Methods for producing exosomes, the resulting exosomes, and their applications

Abstract

This invention relates to a method for producing exosomes, the exosomes obtained therefrom, and their applications. The invention discloses a method for producing exosomes, comprising: culturing avian embryonic mesenchymal stem cells (AMSCs) in a medium containing 2,3,4′,5-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) to obtain a cell culture of AMSCs; and collecting exosomes from the cell culture. The invention also discloses that the exosomes obtained by the above method can be used to improve skin conditions.

Description

Technical Field

[0001] This invention relates to a method for producing exosomes, comprising: culturing avian embryonic mesenchymal stem cells (AMSCs) in a medium containing 2,3,4′,5-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) to obtain a cell culture of AMSCs; and collecting exosomes from the cell culture. This invention also relates to using these exosomes to improve skin conditions. Background Technology

[0002] Exosomes are nanosized vesicles secreted by cells. They are a type of extracellular vesicle (EVs) and are found in various biological fluids, including amniotic fluid, urine, and blood. They are rich in proteins, lipids, mRNAs, and miRNAs.

[0003] Size-selective separation (e.g., filtration, dialysis, and chromatography) and density-selective separation (e.g., centrifugation) have both been used to isolate exosomes from stem cell cultures to facilitate the study of exosome activity. Previous studies have indicated that exosomes produced from stem cells of different origins or types exhibit different biological activities. For example, in Wang Y. et al. (2017), Stem Cell Res. Ther., 8:189, Wang Y. et al. found that exosomes derived from human embryonic stem cell-induced mesenchymal stem cells have an effect in alleviating osteoarthritis. In Kim YJ et al. (2017), Biochem. Biophys. Res. Commun., 493: 1102-1108, Kim YJ et al. found that exosomes derived from human umbilical cord blood mesenchymal stem cells have the effect of promoting skin rejuvenation.

[0004] To the best of the applicant's knowledge, no literature or pre-patent application has to date described the use of exosomes in the production of 2,3,4′,5-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) [an active ingredient found in Polygonum multiflorum and belonging to the polyhydroxystilbene group] and avian embryonic mesenchymal stem cells (AMSCs). Summary of the Invention

[0005] In this invention, the applicant discovered that using 2,3,4′,5-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) to culture avian embryo mesenchymal stem cells (AMSCs) can effectively promote the production of large quantities of exosomes containing a variety of components (including proteins and RNA) by AMSCs cells. Furthermore, these exosomes can effectively promote skin regeneration and wound healing, reduce skin wrinkles, improve hair loss, and reduce skin inflammation. Therefore, they are expected to be applicable to improving various skin conditions.

[0006] Therefore, in a first aspect, the present invention provides a method for producing exosomes, comprising:

[0007] Avian embryonic mesenchymal stem cells (AMSCs) were cultured in a medium containing 2,3,4′,5-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) to obtain AMSC cell cultures; and

[0008] Exosomes were collected from the cell culture.

[0009] Preferably, the culture medium contains THSG at a concentration ranging from 0.1 to 50 μM.

[0010] Ideally, the culture is carried out for 24 to 96 hours.

[0011] Preferably, the exosomes are collected by size-selective or density-selective separation processes.

[0012] More preferably, the separation process is a combination of tangential flow filtration and size-exclusion chromatography.

[0013] In a second aspect, the present invention provides an exosome that is prepared by the method described above.

[0014] Preferably, the exosome has a particle size in the range of 50 to 200 nm.

[0015] Preferably, the exosome has an average particle size in the range of 50 to 100 nm.

[0016] In a third aspect, the present invention provides the use of an exosome supply as described above for preparing a composition for improving skin conditions.

[0017] Preferably, the skin condition includes at least one of the following: wound, aging, hair loss, and inflammation.

[0018] Preferably, the composition is a cosmeceutical composition.

[0019] Preferably, the composition is a pharmaceutical composition.

[0020] More preferably, the pharmaceutical composition is in a dosage form for non-enteral or oral administration.

[0021] In a fourth aspect, the present invention provides a method for improving skin conditions, comprising administering exosomes as described above to an individual in need of such treatment. Attached Figure Description

[0022] Figure 1 The display shows the exosomal protein concentrations obtained according to the method of the present invention and existing methods, where "*" indicates that p < 0.05 when compared with control exosomals;

[0023] Figure 2 The display shows the exosomal RNA concentrations obtained according to the method of the present invention and existing methods, wherein "***" indicates that when compared with control exosomals, p < 0.001;

[0024] Figures 3 to 5 The expression levels of COL1A1, COL3A1, and ELN genes in HSFs cells in each group are shown. The control group represents cells without any treatment; experimental groups 1 to 3 represent cells treated with exosome suspensions at different concentrations (0.07, 0.7, and 70 μg / mL), respectively; and "***" indicates that when compared with the control group, p < 0.001.

