Culture method and application of anti-aging stem cell exosome

Abstract

The invention belongs to the field of biological medicines or health care products, and particularly discloses a culture method and application of an anti-aging stem cell exosome. According to the method, low-oxygen (5%-8% O) pre-culture is combined with rotary three-dimensional dynamic culture, so that the yield of the exosome is remarkably increased to 2.1 * 10 < 11 > parts / mL; tGF-beta1, IFN-gamma and NMN are added in stages to synergistically activate an immunomodulatory pathway, so that the content of TGF-beta1 reaches 8.5 ng / [mu] g. The exosome and active components (vitamin C ethyl ether, Q10 and astaxanthin) are jointly embedded in liposome (HSPC: cholesterol = 7: 3), the gastric juice stability reaches 92.3% after oral administration, and the intestinal absorption rate is increased to 80%. Experiments show that the composition can improve the Treg ratio of the spleen of an old mouse by 35%, reduce the IL-6 level by 48% and improve the skin barrier function (TEWL is reduced by 40%). Long-term toxicity experiments prove that the compound is safe (ALT has no abnormality). The invention has remarkable technical progress and clinical application potential in the anti-aging field.

Description

Technical Field

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

[0002] In recent years, due to the characteristics of carrying active molecules such as miRNAs and cytokines, stem cell exosomes have attracted much attention in the field of immunomodulation. It has been found that exosomes derived from mesenchymal stem cells (MSCs) can improve immunosenescence by regulating the differentiation of T cell subsets (such as 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) can significantly reduce the levels of pro-inflammatory factors IL-2 and IFN-γ, while upregulating the immunosuppressive factors IL-10 and TGF-β, thereby prolonging the survival time of heart transplantation. In addition, exosomes from human umbilical cord MSCs cultured in three dimensions activate Treg cell-mediated immunosuppression by delivering miR-132-3p and miR-125b-5p, and have been proven to alleviate the progression of vitiligo.

[0003] However, the existing technology still has 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 in the treatment of diabetic periodontitis in the patent CN116251126B), while oral delivery faces the problems of gastric acid destruction and low intestinal absorption efficiency, and the existing patents (such as the anti-aging preparation of urinary stem cell exosomes proposed in CN202210738898.7) do not design a specific delivery system for immunosenescence.

[0005] Limited functional activity: Exosomes cultured by traditional two-dimensional culture have a low secretion amount (<10^9 particles / mL), and the technologies of hypoxic pre-culture and dynamic three-dimensional culture are not combined, resulting in insufficient concentrations of immunomodulatory molecules (such as TGF-β1 and NMN) carried by exosomes;

[0006] Lack of synergistic mechanism: The existing technology mostly relies on a single signaling pathway (such as the KEAP1-NRF2 antioxidant pathway 1 or the Wnt / β-catenin anti-fibrotic pathway), and lacks a strategy for synergistically activating Treg differentiation and telomerase activity by adding multiple factors such as TGF-β1, IFN-γ, and NMN in stages. Summary of the Invention

[0007] In view of the above problems, the present invention proposes a culture method and application of stem cell exosomes for anti-aging. Through hypoxic pre-culture (5%-8% O 2)Co-rotational three-dimensional dynamic culture significantly improves the yield of exosomes (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 embedding (hydrogenated soy phosphatidylcholine / cholesterol) significantly improves the oral stability of exosomes. Animal experiments show that it can increase the proportion of splenic Tregs in aged model mice by 30% and enhance the vaccine antibody response by more than 4 times

[0008] The object of the present invention is achieved by the following technical solutions.

