Exosome as well as preparation method and application thereof

Abstract

The invention discloses an exosome as well as a preparation method and application thereof, and relates to the field of biological medicines. The mesenchymal stem cells and the immune cells are co-cultured, the immunomodulatory cell factor IL-2 and the mTOR inhibitor are added, the exosome in the co-culture supernatant is extracted and applied to wound injury repair, wound healing can be effectively improved, and a better repair effect is achieved. Meanwhile, the exosome also has better effects of removing oil, preserving moisture, fading spots, whitening, resisting wrinkles, shrinking pores and increasing elasticity, and can effectively promote skin recovery.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to an exosome, its preparation method, and its application. Background Technology

[0002] As the largest organ in the human body, the skin functions as a physical barrier, chemical defense, and biological protection. It isolates the body from harmful substances and mechanical damage, maintains homeostasis through temperature regulation, participates in sensory perception transmission and tissue regeneration, and its structural integrity is crucial to the body's health. However, in clinical practice, factors such as large-area burns, mechanical trauma, and diabetic complications often lead to acute or chronic skin trauma. According to clinical statistics, approximately 90% of chronic wounds are diabetic wounds, venous ulcers, and pressure sores. Such traumas not only cause physical and mental health problems such as pain and functional impairment but also impose a huge economic burden on the social medical system due to the prolonged course of the disease and long treatment period.

[0003] Non-healing wounds have become a major cause of clinical infections, easily leading to serious complications such as sepsis and tissue necrosis, posing a significant challenge to current surgical and burn care. Skin wound repair is a dynamic process involving multiple stages and coordinated multicellular regulation, typically including the hemostasis phase, inflammatory response phase, cell migration and proliferation phase, and vascular matrix remodeling phase (or simplified as the inflammatory phase, cell proliferation phase, and extracellular matrix storage and reconstruction phase). The orderly progression of this process is the core of wound healing. However, in chronic wounds, this repair process often stalls due to regulatory imbalances (such as excessive or persistent inflammatory responses), further exacerbating the problem of slow wound healing.

[0004] Mesenchymal stem cell exosomes, as cell-free therapeutic agents, can participate in the full-stage regulation of wound repair by carrying active substances such as proteins, nucleic acids, and lipids. However, the current clinical translation of mesenchymal stem cell exosomes faces a core technological bottleneck—their therapeutic efficacy is limited by factors such as exosome yield, active substance loading efficiency, targeting, and in vivo stability.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide an exosome, its preparation method, and its application.

[0007] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide a method for preparing exosomes, comprising the following steps: isolating exosomes from a co-culture product of mesenchymal stem cells and immune cells; wherein, 100-1200 IU / mL of inflammatory regulatory factor and 50 nmol / L-50 μmol / L of mTOR inhibitor are added to the co-culture system of mesenchymal stem cells and immune cells.

[0008] Secondly, embodiments of the present invention provide an exosome, which is prepared by the preparation method described in the foregoing embodiments.

[0009] Thirdly, embodiments of the present invention provide a composition for improving or repairing skin wound damage, comprising: the exosomes described in the foregoing embodiments.

[0010] Fourthly, embodiments of the present invention provide a cosmetic product comprising: the exosomes described in the foregoing embodiments.

[0011] Fifthly, embodiments of the present invention provide the preparation method as described in the foregoing embodiments or the application of exosomes as described in the foregoing embodiments in the preparation of products that improve or repair skin wound damage.

[0012] Sixthly, embodiments of the present invention provide the application of the preparation method as described in the foregoing embodiments or the exosomes as described in the foregoing embodiments in the preparation of products having at least one of the following effects: oil removal, moisturizing, spot fading, whitening, anti-wrinkle, pore shrinking and elasticity increase.

[0013] The present invention has the following beneficial effects: This invention involves co-culturing mesenchymal stem cells with immune cells, adding immunomodulatory cytokines IL-2 and mTOR inhibitors, and extracting exosomes from the co-culture supernatant. These exosomes are then applied to wound repair, effectively improving wound healing and achieving better repair results. Simultaneously, these exosomes also exhibit good oil-removing, moisturizing, spot-fading, whitening, anti-wrinkle, pore-minimizing, and elasticity-enhancing effects, effectively promoting skin recovery. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a schematic diagram of the process for preparing exosomes; Figure 2 The results of a clinical observation on the skin-whitening effect of exosomes; Figure 3 The results of a clinical observation on the efficacy of exosomes in treating wrinkles; Figure 4 Clinical observation results on the effect of exosome therapy in increasing elasticity; Figure 5 This is a clinical observation result on the efficacy of exosome therapy in repairing sunburned skin. Figure 6 Clinical observation results on the efficacy of exosome therapy in improving skin allergies and rosacea; Figure 7 Clinical observation results on the efficacy of exosome therapy in improving acne scars; Figure 8 The results of a clinical observation on the efficacy of exosomes in treating pigmentation. Detailed Implementation

[0016] 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.

[0017] The term "exosome" refers to a type of tiny extracellular vesicle (EV) with a lipid bilayer membrane structure that is actively secreted by living cells. They are important carriers for the transfer of bioactive molecules between cells and mediate intercellular communication.

[0018] The term "skin wound injury" refers to the disruption of the integrity of skin tissue caused by external factors (such as mechanical, physical, chemical, and biological stimuli) or internal pathological conditions (such as diabetes and vascular diseases). It is accompanied by defects or functional impairments of the epidermis, dermis, and even subcutaneous tissue, manifested as loss of skin barrier function and exposure of local tissues. It may be accompanied by inflammation, bleeding, and infection risks, and ultimately requires a repair process to restore tissue continuity (or form scars).