[0025] Figure 6 The percentage of HSFs cells that are viable after treatment with the exosomes of the present invention is shown, where the control group represents cells that have not been treated in any way; the experimental group represents cells that have been treated with an exosome suspension of 70 μg / mL; and “***” indicates that when compared with the control group, p < 0.001.

[0026] Figure 7 The results observed by the cell migration analysis of HSFs cells after treatment with the exosomes of the present invention via wound healing are shown, wherein the control group represents cells without any treatment; and the experimental group represents cells treated with an exosome suspension of 70 μg / mL.

[0027] Figure 8 The percentage of HFDPCs cells that are viable after treatment with the exosomes of this invention is shown, where the control group represents cells that have not been treated; the experimental group represents cells treated with an exosome suspension of 70 μg / mL; and "***" indicates that p < 0.001 when compared with the control group; and

[0028] Figures 9 to 11 The expression levels of IL-6, IL-1β, and TNF-α genes measured in HSFs cells in each group are shown. The normal control group represents cells without any treatment; the pathological control group represents cells treated with 1 μg / mL lipopolysaccharides (LPS); and the experimental group represents cells treated with 1 μg / mL LPS and 0.07 μg / mL exosome suspension. "***" indicates that p < 0.001 compared with the normal control group; and "###" indicates that p < 0.001 compared with the pathological control group. Detailed Implementation

[0029] For the purposes of this instruction manual, it will be clearly understood that the word "comprising" means "including but not limited to", and the word "comprises" has the corresponding meaning.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art. Those skilled in the art will recognize many similar or equivalent methods and materials that can be used to practice the invention. Of course, the invention is by no means limited to the methods and materials described.

[0031] This invention provides a method for producing exosomes, comprising:

[0032] Avian embryonic mesenchymal stem cells (AMSCs) were cultured in a medium containing 2,3,4′,5-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) to obtain AMSC cell cultures; and

[0033] Exosomes were collected from the cell culture.

[0034] Preferably, the content of exosomes obtained by the above method can be 1×10⁻⁶. 8 More than 1000 mg / particle, preferably at 1×10⁻⁶.9 Up to 1×10 10 Within the range of particles / mg. In some specific examples, the content of this exosome was 5.08 × 10⁻⁶. 9 1 capsule / mg.

[0035] According to the present invention, AMSCs cells can have a size of 8.3 × 10⁻⁶. 7 Up to 8.3×10 9 The number within the cell. Preferably, AMSCs cells have 1 × 10⁻⁶ cells. 8 The quantity.

[0036] According to the present invention, the culture medium may contain THSG at a concentration ranging from 0.1 to 50 μM. In some specific examples, the culture medium contains 25 μM of THSG.

[0037] According to the present invention, THSG can be isolated and purified from Polygonum multiflorum using separation and purification methods commonly used in the art. In this regard, reference may be made, for example, Tsai PWet al. (2018), Molecules, 23(3): 571.

[0038] Alternatively, THSG may be obtained from commercially available products, such as Polygonum multiflorum glycoside (stilbene glycoside) (product number BP0039) purchased from Chengdu Biopurify Phytochemicals Ltd.

[0039] As used herein, the terms “culturing” and “cultivation” may be used interchangeably. The procedures and parameters relating to culturing fall within the scope of expertise and routine practice of those skilled in the art. In this regard, see, for example, Gao Y. et al. (2013), Biomed. Res. Int., doi:10.1155 / 2013 / 626258.

[0040] According to the present invention, the culture can be carried out at a temperature ranging from 36°C to 37°C. In some specific examples, the culture is carried out at 37°C.

[0041] According to the present invention, the culture can be carried out for 24 to 96 hours. In some specific examples, the culture is carried out for 36 hours.

[0042] According to the present invention, the operation process and parameter conditions of the separation process can be carried out using techniques well known and commonly used by those skilled in the art. In this regard, reference can be made, for example, to Wang Y. et al. (2017) (as described above).

[0043] According to the present invention, the exosomes can be collected by size-selective or density-selective separation processes.

[0044] Preferably, the size-selective separation process can be selected from the group consisting of: filtration [e.g., ultrafiltration and tangential flow filtration (TFF)], dialysis [e.g., diafiltration], and chromatography [e.g., size-exclusion chromatography (SEC)], and combinations thereof.

[0045] Preferably, the density-selective separation process can be selected from centrifugation processes consisting of the group consisting of ultracentrifugation and density gradient centrifugation.

[0046] In some specific examples, the separation process is a combination of tangential flow filtration (TFF) and size-exclusion chromatography (SEC).

