A method for stably producing exosomes of sheep umbilical cord mesenchymal stem cells regenerative repair and anti-inflammatory molecules and application in skin care products

Abstract

The application discloses a method for stably producing exosomes of regenerative repair and anti-inflammatory molecules of sheep umbilical cord mesenchymal stem cells and application thereof in skin care products, and comprises the following steps: S1, using healthy sheep umbilical cord as raw material, obtaining primary sheep umbilical cord mesenchymal stem cells through aseptic treatment, Wharton's jelly extraction and tissue block adherent culture; S2, introducing an immortalization gene into the primary sheep umbilical cord mesenchymal stem cells through a lentivirus transduction technology, and obtaining an immortalized sheep umbilical cord mesenchymal stem cell line through resistance screening; S3, collecting supernatant after amplification culture of the immortalized sheep umbilical cord mesenchymal stem cell line, and purifying through multi-stage centrifugal impurity removal and PEG precipitation method to obtain stably produced exosomes containing regenerative repair and anti-inflammatory molecules. The exosomes containing regenerative repair and anti-inflammatory molecules provided by the application exhibit significant advantages in yield and quality, and have outstanding application value in the fields of medical beauty anti-aging and skin care products.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for stably producing exosomes containing sheep umbilical cord mesenchymal stem cell regeneration, repair, and anti-inflammatory molecules, and their application in skincare products. Background Technology

[0002] Mesenchymal stem cells (MSCs), as a type of adult stem cell with multi-lineage differentiation potential, immunomodulatory function, and tissue repair capabilities, have had their biological value widely recognized in multiple fields. Among them, sheep umbilical cord mesenchymal stem cells (SUS-MSCs), a type of cell found in sheep umbilical cords, have become a research hotspot in recent years due to their abundant source and non-invasive donor extraction process. Studies have found that in addition to their own diverse biological functions, SUS-MSCs can also exert paracrine effects by secreting a series of vesicle-like structures into the extracellular space. These vesicles mainly include exosomes (30-200 nm in diameter), microvesicles (200-2000 nm in diameter), and apoptotic bodies (500-2000 nm in diameter). Among these secreted products, exosomes, rich in bioactive substances such as proteins, nucleic acids, and lipids, and possessing good biocompatibility and targeting properties, show significant potential, especially in regeneration, repair, and anti-inflammation, providing a new direction for the treatment of inflammation-related diseases and the development of functional cosmetics.

[0003] In the process of realizing the industrial application of SUS-MSCs exosomes, the first challenge is the dual bottleneck of large-scale preparation and efficient purification. Currently, the culture of primary SUS-MSCs generally relies on the traditional 2D adherent culture mode. In this mode, the cell growth space is limited and the density is low, directly resulting in extremely low exosome yields, which is difficult to meet the needs of industrial production. More importantly, in traditional culture systems, serum or serum substitutes are usually added to maintain cell viability. These components can mix with the exosomes secreted by SUS-MSCs, which not only greatly increases the complexity of subsequent purification processes, but may also introduce foreign proteins, viruses and other exogenous contaminants, seriously affecting the purity and safety of exosomes. At the same time, existing laboratory-level exosome purification technologies, such as ultracentrifugation and density gradient centrifugation, generally suffer from drawbacks such as cumbersome operation, long processing cycles and small single-processing volumes, making it impossible to achieve industrial scale-up. Ultimately, this leads to low exosome production efficiency and high costs, which greatly restricts the speed of product updates and cost control in fields such as cosmetics.

[0004] Besides preparation and purification issues, SUS-MSCs exosomes also face multiple challenges, including source stability, scalability, standardization, and safety. Regarding source, SUS-MSCs enter a replication-senescence state after about 10 generations of in vitro culture, making stable and continuous expansion difficult. This directly leads to significant fluctuations in the properties and activity of exosome supernatants from different batches. In terms of scalability, the extraction of SUS-MSCs from sheep umbilical cords and subsequent exosome separation involves complex procedures requiring highly specialized knowledge and technical personnel, and production capacity is insufficient to meet the large-scale demands of the national cosmetics industry. Regarding standardization, the industry currently lacks unified exosome production, characterization, and quality control protocols, resulting in significant batch instability. In terms of safety, although exosomes are generally considered to have good safety profiles, SUS-MSCs exosomes are of xenogeneic origin, and their potential side effects, immunogenicity, and long-term safety require further verification. These issues collectively constitute the main obstacles to the industrialization of SUS-MSCs-derived exosomes, necessitating the construction of a stable, efficient, and safe production system through technological innovation to achieve their sustainable application. Summary of the Invention

[0005] The main objective of this invention is to propose a method for the stable production of sheep umbilical cord mesenchymal stem cell regeneration, repair, and anti-inflammatory molecular exosomes (miR-146a-5p, miR-210-3p, miR-155-5p, let-125-5p, and miR-29a) and their application in skincare products. This aims to solve the problems of low production efficiency, low purity, and high cost of existing sheep umbilical cord mesenchymal stem cell exosomes, which prevent the transformation from laboratory research to market application.

[0006] To achieve the above objectives, this invention proposes a method for the stable production of exosomes from sheep umbilical cord mesenchymal stem cells, comprising the following steps: S1. Primary sheep umbilical cord mesenchymal stem cells were obtained by aseptic treatment, Wharton's jelly extraction and tissue block adherence culture using healthy sheep umbilical cord as raw material. S2. The immortalized gene was introduced into the primary sheep umbilical cord mesenchymal stem cells through lentivirus transduction technology, and an immortalized sheep umbilical cord mesenchymal stem cell line was obtained through resistance screening. S3. After expanding and culturing the immortalized sheep umbilical cord mesenchymal stem cell line, the supernatant is collected, purified by multi-stage centrifugation and PEG precipitation to obtain stable sheep umbilical cord mesenchymal stem cell exosomes.

[0007] Preferably, in step S1, the aseptic treatment of the sheep umbilical cord includes: when the sheep fetus is born, tying both ends of the umbilical cord tightly with high-temperature sterilized cotton thread, cutting off a 10-15cm segment, immersing it in physiological saline containing antibiotics, and repeatedly washing it with ethanol and PBS containing antibiotics 3-5 times until there is no blood.

[0008] Preferably, in step S1, the conditions for the adherent culture of the tissue block are: 37℃, 5% CO2, Wharton's jelly tissue block of 2mm size is spread flat on a 15cm culture dish, 10mL of complete culture medium is added initially, the medium is replenished to 30mL after 24h, the medium is completely replaced on the 7th day, and the primary cells are collected when the cell confluence reaches 70%~80%.

[0009] Preferably, in step S2, the immortalization gene is the hTERT gene or the SV40LT gene; The lentivirus transduction employs a three-plasmid packaging system, which includes a transfer plasmid, an envelope plasmid pMD2.G, and a packaging plasmid psPAX2.

[0010] Preferably, the immortalization gene is the hTERT gene, and the transfer plasmid is constructed using the pLV-IRES-hygro vector as a backbone to contain the hTERT gene and the hygromycin resistance gene. The full length of the hTERT gene is 3399 bp.

[0011] Preferably, in step S2, the conditions for resistance screening are as follows: 72 hours after infection with lentivirus, screening is performed using 1 μg / mL hygromycin for 15 days, with the complete culture medium containing hygromycin being updated every 3-4 days until all primary cells in the control group die.

[0012] Preferably, in step S3, the amplification and culture conditions for the immortalized cells are: 10,000 cells / cm³. 2 The cells were seeded at a density in a cell factory and cultured at 37°C with 5% CO2 for 72 hours, after which the supernatant was collected. The specific steps of the multi-stage centrifugation for impurity removal are as follows: the supernatant is centrifuged sequentially at 300×g at room temperature for 10 min, 2000×g at 4℃ for 10 min, and 10000×g at 4℃ for 30 min to remove cell debris and apoptotic bodies; The specific steps of the PEG precipitation method are as follows: after centrifugation, add 25 mL of 10% PEG solution to the supernatant of every 100 mL, mix gently, incubate at 4°C for >12 h, then centrifuge at 4°C at the maximum speed for 40 min, and resuspend the precipitate in PBS to obtain the exosome solution.

[0013] This invention proposes a method for the stable production of sheep umbilical cord mesenchymal stem cell exosomes. The exosomes are prepared by the method described above. The exosomes of different passages (S1:P5, S2:P20, S3:P40, S4:P70) of sheep umbilical cord mesenchymal stem cell regeneration, repair and anti-inflammatory molecules include miR-146a-5p, miR-210-3p, miR-155-5p, miR-125-5p and miR-29a. miR-146a-5p inhibits the NF-κB inflammatory signaling pathway through a negative feedback mechanism, reducing the production of pro-inflammatory factors (such as TNF-α, IL-1β, and IL-6). It also promotes repair by modulating the inflammatory microenvironment, particularly in osteoarthritis and nerve injury models, where it can alleviate damage and promote tissue remodeling. miR-210-3p: A key "hypoxia-responsive miRNA" that is strongly induced under tissue ischemia / hypoxia conditions, promoting angiogenesis, cell survival, and regulating energy metabolism to adapt to low-oxygen environments. miR-155-5p: a context-dependent regulator that is usually pro-inflammatory in high concentrations or chronic inflammation, but MSC exosomes may exert a fine immunomodulatory effect by delivering low levels of miR-155 or modulating its targets, such as promoting the polarization of M2 macrophages (anti-inflammatory and repair-promoting). miR-125b-5p: Promotes angiogenesis by inhibiting angiogenesis inhibitors, thereby promoting endothelial cell proliferation, migration, and tubular formation, and providing blood supply for tissue repair. The miR-29 family (such as miR-29a, miR-29b, miR-29c) contains powerful anti-fibrotic factors that can inhibit the synthesis of collagen (such as COL1A1, COL3A1), fibronectin and elastin, preventing excessive scar formation. The exosomes of sheep umbilical cord mesenchymal stem cell regeneration, repair and anti-inflammatory molecules from different generations contain miR-146a-5p, miR-210-3p, miR-155-5p, miR-125b-5p and miR-29a with a particle size distribution between 50-200 nm, and highly express CD63, CD81 and CD9 surface markers.

[0014] This invention also proposes a method for the stable production of exosomes from sheep umbilical cord mesenchymal stem cells as described above, or the application of exosomes as described above in the preparation of skin care product raw materials, wherein the exosomes can improve transdermal absorption rate through microneedle or nano-formulation carrier technology.

[0015] Preferably, the exosomes in skincare products can activate the TGF-β / Smad pathway to promote collagen synthesis, inhibit melanin synthesis, and reduce skin moisture loss.

