Preparation method of mesenchymal stem cell exosome based on microcarrier three-dimensional culture and application of mesenchymal stem cell exosome in anti-aging skin care products

Abstract

The invention discloses a preparation method of a mesenchymal stem cell exosome based on microcarrier three-dimensional culture and application of the mesenchymal stem cell exosome in anti-aging skin care products. According to the method, a three-dimensional culture system of mesenchymal stem cells is optimized, and the anti-aging function activity of the subsequently separated and purified exosome is remarkably improved by adopting a microcarrier with recombinant collagen as a main component; subsequently, by optimizing an exosome separation and purification technology, and through a specific separation and purification mode and conditions, the exosome with anti-aging active ingredients is further obtained. Experiments prove that the exosome obtained by the method disclosed by the invention can effectively remove metalloproteinase 1 and promote collagen regeneration, has a remarkable anti-aging effect, is suitable for essence, face cream and other dosage forms, and has a wide market prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to a method for preparing mesenchymal stem cell exosomes based on three-dimensional culture of microcarriers and their application in anti-aging skin care products. Background Art

[0002] Exosomes refer to small membrane vesicles (30 - 150 nm) containing complex RNA and proteins. Currently, they specifically refer to discoidal vesicles with a diameter of 40 - 100 nm. In 1983, exosomes were first discovered in sheep reticulocytes and were named "exosome" by Johnstone in 1987. A variety of cells can secrete exosomes under normal and pathological conditions. They mainly originate from multivesicular bodies formed by the invagination of lysosome particles within cells and are released into the extracellular matrix after the fusion of the outer membrane of multivesicular bodies with the cell membrane.

[0003] All cultured cell types can secrete exosomes, and exosomes naturally exist in body fluids, including blood, saliva, urine, cerebrospinal fluid, and milk. The precise molecular mechanisms regarding their secretion, uptake, composition, "cargo", and corresponding functions have just begun to be studied. Exosomes are regarded as specifically secreted membrane vesicles involved in cell - to - cell communication, and the research interest in exosomes is increasing, whether it is to study their functions or to understand how to use them in the development of minimally invasive diagnostics.

[0004] Exosomes have various effects in cosmetics, mainly including promoting skin repair, anti - aging, moisturizing, anti - inflammation, and improving skin color. Exosomes are tiny extracellular vesicles naturally released by cells into the extracellular environment, carrying bioactive molecules such as proteins, lipids, and RNA, which play important roles in cell - to - cell communication. Exosome skin care is an anti - aging method that fundamentally promotes metabolism, tissue repair, improves immunity, inhibits scarring, etc. It has a function of repairing and regenerating cells, enabling epidermal cells, fibroblast cells, hair follicle cells, adipocytes, etc. to automatically repair and promoting the secretion and generation of collagen, thereby reducing the loss of collagen. Regarding anti - aging, the components in exosomes can promote the proliferation and differentiation of skin cells, stimulate fibroblasts to produce more collagen and elastic fibers, which are the key substances to maintain skin elasticity and firmness. With age, the reduction of collagen and elastic fibers leads to skin relaxation and the appearance of wrinkles, while exosomes help to supplement and enhance their effects, thus reducing wrinkles and delaying skin aging.

[0005] There are many cell culture methods (such as 2D culture, 3D culture, microcarrier types, etc.) and exosome extraction methods (such as ultracentrifugation, density gradient centrifugation, ultrafiltration, immunomagnetic beads, immunoaffinity chromatography, tangential flow filtration, etc.). However, different cell culture methods and exosome extraction methods have certain effects on the functional activity of their exosomes. Therefore, there is an urgent need to provide a method for preparing exosomes with good anti-aging effects. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide a method for preparing mesenchymal stem cell exosomes based on three-dimensional culture with microcarriers.

[0007] The second purpose of the present invention is to provide the application of the mesenchymal stem cell exosomes in the preparation of anti-aging skin care products.