[0047] Preferably, TFF can use a molecular weight cut-off value of 100 to 500 kDa. In some specific examples, TFF uses a molecular weight cut-off value of 500 kDa.

[0048] Preferably, TFF can be performed at a rotation speed of 50 to 200 mL / min. In some specific examples, TFF is performed at a rotation speed of 80 to 120 mL / min.

[0049] Preferably, SEC can be performed at a column temperature of 15 to 25°C. In some specific examples, SEC is performed at a column temperature of 25°C.

[0050] The present invention also provides an exosome, which is prepared by the method described above.

[0051] According to the present invention, the particle size of the exosomes falls within the range of 50 to 245 nm. Preferably, it falls within the range of 50 to 150 nm.

[0052] According to the present invention, the exosomes have an average particle size ranging from 50 to 100 nm. In some specific examples, the average particle size of the exosomes is 72 nm.

[0053] According to the present invention, the exosomes may be further subjected to a drying process selected from the following to obtain a powder: freeze-drying, spray-drying, vacuum drying, and fluidized bed drying. In some specific examples, the exosomes are freeze-dried.

[0054] Furthermore, the present invention also provides the use of the exosome supply described above for preparing compositions for improving skin conditions.

[0055] According to the present invention, the skin condition includes at least one of the following: wound, aging, hair loss, and inflammation.

[0056] As used in this article, the term "aging" is intended to encompass both naturally occurring intrinsic skin aging and extrinsic skin aging caused by environmental factors, particularly ultraviolet radiation. Symptoms of skin aging include, but are not limited to: telangiectasia, thinning of the epidermis, skin atrophy, reduction of collagen and elastic fibers, elastosis, decreased skin elasticity, rougher skin texture, dryness, wrinkle formation, and pigmentary changes [e.g., lentigines, freckles, hypopigmentation, or hyperpigmentation].

[0057] According to the present invention, the composition may be a cosmeceutical composition.

[0058] According to the present invention, the cosmeceutical composition may further comprise a cosmetically acceptable adjuvant widely used in cosmetic manufacturing techniques. For example, the cosmetically acceptable adjuvant may comprise one or more agents selected from the following: solvents, gelling agents, active agents, antioxidants, screening agents, coloring agents, thickening agents, fillers, fragrances, and odor absorbers. The selection and quantity of these agents fall within the scope of expertise and routine practice of those skilled in the art.

[0059] According to the present invention, the cosmeceutical composition can be manufactured in a form suitable for skincare or makeup using techniques well known to those skilled in the art. This includes, but is not limited to: aqueous solutions, aqueous-alcohol solutions, or oily solutions; oil-in-water type, water-in-oil type, or complex emulsions, gels, ointments, creams, masks, patches, packs, bandages, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, dispersions, suspensions, drops, mousses, salves, sunblocks, and toners. Water, foundation, and eyeshadow, etc.

[0060] According to the present invention, the composition may be a pharmaceutical composition.

[0061] According to the present invention, the pharmaceutical composition may be in a dosage form suitable for parenteral administration or oral administration.

[0062] According to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may comprise one or more agents selected from the following: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The selection and quantity of these agents fall within the scope of expertise and practice of those skilled in the art.

[0063] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for non-enteric administration using techniques well known to those skilled in the art [including injection, for example, a sterile aqueous solution or dispersion], and administered via a route selected from the group consisting of: intraperitoneal injection, intramuscular injection, intraepidermal injection, subcutaneous injection, intradermal injection, and intralesional injection.

[0064] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for oral administration using techniques known to those skilled in the art, including, but not limited to: sterile powders, tablets, troche, lozenges, pellets, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0065] The present invention also provides a method for improving skin conditions, comprising administering, to an individual in need, exosomes as described above.

[0066] As used herein, the terms “administration” and “administration” may be used interchangeably and mean introducing, providing, or delivering a predetermined active ingredient to an individual by any suitable means to perform its intended effect.

[0067] As used in this article, the term "subject" refers to any mammal of interest, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, and rats.

[0068] According to the present invention, the dosage and frequency of exosome administration will vary depending on the following factors: the severity of the condition to be improved, the route of administration, and the age, physical condition, and response of the individual to be improved. The selection of the dosage and frequency of administration falls within the scope of expertise and routine practice of those skilled in the art.

[0069] The present invention will be further described with reference to the following embodiments, but it should be understood that these embodiments are for illustrative purposes only and should not be construed as limiting the implementation of the present invention.

[0070] <Example>

[0071] General experimental materials:

[0072] 1. The 2,3,4′,5-Tetrahydroxystilbene-2-O-β-D-glucoside (THSG) used in the following examples was prepared by separation and purification according to Tsai PWet al. (2018) (as above).