[0019] The term "improvement" refers to alleviating the "negative pathological state" of a wound (such as inflammation, infection, pain) or activating / accelerating a "stagnant healing process" (such as moving from the inflammatory phase to the proliferative phase), ultimately bringing the wound closer to a "healable condition." At its core, it is about "reducing harm and advancing the process," encompassing all situations where the wound is "not closed but in a better condition."

[0020] The term "repair" refers to the "structural reconstruction of the damaged tissue" and the "restoration of core functions to meet physiological needs" of the wound, ultimately achieving the "healing endpoint"—wound closure (epidermal coverage or scar filling) with no significant impairment in the local skin's barrier function, sensory function, or motor function. It encompasses all situations "from defect to closure and functional attainment."

[0021] Skin wound repair involves four stages: hemostasis, inflammation, cell migration and proliferation, and vascular matrix remodeling. Each stage is precisely regulated through dynamic interactions between cells, cytokines, and the extracellular matrix. Imbalance in any of these interactions can disrupt the repair balance. More than 50 cytokines (such as VEGF and TGF-β) and 20 cell types (such as immune cells, stromal cells, and epidermal cells) participate in regulation during this process, which is also dynamically influenced by the wound microenvironment (such as oxygen partial pressure, pH, and inflammation levels). This "multi-dimensional and highly synergistic" characteristic dictates that any exogenous intervention (such as cells or exosomes) must be precisely matched to the repair stage and microenvironmental needs; otherwise, it will be ineffective or even cause side effects. Current intervention methods lack precise matching to the wound microenvironment and in-depth analysis of the molecular mechanisms involved.

[0022] This invention involves co-culturing mesenchymal stem cells with immune cells, adding immunomodulatory cytokines IL-2 and mTOR inhibitors, and then extracting exosomes from the co-cultured cells for application in wound repair. This approach effectively improves wound healing and achieves better repair results.

[0023] Furthermore, the exosomes co-cultured with mesenchymal stem cells and immune cells of the present invention have good effects in removing oil, moisturizing, fading spots, whitening, anti-wrinkle, shrinking pores, and increasing elasticity, and can effectively promote skin recovery.

[0024] On one hand, embodiments of the present invention provide a method for preparing exosomes, comprising the following steps: isolating exosomes from a co-culture product of mesenchymal stem cells and immune cells; wherein, 100-1200 IU / mL of an inflammatory regulatory factor and 50 nmol / L-50 μmol / L of an mTOR inhibitor are added to the co-culture system of mesenchymal stem cells and immune cells. (Refer to...) Figure 1 .

[0025] In an optional embodiment, 100-1200 IU / mL represents the concentration of the inflammatory regulatory factor in the co-culture system, specifically 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, and 600 IU / mL. The range of any one or any two of the following: 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 1020, 1040, 1060, 1080, 1100, 1120, 1140, 1160, 1180, and 1200 IU / mL.

[0026] In optional embodiments, 50 nmol / L to 50 μmol / L represents the concentration of the mTOR inhibitor in the co-culture system, specifically 50 nmol / L (nM), 60 nmol / L, 80 nmol / L, 100 nmol / L, 120 nmol / L, 150 nmol / L, 200 nmol / L, 250 nmol / L, 300 nmol / L, 350 nmol / L, 400 nmol / L, 450 nmol / L, 500 nmol / L, 550 nmol / L, 600 nmol / L, 650 nmol / L, 700 nmol / L, 750 nmol / L, 800 nmol / L, 850 nmol / L, 900 nmol / L, 950 nmol / L, 1 The range of any one or any two of the following: μmol / L, 1.5 μmol / L, 2 μmol / L, 2.5 μmol / L, 3 μmol / L, 3.5 μmol / L, 4 μmol / L, 4.5 μmol / L, 5 μmol / L, 5.5 μmol / L, 6 μmol / L, 6.5 μmol / L, 7 μmol / L, 7.5 μmol / L, 8 μmol / L, 8.5 μmol / L, 9 μmol / L, 9.5 μmol / L, 10 μmol / L, 11 μmol / L, 12 μmol / L, 15 μmol / L, 20 μmol / L, 25 μmol / L, 30 μmol / L, 35 μmol / L, 38 μmol / L, 40 μmol / L, 42 μmol / L, 45 μmol / L, and 50 μmol / L.

[0027] In an optional embodiment, the co-culture step includes: seeding the mesenchymal stem cells and the immune cells into a culture medium at a live cell ratio of 1:(5~20), co-culturing for 24~72 h, and co-culturing at a temperature of 25~45℃.

[0028] In optional embodiments, the live cell ratio can specifically be any one or any two of 1:5, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, and 1:20. The co-culture time can be any one or any two of 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, and 72 h. The co-culture temperature can be any one or any two of 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 45°C.

[0029] In an optional embodiment, the culture medium used for the co-culture includes serum-free culture media for immune cell culture, including but not limited to... 15. Culture medium AIM SFM culture medium, HIPP-T009 (Beianji Biotechnology), and HyClone CDM4MAb (HyClone), etc.

[0030] In an optional embodiment, the mesenchymal stem cells may be derived from any one or more of the following: umbilical cord, adipose tissue, amnion, and placenta.

[0031] In an optional embodiment, the immune cells are selected from any one or more of CIK cells, NK cells, CTL cells, and γδT cells derived from umbilical cord blood or peripheral blood.

