 2.1×10 11 particles / mL; TGF-β1, IFN-γ, and NMN were added in stages to synergistically activate immune regulatory pathways, increasing TGF-β1 levels to 8.5 ng / μg. The exosomes and active ingredients (vitamin C ethyl ether, Q10, and astaxanthin) were co-encapsulated in liposomes (HSPC:cholesterol = 7:3). After oral administration, the gastric fluid stability reached 92.3%, and the intestinal absorption rate increased to 80%. Experiments showed that this composition increased the proportion of Tregs in the spleen of elderly mice by 35%, reduced IL-6 levels by 48%, and improved skin barrier function (TEWL decreased by 40%). Long-term toxicity studies confirmed its safety (no abnormalities in ALT). This invention represents a significant technological advancement and clinical application potential in the anti-aging field.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine or health care products, and specifically discloses a culture method of stem cell exosomes for anti-aging and its application. Background Art

[0002] In recent years, stem cell exosomes have attracted considerable attention in the field of immunomodulation due to their ability to carry active molecules such as miRNAs and cytokines. Studies have shown that exosomes derived from mesenchymal stem cells (MSCs) can improve immunosenescence by regulating the differentiation of T cell subsets (e.g., promoting the proliferation of regulatory T cells (Tregs) and inhibiting the activation of CD8+ T cells). For example, exosomes from bone marrow MSCs overexpressing indoleamine 2,3-dioxygenase (IDO) significantly reduce the levels of proinflammatory cytokines IL-2 and IFN-γ, while upregulating the immunosuppressive factors IL-10 and TGF-β, thereby prolonging cardiac transplant survival. Furthermore, exosomes from three-dimensionally cultured human umbilical cord MSCs have been shown to alleviate vitiligo progression by delivering miR-132-3p and miR-125b-5p, activating Treg cell-mediated immunosuppression.

[0003] However, existing technologies still have the following bottlenecks in the application of exosome immunomodulation:

[0004] Insufficient targeting and stability: Most studies use intravenous injection or local administration (such as the use of periodontal ligament stem cell exosomes for the treatment of diabetic periodontitis in patent CN116251126B), while oral delivery faces the problems of gastric acid damage and low intestinal absorption efficiency. Existing patents (such as CN202210738898.7 proposing urine-derived stem cell exosome anti-aging preparations) have not designed specific delivery systems for immune aging.

[0005] Limited functional activity: Traditional two-dimensional culture produces low exosome secretion (<10^9 particles / mL) and lacks the combination of hypoxic pre-culture and dynamic three-dimensional culture techniques, resulting in insufficient concentrations of immunomodulatory molecules (such as TGF-β1 and NMN) carried by exosomes.

[0006] Lack of synergistic mechanism: Existing technologies mostly rely on a single signaling pathway (such as the KEAP1-NRF2 antioxidant pathway 1 or the Wnt / β-catenin anti-fibrosis pathway), and lack strategies to synergistically activate Treg differentiation and telomerase activity by adding multiple factors such as TGF-β1, IFN-γ and NMN in stages. Summary of the Invention

[0007] To address the above issues, the present invention proposes a culture method and application of stem cell exosomes for anti-aging. Through hypoxic pre-culture (5%-8% O2) combined with rotating three-dimensional dynamic culture, the exosome yield is significantly improved (up to 1.2×10^11 particles / mL); TGF-β1, IFN-γ and NMN are added in a phased pulsed manner to synergistically activate the SMAD2 / 3 and Sirt1 pathways, promote Treg expansion and inhibit Th17 polarization; the freeze-drying process combined with liposome encapsulation (hydrogenated soy phosphatidylcholine / cholesterol) significantly improves the oral stability of exosomes. Animal experiments have shown that it can increase the proportion of Treg in the spleen of elderly model mice by 30% and enhance the vaccine antibody response by more than 4 times.

[0008] The purpose of the present invention is achieved through the following technical solutions.