[0008] The above objects of the present invention are achieved by the following technical solutions:

[0009] A method for preparing mesenchymal stem cell exosomes, comprising the following steps:

[0010] S1. Three-dimensionally culture mesenchymal stem cells with a cell culture microcarrier. When the cell confluence is greater than or equal to 80%, harvest the supernatant;

[0011] S2. Filter the supernatant with a filter membrane and perform tangential flow filtration to separate and purify mesenchymal stem cell-derived exosomes;

[0012] The preparation method of the cell culture microcarrier in step S1 is as follows:

[0013] a. Preparation of the aqueous phase: Dissolve recombinant collagen and lecithin in water and stir at 45 - 55 °C for 55 - 65 minutes;

[0014] b. Preparation of the oil phase: Add an emulsifier to olive oil and stir at 25 - 35 °C for 25 - 35 minutes;

[0015] c. Emulsification: Add the aqueous phase to the oil phase, with a stirring speed of 35 - 45 rpm / min, and emulsify for 8 - 12 minutes;

[0016] d. Solidification: Solidify at room temperature for 18 - 22 minutes and solidify in an ice-water bath for 18 - 22 minutes;

[0017] e. Crosslinking: Add a crosslinking agent for crosslinking, quickly freeze in liquid nitrogen, and vacuum freeze for 2 - 4 days; Select recombinant collagen microspheres with a particle size of 150 - 500 microns, sterilize, and obtain;

[0018] The dissolved oxygen content of the three-dimensional culture in step S1 is > 80%, and the culture time is 5 - 7 days;

[0019] The filtration by the filter membrane and the tangential flow filtration described in step S2 are as follows: the supernatant is first filtered through a 0.22 μm filter membrane at a flow rate of 25-30 mL / min, and then the filtered supernatant is concentrated by tangential flow filtration at a flow rate of 90-110 mL / min, a shear force of 3-5 psi, and a treatment time of about 3.5-5.5 h.

[0020] The present invention first optimizes the three-dimensional culture system of mesenchymal stem cells, and uses a microcarrier mainly composed of recombinant collagen to significantly improve the anti-aging functional activity of exosomes separated and purified subsequently; then, by optimizing the exosome separation and purification technology, through specific separation and purification methods and conditions, exosomes with anti-aging active ingredients are further obtained. Experiments have confirmed that the exosomes obtained by the method of the present invention can effectively remove matrix metalloproteinase 1 and promote collagen regeneration, have a significant anti-aging effect, are suitable for dosage forms such as essence and cream, and have broad market prospects.

[0021] Specifically, recombinant collagen, as the main component of the microcarrier for three-dimensional culture of mesenchymal stem cells in the present invention, provides a natural extracellular matrix environment for mesenchymal stem cells, which is conducive to the growth, proliferation, and differentiation of stem cells, enabling them to secrete exosomes with normal functions and high activities. Exosomes may carry more bioactive molecules related to anti-aging, such as growth factors, cytokines, etc., thereby enhancing their anti-aging function. Lecithin is a commonly used emulsifier and surfactant, which not only helps the aqueous phase to disperse better in the oil phase to form a stable emulsion. At the same time, lecithin is an important component of cell membranes, which helps to maintain the stability and fluidity of cell and exosome membranes. In the preparation of microcarriers, it may help to form a more stable microcarrier structure, provide a stable culture environment for stem cells, and indirectly promote the good anti-aging functional activity of exosomes. As the oil phase, olive oil has good biocompatibility and chemical stability, provides a suitable physical environment for the formation of microcarriers, and helps to maintain the morphological and structural integrity of microcarriers. A suitable microcarrier structure is conducive to the three-dimensional culture of stem cells, enabling stem cells to grow and secrete exosomes in an environment closer to the in vivo situation, which may enhance the anti-aging function of exosomes. Recombinant collagen, lecithin, and olive oil are crucial for obtaining exosomes with good anti-aging efficacy in the present invention. When isolating and purifying exosomes, filtration through a 0.22 μm filter membrane can effectively remove impurities such as cell debris and undissolved proteins, preliminarily purify the exosome suspension, reduce the interference of impurities on the exosome function, and preliminarily enrich the exosomes through the filter membrane, increasing the relative concentration of exosomes in the solution, which helps to improve the efficiency of subsequent steps such as tangential flow filtration, ensuring the final acquisition of exosomes with high purity and concentration, and providing a material basis for their anti-aging function. Tangential flow filtration can efficiently concentrate the filtered supernatant while maintaining the biological activity of exosomes, increase the concentration of exosomes, make their anti-aging functional components more concentrated, and thus better exert their anti-aging function.