[0032] In optional embodiments, the mTOR inhibitor includes any one or more of the following: rapamycin (sirolimus), everolimus, tesimolimus, desfolimus, apeliximab, sapacreti, AZD2014, OSI-027, Rapalink-1, LY3023414, curcumin, epigallocatechin gallate (EGCG), resveratrol, sulforaphane, quercetin, chlorogenic acid, anthocyanins, ginsenosides, shogaol, capsaicin, melatonin, allicin, and Omega-3 polyunsaturated fatty acids.

[0033] In an optional embodiment, the inflammatory regulator includes IL-2.

[0034] In an optional embodiment, the step of separating and obtaining exosomes includes: centrifuging the co-culture product or the supernatant of the co-culture product at 0-10°C, 100-500×g for 5-20 min to obtain a first supernatant; centrifuging the first supernatant at 0-10°C, 1500-2500×g for 5-20 min to obtain a second supernatant; filtering the second supernatant and / or centrifuging it at 0-10°C, 8000-12000×g for 10-60 min to obtain a filtrate or a third supernatant; the membrane used for filtration has a pore size of 0.1-0.45 μm; centrifuging the filtrate or the third supernatant at 0-10°C, 80000-120000×g for 30-90 min, and collecting the precipitate.

[0035] In an optional embodiment, the 0~10℃ can specifically be any one or any two of 0, 2, 4, 6, 8 and 10℃.

[0036] In an optional embodiment, the 100~500×g can specifically be any one or any two of 100, 200, 300, 400 and 500×g.

[0037] In an optional embodiment, the 1500~2500×g can specifically be any one or any two of 1500, 1600, 1800, 2000, 2200, 2400 and 2500×g.

[0038] In an optional embodiment, the 8000~12000×g can specifically be any one or any two of 8000, 9000, 10000, 11000 and 12000×g.

[0039] In an optional embodiment, the 80000~120000×g can specifically be any one or any two of 80000, 90000, 100000, 110000 and 120000×g.

[0040] In an optional embodiment, the 5~20 min can specifically be any one or any two of the following: 5, 6, 8, 10, 12, 14, 16, 18 and 20 min.

[0041] In an optional embodiment, the 10~60min can specifically be any one or any two of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60min.

[0042] In an optional embodiment, the 30~90min can specifically be any one or any two of the following: 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and 90min.

[0043] In an optional embodiment, the 0.1~0.45 μm can specifically be any one or any two of 0.1, 0.15, 0.2, 0.22, 0.25, 0.3, 0.35, 0.4 and 0.45 μm.

[0044] On the other hand, embodiments of the present invention provide an exosome prepared by the preparation method described in any of the foregoing embodiments.

[0045] On the other hand, embodiments of the present invention provide a composition for improving or repairing skin wound damage, comprising: exosomes as described in any of the foregoing embodiments.

[0046] In an optional embodiment, the composition includes a pharmaceutically acceptable carrier.

[0047] In an optional embodiment, the pharmaceutically acceptable carrier includes any one or more of the following: pharmaceutically acceptable excipients, carriers, adjuvants, additives, surfactants, desiccants, and diluents.

[0048] On the other hand, embodiments of the present invention provide a cosmetic product comprising: the exosomes described in any of the foregoing embodiments.

[0049] In an optional embodiment, the cosmetic product further includes a cosmetic base.

[0050] In an optional embodiment, the cosmetic base has at least one of the following effects: oil removal, moisturizing, spot fading, whitening, anti-wrinkle, pore minimizing, and increased elasticity.

[0051] In optional embodiments, according to their functions and uses, the cosmetics or cosmetic bases include cleansing products (e.g., facial cleansers, shower gels, makeup removers), care products (e.g., moisturizers, serums, sunscreens), beauty and makeup products (e.g., foundations, lipsticks, eyeshadows, blushes), hair care products (e.g., shampoos, conditioners, hair masks), and special function products (e.g., sunscreens, freckle creams, hair removal creams).

[0052] On the other hand, embodiments of the present invention provide the preparation method as described in any of the foregoing embodiments or the application of exosomes as described in any of the foregoing embodiments in the preparation of products that improve or repair skin wound damage.

[0053] In an optional implementation, the skin wound injury is classified according to the cause of injury, including any one or more of the following: mechanical injury (skin tearing, cutting, or squeezing caused by external force), physical injury (physical factors such as temperature, radiation, and pressure), chemical injury (corrosion or irritation by chemical substances), biological injury (microbial infection or biological bite), and pathological injury (skin tissue necrosis caused by internal diseases).

[0054] In optional embodiments, the skin wounds are classified according to wound depth and healing difficulty, including any one or more of the following: superficial wounds (epidermal layer damage), partial dermal wounds (epidermal + partial dermal damage), full dermal wounds (epidermal + full dermal layer damage), and refractory wounds (chronic wounds).

[0055] Furthermore, embodiments of the present invention provide the application of the preparation method as described in any of the foregoing embodiments or the exosomes as described in any of the foregoing embodiments in the preparation of products having at least one of the effects of oil removal, moisturizing, spot fading, whitening, anti-wrinkle, pore shrinking and elasticity increase.