[0009] A method for culturing stem cell exosomes for anti-aging, comprising the following steps:

[0010] (a) Mesenchymal stem cells were cultured at a rate of 5 × 10 4 -1×10 5 Cells were seeded at a density of 100 cells / cm² in DMEM / F12 medium containing 5%-10wt% serum replacement (preferably KnockOut™ SR) and pre-cultured in a hypoxic environment with an oxygen concentration of 5%-8% for 24-48 hours.

[0011] (b) adding a cytokine combination to the culture medium, wherein the cytokine combination comprises TGF-β1 at a final concentration of 10-20 ng / mL, IFN-γ at a final concentration of 5-15 ng / mL, and nicotinamide mononucleotide (NMN) at a final concentration of 1-5 μM;

[0012] (c) transferring the culture system to a rotary bioreactor and performing dynamic three-dimensional culture at a rotation speed of 0.5-2 rpm for 72-120 hours, controlling the temperature at 37 ± 0.5°C, and maintaining the pH at 7.2-7.4 using a CO2 buffer system;

[0013] (d) The culture supernatant was collected and centrifuged at 2000 × g for 10–20 min to remove cell debris, followed by ultracentrifugation at 100,000 × g for 1–2 h to enrich exosomes. The exosomes were then purified using a size exclusion chromatography column (Sepharose CL-4B) to obtain an exosome suspension with a particle size of 50–150 nm.

[0014] Furthermore, the above-mentioned method for culturing stem cell exosomes for anti-aging further comprises the following steps:

[0015] (e) The exosome suspension obtained in step (d) was mixed with a lyoprotectant in a volume ratio of 1:1, wherein the lyoprotectant comprised 5 wt% trehalose, 2 wt% mannitol, and 0.1 wt% human serum albumin (HSA). After prefreezing (-80°C, 2 hours), the mixture was freeze-dried at a vacuum of ≤10 Pa and a cold trap temperature of -50°C for 24-36 hours to obtain a lyophilized powder of stem cell exosomes. The protein recovery rate after reconstitution was ≥85%.

[0016] Furthermore, in the above-mentioned method for culturing stem cell exosomes for anti-aging, 0.1-0.5 mM of the antioxidant propyl gallate PG is added to the culture medium in step (a).

[0017] Furthermore, in the above-mentioned method for culturing stem cell exosomes for anti-aging, the cytokine combination in step (b) is added in a phased pulse manner, specifically: TGF-β1 is added at the 0th hour, IFN-γ is added at the 24th hour, and NMN is supplemented at the 48th hour.

[0018] Furthermore, in the above-mentioned method for culturing stem cell exosomes for anti-aging, the inner wall of the rotary bioreactor in step (c) is coated with a collagen / hyaluronic acid composite scaffold, and the scaffold porosity is 80-90%.

[0019] Furthermore, in the above-mentioned method for culturing stem cell exosomes for anti-aging, the eluent of the size exclusion chromatography in step (d) is a PBS buffer solution containing 1% human serum albumin HSA, and the elution flow rate is 0.5-1 mL / min.

[0020] The present invention also discloses an anti-aging composition, comprising the stem cell exosome freeze-dried powder prepared by the above method and a carrier acceptable to pharmaceuticals or health products; the content of the stem cell exosome freeze-dried powder in the composition is 0.1-5 mg / unit dose.

[0021] Furthermore, the above-mentioned anti-aging composition contains synergistic anti-aging active ingredients, which are composed of vitamin C ethyl ether, reduced coenzyme Q10 and astaxanthin, and the mass ratio of the three is 2:1:1; the mass ratio of the stem cell exosome freeze-dried powder to the active ingredient is 1:1 to 1:3, and the stem cell exosome freeze-dried powder and the active ingredient are co-encapsulated in liposomes, and the liposome membrane material is hydrogenated soybean phosphatidylcholine and cholesterol, and the mass ratio of the two is 7:3.

[0022] Furthermore, the above anti-aging composition is characterized in that its dosage form is an oral solid preparation, including one of capsules, tablets, pills or granules.

[0023] The invention discloses the application of the anti-aging composition in preparing a preparation for delaying skin aging.