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

[0010] (a) Inoculate mesenchymal stem cells at an inoculation density of 5×10 4 -1×10 5 cells / cm² into DMEM / F12 medium containing 5%-10 wt% serum substitute (preferably KnockOut™ SR), and pre-culture in a hypoxic environment with an oxygen concentration of 5%-8% for 24-48 hours;

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

[0012] (c) Transfer the culture system to a rotary bioreactor and perform dynamic three-dimensional culture at a rotation speed of 0.5-2 rpm for 72-120 hours, control the temperature at 37±0.5°C, and maintain the pH value at 7.2-7.4 through a CO 2 buffer system;

[0013] (d) Collect the culture supernatant, sequentially remove cell debris by centrifugation at 2000×g for 10-20 minutes, enrich exosomes by ultracentrifugation at 100,000×g for 1-2 hours, and then purify through a size exclusion chromatography column Sepharose CL-4B to obtain an exosome suspension with a particle size of 50-150 nm.

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

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

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

[0017] Further, in the above culture method of stem cell exosomes for anti-aging, the cytokine combination in step (b) is added in a staged pulsed 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] Further, in the above culture method of 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 porosity of the scaffold is 80 - 90%.

[0019] Further, in the above culture method of stem cell exosomes for anti-aging, the eluent of the size exclusion chromatography in step (d) is a PBS buffer 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, which contains the freeze-dried stem cell exosomes prepared by the above method and a pharmaceutically or nutraceutically acceptable carrier; the content of the freeze-dried stem cell exosomes in the composition is 0.1 - 5 mg / unit dose.

[0021] Further, the above 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 freeze-dried stem cell exosomes to the active ingredients is 1:1 to 1:3, and the freeze-dried stem cell exosomes and the active ingredients are co-embedded in liposomes. The liposome membrane material is hydrogenated soy phosphatidylcholine and cholesterol, and the mass ratio of the two is 7:3.

[0022] Further, 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 present invention discloses the application of the above anti-aging composition in the preparation of a preparation for delaying skin aging.

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

[0025] 1. Significantly improved exosome production and functional activity:

[0026] Through hypoxic (5%-8% O 2 ) pre-culture combined with three-dimensional dynamic culture, the exosome production reaches 2.1×10 11 particles / mL, which is 7 times higher than that of traditional two-dimensional culture (Comparative Example 1).

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

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

[0029] Liposome embedding (HSPC: cholesterol = 7:3) significantly improves the gastric juice stability of exosomes (92.3% vs 35.7% in the unembedded group), and the retention rate of active ingredients reaches 84.6% (Test Example 1).

[0030] After intestinal absorption, the composition is enriched in the spleen (fluorescence intensity of 20.8×10 6 ) and targets the regulation of immune aging (Test Example 5).

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

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

[0033] 4. 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 (Test Example 4).

[0034] 5. Sufficient safety verification:

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

[0036] Therefore, the present invention has significant technological progress and clinical application potential in the field of anti-aging. Description of the Drawings

[0037] Figure 1For the comparison of the proportion of Treg (%) in Test Example 2;

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

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

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

[0041] Figure 5 For the comparison of ROS level (relative fluorescence intensity) in Test Example 3. Detailed implementation mode

[0042] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts 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, without conflict, the embodiments in the present application and the features in the embodiments 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 embodiments 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 method of stem cell exosomes

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

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

[0051] Step (c): Transfer the cells to a rotating bioreactor (with an uncoated inner wall), and culture them dynamically at a rotation speed of 1 rpm for 96 hours, maintaining the temperature at 37 ± 0.5 °C and the pH at 7.3.

[0052] Step (d): Collect the supernatant, centrifuge at 2000×g for 15 minutes to remove debris, ultracentrifuge at 100,000×g for 1.5 hours to enrich exosomes, and then purify them through a Sepharose CL-4B chromatography column (the eluent is 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] Preparation of exosomes with optimized freeze-drying process.

[0055] Steps (a) - (d): The same as in Example 1.

[0056] Step (e): Mix the exosome suspension with a freeze-drying protectant (5% trehalose, 2% mannitol, 0.1% HSA) at a ratio of 1:1. After pre-freezing (-80 °C, 2 hours), perform freeze-drying at a vacuum degree of 8 Pa and a cold trap temperature of -50 °C for 30 hours. The protein recovery rate after reconstitution is 88%, and the particle size distribution remains stable (detected by DLS).