[0056] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0057] Experimental materials The serum-free culture medium used in this example comprises the following components: hormones, proteins, amino acids, vitamins, lipids, inorganic salts, and additives. The hormones include 10 mg / L recombinant human insulin and 40 μg / L cortisol. The proteins include 5 g / L recombinant human serum albumin and 10 mg / L recombinant human transferrin. The amino acids include 210.0 mg / L L-arginine, 115.0 mg / L L-cysteine, 84.0 mg / L L-histidine, 157.4 mg / L L-isoleucine, 157.4 mg / L L-leucine, 218.5 mg / L L-lysine, 45.0 mg / L L-methionine, 100.0 mg / L L-phenylalanine, 142.6 mg / L L-threonine, 26.0 mg / L L-tryptophan, 460.0 mg / L L-tyrosine, and 564.0 mg / L L-lysine. L-valine, 45.0 mg / L glycine, 43.0 mg / L L-alanine, 55.0 mg / L L-asparagine, 57.0 mg / L L-aspartic acid, 105.0 mg / L L-glutamic acid, 63.0 mg / L L-proline, 62.0 mg / L L-serine, 584 mg / L L-glutamine, 160 mg / L N-acetylcysteine; the vitamins include: 5 mg / L D-calcium pantothenate, 5 mg / L choline chloride, 5 mg / L folic acid, 5 mg / L nicotinamide, 5 mg / L pyridoxal hydrochloride, 0.5 mg / L riboflavin, 5 mg / L thiamine hydrochloride, 8.2 mg / L inositol, 0.013 mg / L vitamin B12, 0.013 mg / L biotin, 10 mg / L vitamin C, 10 The lipids include: 0.02 mg / L arachidonic acid, 2.20 mg / L cholesterol, 0.70 mg / L DL-α-tocopheryl acetate, 0.10 mg / L linoleic acid, 0.10 mg / L linolenic acid, 0.10 mg / L myristic acid, 0.10 mg / L oleic acid, 0.10 mg / L palmitoleic acid, 0.10 mg / L palmitic acid, and 0.10 mg / L stearic acid; the inorganic salts include: 165.3 mg / L CaCl2, 97.67 mg / L MgSO4, 330 mg / L KCl, 3024 mg / L NaHCO3, 4505 mg / L NaCl, 109 mg / L NaH2PO4, 0.017 mg / L Na2SeO3•5H2O, 1.60 μg / L CuSO4•5H2O, and 863.00 μg / L... ZnSO4·7H2O, 1155.10μg / L FeC6H5O7, 0.17μg / L MnSO4·H2O, 140.00μg / LNa2SiO3·9H2O, 1.24 μg / L (NH4)6Mo7O. 24 The additives include: 0.65 μg / L NH4VO3, 0.13 μg / L NiSO4•6H2O, 0.12 μg / L SnCl2, 1.20 μg / L AlCl3•6H2O, 0.17 μg / L AgNO3, 2.55 μg / L Ba(C2H3O2)2, 0.12 μg / L KBr, 2.28 μg / L CdCl2, 2.38 μg / L CoCl2•6H2O, 0.32 μg / L CrCl3, 4.20 μg / L NaF, 0.53 μg / L GeO2, 0.17 μg / L KI, 1.21 μg / L RbCl, and 3.22 μg / L ZrOCl2•8H2O; the additives also include: 900.0 mg / L Pronic F68, 22.0 mg / L Tween 80, 4.30 mg / L 2-mercaptoethanol, 4500 mg / L L-glucose, 5958 mg / L HEPES, 110 mg / L sodium pyruvate, 5000 IU / L heparin.

[0058] The source of mesenchymal stem cells in Example 1: Human umbilical cord specimens were obtained from full-term pregnant women in the Department of Obstetrics at Beijing Friendship Hospital. Fetal malformations, maternal congenital genetic diseases, and infectious disease history were excluded, and informed consent was obtained. Donors were screened for specific human-derived viruses (including HIV, HBV, HCV, HTLV, EBV, and CMV), Treponema pallidum, glucose-6-phosphate dehydrogenase, and alanine aminotransferase. Umbilical cord tissue was collected under sterile conditions in the operating room and processed within 12 hours. The preparation of human umbilical cord mesenchymal stem cells was carried out strictly in accordance with GMP standards and standard operating procedures and management procedures.

[0059] The source of mesenchymal stem cell specimens in Example 2: Human adipose tissue specimens were obtained from healthy donors at the Department of Plastic Surgery, Beijing Friendship Hospital. Donors with congenital genetic diseases or a history of infectious diseases were excluded, and informed consent was obtained. Donors had passed tests for specific human-derived viruses (including HIV, HBV, HCV, HTLV, EBV, CMV, etc.), Treponema pallidum, glucose-6-phosphate dehydrogenase, and alanine aminotransferase. Adipose tissue was collected under sterile conditions in the operating room and processed within 12 hours.

[0060] The source of mesenchymal stem cell specimens in Example 3: Human amniotic membrane specimens were obtained from full-term pregnant women in the Department of Obstetrics at Beijing Friendship Hospital. Fetal malformations, maternal congenital genetic diseases, and infectious disease history were excluded, and informed consent was obtained. Donors had passed tests for specific human-derived viruses (including HIV, HBV, HCV, HTLV, EBV, CMV, etc.), Treponema pallidum, glucose-6-phosphate dehydrogenase, and alanine aminotransferase. Amniotic membrane tissue was collected under sterile conditions in the operating room and processed within 12 hours. The preparation of human amniotic stem cells was carried out in strict accordance with GMP standards, following standard operating procedures and management procedures.