[0022] Further, the emulsifier is Span 80. Span 80 is a commonly used water-in-oil emulsifier, which can reduce the surface tension at the oil-water interface, enable the aqueous phase to be evenly dispersed in the oil phase to form a stable emulsion, and helps to prepare microcarriers with uniform particle size and stable performance. Good microcarrier characteristics are conducive to the culture of stem cells and the secretion of exosomes, and have a positive effect on maintaining the anti-aging functional activity of exosomes.

[0023] Further, the cross-linking agent is glutaraldehyde, which can form a cross-linked structure between collagen molecules, enhance the mechanical strength and stability of the microcarrier, make the microcarrier form a more stable three-dimensional structure, ensure the stability of the microcarrier during cell culture, provide a stable supporting environment for stem cells, and is conducive to the normal secretion and function maintenance of exosomes.

[0024] Furthermore, the dosage ratio of the recombinant collagen to the lecithin is 1-3:1.

[0025] Furthermore, the volume ratio of the aqueous phase to the oil phase is 1:7-9.

[0026] Furthermore, the seeding density of the mesenchymal stem cells in step S1 is 1×10 4 -5×10 4 cells / cm 2 .

[0027] Furthermore, the mesenchymal stem cells are animal umbilical cord mesenchymal stem cells. Animal umbilical cords are usually discarded after the animal gives birth. Obtaining umbilical cord mesenchymal stem cells from them will not cause harm to the animal itself. The operation is relatively simple, and the collection process is also relatively safe. Unlike the collection of bone marrow stem cells, which requires bone marrow puncture and causes certain trauma to the donor animal. At the same time, the umbilical cord contains a large number of mesenchymal stem cells. Compared with other tissue sources, such as adipose tissue, etc., a larger number of stem cells can be obtained from a unit volume of umbilical cord tissue, which can meet the needs of a large number of experiments and clinical applications.

[0028] Preferably, the animal umbilical cord mesenchymal stem cells are bovine umbilical cord mesenchymal stem cells.

[0029] Preferably, the separation method of the bovine umbilical cord mesenchymal stem cells is to remove blood vessels and separate the bovine umbilical cord tissue. After washing the umbilical cord with PBS, Wharton's jelly is separated. Cut it into small pieces of 2-4 cm and adhere them to the tissue. Then, the cut tissue pieces are added to the complete medium for primary culture (37±1°C, 5% CO 2 2), observe the cell growth situation. After the cell confluence reaches 80-90%, perform passage two-dimensional culture for 2 generations, and then bovine umbilical cord mesenchymal stem cells are obtained.

[0030] Furthermore, the three-dimensional culture in step S1 is carried out in a bioreactor.

[0031] Furthermore, the bioreactor is a stirred bioreactor, and the culture rotation speed is 20-50 rpm / min.

[0032] Furthermore, the method for harvesting the supernatant in step S1 is to stop stirring the reactor, let the reaction tank stand still for 5-10 minutes. The microcarriers are not suspended in the supernatant. Connect the silica gel tube to the peristaltic pump on the reactor control cabinet, and open the peristaltic pump to pump out the supernatant (about 90% of the total volume of the culture medium) through the pre-installed liquid extraction tube into a sterile raw material bag. The collected supernatant is used for subsequent exosome isolation and purification.

[0033] Furthermore, step S1 is to harvest the supernatant when the mesenchymal stem cells are cultured in passages P4-P7.

[0034] The present invention also provides mesenchymal stem cell exosomes prepared by any of the above-described preparation methods.

[0035] Experiments of the present invention have confirmed that the mesenchymal stem cell exosomes obtained by the above method can effectively remove matrix metalloproteinase 1, promote collagen regeneration, further enhance the anti-aging effect, are applicable to dosage forms such as essence and cream, and have broad market prospects. Therefore, the present invention also provides the application of the mesenchymal stem cell exosomes in the preparation of anti-aging skin care products.

[0036] The present invention also provides an anti-aging skin care product, which contains any of the above-described mesenchymal stem cell exosomes.

[0037] Further, the weight percentage content of the exosomes is 0.05-10%, and is supplemented with a pharmaceutically or cosmetically acceptable carrier.