[0057] Example 3

[0058] Combined use of staged cytokine addition and antioxidants.

[0059] Based on Example 1:

[0060] Step (a): Add 0.3 mM propyl gallate (PG) to the culture medium, with an inoculation density of 1×10 5 cells / cm², and pre-culture under low oxygen (5% O 2 ) for 48 hours.

[0061] Step (b): Add cytokines in stages: Add TGF-β1 (20 ng / mL) at the 0th hour, add IFN-γ (15 ng / mL) at the 24th hour, and supplement NMN (5 μM) at the 48th hour.

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

[0063] Step (d): The yield of purified exosomes reaches 2.1×10 11 particles / mL, and the purity > 95% (detected by EXOCET)

[0064] Example 4

[0065] Preparation of an oral anti-aging composition.

[0066] Liposome preparation:

[0067] Dissolve hydrogenated soy phosphatidylcholine (HSPC) and cholesterol in chloroform at a mass ratio of 7:3, and rotary evaporate to form a uniform lipid film;

[0068] Add the freeze-dried exosome powder obtained in Example 3 (2 mg) and the active ingredients (ethyl ether of vitamin C: coenzyme Q10: astaxanthin = 2:1:1, total mass 4 mg), hydrate with PBS (pH 7.4), and ultrasonically treat (50 W, 5 min) to form a liposome suspension;

[0069] Extrude through a 0.2 μm polycarbonate membrane three times to obtain liposomes with a particle size of 100 - 150 nm, and the encapsulation efficiency of exosomes and active ingredients > 90% (detected by the combined use of HPLC and BCA method).

[0070] Lyophilization process:

[0071] Mix the liposome suspension and the lyoprotectant (5% trehalose, 2% mannitol, 0.1% HSA) at a volume ratio of 1:1, pre-freeze (-80 °C, 2 hours), and then freeze-dry for 24 hours to obtain a freeze-dried powder of composite exosomes encapsulated in liposomes.

[0072] Formulation molding:

[0073] Mix the freeze-dried powder and microcrystalline cellulose (filler) at a mass ratio of 1:5, and fill into gelatin capsules.

[0074] Comparative Example 1

[0075] Conventional two-dimensional static culture.

[0076] Step (a): Inoculate mesenchymal stem cells in a common culture dish (without a hypoxic environment), and statically culture in DMEM medium containing 10% FBS for 48 hours.

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

[0078] Comparative Example 2

[0079] Single cytokine stimulation

[0080] Step (b): Only TGF-β1 (20 ng / mL) was added, and IFN-γ and NMN were not added in stages. The ability of exosomes to promote Treg cell differentiation decreased by 40% (the proportion of CD4+CD25+Foxp3+ cells detected by flow cytometry).

[0081] Comparative Example 3

[0082] No cryoprotectant was used

[0083] Step (e): The exosome suspension was directly freeze-dried (trehalose / HSA was not added). After reconstitution, the protein recovery rate was only 52%, and the particle size distribution became wider (PDI>0.25).

[0084] Test Example 1

[0085] Verification of oral stability of exosomes

[0086] Experimental group (embedded): Liposome-embedded exosomes + active ingredients (Example 4), sample size n = 6.

[0087] Control group (unembedded): Free exosomes + unembedded active ingredients, sample size n = 6.

[0088] Method steps

[0089] Treatment with simulated gastrointestinal fluids:

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

[0091] Simulated intestinal fluid: 50 mM KH 2 PO 4 + 15 mM NaOH + 150 mM NaCl, pH 6.8, shaken at 37°C for 4 hours.

[0092] Detection indexes:

[0093] Integrity of exosomes: The particle size distribution (proportion of 50 - 150 nm) was detected by nanoparticle tracking analysis (NanoSight NS300).

[0094] Retention rate of active ingredients: The contents of vitamin C ethyl ether, Q10, and astaxanthin were detected by HPLC (C18 column, gradient elution with acetonitrile - water).