[0061] The immune cell specimens used in Examples 1-3 were sourced from healthy human peripheral blood donors. Donors provided informed consent and passed tests for specific human viruses (including HIV, HBV, HCV, HTLV, EBV, CMV, etc.), Treponema pallidum, glucose-6-phosphate dehydrogenase, and alanine aminotransferase. Blood samples were collected using blood collection tubes containing heparin sodium as an anticoagulant. The collection process adhered to medical and health regulations to ensure the sterility of the blood samples. Each tube collected 8-9 mL of blood, for a total volume of approximately 80-100 mL. After collection, the blood sample was gently inverted 8 times to thoroughly mix the anticoagulant with the blood. The blood samples were stored and transported at 18-25°C and transported to the laboratory for culture within 12 hours.

[0062] Example 1 A method for preparing exosomes includes the following steps.

[0063] (1) Preparation of human umbilical cord mesenchymal stem cells The obtained umbilical cord was placed in a sterile glass dish and rinsed with PBS solution at 4°C containing 100 U / ml penicillin and 100 μg / ml streptomycin to remove any residual red substance on the surface. It was cut into appropriate sizes, and hyaluronidase, neutral protease, and collagenase were added. The mixture was incubated overnight at 37°C, centrifuged at 250 g for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in culture medium. The mixture was then placed in a 5% culture medium. Incubate at 37℃, changing the culture medium every 3 days. Perform white blood cell counting according to the "National Clinical Laboratory Operation Procedures" (4th Edition) or use a cell counter.

[0064] (2) Isolation and culture of immune cells Isolation of mononuclear cells: Add 20 mL of human lymphocyte separation medium to the bottom of a 50 mL centrifuge tube using a pipette. Mix the peripheral blood with twice the volume of physiological saline, then slowly add 20 mL of blood sample onto the human lymphocyte separation medium in the centrifuge tube using a pipette. Be very gentle. To avoid disrupting the interface between the two liquids, you can slightly tilt the centrifuge tube and slowly add the blood sample while keeping the pipette against the tube wall. Centrifuge in a horizontal rotor centrifuge (22℃, 800 g, 15 min, ascending speed 1, descending speed 0). Aspirate the white membrane layer and transfer it to another 50 mL centrifuge tube. Add 40 mL of PBS, wash by pipetting, and centrifuge (22℃, 400 g, 10 min, ascending speed 9, descending speed 7). Discard the supernatant; this is the peripheral blood mononuclear cells. Resuspend in RPMI 1640 medium and aspirate a small number of cells for counting.

[0065] Culture of CIK immune cells: Peripheral blood mononuclear cells were collected and the cell concentration was adjusted to 2× using serum-free immune cell culture medium. Add 1000 U / mL of recombinant human IFN-γ; after 24 h, add 100 ng / mL of recombinant mouse anti-human CD3 monoclonal antibody and 1000 IU / mL of recombinant human IL-2, along with 100 U / mL of recombinant human IL-1α. Place in a culture flask; incubate at 37℃ for 5%... The CIK cells were incubated in an incubator to stimulate their growth and proliferation; the medium was changed at half volume or the flask was expanded every 3 days, and 1000 IU / ml of recombinant human IL-2 was added; CIK immune cells were harvested on the 14th day of culture.

[0066] (3) Co-culture of mesenchymal stem cells and immune cells The above-mentioned human umbilical cord mesenchymal stem cells and CIK immune cells were mixed at a live cell ratio of 1:5, and then 1×10⁻⁶ cells were added. 6 Cells / ml were seeded into 100 mL of serum-free culture medium for immune cells supplemented with 1000 IU / ml recombinant human IL-2 and 100 nM rapamycin, and incubated at 5% concentration. They were incubated at 37°C for a total of 48 hours.

[0067] (4) Extraction of cell exosomes Supernatants from mesenchymal stem cell (MSC) culture, CIK immune cell (CIK) cell culture, and co-culture of MSCs and immune cells were collected separately. Each was centrifuged at 300×g for 10 min at 4°C, and the supernatant was collected. The samples were then centrifuged at 2000×g for 10 min at 4°C, and the supernatant was collected. After filtration through 0.22 μm, the samples were centrifuged at 10000×g for 30 min at 4°C, and the supernatant was collected. Finally, the samples were centrifuged at 100000×g for 70 min at 4°C, and the supernatant was carefully removed. The samples were resuspended in PBS, and the centrifugation was repeated once. After centrifugation, the supernatant was carefully removed, and the samples were resuspended in PBS to obtain MSC exosomes, CIK immune cell exosomes, and exosomes from the co-culture of MSCs and immune cells. These were stored at -80°C.

[0068] Effect verification (5) Establishment of a mouse skin wound injury model Twenty C57BL / 6 mice were injected intraperitoneally with 3% sodium pentobarbital at a dose of 35 mg / kg. After satisfactory anesthesia, the fur on the back of the rats was cleaned using an electric shaver. The shaved area on the back was disinfected three times with 75% alcohol. Four circular full-thickness skin wounds with a diameter of 9 mm were symmetrically constructed using a skin punch, with the wound edges spaced approximately 15 mm apart.

[0069] (6) Experimental grouping and exosome therapy Control group: PBS without exosomes was evenly applied to the wound site; Experimental group 1: Mesenchymal stem cell exosomes were evenly applied to the wound site; Experimental group 2: Immune cell exosomes were evenly applied to the wound site; Experimental group 3: Exosomes co-cultured with mesenchymal stem cells and immune cells were evenly applied to the wound site.