[0024] Compared with the existing technology, the present invention has the following advantages and beneficial effects:

[0025] 1. Exosome production and functional activity are significantly improved:

[0026] Through hypoxia (5%-8% O2) pre-culture combined with three-dimensional dynamic culture, the exosome yield reached 2.1×10 11 particles / mL, which is 7 times higher than that of traditional two-dimensional culture (Comparative Example 1).

[0027] TGF-β1, IFN-γ and NMN were added in stages to synergistically activate the SMAD2 / 3 and Sirt1 pathways, and the TGF-β1 content was increased to 8.5 ng / μg (the existing technology is only 5.1 ng / μg).

[0028] 2. Breakthrough innovation in oral delivery systems:

[0029] Liposome encapsulation (HSPC: cholesterol = 7:3) significantly improved the gastric fluid stability of exosomes (92.3% vs 35.7% in the non-encapsulated group), with an active ingredient retention rate of 84.6% (Test Case 1).

[0030] The composition was absorbed by the intestine and accumulated in the spleen (fluorescence intensity 20.8×10 6 ), targeted regulation of immune aging (test case 5).

[0031] 3. Multi-target synergistic anti-aging mechanism:

[0032] Exosomes acted synergistically with vitamin C ethyl ether, Q10, and astaxanthin to increase telomerase activity by 2.1 times and reduce ROS levels by 65% (Test Case 3).

[0033] 4. The oral composition significantly improved skin barrier function (TEWL decreased by 40%), and the effect was better than the topical exosome product (Test Example 4).

[0034] 5. Sufficient security verification:

[0035] After long-term high-dose administration (5 mg / kg) for 90 days, there were no abnormalities in biochemical indicators such as ALT and AST, proving the safety of the preparation (Test Example 6).

[0036] Therefore, the present invention has significant technical advancement and clinical application potential in the anti-aging field. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Comparison of Treg ratio (%) in test case 2;

[0038] Figure 2 Comparison of IL-6 (pg / mL) in Test Example 2;

[0039] Figure 3 Comparison of TNF-α (pg / mL) in Test Example 2;

[0040] Figure 4 is a comparison of telomerase activity (relative fold) in Test Example 3;

[0041] Figure 5 Comparison of ROS levels (relative fluorescence intensity) in Test Example 3. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below. However, it should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the scope of the invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. All raw materials in the embodiments of the present invention can be obtained through commercial channels.

[0043] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0044] The main reagents in the examples of the present invention are shown in Table 1, and the main instruments are shown in Table 2.

[0045]

[0046]

[0047] Example 1

[0048] Basic preparation methods of stem cell exosomes

[0049] Step (a): Human umbilical cord mesenchymal stem cells were cultured at a rate of 8 × 10 4 cells / cm² were seeded in DMEM / F12 medium containing 8% serum replacement (KnockOut™ SR) and pre-cultured in a hypoxic incubator with 6% oxygen concentration for 36 hours.

[0050] Step (b): After pre-culture, add TGF-β1 at a final concentration of 15 ng / mL, IFN-γ at 10 ng / mL, and NMN at 3 μM to the culture medium and mix well.

[0051] Step (c): The cells were transferred to a rotary bioreactor (without inner wall coating) and cultured dynamically at 1 rpm for 96 hours, maintaining a temperature of 37 ± 0.5 °C and a pH of 7.3.

[0052] Step (d): The supernatant was collected and centrifuged at 2000 × g for 15 minutes to remove debris. The exosomes were enriched by ultracentrifugation at 100,000 × g for 1.5 hours and then purified by Sepharose CL-4B column (eluent: PBS) to obtain an exosome suspension with a particle size of 80-120 nm and a concentration of 1.5 × 10 11 particles / mL.

[0053] Example 2

[0054] Optimizing the freeze-drying process for exosome preparation.

[0055] Steps (a)-(d): Same as Example 1.

[0056] Step (e): The exosome suspension was mixed with a lyoprotectant (5% trehalose, 2% mannitol, and 0.1% HSA) in a 1:1 ratio. After prefreezing (-80°C for 2 hours), the mixture was lyophilized at a vacuum of 8 Pa and a cold trap temperature of -50°C for 30 hours. Protein recovery after reconstitution was 88%, and the particle size distribution remained stable (DLS analysis).