[0073] 2. Sources of stem cells:

[0074] The avian embryonic mesenchymal stem cells (AMSCs) and human dental pulp stem cells (DPSCs) used in the following examples were isolated according to Gao Y. et al. (2013) (as above) and Lin CY et al. (2019), J. Endod., 45: 435-441, respectively.

[0075] 3. Origin and culture of human skin fibroblasts (HSFs):

[0076] The HSFs cells (BCRC 08C0011) used in the following examples were purchased from the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan (No. 331, Food Road, Hsinchu City, Taiwan 300). The HSFs cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (…). Cells (Cat. No. 10-017-CM) were cultured in 75T flasks containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin, and were incubated at 37°C with 5% CO2. The culture medium was replaced with fresh medium approximately every 2-3 days. When the cell density reached approximately 80-90% confluence, the cells were subcultured.

[0077] 4. Human follicle dermal papilla cells (HFDPCs):

[0078] The HFDPCs used in the following examples were purchased from PromoCell GmbH (Heidelberg, Germany) (Cat. No. C-12071). HFDPCs were cultured in 75T flasks containing Follicle Dermal Papilla Cell Growth Medium (FDPCGM) (PromoCell GmbH, Cat. No. C-26501) [with 1% Growth Medium Supplement Mix (PromoCell GmbH, Cat. No. C-39625), 100 U / mL penicillin, and 100 μg / mL streptomycin], and in an incubator set at 37°C and 5% CO2. The medium was then replaced with fresh medium approximately every 2-3 days. When the cell density reached approximately 80-90% confluence, the cells were passaged.

[0079] General experimental methods:

[0080] 1. Statistical analysis:

[0081] In the following examples, experimental data for each group are expressed as "mean ± standard error of the mean (SEM)". All data are presented using... Statistical analysis was performed using Statistics version 19.0 (SPSS Inc., IL, USA) and a two-tailed Student's t-test was used to assess differences between groups. A p-value < 0.05 indicates statistical significance.

[0082] Example 1. Effect of THSG addition on exosome production by avian embryonic mesenchymal stem cells (AMSCs)

[0083] Experimental methods:

[0084] First, the AMSCs cells obtained according to item 2 of "General Experimental Materials" above were divided into one control group and one experimental group. The AMSCs cells in each group were then divided into 5 × 10⁻⁶ cells / day. 6 Cells / wells were cultured in 10-cm dishes containing 5 mL of DMEM medium (with 2.5% FBS) and incubated in an incubator (37°C, 5% CO2) for 48 hours.

[0085] Next, the cell cultures from each group (approximately 1 × 10⁻⁶ cells) were... 8 The culture medium was replaced with DMEM containing THSG to achieve a final concentration of 25 μM THSG in each group. The culture was then continued in an incubator (37°C, 5% CO2) for 36 hours, and the culture supernatant of each group was collected.

[0086] Then, all the culture supernatants collected from each group were subjected to the following pretreatment: centrifugation at 1,000g for 10 minutes to remove precipitates, and this was repeated once, followed by filtration through a 0.22μm filter cup.

[0087] Next, the pretreated culture supernatants of each group were subjected to tangential flow filtration (TFF) to obtain the TFF filtrate for each group. The instrument used for TFF was a mini tangential flow filtration system equipped with a pump (rotation speed of 80-120 mL / min) and 500 kDa modified polyethersulfone (mPES) hollow fiber filter modules.

[0088] Next, the TFF filtrates from each group were filtered using sterile filter cups, and the filtrates from each group were collected and separated into exosomes using size-exclusion chromatography (SEC). The column and operating conditions used for SEC were as follows: the analytical column was an SEC qEV column; the eluent was phosphate-buffered saline (PBS); and the column temperature was room temperature (25°C). The resulting eluate was then freeze-dried to obtain the exosomes of this invention as a dried powder (hereinafter referred to as the exosomes of this invention).

[0089] In addition, the culture supernatant of AMSCs cells that were not treated with THSG was also subjected to the same TFF and SEC treatments and freeze-drying to obtain exosomes (hereinafter referred to as control exosomes).

[0090] Example 2. Compositional analysis of exosomes of the present invention

[0091] The components of the exosomes of this invention and control exosomes were analyzed and compared by measuring the concentrations of exosomal protein and exosomal RNA.

[0092] Experimental methods:

[0093] A. Determination of exosome protein concentration:

[0094] The exosome protein concentrations of the two types of exosomes obtained in Example 1 above were determined by taking 100 mg of each and dissolving them in RIPA lysis buffer, and then following the manufacturer's instructions using Pierce. TM The BCA protein analysis kit was tested using bovine serum albumin (BSA) (0-250 μg / mL) as a standard.

[0095] B. Determination of exosomal RNA concentration:

[0096] The exosomal RNA concentrations of the two types of exosomes obtained in Example 1 above were determined by taking 100 mg of each and using the qEV RNA extraction kit according to the manufacturer's instructions.