[0070] Apply once in the morning and once in the evening (each application is 100μl, containing 1 billion exosomes) for 7 consecutive days. On day 28 post-surgery, take photos and mark the size of the wound. Repeat this process three times. Calculate the wound healing index = (wound area on day 1-n / wound area on day 0) × 100.

[0071] Table 1. Wound healing index of mice in each group

[0072] Table notes: * indicates a statistically significant difference compared to the control group (P < 0.05); # indicates a statistically significant difference compared to experimental group 1 (P < 0.05); $ indicates a statistically significant difference compared to experimental group 2 (P < 0.05).

[0073] Table 1 shows the wound healing index of each group on day 28 in Example 1. The results show that each regimen in Example 1 has a healing-promoting effect on mice with wound injuries. The healing-promoting effect is: exosomes co-cultured with mesenchymal stem cells and immune cells > mesenchymal stem cell exosomes > immune cell exosomes.

[0074] Example 2 A method for preparing exosomes includes the following steps.

[0075] (1) Preparation of adipose-derived mesenchymal stem cells The obtained fat was placed in a sterile glass dish and rinsed with PBS solution containing 100 U / ml penicillin and 100 μg / ml streptomycin at 4°C to remove any residual red substance on the surface. The fat was cut into appropriate sizes, and hyaluronidase, neutral protease, and collagenase were added. The mixture was incubated overnight at 37°C, centrifuged at 250 g for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in culture medium. The mixture was then incubated in a 5% CO2 incubator at 37°C, with the culture medium replaced with fresh medium every 3 days. White blood cell counts were performed according to the methods outlined in the "National Clinical Laboratory Operating Procedures" (4th Edition) or using a cell counter.

[0076] (2) Isolation and culture of immune cells Isolation of mononuclear cells: Same as in Example 1.

[0077] Culture of NK immune cells: Peripheral blood mononuclear cells were collected and the cell concentration was adjusted to 2× using serum-free immune cell culture medium. Add 1000 IU / mL of recombinant human IL-2 and 0.01 KE / mL of sabrine injection; place in a culture flask coated with recombinant mouse anti-human CD16 antibody, incubate at 37°C, 5% Incubate in an incubator for 72 h, then transfer to a culture flask uncoated with recombinant mouse anti-human CD16 antibody, add fresh culture medium and 1000 IU / mL recombinant human IL-2 to stimulate NK cell growth and proliferation; change half the medium or expand the flask every 3 days, and add 1000 IU / mL recombinant human IL-2; harvest NK immune cells on day 21 of culture.

[0078] (3) Co-culture of mesenchymal stem cells and immune cells The above-mentioned adipose-derived mesenchymal stem cells and NK immune cells were mixed at a live cell ratio of 1:10, and then mixed at a concentration of 1×10⁻⁶ cells / cells. 6 Cells / ml were seeded into 100 mL of serum-free immune cell culture medium supplemented with 200 IU / ml recombinant human IL-2 and 10 μmol / L epigallocatechin gallate (EGCG), and incubated at 5% concentration. They were incubated at 37℃ for a total of 72 hours.

[0079] (4) Extraction of cell exosomes Exosomes were extracted from the culture supernatant of adipose-derived mesenchymal stem cells, the culture supernatant of NK immune cells, and the supernatant of co-culture of adipose-derived mesenchymal stem cells and NK immune cells, respectively, following the same extraction steps as in Example 1. Mesenchymal stem cell exosomes, CIK immune cell exosomes, and exosomes co-cultured with mesenchymal stem cells and immune cells were obtained and stored at -80°C.

[0080] Effect verification (5) Establishment of a mouse skin wound injury model Same as Example 1.

[0081] (6) Experimental grouping and exosome therapy Control group: PBS without exosomes was evenly applied to the wound site; Experimental group 1: Mesenchymal stem cell exosomes were evenly applied to the wound site; Experimental group 2: Immune cell exosomes were evenly applied to the wound site; Experimental group 3: Exosomes co-cultured with mesenchymal stem cells and immune cells were evenly applied to the wound site.

[0082] Apply once in the morning and once in the evening (each application is 100μl, containing 1 billion exosomes) for 7 consecutive days. On day 28 post-surgery, take photos and mark the size of the wound. Repeat this process three times. Calculate the wound healing index = (wound area on day 1-n / wound area on day 0) × 100.

[0083] Table 2. Wound healing index of mice in each group

[0084] Table notes: * indicates a statistically significant difference compared to the control group (P < 0.05); # indicates a statistically significant difference compared to experimental group 1 (P < 0.05); $ indicates a statistically significant difference compared to experimental group 2 (P < 0.05).

[0085] Table 2 shows the wound healing index of each group on day 28 in Example 2. The results show that each regimen promotes wound healing in mice with wound injury. The order of promoting healing effect is: exosomes co-cultured with mesenchymal stem cells and immune cells > mesenchymal stem cell exosomes > immune cell exosomes.

[0086] Example 3 A method for preparing exosomes includes the following steps.

[0087] (1) Preparation of amniotic mesenchymal stem cells The obtained amniotic membrane was placed in a sterile glass dish and rinsed with PBS solution containing 100 U / ml penicillin and 100 μg / ml streptomycin at 4°C to remove any residual red substance on the surface. It was cut into appropriate sizes, and trypsin / EDTA solution was added. After digestion at 37°C for 15 minutes, the remaining amniotic membrane was removed and placed in a centrifuge tube. Hyaluronidase, neutral protease, and collagenase were added, and the mixture was incubated overnight at 37°C. After centrifugation at 250 g for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in culture medium. The mixture was then placed in a 5%... Incubate at 37℃, changing the culture medium every 3 days. Perform white blood cell counting according to the "National Clinical Laboratory Operation Procedures" (4th Edition) or use a cell counter.