[0038] Further, the dosage form of the anti-aging skin care product is essence, cream or mask.

[0039] Further, the anti-aging skin care product comprises the following components in weight percentages: sodium hyaluronate oligosaccharide (0.1-5%), glutathione (0.05-10%), niacinamide (0.2-15%), vitamin C ethyl ether (0.1-15%), glabridin (0.1-10%), asiatic pennywort extract (0.05-10%), glycerol (0.5-20%), aloe extract (0.05-10%), portulaca extract (0.05-10%), hydroxyproline (0.05-10%), phenoxyethanol (0.01-5%), exosomes (0.05-10%), and the balance is water, and they are mixed.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention provides a kind of mesenchymal stem cell exosomes based on three-dimensional culture with microcarriers. The present invention first optimizes the three-dimensional culture system of mesenchymal stem cells, and uses microcarriers mainly composed of recombinant collagen to significantly enhance the anti-aging functional activity of the subsequent isolated and purified exosomes; then further optimizes the exosome isolation and purification technology, and through specific isolation and purification methods and conditions, exosomes with anti-aging active ingredients are further obtained. Experiments have confirmed that the exosomes obtained by the method of the present invention can effectively remove matrix metalloproteinase 1, promote collagen regeneration, have significant anti-aging effects, are applicable to dosage forms such as essence and cream, and have broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Results of matrix metalloproteinase 1 (MMP-1) determination. Experimental group: Example 1, Control group 1: Comparative Example 1, Control group 2: Comparative Example 2.

[0043] Figure 2 It is the test result of type I collagen content. Experimental group: Example 1, Control group 1: Comparative Example 1, Control group 2: Comparative Example 2. Detailed implementation manners

[0044] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0046] Example 1

[0047] (1) Isolation and culture of bovine umbilical cord mesenchymal stem cells

[0048] The bovine umbilical cord tissue was de-vascularized and separated. After washing the umbilical cord with PBS, Wharton's jelly was separated, cut into small pieces and adhered to the tissue. The Wharton's jelly was cut into small sections of 2-4 cm. The cut tissue blocks were added to the complete medium for primary culture (37±1°C, 5% CO 2 2), and the cell growth was observed. After the cell confluence reached 80-90%, subculture in two dimensions was carried out for 2 generations to obtain bovine umbilical cord mesenchymal stem cells.

[0049] (2) Three-dimensional culture of mesenchymal stem cells

[0050] The bovine umbilical cord mesenchymal stem cells were three-dimensionally cultured in a stirred bioreactor using cell culture microcarriers (inoculation density: 3×10 4 cells / cm 2 , rotation speed: 35 rpm / min, dissolved oxygen content > 80%, cultured for 6 days). When the cell confluence was ≥80%, at the time of culturing the P5 generation, preparations were made for harvesting the supernatant and harvesting the cells. The stirring of the reactor was stopped, and the reaction tank was left stationary for 5-10 minutes. The microcarriers were not suspended in the supernatant. The connecting silica tube was connected to the peristaltic pump of the reactor control cabinet, and the peristaltic pump was turned on to pump out the supernatant (about 90% of the total volume of the culture solution) through the pre-installed liquid extraction tube into a sterile raw material bag. The collected supernatant was used for subsequent exosome isolation and purification. The harvested supernatant was sealed and stored at -80°C. The preparation method of the cell culture microcarriers:

[0051] a. Preparation of the aqueous phase: 4 g of recombinant collagen and 2 g of lecithin were dissolved in 50 mL of water and stirred in a water bath at 50°C for 60 minutes.

[0052] b. Preparation of the oil phase: 0.5 g of emulsifier Span 80 was added to 400 mL of olive oil and stirred at 30°C for 30 minutes.

[0053] c. Emulsification: The aqueous phase was added to the oil phase, and the stirring speed was 40 rpm / min for 10 minutes of emulsification.

[0054] d. Solidification: Solidify at room temperature for 20 minutes and in an ice-water bath for 20 minutes.

[0055] e. Crosslinking: Add 0.9 mL of glutaraldehyde, stir for 3 minutes, quickly freeze in liquid nitrogen for 8 minutes, freeze in a freeze dryer for 3 days, select recombinant collagen microspheres with a particle size of 150 - 500 microns, and sterilize with an electron beam.