[0057] Example 3

[0058] Phased cytokine addition combined with antioxidants.

[0059] On the basis of Example 1:

[0060] Step (a): 0.3 mM propyl gallate (PG) was added to the culture medium and the inoculation density was 1 × 10 5 cells / cm², pre-cultured in hypoxia (5% O2) for 48 hours.

[0061] Step (b): Cytokines were added in stages: TGF-β1 (20 ng / mL) was added at 0 h, IFN-γ (15 ng / mL) was added at 24 h, and NMN (5 μM) was supplemented at 48 h.

[0062] Step (c): The inner wall of the rotating bioreactor was coated with a collagen / hyaluronic acid composite scaffold (porosity 85%) and cultured at a rotation speed of 2 rpm for 120 hours.

[0063] Step (d): The exosome yield after purification reached 2.1×10 11 particles / mL, purity>95% (EXOCET test)

[0064] Example 4

[0065] Preparation of oral anti-aging composition.

[0066] Liposome preparation:

[0067] Hydrogenated soybean phosphatidylcholine (HSPC) and cholesterol were dissolved in chloroform at a mass ratio of 7:3 and rotary evaporated to form a uniform lipid film;

[0068] The lyophilized exosome powder (2 mg) obtained in Example 3 and the active ingredient (vitamin C ethyl ether: coenzyme Q10: astaxanthin = 2:1:1, total mass 4 mg) were added, hydrated with PBS (pH 7.4), and sonicated (50 W, 5 min) to form a liposome suspension;

[0069] Liposomes with a particle size of 100-150 nm were obtained by extrusion through a 0.2 μm polycarbonate membrane three times, and the encapsulation efficiency of exosomes and active ingredients was >90% (detected by HPLC combined with BCA method).

[0070] Freeze-drying process:

[0071] The liposome suspension was mixed with a lyoprotectant (5% trehalose, 2% mannitol, and 0.1% HSA) in a volume ratio of 1:1, pre-frozen (-80°C, 2 hours), and then freeze-dried for 24 hours to obtain liposome-encapsulated composite exosome freeze-dried powder.

[0072] Preparation molding:

[0073] The freeze-dried powder was mixed with microcrystalline cellulose (filler) in a mass ratio of 1:5 and filled into gelatin capsules.

[0074] Comparative Example 1

[0075] Conventional two-dimensional static culture.

[0076] Step (a): Mesenchymal stem cells were seeded in a normal culture dish (without hypoxia) and statically cultured in DMEM medium containing 10% FBS for 48 hours.

[0077] Steps (b)-(d): Same as Example 1. The results showed that the exosome yield was only 3×10 10 particles / mL, and the TGF-β1 content was reduced by 60% compared with Example 1 (ELISA test)

[0078] Comparative Example 2

[0079] Single cytokine stimulation

[0080] Step (b): Only TGF-β1 (20 ng / mL) was added, without the staged addition of IFN-γ and NMN. The ability of exosomes to promote Treg cell differentiation decreased by 40% (as measured by flow cytometry, as measured by the proportion of CD4+CD25+Foxp3+ cells).

[0081] Comparative Example 3

[0082] No lyoprotectant was used

[0083] Step (e): Direct lyophilization of the exosome suspension (without the addition of trehalose / HSA). After reconstitution, the protein recovery rate was only 52%, and the particle size distribution became broad (PDI>0.25).

[0084] Test Example 1

[0085] Verification of oral stability of exosomes

[0086] Experimental group (embedded): liposome-embedded exosomes + active ingredient (Example 4), sample number n=6.

[0087] Control group (unencapsulated): free exosomes + unencapsulated active ingredients, sample number n=6.

[0088] Method steps

[0089] Simulated gastrointestinal fluid treatment:

[0090] Simulated gastric fluid: 0.1 M HCl + 34 mM NaCl, pH 1.2, 37°C with shaking (100 rpm) for 2 h.

[0091] Simulated intestinal fluid: 50 mM KH2PO4 + 15 mM NaOH + 150 mM NaCl, pH 6.8, shaken at 37°C for 4 hours.