[0088] (2) Isolation and culture of immune cells Isolation of mononuclear cells: Same as in Example 1.

[0089] Culture of γδT immune cells: Peripheral blood mononuclear cells were collected and the cell concentration was adjusted to 2× using serum-free immune cell culture medium. Add 1000 IU / mL recombinant human IL-2 and 5 μM zoledronic acid, along with 5% autologous plasma; place in a culture flask and incubate at 37°C for 5%... Incubate in an incubator for 72 hours, then add fresh culture medium and 1000 IU / mL recombinant human IL-2 to stimulate the growth and proliferation of γδ T cells; change half the medium or expand the flask every 3 days, and add 1000 IU / mL recombinant human IL-2; harvest γδ cells on day 14 of culture.

[0090] (3) Co-culture of mesenchymal stem cells and immune cells Amniotic mesenchymal stem cells and γδ immune cells were mixed at a cell ratio of 1:15, and then 1×10⁻⁶ cells were added. 6 Cells / ml were seeded into 100mL of serum-free culture medium for immune cells supplemented with 1000 IU / ml recombinant human IL-2 and 10 μmol / L curcumin, and incubated at 5% concentration. They were incubated at 37℃ for a total of 72 hours.

[0091] (4) Extraction of cell exosomes The supernatant of amniotic mesenchymal stem cell culture, the supernatant of γδT immune cells, and the supernatant of co-culture of amniotic mesenchymal stem cells and γδT immune cells were collected and exosomes were extracted respectively. The extraction steps were the same as those in Example 1. Mesenchymal stem cell exosomes, γδT immune cell exosomes, and exosomes co-cultured with mesenchymal stem cells and immune cells were obtained and stored at -80°C.

[0092] Effect verification (5) Establishment of a mouse skin wound injury model Same as Example 1.

[0093] (6) Experimental grouping and exosome therapy Control group: PBS without exosomes was evenly applied to the wound site; Experimental group 1: Mesenchymal stem cell exosomes were evenly applied to the wound site; Experimental group 2: Immune cell exosomes were evenly applied to the wound site; Experimental group 3: Exosomes co-cultured with mesenchymal stem cells and immune cells were evenly applied to the wound site.

[0094] Apply once in the morning and once in the evening (each application is 100μl, containing 1 billion exosomes) for 7 consecutive days. On day 28 post-surgery, take photos and mark the size of the wound. Repeat this process three times. Calculate the wound healing index = (wound area on day 1 - wound area on day 0) × 100.

[0095] Table 3. Wound healing index of mice in each group

[0096] Table notes: * indicates a statistically significant difference compared to the control group (P < 0.05); # indicates a statistically significant difference compared to experimental group 1 (P < 0.05); $ indicates a statistically significant difference compared to experimental group 2 (P < 0.05).

[0097] Table 3 shows the wound healing index of each group on day 28 in Example 3. The results show that each regimen promotes wound healing in mice with wound injury. The order of promoting healing effect is: exosomes co-cultured with mesenchymal stem cells and immune cells > mesenchymal stem cell exosomes > immune cell exosomes.

[0098] The above results demonstrate the effectiveness of this invention in promoting skin wound healing and repair.

[0099] Example 4 A method for preparing exosomes includes the following steps.

[0100] (1) Preparation of human umbilical cord mesenchymal stem cells: Same as in Example 1.

[0101] (2) Isolation and culture of immune cells: Same as in Example 1.

[0102] (3) Co-culture of mesenchymal stem cells and immune cells Experimental group: Adipose-derived mesenchymal stem cells and NK immune cells were mixed at a cell ratio of 1:10, and then added at a concentration of 1×10⁻⁶ cells / mL. 6 Cells / ml were seeded into 100mL of serum-free culture medium for immune cells supplemented with 1000 IU / ml recombinant human IL-2 and 40 µmol / L sulforaphane, and incubated at 5% concentration. They were incubated at 37℃ for a total of 72 hours.

[0103] Control group: Adipose-derived mesenchymal stem cells and NK immune cells were mixed at a cell ratio of 1:10, at a concentration of 1×10⁻⁶ cells / cells. 6 Cells / ml were seeded into 100mL of serum-free immune cell culture medium and incubated at 5% concentration. They were incubated at 37℃ for a total of 72 hours.

[0104] (4) Extraction of cell exosomes The co-culture supernatant of the experimental group and the control group was collected, and exosomes were extracted from them respectively. The extraction steps were the same as those in Example 1. Mesenchymal stem cells and immune cells co-cultured exosomes were obtained from the experimental group and the control group respectively.

[0105] Effect verification Three female subjects aged 35-60 years were selected to use the exosomes described in Example 4. Each subject used the experimental group's exosomes on one side of their face and the control group's exosomes on the other side. The exosomes were applied once every three days (10 billion exosomes / 1ml). After three applications, the skin's moisturizing, oil-controlling, whitening, and elasticity functions were assessed using a skin analyzer, scored from 0 to 100 (the values ​​displayed on the analyzer). A higher score for oil control indicated poorer oil removal; higher scores for moisturizing, whitening, and elasticity indicated better performance in those areas.

[0106] Table 4. Scores of moisturizing, whitening, oil-reducing, and elasticity functions for each group

[0107] The results showed that no allergic reactions occurred. After three uses, the experimental group's scores for moisturizing, whitening, and elasticity were all higher than those of the control group, and the experimental group's oil-removing function was also superior to that of the control group.