[0016] Preferably, skin care products containing the exosomes are biosafety and have anti-wrinkle, firming, moisturizing, soothing and repairing effects.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The exosomes provided by this invention exhibit significant advantages in terms of yield and quality. By introducing the hTERT or SV40LT immortalization gene into primary sheep umbilical cord mesenchymal stem cells through lentiviral transduction technology, the resulting immortalized cell lines can overcome the limitation of primary cells entering replication and senescence after about 10 generations, achieving continuous and stable expansion. This ensures the consistency and quality stability of the exosome source cells from the source, effectively solving the problem of fluctuations in the properties and activity of exosomes from different batches in traditional processes. At the same time, the extraction of exosomes from immortalized sheep umbilical cord mesenchymal stem cells by PEG precipitation method has a high recovery rate, while maintaining the integrity of the structure and function of sheep umbilical cord mesenchymal stem cell exosomes, laying a stable and reliable material basis for subsequent applications.

[0018] (2) The exosomes provided by this invention have outstanding application value in the fields of medical aesthetics, anti-aging, and skincare products. Rich in various anti-inflammatory and repair-functional miRNAs, they can enhance transdermal absorption efficiency through carrier technologies such as microneedles and nano-formulations. After entering the skin, they can precisely activate the TGF-β / Smad pathway to promote collagen synthesis, while simultaneously inhibiting melanin production and reducing water loss, thus achieving multiple effects of anti-inflammation, anti-aging, and repair. Furthermore, the application for cosmetic raw material submission codes is strictly in accordance with the stem cell exosome extraction process, and they are uniformly referred to as animal umbilical cord extract in the cosmetic catalog. Corresponding efficacy tests (repair, anti-wrinkle, soothing, and moisturizing) and complete toxicological and safety assessment experiments were also conducted, verified by acute and 28-day repeated-dose oral toxicity tests. The results show that the exosomes have excellent repair, anti-wrinkle, soothing, and moisturizing effects, with no harmful effects or mutagenicity, demonstrating significant safety advantages, and have already obtained cosmetic registration. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present 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 only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a flowchart illustrating the extraction process of sheep umbilical cord mesenchymal stem cells according to the present invention.

[0021] Figure 2 This is a flowchart illustrating the construction process of the immortalized sheep umbilical cord mesenchymal stem cell line of this invention.

[0022] Figure 3 shows the results of flow cytometry detection of immortalized sheep umbilical cord mesenchymal stem cells P70 according to the present invention; (A) is a distribution diagram of cell populations; (B) is a diagram distinguishing between single cells and cell clusters; (C) is a diagram of CD19 expression; (D) is a diagram of CD34 expression; (E) is a diagram of CD45 expression; (F) is a diagram of CD73 expression; (G) is a diagram of CD90 expression; (H) is a diagram of CD105 expression.

[0023] Figure 4 shows a microscopic observation of the immortalized sheep umbilical cord mesenchymal stem cells of the present invention; (A): ISMSC-001 P5; (B): ISMSC-001 P70.

[0024] Figure 5 This is a flowchart illustrating the process of expanding and culturing immortalized sheep umbilical cord mesenchymal stem cells, collecting and purifying the supernatant according to the present invention.

[0025] Figure 6 This is a flowchart illustrating the entire preparation process of sheep umbilical cord mesenchymal stem cell exosomes according to the present invention.

[0026] Figure 7 is a bar chart of the relative quantitative analysis results of exosome qPCR in this invention; (A): relative expression level of miR-146a-5p; (B): relative expression level of miR-210-3p; (C): relative expression level of miR-155-5p; (D): relative expression level of miR-125b-5p; (E): relative expression level of miR-29a.

[0027] Figure 8 shows the NTA detection of exosomes from the immortalized sheep umbilical cord mesenchymal stem cells of this invention; (A): P5; (B): P70.

[0028] Figure 9 This is a schematic diagram showing the location of the hair removal area on the guinea pig skin in this invention.

[0029] Figure 10 This is a schematic diagram of the application of the test substance during the photoexcitation stage of this invention.

[0030] Figure 11 This is a test image of the in vitro moisturizing efficacy of the ISMSC-001 related sample of this invention.

[0031] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0034] Example 1: Extraction and Identification of Primary Sheep Umbilical Cord Mesenchymal Stem Cells (1) When the sheep fetus is born, tie the two ends of the umbilical cord of the sheep fetus, namely the side closer to the mother and the side closer to the fetus, with high-temperature sterilized cotton thread. Cut the umbilical cord with high-temperature sterilized scissors on the outside of the cotton thread, with a length of 12cm. Then immerse the cut umbilical cord completely in the pre-prepared physiological saline containing antibiotics. The physiological saline should be kept warm at 37°C. After disinfecting the outer surface of the physiological saline bottle containing the umbilical cord with alcohol, place it in a biosafety cabinet for later use.

[0035] (2) Open the pre-sterilized instrument box, place the umbilical cord on the sterile tray with tweezers, soak it in 75% ethanol for 60 seconds to sterilize and disinfect it, then clean the umbilical cord with PBS containing antibiotics to remove blood stains from the outside of the umbilical cord, cut the cotton thread on both sides of the umbilical cord, and clean the umbilical cord with PBS containing antibiotics again. Repeat the cleaning 4 times until the PBS is clear and free of blood stains.

[0036] (3) Cut the cleaned umbilical cord into small segments about 2cm long with scissors. Cut each segment longitudinally from the middle, remove the two veins and one artery inside, and peel off the skin on the surface of the segment to obtain Wharton's jelly. After cleaning the obtained Wharton's jelly, cut it into small square pieces about 2mm in size with a certain thickness using a sterile scalpel. Then, evenly and neatly spread the tissue blocks in a 15cm cell culture dish. Usually, the Wharton's jelly tissue block extracted from one 2cm long umbilical cord segment can be inoculated into one 15cm culture dish.

[0037] (4) Add 10 mL of complete culture medium along the side wall of the culture dish, being careful not to let the tissue block float; place the cell culture dish in a cell culture incubator, culture at 37°C, 5% CO2, and add the medium to 30 mL after 24 hours; change the medium completely on the 7th day of culture. Under normal circumstances, cells can be seen crawling out after about 5 to 9 days of culture, and fibroblast-like cell groups can be seen around the tissue block. When the cell confluence reaches about 70% to 80%, wash the tissue block with PBS and digest it with trypsin to collect the cells, which are primary cells.

[0038] (5) After passage to the second generation, a portion of the cells were preserved using liquid nitrogen cryopreservation. Simultaneously, a portion was used for flow cytometry to detect the expression of surface marker molecules of sheep umbilical cord mesenchymal stem cells: CD14, CD43, CD45, CD44, and CD105, for cell identification. The extraction process of sheep umbilical cord mesenchymal stem cells is as follows: Figure 1 As shown.

[0039] Example 2: Construction of an immortalized sheep umbilical cord mesenchymal stem cell line by lentiviral transduction of the hTERT gene. 1. Construction of hTERT lentiviral expression vector: This study used the pLV-IRES-hygro vector as a backbone, designed and synthesized hTERT-specific primers containing the BamHI (5'-ttccatttcaggtgtcgtgaggatccGCCACCatggataaagttttaaacagagag-3) and EcoRI tagagcggccgccctcgaggaattcttatgtttcaggttcaggggg-3) restriction sites. The hTERT target fragment (a transfer plasmid overexpressing the hTERT gene synthesized by Quanyang Biotechnology Co., Ltd.) was obtained by PCR amplification. The amplified hTERT fragment was ligated into the enzyme-digested vector, subsequently transformed into DH5α competent cells, and positive clones were screened and plasmids were extracted, successfully constructing a lentiviral expression vector containing the hTERT gene (pLV-hTERT-IRES-hygr). The full-length hTERT gene in this vector is 3399 bp and contains the hygromycin (HYGR) resistance gene.

[0040] 2. Lentiviral packaging and titer determination: (1) Packaging system and materials: Lentiviral packaging was performed in 293T cells using a three-plasmid system (psPAX2, pMD2.G and pLV-hTERT-IRES-hygr). The 293T cell line and the lentiviral packaging plasmids psPAX2 Vector (Addgene, ID # 12260) and PMD2.G Vector (Addgene, ID # 12259) were provided by Shanghai Bio-Pharmaceutical Technology Co., Ltd.

[0041] (2) Packaging procedure: During virus packaging, 6.5 μg of envelope plasmid (pMD2.G) and 8.5 μg of packaging plasmid (psPAX2) were added to every 10 μg of transfer plasmid (pLV-hTERT-IRES-hygr). The amounts of relevant reagents are shown in Table 1 below. After co-transfecting the above plasmids into 293T cells, the cell culture supernatant was collected after culturing for 48-72 hours, filtered through a 0.45 μm filter, and the virus titer was determined.

[0042] Table 1. Dosage of reagents related to virus packaging Reagent Name Volume usage OPTI-MEM (1×) 200μL hTERT vector plasmid 10μL PMD2.G 6.5μL psPAX2 8.5μL Transfection reagent PEI 75μL (3) Titer calculation: The viral titer was calculated by detecting the number of integrated viral genome copies in target cells using qPCR, and then using the following formula: IU / mL = (C × N × D × 1000) / V, where C is the average number of integrated copies and N is the number of infected cells (2.5 × 10⁻⁶). 5 (D represents the viral dilution factor, and V represents the volume of virus used for infection). The viral titer, calculated using qPCR, is 3 × 10⁻⁶. 8 The concentration of TU / mL was measured, and experiments showed that the infection efficiency of the target cells reached 80%.

[0043] 3. Target cell infection and screening: (1) Infection procedure: Before viral infection of the target cells, primary sheep umbilical cord mesenchymal stem cells were seeded into six-well plates and passaged twice to achieve optimal cell growth. Each 3 × 10⁻⁶ cell line was used. 6 Each cell was infected with 500 μL of recombinant lentivirus, thoroughly shaken, and incubated at 37°C for 20 h. Then, the cells were replaced with fresh MSC medium and incubated for another 20 h.

[0044] (2) Resistance screening: 72 hours after infection, resistance genes were screened using complete culture medium containing 1 μg / mL hygromycin. Uninfected primary cells served as the control group. The screening process lasted 15 days, with the complete culture medium containing hygromycin being refreshed every 3 days. After all control group cells died, the surviving cells were identified as immortalized sheep umbilical cord mesenchymal stem cells stably integrating the hTERT gene, named ISMSC-001, and used for subsequent experiments. The construction process of the immortalized sheep umbilical cord mesenchymal stem cell line is as follows: Figure 2 As shown.