[0097] result:

[0098] A. Determination of exosome protein concentration:

[0099] Figure 1 This displays the concentrations of exosome proteins obtained according to the method of the present invention and existing methods. From Figure 1 It is evident that the protein concentration in the exosomes of this invention is significantly higher than that in the control exosomes.

[0100] B. Determination of exosomal RNA concentration:

[0101] Figure 2 This shows the exosomal RNA concentrations obtained according to the method of the present invention and existing methods. Figure 2 It is evident that the RNA concentration in the exosomes of this invention is significantly higher than that in the control exosomes.

[0102] Example 3. Effects of THSG addition on exosome production from different stem cells

[0103] To assess the impact of THSG on the efficacy of different stem cells in exosome production, the applicant also selected human dental pulp stem cells (DPSCs) for the experiment.

[0104] Experimental methods:

[0105] First, the DPSCs cells obtained in item 2 of the "General Experimental Materials" section above were subjected to THSG treatment, exosome isolation, and freeze-drying according to the method described in Example 1 above, thereby obtaining exosomes derived from DPSCs cells (hereinafter referred to as control exosomes).

[0106] Next, 18 mg of each of the control exosome and the exosome of the present invention obtained in Example 1 above were taken and suspended in distilled water. Then, the contents and particle size of the two exosomes were measured by tunable resistive pulse sensing (TRPS) using the qNano platform (Izon Science Ltd.).

[0107] result:

[0108] Table 1 below shows the content and particle size of various exosomes. As can be seen from Table 1, compared to treating DPSCs with THSG, treating AMSCs with THSG produces significantly more exosomes. In particular, the present invention also produces exosomes with a particle size smaller than 76 nm. Furthermore, the average particle size of the exosomes from the present invention is significantly smaller than that of the control exosomes. This experimental result demonstrates that the method of the present invention can yield a greater number of exosomes with smaller particle sizes.

[0109] Table 1. Content and particle size of various exosomes

[0110] Content (capsules / mg) <![CDATA[5.08×10 9 ]]> <![CDATA[4.16×10 7 ]]> Particle size range (nm) 53-245 76-436 Average particle size (nm) 72 102

[0111] Example 4. Evaluation of the anti-wrinkle effect of the exosomes of the present invention

[0112] In this embodiment, the applicant used the exosomes of the present invention to treat HSFs cells and observed the expression of type I collagen α1 (COL1A1), type III collagen α1 (COL3A1), and elastin (ELN) in the cells to evaluate the anti-wrinkle efficacy of the exosomes of the present invention.

[0113] Experimental materials:

[0114] 1. Preparation of exosome suspensions with different concentrations:

[0115] An appropriate amount of the exosomes of the present invention obtained according to Example 1 above was suspended in sterile ultrapure water and diluted with sterile ultrapure water to obtain exosome suspensions with different concentrations (0.07, 0.7 and 70 μg / mL, respectively).

[0116] Experimental methods:

[0117] First, the HSFs cells, which were subcultured according to item 3 of the "General Experimental Materials" section above, were divided into one control group and three experimental groups (i.e., experimental groups 1 to 3). The HSFs cells in each group were then cultured at a rate of 1 × 10⁻⁶ cells / mL. 5 Cells / wells were cultured separately in 6-well plates containing 2 mL of DMEM medium (with 10% FBS added) per well and incubated in an incubator (37°C, 5% CO2) for 24 hours.

[0118] Next, the culture medium for each experimental group was replaced with DMEM medium containing 10 mL of the exosome suspension prepared according to item 1 of "Experimental Materials" above (concentrations of 0.07, 0.7, and 70 μg / mL, respectively) [with 0.25% charcoal stripped fetal bovine serum (CS-FBS) (ThermoFisher Scientific Inc, Cat. No. 12676029)]. The concentrations of the exosome suspension in the DMEM medium for experimental groups 1 to 3 were 0.07, 0.7, and 70 μg / mL, respectively. The culture medium for the control group was replaced with DMEM medium without exosome suspension.

[0119] After culturing in an incubator (37℃, 5% CO2) for 48 hours, the resulting cell cultures were centrifuged at 5,000 rpm for 5 minutes. The resulting cell pellets were then collected and quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the expression levels of COL1A1, COL3A1, and ELN genes in HSFs cells.

[0120] The steps for qRT-PCR are as follows: First, take appropriate amounts of cell pellets from each group, then use GENEzol... TMThe TriRNA purification kit was used to extract total RNAs according to the manufacturer's instructions. The obtained total RNAs were then subjected to reverse transcription using the RevertAid H Minus First-Strand cDNA Synthesis Kit, following the manufacturer's instructions, to synthesize first-strand cDNA.