[0108] Example 5 A method for preparing exosomes includes the following steps.

[0109] (1) Preparation of human umbilical cord mesenchymal stem cells: Same as in Example 1.

[0110] (2) Isolation and culture of immune cells: Same as in Example 1.

[0111] (3) Co-culture of mesenchymal stem cells and immune cells: roughly the same as in Example 1, except that 100 nM rapamycin is replaced with 100 nM resveratrol; (4) Extraction of cell exosomes: Same as in Example 1.

[0112] Effect verification Forty general subjects aged 35-60 and 50 subjects with various skin problems were recruited. Exosomes as described in Example 5 were used once every three days (1 mL each time, 10 billion exosomes / mL). After 1-3 applications (the efficacy evaluation for skin repair after sunburn was performed after only one application, while other efficacy evaluations were performed after three applications), the 40 general subjects underwent skin analysis to assess moisturizing, oil production, whitening, and elasticity. The 50 subjects with various skin problems underwent visual observation to observe changes in their skin condition. The test results are shown in the table below.

[0113] Table 5. Test Results

[0114] 1. Whitening effect Figure 2 An example demonstrates that after a subject used exosomes three times, the skin whitening effect was significantly increased.

[0115] 2. Improves wrinkles Figure 3 An example demonstrates that after a subject used exosomes three times, wrinkles around the eyes were significantly improved.

[0116] 3. Increase flexibility Figure 4 An example demonstrates that after a subject used exosomes three times, skin elasticity was significantly increased.

[0117] 4. Repairing sunburned skin Figure 5 For example, after a subject used exosomes once, the symptoms of extensive redness, itching, and even peeling of the skin after sun exposure were significantly improved.

[0118] 5. Improves skin allergies and redness Figure 6 An example is shown where a patient experienced significant improvement in facial allergy discomfort and bumps after using exosomes three times, and skin redness was significantly reduced.

[0119] 6. Improves acne scars Figure 7 An example is shown where a patient experienced significant improvement in facial discomfort caused by acne after using exosomes three times. After the acne healed, there were no obvious pigmentation spots or scars, and the skin showed significant improvement.

[0120] 7. Lighten freckles Figure 8 An example is shown where a patient's pigmentation spots were significantly improved after three applications of exosomes, resulting in a marked improvement in skin condition.

[0121] The above results demonstrate that the present invention promotes the repair and healing of skin wounds, and can also increase whitening and elasticity, improve wrinkles, repair sunburn, treat skin allergies, redness, acne scars, and lighten spots.

[0122] The above description is merely a preferred embodiment of the present invention and is not intended to limit the 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 scope of protection of the present invention.

Claims

1. A method for preparing exosomes, characterized in that, It includes the following steps: Exosomes were isolated from the co-culture products of mesenchymal stem cells and immune cells; wherein 100~1200 IU / mL of inflammatory regulatory factor and 50 nmol / L~50 μmol / L mTOR inhibitor were added to the co-culture system of mesenchymal stem cells and immune cells.

2. The method for preparing exosomes according to claim 1, characterized in that, The co-culture step includes: seeding the mesenchymal stem cells and the immune cells into a culture medium at a live cell ratio of 1:(5~20), co-culturing for 24~72 h, co-culturing at a temperature of 25~45℃, and using a serum-free culture medium for immune cell culture.

3. The preparation method according to claim 1, characterized in that, The mesenchymal stem cells are derived from any one or more of the following: umbilical cord, adipose tissue, amnion, and placenta. The immune cells are selected from any one or more of CIK cells, NK cells, CTL cells, and γδT cells derived from umbilical cord blood or peripheral blood.

4. The preparation method according to claim 1, characterized in that, The mTOR inhibitors include any one or more of the following: rapamycin, everolimus, tesirobolimus, desfolimus, apelimus, sapacreti, AZD2014, OSI-027, Rapalink-1, LY3023414, curcumin, epigallocatechin gallate, resveratrol, sulforaphane, quercetin, chlorogenic acid, anthocyanins, ginsenosides, shogaol, capsaicin, melatonin, allicin, and Omega-3 polyunsaturated fatty acids; The inflammatory regulatory factors include IL-2.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The steps for separating and obtaining exosomes include: centrifuging the co-culture product or the supernatant of the co-culture product at 0-10℃ and 100-500×g for 5-20 min to obtain a first supernatant; centrifuging the first supernatant at 0-10℃ and 1500-2500×g for 5-20 min to obtain a second supernatant; filtering the second supernatant and / or centrifuging it at 0-10℃ and 8000-12000×g for 10-60 min to obtain a filtrate or a third supernatant; the membrane used for filtration has a pore size of 0.1-0.45 μm; centrifuging the filtrate or the third supernatant at 0-10℃ and 80000-120000×g for 30-90 min to collect the precipitate.

6. An exosome, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 5.

7. A composition for improving or repairing skin wounds, characterized in that, It includes: The exosomes as described in claim 6.

8. A cosmetic product, characterized in that, It includes: The exosomes as described in claim 6.

9. The preparation method according to any one of claims 1 to 5 or the use of the exosomes according to claim 6 in the preparation of products that improve or repair skin wound damage.

10. The preparation method according to any one of claims 1 to 5 or the exosomes according to claim 6 in the preparation of products having at least one of the following effects: oil removal, moisturizing, spot fading, whitening, anti-wrinkle, pore shrinking and elasticity enhancement.

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