[0092] Detection indicators:

[0093] Exosome integrity: Nanoparticle tracking analysis (NanoSight NS300) was used to detect the particle size distribution (percentage of 50-150 nm).

[0094] Retention rate of active ingredients: HPLC detection of vitamin C ethyl ether, Q10, and astaxanthin content (C18 column, acetonitrile-water gradient elution).

[0095] Data results, see Table 3

[0096]

[0097] As shown in Table 3, liposome encapsulation significantly improved the stability of exosomes in gastrointestinal fluid (p<0.001), the particle size qualification rate increased by 2.5 times compared with the non-encapsulated group, and the active ingredient retention rate increased by 3.8 times, proving the effectiveness of the oral delivery system.

[0098] Test Example 2

[0099] Immunosenescence improvement effect

[0100] Experimental group: Composition of Example 4, dose 0.5 mg / kg / day, sample number n=10

[0101] Positive control group: exosome freeze-dried powder alone, dose 0.5 mg / kg / day, sample number n=10

[0102] Negative control group: normal saline, sample number n=10.

[0103] Method steps

[0104] Animal model: 24-month-old C57BL / 6 mice, half male and half female, were fed ad libitum.

[0105] Dosage regimen: Daily oral administration for 8 weeks.

[0106] Detection indicators:

[0107] Splenic Treg ratio: flow cytometry (Anti-CD4-FITC / CD25-APC / Foxp3-PE).

[0108] Serum inflammatory factors: Luminex multifactor detection (IL-6, TNF-α).

[0109] Vaccine response: Hemagglutination inhibition test (HI titer) 14 days after influenza vaccine (H1N1 strain) immunization.

[0110] The data results are shown in Table 4.

[0111]

[0112] As can be seen from the data in Table 4, the experimental group (combination) significantly increased the proportion of Tregs (increased by 35%) and reduced the level of inflammatory factors (IL-6 decreased by 48%) compared with the exosomes alone group, indicating that the active ingredients and exosomes have a synergistic immune regulatory effect.

[0113] Test Example 3

[0114] Telomerase activity and oxidative stress

[0115] Experimental design

[0116] Experimental group: exosomes + active ingredient liposomes (Example 4), concentration 0.1 mg / mL, sample number n=6

[0117] Control group 1: exosomes alone, concentration 0.1 mg / mL, sample number n=6

[0118] Control group 2: blank culture medium, sample number n=6.

[0119] Method steps

[0120] Cell treatment: Human PBMCs were isolated from the peripheral blood of healthy volunteers, cultured in RPMI 1640 medium, and treated for 72 hours.

[0121] Detection indicators:

[0122] Telomerase activity: TRAP-PCR method (Telomeric repeat amplification procedure).

[0123] ROS level: DCFH-DA fluorescent probe (mean fluorescence intensity was measured by flow cytometry).

[0124] The data results are shown in Table 5

[0125]

[0126] From the above results, it can be seen that exosomes combined with active ingredients can synergistically enhance telomerase activity (50% higher than using exosomes alone) and significantly reduce ROS levels (by 65%), proving that the combination delays cell aging through multiple pathways.

[0127] Test Example 4

[0128] Improved skin barrier function

[0129] Experimental design

[0130] Experimental group: Composition of Example 4, dose 0.5 mg / kg / day, sample number n=8

[0131] Positive control group: commercially available exosome skin care products (topical application), equivalent dose, sample number n=8

[0132] Negative control group: normal saline, sample number n=8.

[0133] Method steps:

[0134] Animal model: UVB-induced skin photoaging mice (BALB / c, 8 weeks old).

[0135] Detection indicators:

[0136] Epidermal thickness: H&E staining was used to measure the thickness of the stratum corneum.

[0137] Collagen content: Masson staining was used to calculate the percentage of dermal collagen area.

[0138] TEWL (transepidermal water loss): VapoMeter test.

[0139] The results are shown in Table 6

[0140]

[0141] From the above results, it can be seen that the oral composition significantly improves the skin barrier function (TEWL is reduced by 40%), and the effect is better than that of local exosome products, suggesting that the present invention has systemic anti-aging advantages.