[0045] Example 3: Functional and Phenotypic Detection of ISMSC-001 To investigate whether SUC-MSCs changed their cell surface markers after infection and selection, P5 (early passage) and P70 (long-term passage) cells of ISMSC-001 were selected, and the expression of cell surface markers CD19, CD34, CD45, CD73, CD90 and CD105 was measured by flow cytometry. The results of flow cytometry detection of immortalized sheep umbilical cord mesenchymal stem cells P70 are shown in Figure 3.

[0046] (1) Detection procedure: When the cells are in the logarithmic growth phase, they are digested and separated with 0.25% trypsin, gently washed three times with PBS, and then prepared into 1×10⁻⁶ cells. 6 Prepare a cell suspension of 10 cells / mL. Add an antibody incubation solution containing 1% fetal bovine serum to the cell suspension at a concentration of 5µL / 10 mL. 6 Specific antibodies for each of the above cell surface markers were added to the cell suspension at a specific ratio (all antibodies were purchased from BioLegend, San Diego, CA, USA). Each antibody and cell suspension were incubated together at room temperature in the dark for 20 min. After incubation, the cell suspension was washed twice with PBS and resuspended. Data on the expression ratio of cell surface markers were collected using flow cytometry (BD FACS Celesta, BD, USA). Experimental results were recorded and analyzed using FlowJo software.

[0047] (2) Detection results and conclusions: The results showed that in the P5 and P70 generations of ISMSC-001 cells, the expression rate of mesenchymal stem cell positive markers (CD90, CD73, CD105) was as high as 99%, while the expression level of negative markers (CD19, CD34, CD45) was less than 1%. This indicates that ISMSC-001 (immortalized sheep umbilical cord mesenchymal stem cells) exhibits the same surface marker pattern as the parent cells. During continuous proliferation, the surface phenotype expression of immortalized cells is not affected by the exogenously expressed hTERT gene, and the stem cell characteristics of the parent cells are still retained.

[0048] Example 4: Genetic stability of ISMSC-001 - karyotype detection Testing steps: (1) Primary cells were cultured to P3 and ISMSC-001 cells were cultured to P70. Cells in the logarithmic growth phase were selected and colchicine was added to a final concentration of 0.2 μg / ml. The cells were incubated at 37°C for 1.5 hours to arrest cell division.

[0049] (2) The cells were then digested with 0.25% trypsin, centrifuged at 1200 r / min to collect the cells, and resuspended in preheated 0.075 M KCl hypotonic solution. The cells were then treated with hypotonic solution at 37 °C for 40 min. Next, 2 ml of methanol: glacial acetic acid (3:1) fixative was added and mixed. The cells were then centrifuged after standing at room temperature for 10 min. The fixation was repeated 3 times to remove the cytoplasm completely.

[0050] (3) The cell pellet obtained at the end was resuspended with a small amount of fixative to make a cell suspension and dropped onto a pre-cooled glass slide. After natural drying, it was placed in a 75°C oven for 2.5 hours and stained with Giemsa for 8 minutes before observing the karyotype under a microscope.

[0051] Results and conclusions: The results showed that the karyotype of ISMSC-001 (P70 generation) was diploid (2n=46, XY), with normal chromosome number and structure, indicating that immortalization treatment did not cause genetic variation and the newly established cell line had stable karyotype characteristics.

[0052] Example 5: Amplification and culture of ISMSC001 and purification and identification of exosome ISMSC001-EXO 1. Amplification culture of ISMSC001 and collection of supernatant Immortalized sheep umbilical cord mesenchymal stem cells selected from P5 and P70 generations were infected at a rate of 10,000 cells / cm³. 2 Cells were seeded at various densities in a cell factory and cultured under the following conditions: 37℃, 5% CO2. After 72 hours, the supernatant was collected and centrifuged sequentially at 300×g (10 min, room temperature), 2000×g (10 min, 4℃), and 10000×g (30 min, 4℃) to remove cell debris and apoptotic bodies. The supernatant was stored at -80℃ for later use. Immortalized sheep umbilical cord mesenchymal stem cells from passages P5 and P70 were observed under a microscope, and the results are shown in Figure 4.

[0053] 2. Purification of ISMSC001 Add 25 mL of 10% PEG solution to every 100 mL of supernatant, mix gently 5 times, and incubate at 4°C for at least 12 h. After incubation, centrifuge at 4°C at maximum speed for 40 min, discard the supernatant, and resuspend the precipitate in PBS to obtain ISMSC-001-derived exosomes (ISMSC-001-Exo) solution.

[0054] The process flow for the expansion culture, collection, and purification of immortalized sheep umbilical cord mesenchymal stem cells is as follows: Figure 5 As shown in the figure. The entire preparation process of sheep umbilical cord mesenchymal stem cell exosomes is as follows. Figure 6 As shown.

[0055] 3. Detection of microRNA content in exosomes 3.1 Experimental Apparatus Table 3 Experimental Instruments name brand nation Biosafety cabinet Haier China centrifuge Hersey Instruments China refrigerator Haier China Ultra-low temperature freezer Haier China Ultraviolet spectrophotometer Hangzhou Suizhen China Electronic balance METTLER China Pure water system Xiamen Ruisijie China Real-time PCR instrument Bioer China Conventional gradient PCR instrument Bioer China 3.2 Experimental Methods 3.2.1 Exosome extraction and total RNA extraction 3.2.1.1 Sample Concentration: Collect cell supernatant samples and divide them into 50 mL ultrafiltration tubes. Centrifuge at 5000 rpm for 15 min. Wash once with cold PBS, then centrifuge at 5000 rpm for 15 min. Transfer approximately 1 mL of the concentrate into a 1.5 mL centrifuge tube for later use.

[0056] 3.3.1.2 Exosome Extraction Remove the exosome extraction kit, let it stand at room temperature for 20 minutes, and then mix all the reagents. Pretreatment of the concentrated solution with reagent A (2:1) according to the sample: vortex for 1 min and place on ice for 8 h; Centrifuge at 10000g for 1 hour at low temperature and collect the precipitate; Centrifuge for another 2 minutes, and discard the supernatant residue, being careful not to touch the precipitate at the bottom. Resuspend the precipitate in 100 μL PBS, centrifuge at 12000g for 2 min, collect the supernatant and store it at low temperature for later use (this step is omitted for subsequent RNA extraction).

[0057] 3.3.1.3 Total RNA Extraction Take out the RNA extraction kit, add 200 μL of lysis buffer to the exosome precipitate prepared above, and lyse thoroughly for 2 min; Add 200 μL of binding solution I, mix by inverting, and let stand for 2 min; Add 500 μL of binding solution II, mix by inversion, and let stand for 2 min; Transfer the mixture to a centrifuge column and centrifuge at 12000g for 30 seconds at low temperature; Add 600 μL of washing buffer I and centrifuge at 12000g for 30 seconds; Add 600 μL of Wash II and centrifuge at 12000 g for 30 seconds; Repeat the previous step; Add 30-40 μL of elution buffer to the column, making sure to drop it into the center of the column as much as possible, and let it stand at room temperature for 2 minutes. Centrifuge at 12000g for 2 minutes and collect the eluent; The total RNA amount was determined by ultraviolet spectrophotometer and stored at -80℃ for later use.

[0058] 3.3.2 Oligonucleotide Primer Design and Synthesis The miRNA sequence information to be tested was obtained from the NCBI-GENE database, and PCR primers were designed and chemically synthesized.

[0059] 3.3.3 Real-time quantitative PCR (qPCR) 3.3.3.1 First-strand cDNA synthesis a. Following the instructions of the cDNA first-strand synthesis kit, take 0.5 μg of the extracted RNA sample and reverse transcribe it using stem-loop primers to synthesize the first strand of cDNA. Store at -20℃ for later use.

[0060] b. The reaction system is as follows: 0.5-1 μg RNA; 1 μL RNase inhibitor; 2 μL dNTP mix; 4 μL reaction buffer; 1 μL reverse transcriptase; 1 μL stem-loop primer; and DEPC-water to a final volume of 20 μL. c. The reaction procedure is as follows: 25℃, 10min; 45℃, 50min; 80℃, 5min.

[0061] 3.3.3.2 qPCR detection a. The expression level of the target gene was detected using the SYBR Green Mix kit; b. Take 1 μL of the synthesized first-strand cDNA product, add 10 μL of mix, 1 μL of each of the target gene primers, and make up to 20 μL with sterile water. c. Place the sample in a real-time PCR instrument for detection. The reaction procedure is as follows: 95℃,5min; (95℃,15sec; 60℃,15sec)*40cycle d. Finally, the relative expression level of the target gene is calculated using the 2-ΔΔCt method.

[0062] 3.4 Experimental Results The parameters shown in Table 4 were used for determination. UV detection revealed that the extracted RNA was of good quality and suitable for further qPCR detection, as shown in Figure 7. This figure illustrates the expression differences of five specific miRNA genes detected by qPCR in four different samples, confirming the good quality of the extracted RNA.

[0063] Table 4 RNA Concentration Measurement

[0064] The primers used in qPCR testing are as follows: MiR-pR:5-CAGCCACAAAAGAGCACAAT-3 (1)miR-21-5p: miR-21-5p-pF:5-cgggcAAGCAAAACAGACAGAA-3 miR-21-5p-stem: CCTGTTGTCTCCAGCCAAAAGAGCCATATTTCAGGAGAACAGGgtcttc (2)miR-125b-5p: miR-125b-pF:5-cgggcACCCAGAGACCCAAA-3 miR-125b-5p-stem: CCTGTTGTCTCCAGCCAAAAGAGCCATATTTCAGGAGAACAGGctcttg (3) miR-29a: miR-29a-pF:5-cggggcAAGCACCAACAAA-3 miR-29a-stem: CCTGTTGTCTCCAGCCAAAAGAGCCAATTTTCAGGAGACAAGGttccgt (4)miR-29b: miR-29b-pF:5-cggggcAAGCACCAAAAAAAAA-3 miR-29b-stem: CCTGTTGTCTCCAGCCAAAAGAGCCATATTTCAGGAGAACAGGtctctg (5)miR-26a-5p: miR-26a-5p-pF:5-cggggcAACAAAAACCAGGA-3 miR-26a-5p-stem: CCTGTTGTCTCCAGCCAAAAGAGCCAATTTTCAGGAGAACAGGtgcctt (6)miR-210-3p: miR-210-3p-pF:5-cgggcCAGAGCGAGAGACAGC-3 miR-210-3p-stem: CCTGTTGTCTCCAGCCACAAAAGAGCACAATATTTCAGGAGACAACAGGtctgcc (7) miR-155-5p: miR-155-5p-pF:5-cgggcAAAAAGCAAAACGAGAAA-3 miR-155-5p-stem: CCTGTTGTCTCCAGCCACAAAAGAGCACAATATTTCAGGAGACAACAGGttcccc (8) miR-181c-5p: miR-181c-5p-pF:5-cgggcAACAAACAACCAGACG-3 miR-181c-5p-stem: CCTGTTGTCTCCAGCCACAAAAGAGCACAATATTTCAGGAGACAACAGGtctctc 4. Performance evaluation of ISMSC-001-Exo Morphological, phenotypic and particle size detection of exosomes: Morphological observation: The morphology of exosomes purified by PEG method was observed using FEI Tecnai G2 transmission electron microscope (TEM) to verify their typical structural characteristics. The results showed that the isolated ISMSC-001-derived exosomes (ISMSC-001-Exo) P5 and P70 generations both had typical cup-shaped morphology.