[0056] (3) Purification of mesenchymal stem cell exosomes

[0057] Filter membrane filtration and tangential flow filtration were used. Specifically, 500 mL of the cell supernatant obtained in step (2) was first filtered through a 0.22 μm filter membrane at a flow rate of 27 mL / min, and then the filtered cell supernatant was concentrated to 25 mL by tangential flow filtration (TFF) at a flow rate of 100 mL / min, a shear force of 4 psi, and a treatment time of about 4.5 h to separate and purify bovine umbilical cord mesenchymal stem cell-derived exosomes.

[0058] Example 2

[0059] (1) Isolation and culture of bovine umbilical cord mesenchymal stem cells

[0060] Same as Example 1.

[0061] (2) Three-dimensional culture of mesenchymal stem cells

[0062] The bovine umbilical cord mesenchymal stem cells were three-dimensionally cultured in a stirred bioreactor using a cell culture microcarrier (seeding density was 1×10 4 cells / cm 2 , rotation speed 50 rpm / min, dissolved oxygen > 80%, cultured for 5 days). When the cell confluence was ≥ 80%, at passage P4, preparation for supernatant harvesting and cell harvesting was carried out. The reactor was stopped from stirring, and the reaction tank was left stationary for 5 - 10 min. The microcarriers were not suspended in the supernatant. The connecting silica tube was connected to the peristaltic pump of the reactor control cabinet, and the peristaltic pump was turned on to pump out the supernatant (about 90% of the total volume of the culture medium) through the pre-installed liquid extraction tube into a sterile raw material bag. The collected supernatant was used for subsequent exosome separation and purification. The harvested supernatant was sealed and stored at -80°C. The preparation method of the cell culture microcarrier:

[0063] a. Aqueous phase preparation: Dissolve 4 g of recombinant collagen and 2 g of lecithin in 50 mL of water and stir in a 45°C water bath for 65 minutes.

[0064] b. Oil phase preparation: Add 0.5 g of emulsifier Span 80 to 400 mL of olive oil and stir at 25°C for 35 minutes.

[0065] c. Emulsification: The aqueous phase was added to the oil phase, and the stirring speed was 35 rpm / min for 12 minutes of emulsification.

[0066] d. Solidification: Solidify at room temperature for 18 minutes and in an ice-water bath for 18 minutes.

[0067] e. Crosslinking: Add 0.9 mL of glutaraldehyde, stir for 3 minutes, quick-freeze in liquid nitrogen for 5 minutes, freeze-dry for 2 days, select recombinant collagen microspheres with a particle size of 150 - 500 microns, and sterilize with electron beam.

[0068] (3) Purification of mesenchymal stem cell exosomes

[0069] Take 500 mL of the cell supernatant obtained in step (2), first filter it through a 0.22 μm filter membrane at a flow rate of 25 mL / min, and then concentrate the filtered cell supernatant to 30 mL by tangential flow filtration (TFF) at a flow rate of 90 mL / min, a shear force of 5 psi, and a treatment time of about 3.5 h to isolate and purify bovine umbilical cord mesenchymal stem cell-derived exosomes.

[0070] Example 3

[0071] (1) Isolation and culture of bovine umbilical cord mesenchymal stem cells

[0072] Same as Example 1.

[0073] (2) Three-dimensional culture of mesenchymal stem cells

[0074] The bovine umbilical cord mesenchymal stem cells were three-dimensionally cultured in a stirred bioreactor using a cell culture microcarrier (seeding density: 5×10 4 cells / cm 2 , rotation speed 20 rpm / min, dissolved oxygen > 80%, cultured for 7 days). When the cell confluence was ≥ 80% and at passage P7, the supernatant was harvested and the cells were harvested. The reactor stopped stirring, and the reaction tank was left stationary for 5 - 10 min. The microcarriers were no longer suspended in the supernatant. The connecting silica tube was connected to the peristaltic pump of the reactor control cabinet, and the peristaltic pump was turned on to pump out the supernatant (about 90% of the total volume of the culture medium) through the pre-installed liquid extraction tube into a sterile raw material bag. The collected supernatant was used for subsequent exosome isolation and purification. The harvested supernatant was sealed and stored at -80°C. The preparation method of the cell culture microcarrier:

[0075] a. Preparation of the aqueous phase: Dissolve 4 g of recombinant collagen and 2 g of lecithin in 50 mL of water and stir in a water bath at 55 °C for 55 minutes.