[0142] Test Example 5

[0143] Intestinal absorption and biodistribution

[0144] Experimental design

[0145] Experimental group: Cy7-labeled exosomes (Example 4) were orally administered, with detection time points of 2, 6, and 24 hours, sample number n=5, C57BL / 6 mice.

[0146] Method steps

[0147] Fluorescent labeling: Exosomes were incubated with Cy7 NHS ester (37°C, 30 min), and free dye was removed by ultrafiltration.

[0148] In vivo imaging: The fluorescence signal distribution was detected using the IVIS Spectrum small animal imaging system.

[0149] The data results are shown in Table 7.

[0150]

[0151] From the data in Table 7, it can be seen that after the preparation of Example 4, the exosomes are mainly absorbed through the intestine (peaking at 2 hours) and gradually enriched in the immune organ (spleen), providing a pharmacokinetic basis for their oral immunomodulatory effect.

[0152] Test Example 6

[0153] Long-term safety assessment

[0154] Experimental design

[0155] Experimental group: composition of Example 4, dosage 5.0 mg / kg / day (10 times the effective dose), sample number n=10, C57BL / 6 mice.

[0156] Negative control group: normal saline, sample number n=10.

[0157] Method steps

[0158] Dosage cycle: continuous gavage for 90 days.

[0159] Detection indicators:

[0160] Blood biochemistry: ALT, AST, BUN, Cr (fully automatic biochemical analyzer).

[0161] The data results are shown in Table 8.

[0162]

[0163] From the data in Table 8, it can be seen that long-term administration of high doses does not cause liver and kidney toxicity, which proves that the composition has good safety.

[0164] Summary of Examples

[0165] It can be seen from the above embodiments and test examples that, compared with the prior art, the present invention has the following improvements.

[0166] 1. Increased exosome production and functional activity

[0167]

[0168] From the data, it can be seen that the present invention has the following advantages:

[0169] Hypoxia combined with three-dimensional dynamic culture: Activate the HIF-1α pathway through 5%-8% O2 pre-culture, promoting exosome secretion (yield increased by 7 times compared to traditional methods); collagen / hyaluronic acid scaffold provides a three-dimensional microenvironment, enhances intercellular signaling, and increases TGF-β1 content by 2.7 times.

[0170] Phased pulse addition of factors: TGF-β1 induces Smad2 / 3 phosphorylation, IFN-γ activates the STAT1 pathway, and NMN increases NAD+ levels and synergistically enhances Sirt1 activity, forming a multi-pathway regulatory network (compared to single factor stimulation, Treg differentiation ability is increased by 40%).

[0171] Freeze-drying process optimization: Trehalose protects the exosome membrane structure through glass transition, and HSA reduces protein aggregation, thereby increasing the activity retention rate after reconstitution by 35% compared with existing technologies.

[0172] 2. Oral delivery system and synergistic anti-aging effects

[0173]

[0174] From the data, it can be seen that the present invention has the following advantages:

[0175] Liposome encapsulation technology: Hydrogenated soybean phosphatidylcholine / cholesterol (7:3) forms a rigid membrane structure to resist gastric acid erosion, and the intestinal absorption rate of exosomes is increased to 80% (commercially available products ≤50%).

[0176] Synergistic ingredients: Vitamin C ethyl ether (transdermal antioxidant), Q10 (mitochondrial function), astaxanthin (inhibits NF-κB) and exosomes form a three-level anti-aging mechanism of "scavenging free radicals-repairing damage-regulating immunity" (ORAC value increased by 63%).

[0177] 3.Immune regulation and safety advantages

[0178]

[0179] It can be seen from Table 11 that the present invention has the following advantages:

[0180] Targeted regulation of immune aging: TGF-β1 and IFN-γ carried by exosomes induce the expression of Treg-specific transcription factor Foxp3 through spatiotemporal release (staged addition), while inhibiting RORγt (a key factor for Th17 differentiation) to achieve immune balance reconstruction.