[0065] Phenotypic analysis and particle size determination: Single-particle interferometric reflection imaging sensing analysis (SP-IRIS) system was used for detection, and samples were processed according to the requirements of the ExoView exosome detection kit (#EV-TE-TRA-C, Sigma, Germany). The results showed that ISMSC-001-Exo samples from different passages highly expressed the exosome-specific transmembrane protein markers CD63, CD81, and CD9, while the negative controls CD9 and MIgG showed no expression. The exosome particle size distribution ranged from 50 to 200 nm, meeting the particle size standard for exosomes.

[0066] NTA detection: Nano Tracking Analysis (NTA) was used for detection. The results are shown in Figure 8. For immortalized ovine umbilical cord stem cells of either P5 or P70, both their quantity and particle size showed uniform stability and were not affected even after continuous passage to more than P70 generations. These results indicate that the ISMSC-001 cell line can effectively solve problems existing in traditional primary cells, such as large differences in raw material batches, low amplification multiples, and difficulties in large-scale production.

[0067] Example 6 Toxicity Test of ISMSC-001-Related Samples According to the "Technical Specifications for Cosmetics Safety" (2015 Edition), acute oral toxicity tests and 28-day repeated-dose oral toxicity tests were conducted on the submitted samples.

[0068] I. Acute Oral Toxicity Test (I) Materials and Methods 1. Information on Tested Samples Test Substance: Lyophilized powder of exosomes derived from ISMSC-001 (ISMSC-001-Exo), white or light yellow powder; tested as received.

[0069] 2. Experimental Animals and Rearing Environment Experimental Animals: 10 healthy adult SD rats, SPF grade, weighing 180 - 220 g, half males and half females, purchased from China Three Gorges University. The production license number of experimental animals: SCXK(E)2022 - 0012, and the quality certificate number of experimental animals: No.422542221791200315.

[0070] Rearing Environment: After purchase, the animals were reared in the animal house. The temperature of the rearing room was 25°C, and the relative humidity was 50%. The license number for the use of experimental animal environment: SYXK(E)2023 - 0096.

[0071] Feed: The feed was purchased from Jiangsu Medison Biomedical Co., Ltd., license number: Su Feed License (2023)10030, and the quality certificate number of experimental animal feed: NO.120250223015.

[0072] 3. Test Methods Dose Design and Grouping: Based on the main components of the provided tested samples, literature was consulted, indicating that there are no obvious toxic components in the tested samples. Therefore, the single-limit method was selected. According to the acute oral toxicity test method in the "Technical Specifications for Cosmetics Safety" (2015 Edition), the tested sample as received was used for gavage at a dose of 5000 mg / kg body weight, with a single gavage.

[0073] Sample Preparation Method: Taking the preparation of 100 mL as an example: Take 50 g of the tested sample as received and make up to 100 mL with ultrapure water.

[0074] Route of administration of test sample: Acute oral toxicity test method in the "Cosmetic Safety Technical Specifications" (2015 edition): After purchasing rats, they were quarantined and confirmed for 4 days. They were fasted overnight before being given the test sample, but their drinking water was not restricted. Then, the prepared solution of the test sample was administered orally by gavage at a dose of 1 mL / 100g body weight once a day to the animals.

[0075] Detection frequency and methods for various indicators: After drug administration, a comprehensive record should be kept for each animal. On the first day after administration, the experimental animals should be observed regularly for signs of poisoning and mortality. Thereafter, a thorough examination should be conducted at least daily. The observation period generally should not exceed 14 days, but this is not fixed and should be determined based on the severity of the animal's poisoning reaction, the speed of symptom onset, and the length of the recovery period. A gross anatomical examination should be performed on the experimental animals, and all gross pathological changes should be recorded. Histopathological examination should be performed on organs from animals that have died or survived for more than 24 hours and exhibit gross pathological changes.

[0076] 4. Data Statistical Processing Methods The number of animals that died and the number of animals that survived within 14 days after administration of the test sample were statistically analyzed, and the LD50 was calculated. 50 (Median lethal dose). Based on the calculated LD50. 50 The toxicity of the test sample was evaluated according to the oral toxicity classification standard.

[0077] (II) Test Results After administration of the test substance, none of the 10 animals showed any abnormalities or died.

[0078] (III) Experimental Conclusions Acute oral toxicity test of the test substance in male and female rats: Under the conditions of this test, LD50 50 Value > 5000 mg / kg, practically non-toxic.

[0079] II. 28-day repeated-dose oral toxicity test (I) Materials and Methods 1. Test Sample Information Test substance: ISMSC-001-derived exosomes (ISMSC-001-Exo) lyophilized powder, white or pale yellow powder; negative control / solvent: ultrapure water; all tested as is.

[0080] 2. Laboratory animals and their housing environment 2.1 SPF-grade SD rats, 48 in total, with 24 males and 24 females, healthy adults. Female animals should be non-pregnant and non-lactating. They were provided by Hunan Slack Jingda Laboratory Animal Co., Ltd., with the license number: SCXK(Xiang)2021-0002, and the quality certificate numbers: No.422567359287200441, No.422567359287200442.

[0081] 2.2 Rearing environment: The temperature in the rearing room is 25°C, and the relative humidity is 50%.

[0082] 2.3 License number for the use of experimental animal environment: SYXK(E)2023-0096.

[0083] 2.4 Feed: The feed was purchased from Jiangsu Medison Biopharmaceutical Co., Ltd., with the license number Su Feed License (2023)10030, and the quality certificate numbers for experimental animal feed: No.250519, No.250630, No.250710.

[0084] 3. Test methods 3.1 Test basis: "Announcement of the National Medical Products Administration (No. 12, 2024) on Incorporating 19 Revised Items such as the General Rules for Sample Pretreatment in Cosmetics Toxicology Tests into the Cosmetics Safety Technical Specifications (2015 Edition), Annex 8: 28-Day Repeated Dose Oral Toxicity Test Method".

[0085] 3.2 Operating procedures 3.2.1 Dose design and grouping: According to the requirements of "Announcement of the National Medical Products Administration (No. 12, 2024) on Incorporating 19 Revised Items such as the General Rules for Sample Pretreatment in Cosmetics Toxicology Tests into the Cosmetics Safety Technical Specifications (2015 Edition), Annex 8: 28-Day Repeated Dose Oral Toxicity Test Method", the test group (T group) and the negative control group (NC group) were carried out at a dose of 1000 mg / kg bw / day. A negative control satellite group (NS group) and a test group satellite group (TS group) were added. Each group had 10 animals, with 5 males and 5 females. Among them, 8 animals were for backup. The animals should be weighed at the beginning of the test, and the weight difference of animals of the same sex should not exceed ±20% of the average value.

[0086] 3.2.2 Sample preparation method Test group (T group: 1000 mg / kg bw / day): Taking the preparation of 100 mL as an example: Take 10 g of the original test sample and make up to 100 mL with ultrapure water, and mix well to obtain the test sample of 100 mL for the test group (T group).

[0087] Experimental satellite group (TS group: 1000 mg / kg bw / day): Taking 100 mL as an example: Take 10 g of the original test sample, dilute to 100 mL with ultrapure water, mix well to obtain 100 mL of the test sample for the experimental satellite group (TS group). The negative control group (NC group) and the negative control satellite group (NS group) were given ultrapure water.

[0088] 3.2.3 The test was conducted according to Annex 8 of the "Announcement of the National Medical Products Administration on Incorporating 19 Revision Items, including the General Rules for Sample Pretreatment of Cosmetic Toxicological Test Methods, into the Cosmetic Safety Technical Specifications (2015 Edition) (No. 12 of 2024): 28-Day Repeated-Dose Oral Toxicity Test Method": After purchasing rats, they underwent 5 days of quarantine confirmation. The experimental animals were randomly grouped and administered the drug via gavage. The daily administration time should be the same. During the administration period, the animals were weighed twice a week, and the dosage was adjusted according to body weight to maintain a constant dosage level per unit body weight. During the 28-day administration period, the experimental animals were administered the drug 7 days a week. The dosage for each group was 1 mL / 100g body weight.

[0089] The negative control group was the solvent control group. Except for not coming into contact with the test samples, the treatment methods were exactly the same as those for the experimental group animals.

[0090] 3.2.4 Clinical observation 3.2.4.1 The observation period is 28 days. The satellite group will continue to be observed for at least 14 days after the experiment, without any treatment, to understand the reversibility, persistence, and delayed onset of the toxic effects. Animals should be observed at least once daily, with observation periods as consistent as possible, or during the expected timeframe for toxic reactions. The symptoms, onset time, and duration of the toxic reaction should be recorded. If a toxic reaction occurs, the frequency of daily observations should be increased appropriately.

[0091] 3.2.4.2 Observations include the following: changes in skin and hair, changes in eyes and mucous membranes, respiration, circulation, autonomic and central nervous systems, limb movement and behavioral activities, etc.

[0092] 3.2.4.2 Record and calculate weekly changes in weight and food intake.

[0093] 3.2.5 Clinical Examination 3.2.5.1 Blood tests: After the administration of medication and the observation of the satellite group, the white blood cell count and differential, red blood cell count, hematocrit, hemoglobin concentration, platelet count, reticulocyte count, and coagulation function should be measured.

[0094] 3.2.5.2 Blood biochemistry tests: These were performed after the administration of medication and after the observation of the satellite group. The indicators included: sodium ions, potassium ions, glucose, total cholesterol, urea, creatinine, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, and bile acids.

[0095] 3.2.5.3 Blood samples were collected in accordance with PLTC-DL-SOP-037, "Standard Operating Procedures for Rat Blood Sample Collection".