[0121] Next, using the obtained first-strand cDNA as a template, and with primer pairs for the COL1A1, COL3A1, and ELN genes as shown in Table 2 below, CFX Connect was used. TM A real-time PCR detection system was used, and qRT-PCR was performed according to the manufacturer's operating instructions. Additionally, 18S gene expression was used as an internal control. The operating and reaction conditions for qRT-PCR are shown in Table 3 below.

[0122] Table 2. Primers used for qRT-PCR

[0123]

[0124] Table 3. Reaction conditions for qRT-PCR

[0125]

[0126] The PCR products obtained were detected using the fluorescence of SYBR Green (a double-stranded DNA binding dye), and the cycle threshold (C) of each PCR product was determined. t The relative mRNA expression level was calculated from the cycle threshold of each PCR product and compared using a comparative C-value. t Method (comparative C) t The method was standardized using the cycle threshold of the PCR product obtained from the 18S gene. Then, the expression levels of COL1A1, COL3A1, and ELN genes in each experimental group were calculated as folds relative to the control group.

[0127] Then, the obtained experimental data were analyzed according to the method described in item 1, "Statistical Analysis," of the "General Experimental Methods" section above.

[0128] result:

[0129] Figures 3 to 5 The expression levels of COL1A1, COL3A1, and ELN genes, as measured in HSFs cells of each group, are displayed. Figures 3 to 5 As can be seen, compared with the control group, the expression levels of COL1A1, COL3A1, and ELN genes in each experimental group were significantly increased, and this increase became more pronounced with increasing exosome concentration. This experimental result shows that the exosomes of this invention promote the secretion of collagen and elastin by dermal fibroblasts in a dose-dependent manner. Based on this, the applicant believes that the exosomes obtained according to the method of this invention can achieve an anti-wrinkle effect by promoting the secretion of collagen and elastin by dermal fibroblasts.

[0130] Example 5. Evaluation of the efficacy of the exosomes of the present invention in promoting skin regeneration.

[0131] In this embodiment, the applicant evaluates the efficacy of the exosomes of the present invention in promoting skin regeneration by assessing the proliferation of dermal fibroblasts.

[0132] Experimental methods:

[0133] First, the HSFs cells, which were subcultured according to item 3 of the "General Experimental Materials" section above, were divided into one control group and one experimental group. The HSFs cells in each group were then cultured at a rate of 5 × 10⁻⁶ cells / year. 3 Cells / wells were cultured separately in 96-well plates containing 100 μL of DMEM medium (with 10% FBS added) per well and incubated in an incubator (37°C, 5% CO2) for 24 hours.

[0134] Next, the culture medium for the experimental group was replaced with DMEM medium (with 0.25% CS-FBS) containing 100 μL of the exosome suspension (70 μg / mL) prepared according to item 1 of “Experimental Materials” in Example 4 above. The culture medium for the control group was replaced with DMEM medium without exosome suspension.

[0135] Each group was incubated in an incubator (37℃, 5% CO2) for 72 hours, after which the solution was replaced with CellTiter solution containing 20% ​​MTS. The cells were incubated in the growth media of the AQueous One Solution Cell Proliferation Assay Kit for 2 hours. Afterwards, the absorbance (OD) of each well was read at 490 nm using a VersaMax ELISA microdisc reader. 490 ).

[0136] The percentage of cells that are still viable (%) is determined by measuring the absorbance (OD) value. 490 Substituting into the following formula (1) yields the result:

[0137] Formula (1): A = (B / C) × 100

[0138] Where: A = percentage of cell viability (%)

[0139] B = OD measured in each group 490 absorbance

[0140] C = OD measured in the control group 490 absorbance

[0141] Then, the obtained experimental data were analyzed according to the method described in item 1, "Statistical Analysis," of the "General Experimental Methods" section above.

[0142] result:

[0143] Figure 6 This displays the percentage of HSFs cells that are viable after treatment with the exosomes of this invention. Figure 6 As can be seen, compared with the control group, the percentage of cell viability in the experimental group was significantly increased. This experimental result shows that the exosomes of this invention can promote the proliferation of dermal fibroblasts. Based on this, the applicant believes that the exosomes obtained according to the method of this invention can achieve the effect of promoting skin regeneration by promoting the proliferation of dermal fibroblasts.

[0144] Example 6. Evaluation of the efficacy of the exosomes of the present invention in promoting wound healing.

[0145] In this embodiment, the applicant used a wound-healing cell migration assay to evaluate the efficacy of the exosomes of the present invention in promoting wound healing.