[0095] Data results are shown in Table 3

[0096]

[0097] As can be seen from the data in Table 3, liposome encapsulation significantly improved the stability of exosomes in gastrointestinal fluids (p < 0.001), with the passing rate of particle size increasing by 2.5 times and the retention rate of active ingredients increasing by 3.8 times compared to the non-encapsulated group, demonstrating the effectiveness of the oral delivery system.

[0098] Test Example 2

[0099] Effect of improving immune senescence

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

[0101] Positive control group: Freeze-dried exosomes alone, dose 0.5 mg / kg / day, sample size n = 10

[0102] Negative control group: Normal saline, sample size n = 10.

[0103] Method steps

[0104] Animal model: 24-month-old C57BL / 6 mice, half male and half female, with free access to food and water.

[0105] Drug administration regimen: Gavage administration daily for 8 weeks.

[0106] Detection indicators:

[0107] Proportion of splenic Tregs: Flow cytometry (Anti-CD4-FITC / CD25-APC / Foxp3-PE).

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

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

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

[0111]

[0112] As can be seen from the data in Table 4, the experimental group (composition) significantly increased the proportion of Tregs (by 35%) and decreased the level of inflammatory factors (the decrease in IL-6 reached 48%) compared to the group using exosomes alone, indicating that the active ingredient and exosomes have a synergistic immunomodulatory 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 size n = 6

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

[0118] Control group 2: Blank medium, sample size 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 (Telomere Repeat Amplification Protocol).

[0123] ROS level: DCFH-DA fluorescent probe (Flow cytometry to detect the mean fluorescence intensity).

[0124] The data results are shown in Table 5

[0125]

[0126] It can be seen from the above results that the combination of exosomes and active ingredients can synergistically enhance telomerase activity (50% higher than that of exosomes alone), and significantly reduce the ROS level (a decrease of 65%), proving that the composition delays cell aging through multiple pathways.

[0127] Test example 4

[0128] Improvement of skin barrier function

[0129] Experimental design

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

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

[0132] Negative control group: Normal saline, sample size n = 8.

[0133] Method steps:

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

[0135] Detection indicators:

[0136] Epidermal thickness: Measure the stratum corneum thickness by H&E staining.

[0137] Collagen content: The proportion of dermal collagen area was calculated by Masson staining.

[0138] TEWL (trans-epidermal water loss): Measured by VapoMeter.

[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 (a 40% reduction in TEWL), and the effect is better than that of the local exosome product, indicating that the present invention has systematic anti-aging advantages.

[0142] Test Example 5

[0143] Intestinal Absorption and Biodistribution

[0144] Experimental Design

[0145] Experimental group: Oral administration of Cy7-labeled exosomes (Example 4), detection time points at 2, 6, and 24 hours, sample size n = 5, C57BL / 6 mice.

[0146] Method Steps

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

[0148] In vivo imaging: The distribution of fluorescent signals was detected by a small animal imaging system (IVIS Spectrum).

[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, exosomes were mainly absorbed through the intestine (reached the peak at 2 hours) and gradually accumulated in the immune organ (spleen), providing a pharmacokinetic basis for its oral immunomodulatory effect.

[0152] Test Example 6

[0153] Long-term Safety Assessment

[0154] Experimental Design

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

[0156] Negative control group: Normal saline, sample size n = 10.

[0157] Method Steps

[0158] Administration period: continuous intragastric administration for 90 days.

[0159] Detection indexes:

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

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

[0162]

[0163] It can be seen from the data in Table 8 that long-term administration at high dose did not cause liver and kidney toxicity, proving that the composition has good safety.

[0164] Summary of examples

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

[0166] 1. Improvement in exosome production and functional activity

[0167]

[0168] It can be seen from the data that the present invention has the following advantages:

[0169] Hypoxia combined with three-dimensional dynamic culture: Pre-culture activates the HIF-1α pathway through 5%-8% O 2 to promote exosome secretion (the production is increased by 7 times compared with the traditional method); the collagen / hyaluronic acid scaffold provides a three-dimensional microenvironment, enhances cell-to-cell signal transmission, and increases the TGF-β1 content by 2.7 times.