[0096] 3.2.6 Pathological examination 3.2.6.1 Gross Autopsy: All animals should undergo a systematic gross dissection, including the body surface, skull, thoracic cavity, abdominal cavity, and their organs. Organs requiring autopsy include: heart, liver, spleen, lungs, kidneys, adrenal glands, testes, epididymis, uterus (including cervix), ovaries, prostate + seminal vesicles (including coagulating glands), thymus, eyes, brain (including cerebrum, cerebellum, medulla oblongata / pons), thyroid / parathyroid glands, trachea, stomach, small intestine and large intestine (duodenum, jejunum, ileum, cecum, colon, rectum) and mesenteric lymph nodes, bladder, vagina, sternum (including bone marrow), skeletal muscles, sciatic nerve, spinal cord (including cervical, thoracic, and lumbar segments), and pathological tissues. These tissues and organs should be preserved in fixative for histopathological examination. The organs that need to be weighed are: heart, liver, spleen, kidney, adrenal gland, testis, epididymis, uterus, ovary, prostate + seminal vesicle (including coagulation gland), thymus, brain, and thyroid gland. Calculate the organ coefficient (expressed as g / 100g BW or mg / 100g BW). The above organs should be weighed as soon as possible after separation to prevent moisture loss.

[0097] 3.6.2.2 Histopathology: Examine the above-mentioned tissues and organs, and provide histopathological photographs of the diseased tissues.

[0098] 3.6.2.2.1 The tissues and organs dissected in the control group and experimental group need to undergo pathological examination.

[0099] 3.6.2.2.2 For tissues and organs that exhibit toxic effects at the test substance dosage group, the satellite group shall conduct pathological examinations.

[0100] 4. Data Statistics 4.1 All data should be analyzed using appropriate statistical methods. Clinical observations and histopathological examination results, combined with statistical results of body weight, food intake, and clinical examination data, should be comprehensively analyzed to preliminarily determine the toxicological characteristics, dose-response relationship, reversibility, and delayed effects of the test substance, identify the target organ and NOAEL of the test substance, and preliminarily evaluate the oral safety of the test substance, providing reference information for further toxicity testing.

[0101] 4.2 The 28-day repeated-dose oral toxicity test can provide data on the toxic effects caused by repeated oral exposure to the test substance within a relatively short period. Due to species differences between animals and humans, extrapolating the test results to humans has certain limitations, but it can provide valuable information for preliminarily estimating the allowable exposure level for the population.

[0102] (II) Test conclusions The test substance was administered to SD rats via oral gavage for 28 consecutive days, and the satellite group was observed for an additional 14 days after the dosing ended. During the dosing period, no abnormal conditions or animal deaths were observed in each dose group, and no toxicologically significant changes were found in the test values of each index in each dose group. After the dosing ended, histopathological examinations were performed, and no histopathological changes related to the test substance were seen. No delayed toxic reactions occurred in the satellite group. Therefore, it is considered that: under the conditions of this experiment, the No Observed Adverse Effect Level (NOAEL) of the test substance for the 28-day repeated-dose oral toxicity test in SD rats is 1000 mg / kg bw / day.

[0103] Example 7 Irritation test of ISMSC-001-related samples I. Acute eye irritation test (I) Materials and methods 1. Information on the test samples Test substance: Freeze-dried powder of exosomes derived from ISMSC-001 (ISMSC-001-Exo), white or light yellow powder; negative control / solvent: ultrapure water; both were tested as received.

[0104] 2. Experimental animals and rearing environment Experimental animals: 3 healthy adult New Zealand rabbits, ordinary grade, weighing 2.0 - 2.5 kg, all female, purchased from Hunan Taiping Biotechnology Co., Ltd., production license number of experimental animals SCXK(Xiang)2023 - 0011, quality certificate number of experimental animals NO.430730250100142036.

[0105] Rearing environment: After the animals were purchased, they were reared in the animal house. The temperature of the rearing room was 25°C, and the relative humidity was 50%. The license number for the use of the experimental animal environment was: SYXK(E)2022 - 0132.

[0106] Feed: The feed was purchased from Jiangsu Medison Biopharmaceutical Co., Ltd., license number: Su Feed License (2023)10030, quality certificate number of experimental animal feed NO.250520.

[0107] 3. Test methods Before the experiment, the animals were acclimated to the environment of the experimental animal house for 3 days. Within 24 hours before the start of the experiment, the eyes of each rabbit were examined directly using a 2% sodium fluorescein solution and a hand-held slit lamp. Rabbits with eye irritation symptoms, corneal defects, and conjunctival injuries were not used in the experiment. Gently pull down the lower eyelid of one eye of the rabbit, and place 0.1 g of the test substance as it is into the conjunctival sac, and passively close the upper and lower eyelids for 1 s to prevent the loss of the test substance. The other eye was not treated as a self-control.

[0108] Examination and scoring: The eyes of the animals were examined at 1 h, 24 h, 48 h, 72 h after instillation of the test substance, and on the 4th and 7th days. If no irritation reaction occurred within 72 h, the experiment could be terminated. The integral of the eye irritation reaction was recorded according to the scoring standard for eye damage in the "Technical Specifications for Cosmetic Safety" (2015 Edition).

[0109] (II) Test conclusion The acute eye irritation of the test substance to rabbits: Under the condition of not rinsing, it was non-irritating, and the experimental results are shown in Table 5 below.

[0110] Table 5 Experimental results of the acute eye irritation of the test substance to rabbits

[0111] II. Repeated skin irritation test (I) Materials and methods 1. Information of the test samples Test substance: Freeze-dried powder of exosomes derived from ISMSC-001 (ISMSC-001-Exo), white or light yellow powder; all tested as it is.

[0112] 2. Experimental animals and rearing environment Experimental animals: 4 healthy adult New Zealand rabbits, ordinary grade, weighing 2.0 - 2.5 kg, all female, purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd., production license number of experimental animals SCXK (E) 2021-0011, quality certificate number of experimental animals NO.422555503328700331.

[0113] Rearing environment: After the animals were purchased, they were reared in the animal house. The temperature of the rearing room was 25 °C, and the relative humidity was 50%. The license number for the use of the experimental animal environment was: SYXK (E) 2022-0132.

[0114] Feed: The feed was purchased from Jiangsu Medison Biomedical Co., Ltd., license number: Su Feed License (2023) 10030, quality certificate number of experimental animal feed: NO.250520.

[0115] 3. Test method Before the experiment, the animals were acclimated to the experimental animal house environment for 3 days. Four rabbits were selected, and the hair on both sides of the back spine was removed, with the hair removal area on each side being approximately 3 cm × 3 cm. After observing for 24 hours, on one side of the depilated area of a non-damaged rabbit, 0.5 g of the test substance as it was, moistened with ultrapure water, was applied, and the application area was 2.5 cm × 2.5 cm. When the test substance was prepared with a non-irritating solvent, on the other side, the solvent was applied as a control. It was applied once a day for 14 consecutive days. Starting from the second day, the hair should be cut before each application, and the residual test substance should be removed with warm water. The skin reaction at the application site was observed 1 hour after removing the test substance, and the skin irritation reaction was scored according to the relevant regulations in the "Technical Specifications for Cosmetics Safety" (2015 Edition). After the experiment, the total score for 14 days, the average score per animal for 14 days, and the average score per animal per day were calculated respectively, and the skin irritation intensity was classified.

[0116] (II)Test Conclusion The repeated skin irritation of the test substance to rabbits was: non-irritating, and the experimental results are shown in Table 6 below.

[0117] Table 6 Experimental Results of the Repeated Skin Irritation of the Test Substance to Rabbits

[0118]

[0119] III. Skin Phototoxicity Test (I)Materials and Methods 1. Information of the Test Samples Test substance: Freeze-dried powder of exosomes from ISMSC-001 source (ISMSC-001-Exo), white or light yellow powder; Preparation method (concentration used): The original test sample moistened with ultrapure water was directly used for testing.

[0120] 2. Name of the positive control: 8-Methoxypsoralen, batch number: C10270183, manufacturer: Shanghai Macklin Biochemical Co., Ltd.; Solvent: Absolute ethanol, concentration: 0.05%, dosage: 0.2 mL.

[0121] 3. Experimental Animals and Breeding Environment: Experimental animals: 6 healthy adult Hartley guinea pigs, ordinary grade, weighing 350 - 500 g, half male and half female, purchased from Hunan Taiping Biotechnology Co., Ltd., production license number of experimental animals SCXK(Xiang)2023-0011, quality certificate number of experimental animals NO.430730250100142373.

[0122] Breeding environment: After the animals were purchased, they were bred in the animal house. The temperature of the breeding room was 25°C, and the relative humidity was 50%. The license number for the use of the experimental animal environment was: SYXK(E)2022-0132.

[0123] Feed: The feed was purchased from Jiangsu Medison Biomedical Co., Ltd., license number: Su Feed Certificate (2023) 10030, laboratory animal feed quality certificate number: NO.250520.

[0124] UV light source: Manufacturer is Tianjin Economic & Technological Development Area Hepu Industry & Trade Co., Ltd.; Model is HOPE-MED8130C.

[0125] 4. Test Methods The submitted samples were subjected to skin phototoxicity tests in accordance with the "Cosmetic Safety Technical Specifications" (2015 edition).

[0126] Measurement of UV light source intensity: Before the experiment, the light intensity (mW / cm²) was measured at 6 points on the irradiated area of ​​the experimental animal's back using a radiation meter. 2 (The values ​​are average.) Calculation of UV light source irradiation time: Irradiation dose is 10 J / cm². 2 The irradiation time is calculated using the following formula.

[0127]

[0128] Average light intensity and irradiation time: Average UVA (wavelength 365nm) irradiation intensity 6.60mW / cm² 2 The irradiation time was 25 minutes and 15 seconds.

[0129] Experimental Procedure: Animals were allowed to acclimatize to the experimental animal facility for 3 days prior to the experiment. 24 hours before the experiment, the fur on the backs of the guinea pigs was removed, and four intact skin samples were selected (see...). Figure 9 Each dehaired area was approximately 2cm × 2cm, with areas 1 and 3 on the left and areas 2 and 4 on the right. The animal was fixed in place, and 0.2g of the original test substance, moistened with ultrapure water, was applied to areas 1 and 2. After 30 minutes, areas 1 and 3 were covered with aluminum foil and secured with tape. The right side was irradiated with UVA. Skin reactions were observed at 1, 24, 48, and 72 hours after the procedure. The positive control group underwent the same test. Skin reactions were scored according to Chapter 6, Section 7, Skin Phototoxicity Tests, of the "Cosmetic Safety Technical Specifications" (2015 edition).

[0130] (II) Experimental Conclusions Results of the phototoxicity test on guinea pig skin: No skin phototoxicity was observed. The experimental results are shown in Table 7.