[0146] Experimental methods:

[0147] First, the HSFs cells, which were subcultured according to item 3 of the "General Experimental Materials" section above, were divided into one control group and one experimental group. The HSFs cells in each group were then cultured at a rate of 2.5 × 10⁻⁶ cells / year. 5 Cells / wells were cultured separately in 24-well plates containing 500 μL of DMEM medium (with 10% FBS added) per well and incubated in an incubator (37°C, 5% CO2) for 24 hours.

[0148] Next, the culture medium for each group was replaced with fresh DMEM medium without serum, and the cells were cultured in an incubator (37°C, 5% CO2) for 24 hours. Then, HSFs cells were scraped off along the diameter of each well using sterile forceps to create a cell-free wound area approximately 500 μm wide along the diameter of the culture plate. Next, the culture medium for the experimental groups was replaced with DMEM medium (with 0.25% CS-FBS) containing 500 μL of the exosome suspension (70 μg / mL) prepared according to item 1 of “Experimental Materials” in Example 4 above. The culture medium for the control group was replaced with DMEM medium without the exosome suspension.

[0149] Afterwards, each group was incubated in an incubator (37℃, 5% CO2) for 24 hours. Before incubation and at 24 hours after the start of incubation, the wound areas of each group were observed using an inverted microscope (model CKX53, brand OLYMPUS) at 40x magnification, and photographs were taken using a digital camera (model EP50, brand OLYMPUS).

[0150] result:

[0151] Figure 7 This shows the results observed in HSFs cells after treatment with the exosomes of the present invention via cell migration analysis during wound healing. Figure 7 As can be seen, compared with the control group, wound closure was clearly observed in the experimental group. This experimental result shows that the exosomes of the present invention can effectively promote fibroblast-mediated wound closure. Based on this, the applicant believes that the exosomes obtained according to the method of the present invention can achieve the effect of promoting wound healing by promoting fibroblast-mediated wound closure.

[0152] Example 7. Evaluation of the efficacy of the exosomes of the present invention in improving hair loss.

[0153] In this embodiment, the applicant evaluates the efficacy of the exosomes of the present invention in improving hair loss by measuring the proliferation of dermal papillary cells in hair follicles.

[0154] Experimental methods:

[0155] First, the HFDPCs cells, which were subcultured according to item 4 of the "General Experimental Materials" section above, were divided into one control group and one experimental group. The HFDPCs cells in each group were then cultured at a rate of 5 × 10⁻⁶ cells / year. 3Cells / wells were cultured separately in 96-well plates containing 100 μL of FDPCGM medium (with 1% growth medium supplement mixture) per well and incubated in an incubator (37°C, 5% CO2) for 24 hours.

[0156] Next, the culture medium for the experimental group was replaced with FDPCGM medium (without 1% growth medium supplement mixture) containing 100 μL of the exosome suspension (70 μg / mL) prepared according to item 1 of “Experimental Materials” in Example 4 above. The culture medium for the control group was replaced with FDPCGM medium without exosome suspension.

[0157] Each group was incubated in an incubator (37℃, 5% CO2) for 96 hours, then the medium was replaced with growth medium containing 20% ​​MTS solution and incubated for another 2 hours. Afterwards, the absorbance (OD) of each well was read at a wavelength of 490 nm using a VersaMax ELISA microdisc reader. 490 Next, the measured absorbance value (OD) 490 Substitute into the above formula (1) to calculate the percentage of cells that are still viable (%).

[0158] Then, the obtained experimental data were analyzed according to the method described in item 1, "Statistical Analysis," of the "General Experimental Methods" section above.

[0159] result:

[0160] Figure 8 This displays the percentage of HFDPCs cells that are viable after treatment with the exosomes of this invention. Figure 8 As can be seen, compared with the control group, the percentage of cell viability in the experimental group was significantly increased. This experimental result shows that the exosomes of this invention can promote the proliferation of dermal papillary cells in hair follicles. Based on this, the applicant believes that the exosomes obtained according to the method of this invention can improve hair loss by promoting hair follicle regeneration.

[0161] Example 8. Evaluation of the anti-inflammatory effect of the exosomes of the present invention

[0162] In this embodiment, the applicant used the exosomes of the present invention to treat HSFs cells with lipopolysaccharides (LPS)-induced skin inflammation and observed the expression of proinflammatory cytokines [such as interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α)] in the cells to evaluate the anti-inflammatory efficacy of the exosomes of the present invention.

[0163] Experimental methods:

[0164] First, the HSFs cells, which were subcultured according to item 3 of the "General Experimental Materials" section above, were divided into one normal control group, one pathological control group, and one experimental group. The HSFs cells in each group were then cultured at a rate of 1 × 10⁻⁶ cells / mL. 5 Cells / wells were cultured separately in 6-well plates containing 2 mL of DMEM medium (with 10% FBS added) per well and incubated in an incubator (37°C, 5% CO2) for 24 hours.