[0076] b. Preparation of the oil phase: Add 0.5 g of emulsifier Span 80 to 400 mL of olive oil and stir at 35 °C for 25 minutes.

[0077] c. Emulsification: The aqueous phase was added to the oil phase, and the stirring speed was 45 rpm / min for 8 minutes of emulsification.

[0078] d. Solidification: Solidify at room temperature for 22 minutes and in an ice-water bath for 22 minutes.

[0079] e. Crosslinking: Add 0.9 mL of glutaraldehyde, stir for 3 minutes, quickly freeze in liquid nitrogen for 10 minutes, freeze-dry for 4 days, select recombinant collagen microspheres with a particle size of 150 - 500 microns, and sterilize with an electron beam.

[0080] (3) Purification of mesenchymal stem cell exosomes

[0081] Take 500 mL of the cell supernatant obtained in step (2), first filter it through a 0.22 μm filter membrane at a flow rate of 30 mL / min, and then concentrate the filtered cell supernatant to 20 mL by tangential flow filtration (TFF) at a flow rate of 110 mL / min, a shear force of 3 psi, and a treatment time of approximately 5.5 h to obtain exosomes derived from bovine umbilical cord mesenchymal stem cells by separation and purification.

[0082] Comparative Example 1

[0083] A method for preparing bovine umbilical cord mesenchymal stem cell exosomes is basically the same as that in Example 1, except that a commercially available microcarrier is used for three-dimensional culture, and the microcarrier does not contain recombinant collagen peptide, lecithin, and olive oil.

[0084] Comparative Example 2

[0085] A method for preparing bovine umbilical cord mesenchymal stem cell exosomes is basically the same as that in Example 1, except that another commercially available microcarrier is used for three-dimensional culture, and the microcarrier does not contain recombinant collagen peptide, lecithin, and olive oil.

[0086] Comparative Example 3

[0087] A method for preparing bovine umbilical cord mesenchymal stem cell exosomes is basically the same as that in Example 1, except that only filtration through a filter membrane is used for exosome purification.

[0088] Comparative Example 4

[0089] A method for preparing bovine umbilical cord mesenchymal stem cell exosomes is basically the same as that in Example 1, except that only tangential flow filtration is used for exosome purification.

[0090] Comparative Example 5

[0091] A method for preparing bovine umbilical cord mesenchymal stem cell exosomes is basically the same as that in Example 1, except that the tangential flow filtration rate is 120 mL / min, the shear force is 6 psi, and the treatment time is about 6 h.

[0092] Test Example: Evaluation of Anti-aging Efficacy

[0093] To evaluate whether the above-mentioned bovine umbilical cord mesenchymal stem cells exhibit anti-aging effects, the contents of matrix metalloproteinase 1 (MMP-1) and type I collagen were determined by ELISA method.

[0094] (1) Determination of type I collagen content: At 37 °C and 5% carbon dioxide, HEFs were cultured with growth medium until the cells reached 80% confluence. Then, the medium was removed and the cells were washed with PBS. Human epidermal fibroblasts (HEFs) were exposed to ultraviolet radiation B (UVB) light at a dose of 0.2 J. Then the cells were co-cultured with the growth medium for 24 hours. The above process was repeated three times. Subsequently, the cells were washed 3 times with PBS, and the exosomes of Example 1 were used, and 10 10 particles / mL of the medium was used as the experimental group, and the exosomes of Comparative Examples 1-5 were used as Control Group 1 - Control Group 5 respectively. At the same time, the cells in the normal group were not irradiated with UVB light as the negative control, and those only irradiated with UV were used as the positive control. The content of type I collagen was determined according to the ELISA method.