[0131] Table 7 Results of the test substance's phototoxicity test on guinea pig skin

[0132] Example 8: Skin Allergy Detection Test of ISMSC-001 Related Samples I. Skin allergy test (I) Materials and Methods 1. Information of Test Samples Test Substance: Freeze-dried powder of exosomes derived from ISMSC-001 (ISMSC-001-Exo), white or light yellow powder; Preparation Method (Concentration Used): Test the original test sample moistened with ultrapure water directly.

[0133] 2. Name of Positive Control: 2,4-Dinitrochlorobenzene, Batch Number: 202203271, Manufacturer: Chengdu Boruite Chemical Technology Co., Ltd.; Solvent: Acetone, Induction Concentration: 1.0%, Excitation Concentration: 0.1%, Dosage: 0.2 mL.

[0134] 3. Experimental Animals and Rearing Environment Experimental Animals: 30 healthy adult Hartley guinea pigs, ordinary grade, weighing 300 - 500 g, all female, purchased from Hunan Taiping Biotechnology Co., Ltd., Production License Number of Experimental Animals SCXK(Xiang)2023 - 0011, Quality Certificate Number of Experimental Animals NO.430730250100142251.

[0135] Rearing Environment: After the animals are purchased, they are reared in the animal house. The temperature of the rearing room is 25°C, and the relative humidity is 50%. License Number of Experimental Animal Environment SYXK(E)2022 - 0132.

[0136] Feed: The feed is purchased from Jiangsu Medison Biomedical Co., Ltd., License Number: Su Feed License (2023)10030, Quality Certificate Number of Experimental Animal Feed NO.250520.

[0137] 4. Test Methods The skin allergy test is conducted on the submitted samples in accordance with the "Technical Specifications for Cosmetics Safety" (2015 Edition). Before the test, the animals are acclimated in the experimental animal house environment for 3 days. Inductive Contact: Depilate the left side of the guinea pigs' backs 24 hours before the test. The depilated area is about 4 cm 2During the experiment, 0.2 g of the test substance, moistened with ultrapure water, was applied to the shaved area of ​​the experimental animal. It was covered with two layers of gauze and one layer of cellophane, then fixed with non-irritating adhesive tape for 6 hours. This process was repeated on days 7 and 14. The negative control group was treated with distilled water, and the positive control group was treated with 1% 2,4-dinitrochlorobenzene. Challenge contact: 14 days after the last induction, 0.2 g of the test substance, moistened with ultrapure water, was applied to the right shaved area of ​​the experimental animal (shaved 24 hours prior to contact). It was covered with two layers of gauze and one layer of cellophane, then fixed with non-irritating adhesive tape for 6 hours. The negative control group underwent the same treatment with the test substance during challenge contact, and the positive control group was treated with 0.1% 2,4-dinitrochlorobenzene. Skin allergic reactions were observed 24 hours and 48 hours after stimulation. Skin reaction scores and sensitization intensity were determined according to Chapter 6, Section 6 of the "Cosmetic Safety Technical Specifications" (2015 edition).

[0138] (II) Experimental Conclusions The results of the guinea pig skin allergy test were as follows: no skin allergy was observed. The experimental results are shown in Table 8 below.

[0139] Table 8 Results of the test substance's effect on guinea pig skin allergy test (BT method)

[0140] II. Skin photoallergy test (I) Materials and Methods 1. Test substance: Physical state: ISMSC-001-derived exosomes (ISMSC-001-Exo) lyophilized powder, white or pale yellow powder; Preparation method (concentration used): Test the original sample directly using ultrapure water.

[0141] 2. Positive control name: 6-methylcoumarin, batch number: RH314728, manufacturer: Shanghai Yi'en Chemical Technology Co., Ltd., solvent: acetone, induction concentration: 5.0%, volume: 0.1mL; activation concentration: 5.0%, volume: 0.02mL.

[0142] 3. Laboratory animals and their housing: Experimental animals: 30 healthy adult Hartley guinea pigs, ordinary grade, weighing 350 - 500 g, all female, purchased from Hunan Taiping Biotechnology Co., Ltd., with the experimental animal production license number SCXK(Xiang)2023 - 0011 and the experimental animal quality certificate number NO.430730250100142373. Feeding environment: After the animals were purchased, they were raised in the animal house. The temperature of the breeding room was 25°C and the relative humidity was 50%. The experimental animal environment use license number was SYXK(E)2022 - 0132. Feed: The feed was purchased from Jiangsu Medison Biomedical Co., Ltd., with the license number: Su Feed License (2023)10030, and the experimental animal feed quality certificate number: NO.250520.

[0143] UV light source: The manufacturer is Tianjin Development Zone Hepu Industry and Trade Co., Ltd., and the model is HOPE - MED8130C.

[0144] 4. Test methods The skin phototoxicity test was conducted on the submitted samples in accordance with the "Cosmetics Safety and Technology Specifications" (2015 edition). Measurement of the UV light source intensity: Before the test, 6 points were set in the irradiation area on the back of the experimental animals with a radiation meter to measure the light intensity (mW / cm 2 ), and the average value was used. Calculation of the UV light source irradiation time: The irradiation dose was 10.2 J / cm 2 , and the irradiation time was calculated according to the following formula.

[0145]

[0146] Before the test, the animals were acclimated in the experimental animal house environment for 3 days. 18 hours before the formal test, the hair on the neck skin of the animals was removed, and the hair removal area was approximately 2 cm × 4 cm. The skin at the test site should be intact, without damage or abnormality. Photoinduction stage: At the four corners of the hair - removed area on the animal's neck, 0.1 mL of a 1:1 (v / v) emulsion mixture of FCA and physiological saline was intradermally injected (only injected once), and then the area was adhered and uncovered with transparent tape several times to remove part of the epidermal stratum corneum. Then, 0.1 g of the test substance moistened with ultrapure water (for the positive control group and the negative control group, about 0.1 mL of the positive substance or negative (solvent) control substance was applied) was evenly and openly applied to the central hair - removed area on the neck skin preparation. After 30 minutes, it was irradiated with UVA, and the irradiation dose was 10.2 J / cm 2 , and the UVB dose should not exceed 0.1 J / cm 2 [Before the test, the light intensity (mW / cm 2 ) was measured in the irradiation area on the neck and back of the experimental animals with an ultraviolet irradiometer, and the irradiation time was calculated], see Figure 10The exfoliation, open application of the test substance, and UV irradiation processes were performed once daily for a total of 5 times. Photoexcitation phase: Two weeks after the completion of photoinduction treatment, 18 hours prior, the skin on both sides of the spine on the back of the guinea pig was shaved. Each shaved area was approximately 2cm × 2cm, and the actual application area was 1.5cm × 1.5cm, for a total of 4 shaved areas. The animal was fixed, and 0.02g of the original test substance moistened with ultrapure water was applied to shaved areas 3 and 4 (for the positive control group and negative control group, 0.02mL of the positive substance or negative (solvent) control was applied). (See the application areas for details). Figure 10 After 30 minutes, the hair removal areas on the neck and left side (1 and 3) were covered with aluminum foil and secured with non-irritating adhesive tape, while the hair removal areas on the right side (2 and 4) were irradiated with a UVA light source. 0.2 g of the original test sample, moistened with ultrapure water, was applied as a coating. The light source was UVA, and the irradiation dose was 10.2 J / cm². 2 During the photoexcitation phase, 24 hours and 48 hours after UV irradiation, the local skin reaction at the application site was observed visually and scored. During the photoinduction phase, 24 hours after UV irradiation, the local skin reaction at the application site was observed visually and scored. The animal's general condition, behavior, and physical signs were also observed. If necessary, histopathological examination of the skin at the application site could be performed. The skin reaction score and sensitization intensity were graded according to Chapter 6, Section 20 of the "Cosmetic Safety Technical Specifications" (2015 edition).

[0147] (II) Experimental Conclusions The results of the photoallergic reaction test on guinea pig skin were as follows: no photoallergic reaction was observed. The experimental results are shown in Table 9 below.

[0148] Table 9 Results of the test substance's photoallergic reaction on guinea pig skin

[0149] Example 9: Genotoxicity test of ISMSC-001 related samples I. Bacterial Reversion Mutation Test (I) Materials and Methods 1. Test strains: Salmonella Typhimurium TA1535, TA97a, TA98, TA100, TA102, source: Moltox, USA. See Table 10 for strain descriptions.

[0150] 2. Metabolite Activation System: Liver homogenate (S9) was prepared from rats induced by the combination of phenobarbital sodium and β-naphthylflavonoid. Source: Wuxi Xinrun Biotechnology Co., Ltd., batch number: 20240924. The S9 mixture contained 10% of the S9 component and supplementary cofactors, and was used as the in vitro metabolic activation system in this experiment.

[0151] Table 10 Strain Description strain Positive material source batch number solvent dose +S9 TA97a, TA98, TA100, TA1535TA102 2-Aminofluorene 1,8-Dihydroxyanthraquinone Ron Reagents Shanghai McLean Biochemical Technology Co., Ltd. RH734168C15900937 DMSODMSO 10g / dish 50 / dish -S9 TA100, TA1535TA97a, TA98, TA102 Sodium azide dichlorvos Northern Weiye Metrology Group Co., Ltd. Tanmo Quality Inspection Technology Co., Ltd. 2024071275ALT602978 Sterile ultrapure water 2g / dish 50g / dish 3. Positive sample: 4. Test Substance: ISMSC-001-derived exosomes (ISMSC-001-Exo) lyophilized powder, white or pale yellow powder. During the experiment, sterile ultrapure water was used as the solvent to prepare a series of test solutions with corresponding dosages. The preliminary test used 10000 g / plate as the highest dosage, with four dosage groups: 5000 g / plate, 2500 g / plate, and 1250 g / plate. Preliminary test results showed that the test substance had good solubility at dosages of 10000 g / plate and below; under the condition of adding a metabolic activation system, antibacterial activity began to appear at a dosage of 5000 g / plate, with clear background bacterial changes in TA97a and TA102, and a significant reduction in the number of revertant colonies in TA100; under the condition of not adding a metabolic activation system, antibacterial activity began to appear at a dosage of 2500 g / plate, with clear background bacterial changes in TA100 and TA97a. The minimum inhibitory doses (MICs) with and without S9 were 5000 g / plate and 2500 g / plate, respectively. Based on the bacterial toxicity and solubility of the test analyte, the dosages for the formal test were set as follows: +S9 conditions: 5000 g / plate, 2500 g / plate, 1250 g / plate, 625 g / plate; -S9 conditions: 2500 g / plate, 1250 g / plate, 625 g / plate, 312.5 g / plate. A blank control (spontaneous reversion), a solvent control (sterile ultrapure water), a positive control, and a sterile control were also included in the test.