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

[0171] Optimization of the freeze-drying process: Trehalose protects the exosome membrane structure through vitrification transition, and HSA reduces protein aggregation, resulting in a 35% increase in the activity retention rate after reconstitution compared with the prior art.

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

[0173]

[0174] It can be seen from the data that the present invention has the following advantages:

[0175] Liposome embedding technology: Hydrogenated soy 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% (≤50% for commercially available products).

[0176] Synergistic ingredient formulation: Ethyl ether of vitamin C (transdermal antioxidant), Q10 (mitochondrial function), astaxanthin (inhibiting 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] Immune aging targeted regulation: TGF-β1 and IFN-γ carried by exosomes induce the expression of the Treg-specific transcription factor Foxp3 through temporal and spatial release (added in stages), and at the same time inhibit RORγt (a key factor for Th17 differentiation), realizing the reconstruction of immune balance.

[0181] Safety guarantee: The optimized freeze-drying process avoids the use of toxic cross-linking agents (such as glutaraldehyde), and the liposome embedding of active ingredients reduces gastrointestinal irritation. The 90-day long-term toxicity experiment shows that the liver and kidney indicators are normal.

[0182] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Therefore, based on the innovative concept of the present invention, the changes and modifications made to the embodiments described herein, or the equivalent structural or equivalent process transformations made using the content of the specification of the present invention, directly or indirectly applying the above technical solutions to other related technical fields, are all included in the protection scope of the present invention patent.

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 5×10 4 -1×10 5 The cells were inoculated at a density of 10 cells / cm² in DMEM / F12 medium containing 5%-10wt% 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 value at 7.2-7.4 by a CO2 buffer system; (d) The culture supernatant was collected and centrifuged at 2000 × g for 10-20 min to remove cell debris and then ultracentrifuged 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.

2. The culture method according to claim 1, characterized in that The following steps are also included: (e) The exosome suspension obtained in step (d) is mixed with a lyophilization protective agent in a volume ratio of 1:1, wherein the lyophilization protective agent comprises 5wt% trehalose, 2wt% mannitol and 0.1wt% human serum albumin HSA, and is pre-frozen at -80°C for 2 hours, and then freeze-dried for 24-36 hours under the conditions of vacuum degree ≤10 Pa and cold trap temperature -50°C to obtain stem cell exosome lyophilized powder, and the protein recovery rate after reconstitution is ≥85%.

3. The culture method according to claim 2, characterized in that: The culture medium in step (a) is supplemented with 0.1-0.5 mM of the antioxidant propyl gallate PG.

4. The culture method according to claim 3, characterized in that: The cytokine combination in step (b) is added in a phased pulse manner, specifically: TGF-β1 is added at 0 hour, IFN-γ is added at 24 hours, and NMN is supplemented at 48 hours.

5. The culture method according to claim 4, characterized in that: 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%.

6. The culture method according to claim 5, characterized in that: The eluent of the size exclusion chromatography in step (d) is a PBS buffer containing 1% human serum albumin HSA, and the elution flow rate is 0.5-1 mL / min.

7. An anti-aging composition, characterized in that: The composition comprises a stem cell exosome freeze-dried powder prepared by the method of claim 6, and a pharmaceutically or health-care product acceptable carrier; the content of the stem cell exosome freeze-dried powder in the composition is 0.1-5 mg / unit dose.

8. The anti-aging composition according to claim 7, characterized in that: It 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.

9. The anti-aging composition according to claim 8, characterized in that: The dosage form is an oral solid preparation, including one of capsules, tablets, pills or granules.

10. Use of the anti-aging composition according to any one of claims 7 to 9 in the preparation of a preparation for delaying immunosenescence.

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