[0181] Safety assurance: The optimized freeze-drying process avoids the use of toxic cross-linking agents (such as glutaraldehyde), and the active ingredients are encapsulated in liposomes to reduce gastrointestinal irritation. A 90-day long-term toxicity study showed no abnormalities in liver and kidney indicators.

[0182] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications to the embodiments described herein, or equivalent structural or equivalent process transformations made using the contents of the present invention specification, directly or indirectly applying the above technical solutions to other related technical fields are all included in the scope of protection of the patent of the present invention.

Claims

1. A method for culturing stem cell exosomes for anti-aging, characterized in that: The following steps are involved: (a) Mesenchymal stem cells were cultured at a rate of 5 × 10 4 -1×10 5 cells / cm 2 The cells were inoculated at a density of 100 μg / mL in DMEM / F12 medium containing 5%-10% serum replacement and pre-cultured in a hypoxic environment with an oxygen concentration of 5%-8% for 24-48 hours. (b) adding a cytokine combination to the culture medium, wherein the cytokine combination comprises TGF-β1 at a final concentration of 10-20 ng / mL, IFN-γ at a final concentration of 5-15 ng / mL, and nicotinamide mononucleotide (NMN) at a final concentration of 1-5 μM; (c) transferring the culture system to a rotary bioreactor and performing dynamic three-dimensional culture at a rotation speed of 0.5-2 rpm for 72-120 hours, controlling the temperature at 37 ± 0.5°C, and maintaining the pH at 7.2-7.4 using a CO2 buffer system; (d) The culture supernatant was collected and centrifuged at 2000 × g for 10–20 minutes to remove cell debris, followed by ultracentrifugation at 100,000 × g for 1–2 hours to enrich exosomes. The exosomes were then purified using a size exclusion chromatography column (Sepharose CL-4B) to obtain an exosome suspension with a particle size of 50–150 nm. (e) mixing the exosome suspension obtained in step (d) with a lyoprotectant in a volume ratio of 1:1, wherein the lyoprotectant is 5 wt% trehalose, 2 wt% mannitol, and 0.1 wt% human serum albumin (HSA); pre-freezing the mixture at -80°C for 2 hours, and then freeze-drying the mixture at a vacuum of ≤10 Pa and a cold trap temperature of -50°C for 24-36 hours to obtain a lyophilized powder of stem cell exosomes, wherein the protein recovery rate after reconstitution is ≥85%; The culture medium in step (a) is supplemented with 0.1-0.5 mM of the antioxidant propyl gallate PG; The cytokine combination in step (b) is added in a phased pulsed manner, specifically: TGF-β1 is added at hour 0, IFN-γ is added at hour 24, and NMN is supplemented at hour 48; The inner wall of the rotary bioreactor in step (c) is coated with a collagen and hyaluronic acid composite scaffold, and the scaffold has a porosity of 80-90%; The eluent for the size exclusion chromatography in step (d) is PBS buffer containing 1% human serum albumin HSA, and the elution flow rate is 0.5-1 mL / min.

2. An anti-aging composition, characterized in that: The composition comprises a stem cell exosome lyophilized powder prepared by the method of claim 1, and a pharmaceutically acceptable carrier; the content of the stem cell exosome lyophilized powder in the composition is 0.1-5 mg / unit dose.

3. The anti-aging composition according to claim 2, characterized in that: It contains synergistic anti-aging active ingredients, which are composed of vitamin C ethyl ether, reduced coenzyme Q10 and astaxanthin, with a mass ratio of 2:1:1; the mass ratio of the stem cell exosome freeze-dried powder to the active ingredient is 1:1 to 1:3, and the stem cell exosome freeze-dried powder and the active ingredient are co-encapsulated in liposomes, and the liposome membrane material is hydrogenated soybean phosphatidylcholine and cholesterol, with a mass ratio of 7:

3.

4. The anti-aging composition according to claim 3, characterized in that: The dosage form is an oral solid preparation, including one of capsules, tablets, pills or granules.

5. Use of the anti-aging composition according to any one of claims 2 to 4 in the preparation of a preparation for delaying immunosenescence.

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