[0152] 5. Experimental Method: Plate Incorporation Method. Dispense 2.0 mL of top agar medium containing 0.5 mmol / L histidine-0.5 mmol / L biotin solution into test tubes and incubate at 45°C. Then, add 0.1 mL of enrichment broth for the test strain, 0.1 mL of the test solution (or solvent, positive control; no addition for the blank control group), and 0.5 mL of phosphate buffer (without S9 conditions) or 0.5 mL of S9 mixture (with S9 conditions) to each tube sequentially. Pour the mixture onto the bottom agar plate, with three parallel plates for each test point. After the plates solidify, incubate them upside down at 37°C for 48 hours, and count the number of revertant colonies per plate.

[0153] 6. Result Interpretation: If the number of revertant colonies of test substance TA1535 is three times or more than three times the number of revertant colonies of the solvent control, and the number of revertant colonies of test substances TA97a, TA98, TA100, and TA102 is two times or more than two times the number of revertant colonies of the solvent control, and a dose-response relationship is shown or a positive reaction is observed under any dose condition with repeatability, then the test substance is determined to be mutagenic positive. If, after testing the test sample with five test strains, any one test strain is positive regardless of whether S9 is added or not, the bacterial reversion test of the test substance can be determined to be mutagenic positive. If all five test strains are negative under both S9 and non-S9 conditions, the test substance can be determined to be mutagenic negative.

[0154] II) Experimental Conclusions The number of revertant colonies in the blank control group, solvent control group, and positive control group were all within the normal range of our laboratory, indicating that the test system was effective. Under both S9-added and S9-free conditions, the number of revertant colonies of the test strains TA97a, TA98, TA100, and TA102 in each dose group of the test substance did not reach twice the number of revertant colonies in the corresponding solvent control group, and the number of revertant colonies of TA1535 did not reach three times the number of revertant colonies in the corresponding solvent control group, and no dose-response relationship was observed. Results of the bacterial reversion mutation test: Under the conditions of our laboratory, no mutagenicity was observed with or without the addition of the metabolic activation system. The experimental results are shown in Table 11 below.

[0155] Table 11. Number of reverting colonies of ANES test strains (mean ± standard deviation)

[0156] II. In vitro mammalian cell chromosome aberration test (I) Materials and Methods 1. Cell line: Chinese hamster lung (CHL) cell line, purchased from Cybio (Shanghai) Biotechnology Co., Ltd.

[0157] 2. Metabolite Activation System: Liver homogenate (S9) was prepared from rats induced by the combination of phenobarbital sodium and β-naphthylflavonoid. Source: Wuxi Xinrun Biotechnology Co., Ltd., batch number: 20240924. The S9 mixture contained 10% of the S9 component and supplementary cofactors, and was used as the in vitro metabolic activation system in this experiment.

[0158] 3. Positive sample: +S9: Aqueous solution of cyclophosphamide (provided by Shanghai Yuanye Biotechnology Co., Ltd., batch number: KS354729). Take 15mg of cyclophosphamide and add it to sterile ultrapure water to make up to 10mL to prepare a solution with a concentration of 1.50mg / mL. Filter to sterilize and use immediately according to the dosage of 1500g / mL.

[0159] -S9: Mitomycin C (provided by Jiangxi Ruiwei Biotechnology Co., Ltd., batch number: 174166415159491) aqueous solution. Take 5mg of mitomycin C (MMC) and add it to sterile ultrapure water to make up to 100mL to prepare a solution with a concentration of 0.05mg / mL. Filter to remove bacteria and use immediately at a dosage of 50g / mL.

[0160] 4. Test substance: ISMSC-001-derived exosome (ISMSC-001-Exo) lyophilized powder, white or pale yellow powder, with intact packaging. Under aseptic conditions, it is prepared directly as the test substance with sterile ultrapure water to prepare test solutions of different concentrations. No visible insoluble matter is present.

[0161] 5. Cytotoxicity assay: Well-grown CHL cells were selected, digested with 0.25% trypsin solution, and then cultured in medium containing 10% fetal bovine serum to prepare 1.0 × 10⁶ cells / mL. 5 Cells were cultured at a density of 10 cells / mL, and 5 mL of the suspension was seeded into T25 culture flasks. The flasks were then incubated at 37°C for 24 h in a 5% CO2 incubator. After 24 h, the culture medium was discarded, and 0.1 mL of different concentrations of test solution or negative control solution, 0.5 mL of S9 mixture (if S9 mixture was not added, the volume was adjusted with serum-free culture medium), and serum-free culture medium were added to a final volume of 5 mL. The flasks were then incubated in a CO2 incubator for 4 h. After this period, the culture medium in the T25 flasks was discarded, and the cells were washed twice with PBS. 5 mL of culture medium containing 10% fetal bovine serum was added, and the flasks were incubated in a CO2 incubator for further culture. Cells were harvested within 24 h. After culture, the cells were digested with 0.25% trypsin solution and a cell suspension was prepared for cell counting.

[0162] Dosage design and maximum dose design basis: Preliminary test results showed that, with the addition of the metabolic activation system, the cell survival rate reached 50% at a test substance concentration of 5000 g / mL, and without the addition of the metabolic activation system, the cell survival rate reached 50% at a test substance concentration of 5000 g / mL. Based on the toxicity and solubility of the test substance, the formal test doses were: with S9: 5000 g / mL, 2500 g / mL, 1250 g / mL; without S9: 5000 g / mL, 2500 g / mL, 1250 g / mL.

[0163] 6. Chromosomal aberration assay: Select well-grown CHL cells, digest them with 0.25% trypsin solution, and then prepare 1.0×10⁶ cells using culture medium containing 10% fetal bovine serum. 6Cells were cultured at a concentration of 10 cells / mL, and 5 mL of the suspension was seeded into T25 culture flasks. The flasks were then incubated at 37°C for 24 h in a 5% CO2 incubator. After 24 h, the culture medium was discarded, and 0.1 mL of different concentrations of the test substance solution, 0.1 mL of negative control solution, or 0.05 mL of positive control solution (cyclophosphamide, mitomycin C), 0.5 mL of S9 mixture, and serum-free culture medium were added to bring the total volume to 5 mL. After mixing, the flasks were incubated at 37°C in a 5% CO2 incubator. After 4 h of contact, the culture medium was discarded, and the cells were washed three times with Hanks' solution. 5 mL of serum-containing culture medium was added, and the flasks were incubated at 37°C in a 5% CO2 incubator for another 24 h. Four hours before harvesting the cells, colchicine (final concentration 1 g / mL) was added to inhibit metaphase of mitosis. After culture, the culture medium was discarded, and the cells were harvested. The cells were then subjected to hypotonic fixation, slide preparation, and staining according to standard methods. Two hundred well-dispersed metaphase cells from each group were selected for chromosome aberration analysis.

[0164] 7. Statistical analysis: The chi-square test was used on cells with chromosomal aberrations to evaluate the mutagenicity of the test substance.

[0165] 8. Result evaluation: The test substance can be determined to be mutagenic in this test system under the following two conditions.

[0166] (1) The number of chromosomal structural aberrations induced by the test substance was statistically significant and dose-dependent.

[0167] (2) The test substance causes a statistically significant increase under any dosage condition, and the increase is reproducible.

[0168] Biological and statistical significance should be considered together in the evaluation.

[0169] (II) Experimental Conclusions Results of in vitro mammalian cell chromosome aberration assay: Under the conditions of this laboratory, no mutagenicity was observed in the in vitro mammalian cell chromosome aberration assay of this test substance, with or without the addition of a metabolic activation system. The experimental results are shown in Tables 12 and 13.

[0170] Table 12. Cell toxicity results of the test substances

[0171] Table 13 Results of in vitro mammalian cell chromosome aberration assay

[0172] Example 10: Evaluation of the skin repair efficacy of ISMSC-001 related samples 1. Test Sample Information Test substance: ISMSC-001-derived exosome (ISMSC-001-Exo) lyophilized powder + solvent. The stock solution was prepared by mixing 50mg of lyophilized powder with 10ml of solvent and used as the test sample.

[0173] 2. Test Methods According to the "PLTC-GX-SOP-017 Skin Nourishing / Repairing / Moisturizing Efficacy Evaluation Test Operating Instructions", 30 participants with TEWL > 15 g / m 2 All volunteers with dry, reddish skin (approximately 1 hour) signed informed consent forms before the test. Prior to signing, volunteers were informed of the purpose of the test, potential benefits, risks and issues, and their rights and obligations. After signing, volunteers cleaned their faces and sat quietly for 30 minutes in a laboratory at 21±1℃ and 50±10% humidity. The basal transepidermal water loss (TEWL) value and skin hemoglobin content at a depth of 2 cm from the nose were measured (each measured three times). Volunteers were instructed to apply the test sample. Throughout the test, no other similar cosmetics could be used on the test area (face) except for the provided test sample. A follow-up visit was conducted 14 days later, and the cleansing, resting, and indicator tests were repeated. Paired statistical analysis was performed using SPSS software to calculate the TEWL improvement rate. Subjective self-assessment questionnaires were also collected from volunteers to calculate the satisfaction rate.

[0174] 3. Statistical methods and test results SPSS software was used to perform relevant statistical analysis on the results data.

[0175] The continuous data underwent a normality test. For data conforming to a normal distribution, paired t-tests were used; for data not conforming to a normal distribution or ordinal data, Wilcoxon paired tests were used. The results are shown in Tables 14-16 below.

[0176] Table 14 Results of transdermal water loss test

[0177] Note: Improvement rate (%) = (Base value - Test value after N days of product use) / Base value × 100% Table 15 Results of Skin Hemoglobin Content Test

[0178] Note: Improvement rate (%) = (Base value - Test value after N days of product use) / Base value × 100% Table 16 Subject self-report results

[0179] Note: For significance, “ns” indicates no statistical difference, p≥0.05; “*” indicates a significant difference, 0.01≤p<0.05; “**”, 0.001≤p<0.01; “***”, p<0.001.

[0180] The results showed that after 14 days of product use, the average transepidermal water loss of volunteers decreased from the baseline value of 18.63 g / m³. 2 / h decreased to 14.71g / m 2 The improvement rate was 21.03% per hour, with a statistically significant difference compared to the baseline (P < 0.001). Skin hemoglobin levels decreased significantly, with an improvement rate of 16.73%, indicating that the skin barrier function was repaired and moisture loss was significantly reduced. Subject self-reports showed that 100.0% of volunteers reported improved skin hydration, relief from dryness, and reduced redness, while 96.7% reported improved skin roughness, confirming that the product can reduce moisture loss and improve dryness and roughness by repairing the skin barrier.