[0095] (2) Determination of matrix metalloproteinase 1 (MMP-1): At 37 °C and 5% carbon dioxide, HEFs were cultured with growth medium until the cells reached 80% confluence. Then, the medium was removed and the cells were washed with PBS. Human epidermal fibroblasts (HEFs) were exposed to ultraviolet radiation B (UVB) light at a dose of 0.2 J. Then the cells were co-cultured with the growth medium for 24 hours. The above process was repeated three times. Subsequently, the cells were washed 3 times with PBS, and the exosomes of Example 1 were used, and 10 10 particles / mL of the medium was used as the experimental group, and the exosomes of Comparative Examples 1-5 were used as Control Group 1 - Control Group 5 respectively. At the same time, the cells in the normal group were not irradiated with UVB light as the negative control, and those only irradiated with UV were used as the positive control. The matrix metalloproteinase 1 (MMP-1) was determined according to the ELISA method.

[0096] The results showed that after the human epidermal fibroblasts (HEFs) were treated with UV irradiation, the expression of type I collagen content decreased and the MMP-1 content increased, and the cells were damaged. When the exosome medium containing Example 1 and Comparative Examples 1-5 was used for treatment, the type I collagen content increased and the MMP-1 content decreased. Specifically, as Figure 1 and Figure 2As shown, the exosomes obtained after three-dimensional culture using the microcarriers prepared from the recombinant collagen peptide, lecithin, and olive oil of the present invention have a better anti-aging effect compared to the exosomes obtained after three-dimensional culture using two commercially available microcarriers that do not contain recombinant collagen peptide, lecithin, and olive oil. In addition, the exosomes obtained under the purification conditions of Example 1 of the present invention have a better anti-aging effect compared to the exosomes obtained in Comparative Examples 3 to 5.

Claims

1. A method for preparing mesenchymal stem cell exosomes, characterized in that: The steps include: S1. The mesenchymal stem cells are cultured in three dimensions using cell culture microcarriers, and when the cell confluence is greater than or equal to 80%, the supernatant is harvested; S2. filtering the supernatant by membrane filtration and tangential flow filtration to separate and purify the mesenchymal stem cell-derived exosomes; The preparation method of the cell culture microcarrier in step S1 is: a. Preparation of aqueous phase: Dissolve recombinant collagen and lecithin in water and stir at 45-55°C for 55-65 minutes; b. Preparation of oil phase: Add emulsifier to olive oil and stir at 25-35°C for 25-35 minutes; c. Emulsification: Add the water phase to the oil phase, stir at a speed of 35-45 rpm / min, and emulsify for 8-12 minutes; d. Curing: Curing at room temperature for 18 to 22 minutes, curing in an ice water bath for 18 to 22 minutes; e. Cross-linking: adding a cross-linking agent for cross-linking, quick freezing in liquid nitrogen, and vacuum freezing for 2 to 4 days; selecting recombinant collagen microspheres with a particle size of 150 to 500 microns, sterilizing, and obtaining; The dissolved oxygen content of the three-dimensional culture in step S1 is >80%, and the culture time is 5 to 7 days; The membrane filtration and tangential flow filtration in step S2 are to first filter the supernatant through a 0.22 μm membrane at a flow rate of 25 to 30 mL / min, and then concentrate the filtered supernatant through tangential flow filtration at a flow rate of 90 to 110 mL / min, a shear force of 3 to 5 psi, and a processing time of about 3.5 to 5.5 h.

2. The preparation method according to claim 1, characterized in that: The seeding density of mesenchymal stem cells in step S1 is 1×10 4 ~5×10 4 cells / cm 2 .

3. The preparation method according to claim 1, characterized in that: The mesenchymal stem cells are animal umbilical cord mesenchymal stem cells.

4. The preparation method according to claim 3, characterized in that: The animal umbilical cord mesenchymal stem cells are bovine umbilical cord mesenchymal stem cells.

5. The preparation method according to claim 1, characterized in that: Step S1 is to harvest the supernatant when the mesenchymal stem cells are cultured at passages P4 to P7.

6. Mesenchymal stem cell exosomes prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the mesenchymal stem cell exosomes according to claim 6 in the preparation of anti-aging skin care products.

8. An anti-aging skin care product, characterized in that: Comprising the mesenchymal stem cell exosomes according to claim 6.

9. The anti-aging skin care product according to claim 8, characterized in that: The exosomes of the mesenchymal stem cells have a weight percentage of 0.05-10%, and are supplemented with a pharmaceutically or cosmetically acceptable carrier.

10. The anti-aging skin care product according to claim 8, characterized in that: The anti-aging skin care product is in the form of essence, cream or mask.

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