[0181] Example 11: Determination of the soothing efficacy of ISMSC-001 related samples 1. Test Sample Information Test substance: ISMSC-001-derived exosome (ISMSC-001-Exo) lyophilized powder + solvent. The stock solution was prepared by mixing 50mg of lyophilized powder with 10ml of solvent and used as the test sample.

[0182] 2. Test Methods This method is an in vitro method that reflects the correlation between the sample and the activity of hyaluronidase. Inhibiting hyaluronidase activity can ensure the normal content and function of hyaluronic acid. Therefore, whether the test sample has a soothing effect can be determined by the inhibition rate of hyaluronidase activity. If the test sample has a certain inhibition rate of hyaluronidase, it can be considered that the test sample has a certain soothing effect.

[0183] (1) Sample preparation: Take the prepared test sample stock solution and perform standardized pretreatment according to the test requirements to ensure that the sample concentration is uniform and stable; (2) Color reaction: The pretreated test sample is mixed with hyaluronidase solution, substrate solution, etc. in a set ratio to carry out the color reaction; (3) Absorbance test: After the reaction is completed, the absorbance value of the reaction system is measured by a spectrophotometer at a wavelength of 530 nm; (4) Blank control setting: A blank control group without the test sample was set up at the same time. The other reaction conditions were completely the same as those of the test group, which was used to calculate the baseline value of the inhibition rate.

[0184] 3. Inhibition rate calculation Based on the difference in absorbance between the blank control group and the test sample group, the hyaluronidase activity inhibition rate was calculated using the formula: Inhibition rate (%) = (Absorbance of blank group - Absorbance of sample group) / Absorbance of blank group × 100%.

[0185] 4. Test Results The absorbance of the blank control group was A0, and the absorbance of the test sample group was A1. Substituting these values ​​into the formula, the calculated inhibition rate of ISMSC-001-Exo lyophilized powder + solvent stock solution on hyaluronidase activity was 49.6%.

[0186] 5. Experimental Conclusions The test sample showed significant inhibitory activity against hyaluronidase, with an inhibition rate of 49.6%. This indicates that the product can reduce the degradation of hyaluronic acid in the skin by inhibiting hyaluronidase activity, maintain the skin barrier function, and thus exert a certain soothing effect, relieving discomfort symptoms related to skin inflammation and allergies.

[0187] Example 12 Evaluation of the anti-wrinkle and firming effects of ISMSC-001 related samples 1. Test Sample Information Test substance: ISMSC-001-derived exosomes (ISMSC-001-Exo) lyophilized powder was prepared into a stock solution using ultrapure water. 2. Test Methods This method is an in vitro method that reflects the correlation between the sample and elastase activity. Related literature shows that the trends of in vivo methods are somewhat correlated with those of in vitro methods. The absorption changes due to the inhibition of elastase activity by the sample; therefore, whether a test sample has anti-wrinkle and firming effects can be determined by the elastase activity inhibition rate. If the test sample has a certain inhibition rate on elastase, it can be considered that the test sample has certain anti-wrinkle and firming effects.

[0188] Using N-Succinyl-Ala-Ala-Ala-p-nitroanilide as a substrate, porcine pancreatic elastase was catalyzed to react with the substrate, and test samples were added simultaneously. After incubation at 25°C for a specified time, the absorbance of the reaction system was measured. The elastase inhibition rate was calculated by measuring the change in absorbance to determine the anti-wrinkle potential of the product.

[0189] 3. Experimental Results The test sample showed an inhibition rate of 45.1% against elastase, indicating that it can effectively inhibit elastase activity, slow down the degradation of skin elastin, and has certain anti-wrinkle and firming effects.

[0190] Example 13: Evaluation of the in vitro moisturizing efficacy of ISMSC-001 related samples 1. Test Sample Information Test substance: ISMSC-001-derived exosome (ISMSC-001-Exo) lyophilized powder + solvent. The stock solution was prepared by mixing 50mg of lyophilized powder with 10ml of solvent and used as the test sample.

[0191] 2. Test Methods This method is an in vitro method, reflecting the true state of the sample during testing. Relevant literature shows that DL-glutamate has good moisturizing effects, and the trend of the in vivo method is basically consistent with the results of the in vitro method, indicating a certain correlation between the two methods. A blank control, a positive control (DL-glutamate), and an experimental sample group (ISMSC-001-derived exosomes (ISMSC-001-Exo) lyophilized powder + solvent, lyophilized powder: solvent = 50 mg: 10 ml) were set up. After preparing each control and experimental sample, all three were placed in a constant humidity environment, and weighed at 1 h, 2 h, and 4 h, respectively, to calculate the moisturizing rate and perform comparative analysis.

[0192] 3. Experimental Results The results of the moisturizing rate test at different time points are shown in Table 17 below. Figure 11 As shown in the figure. The results showed that the moisturizing rate of the test samples at each time point was significantly higher than that of the blank control and the positive control, indicating that the product has a significant moisturizing effect.

[0193] Table 17 Moisturizing Rate Test Results Test time 1h 2h 4h Moisturizing rate (% of blank control) 32.34 14.56 6.39 Positive control hydration rate, % 38.58 18.28 8.66 Moisture retention rate of test samples, % 70.55 38.32 14.61 The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A method for stably producing exosomes containing sheep umbilical cord mesenchymal stem cell regeneration, repair, and anti-inflammatory molecules, characterized in that, Includes the following steps: S1. Primary sheep umbilical cord mesenchymal stem cells were obtained by aseptic treatment, Wharton's jelly extraction and tissue block adherence culture using healthy sheep umbilical cord as raw material. S2. The immortalized gene was introduced into the primary sheep umbilical cord mesenchymal stem cells through lentivirus transduction technology, and an immortalized sheep umbilical cord mesenchymal stem cell line was obtained through resistance screening. S3. After expanding and culturing the immortalized sheep umbilical cord mesenchymal stem cell line, the supernatant was collected, purified by multi-stage centrifugation and PEG precipitation to obtain exosomes containing regenerative repair and anti-inflammatory molecules that are stably produced.

2. The method for stably producing exosomes containing sheep umbilical cord mesenchymal stem cell regeneration, repair, and anti-inflammatory molecules according to claim 1, characterized in that, In step S1, the aseptic treatment of the sheep umbilical cord includes: when the sheep fetus is born, tie both ends of the umbilical cord tightly with high-temperature sterilized cotton thread, cut off a 10-15cm segment, immerse it in physiological saline containing antibiotics, and repeatedly wash it with ethanol and PBS containing antibiotics 3-5 times until there is no blood.

3. The method for stably producing exosomes containing regenerative, repair, and anti-inflammatory molecules from sheep umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step S1, the conditions for the adherent culture of the tissue block are: 37℃, 5% CO2. The 2mm Wharton's jelly tissue block is spread flat on a 15cm culture dish, 10mL of complete culture medium is added initially, and the medium is replenished to 30mL after 24h. The medium is completely replaced on the 7th day. Primary cells are collected when the cell confluence reaches 70%~80%.

4. The method for stably producing exosomes containing regenerative, repair, and anti-inflammatory molecules from sheep umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step S2, the immortalization gene is either the hTERT gene or the SV40LT gene; The lentivirus transduction employs a three-plasmid packaging system, which includes a transfer plasmid, an envelope plasmid pMD2.G, and a packaging plasmid psPAX2.

5. The method for stably producing exosomes containing sheep umbilical cord mesenchymal stem cell regeneration, repair, and anti-inflammatory molecules according to claim 4, characterized in that, The immortalization gene is the hTERT gene. The transfer plasmid is constructed using the pLV-IRES-hygro vector as a backbone and contains the hTERT gene and the hygromycin resistance gene. The full length of the hTERT gene is 3399 bp.

6. The method for stably producing exosomes containing regenerative, repair, and anti-inflammatory molecules from sheep umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step S2, the conditions for resistance screening are as follows: 72 hours after infection with lentivirus, screening is performed using 1 μg / mL hygromycin for 15 days, with the complete culture medium containing hygromycin being updated every 3-4 days until all primary cells in the control group die.

7. The method for stably producing exosomes containing regenerative, repair, and anti-inflammatory molecules from sheep umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step S3, the amplification and culture conditions for the immortalized cells are: 10,000 cells / cm³. 2 The cells were seeded at a density in a cell factory and cultured at 37°C with 5% CO2 for 72 hours, after which the supernatant was collected. The specific steps of the multi-stage centrifugation for impurity removal are as follows: the supernatant is centrifuged sequentially at 300×g at room temperature for 10 min, 2000×g at 4℃ for 10 min, and 10000×g at 4℃ for 30 min to remove cell debris and apoptotic bodies; The specific steps of the PEG precipitation method are as follows: after centrifugation, add 25 mL of 10% PEG solution to the supernatant of every 100 mL, mix gently, incubate at 4°C for >12 h, then centrifuge at 4°C at the maximum speed for 40 min, and resuspend the precipitate in PBS to obtain the exosome solution.

8. An exosome that stably produces regenerative, repair, and anti-inflammatory molecules from sheep umbilical cord mesenchymal stem cells, characterized in that... The exosomes are prepared by the method described in any one of claims 1 to 7, and the exosomes of sheep umbilical cord mesenchymal stem cell regeneration, repair and anti-inflammatory molecules of different generations include miR-146a-5p, miR-210-3p, miR-155-5p, miR-125-5p and miR-29a; The exosomes of sheep umbilical cord mesenchymal stem cell regeneration, repair and anti-inflammatory molecules from different generations contain miR-146a-5p, miR-210-3p, miR-155-5p, miR-125b-5p and miR-29a with a particle size distribution between 50-200 nm, and highly express CD63, CD81 and CD9 surface markers.

9. The exosomes prepared by the method for stably producing exosomes of sheep umbilical cord mesenchymal stem cell regeneration, repair, and anti-inflammatory molecules as described in any one of claims 1 to 7, or the application of the exosomes as described in claim 8 in the preparation of skin care product raw materials, characterized in that, The exosomes can enhance transdermal absorption through microneedle or nanoparticle carrier technology.

10. The application according to claim 9, characterized in that, The exosomes can activate the TGF-β / Smad pathway in skincare products to promote collagen synthesis, inhibit melanin synthesis, and reduce skin moisture loss.

11. The application according to claim 9, characterized in that, Skin care products containing the exosomes are biosafety and have anti-wrinkle, firming, moisturizing, soothing and repairing effects.

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