[0165] Next, the culture medium for the experimental group was replaced with DMEM medium (with 0.25% CS-FBS) containing 1 μg / mL LPS [Escherichia coli serotype 0111:B4, Sigma] and 1 mL of exosome suspension (concentration 0.07 μg / mL) prepared according to item 1 of "Experimental Materials" in Example 4 above. The culture medium for the pathological control group was replaced with DMEM medium containing 1 μg / mL LPS but without the exosome suspension, and the culture medium for the normal control group was replaced with DMEM medium without LPS and exosome suspension.

[0166] After culturing in an incubator (37℃, 5% CO2) for 6 hours, the cell pellets of each group were collected and quantitative real-time polymerase chain reaction (qRT-PCR) was performed in accordance with the method described in Example 4 above. The expression levels of IL-6, IL-1β, and TNF-α genes in the pathological control group and the experimental group were calculated as folds relative to the normal control group. The difference was that the cell pellets of each group were used for qRT-PCR targeting IL-6, IL-1β, and TNF-α, and the primer pairs used are shown in Table 4 below.

[0167] Table 4. Primers used for qRT-PCR

[0168]

[0169] Then, the obtained experimental data were analyzed according to the method described in item 1, "Statistical Analysis," of the "General Experimental Methods" section above.

[0170] result:

[0171] Figures 9 to 11 The expression levels of IL-6, IL-1β, and TNF-α genes, as measured in HSFs cells from each group, are displayed. Figures 9 to 11 As can be seen, compared with the normal control group, the expression levels of IL-6, IL-1β, and TNF-α genes in the pathological control group were significantly increased, indicating that LPS successfully induced inflammation in HSFs cells. Conversely, compared with the pathological control group, the expression levels of IL-6, IL-1β, and TNF-α genes in the experimental group were significantly decreased. These experimental results demonstrate that the exosomes of this invention can effectively inhibit the expression of pro-inflammatory cytokines. Based on this, the applicant believes that the exosomes obtained according to the method of this invention can achieve anti-inflammatory effects by inhibiting the expression of pro-inflammatory cytokines.

[0172] Based on the above experimental results, the method of this invention can effectively promote the production of large quantities of exosomes containing a variety of components (including proteins and RNA) from avian embryonic mesenchymal stem cells (AMSCs), and these exosomes exhibit excellent efficacy in promoting skin regeneration and wound healing, reducing wrinkles, improving hair loss, and combating skin inflammation. Therefore, the applicant believes that the exosomes obtained according to the method of this invention have high potential to be developed into products for improving skin conditions (including wounds, aging, hair loss, and inflammation).

[0173] All patents and documents cited in this specification are incorporated herein by reference in their entirety. In the event of any conflict, the detailed description of this application (including limitations) shall prevail.

[0174] Although the invention has been described with reference to the specific examples described above, it is apparent that many modifications and variations can be made without departing from the scope and spirit of the invention. Therefore, it is intended that the invention be limited only to those shown in the appended claims. sequence list <110> Shengyi Biotechnology Co., Ltd. <120> Methods for producing exosomes, the resulting exosomes, and their applications <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 16 <212> DNA <213> Artificial sequences <220> <223> Forward primer COL1A1-F for qRT-PCR of the COL1A1 gene <400> 1 gtcagatggg cccccg 16 <210> 2 <211> 20 <212> DNA <213> Artificial sequences <220> <223> The reverse primer for the COL1A1 gene used in qRT-PCR: COL1A1-R <400> 2 caccatcatt tccacgagca 20 <210> 3 <211> twenty one <212> DNA <213> Artificial sequences <220> <223> Forward primer for the COL3A1 gene used in qRT-PCR: COL3A1-F <400> 3 gaggatggtt gcacgaaaca c 21 <210> 4 <211> twenty one <212> DNA <213> Artificial sequences <220> <223> The reverse primer for the COL3A1 gene used in qRT-PCR: COL3A1-R <400> 4 cagccttgcg tgttcgatat t 21 <210> 5 <211> 18 <212> DNA <213> Artificial sequences <220> <223> Forward primers for the ELN gene used in qRT-PCR: ERN-F <400> 5 caggtgcggt ggttcctc 18 <210> 6 <211> 20 <212> DNA <213> Artificial sequences <220> <223> The reverse primer ELN-R for the ELN gene used in qRT-PCR <400> 6 ctgggtatac acctggcagc 20 <210> 7 <211> 20 <212> DNA <213> Artificial sequences <220> <223> Forward primer 18S-F for qRT-PCR of the 18S gene <400> 7 gtaacccgtt gaaccccatt 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequences <220> <223> 18S-R reverse primer for the 18S gene used in qRT-PCR <400> 8 ccatccaatc ggtagtagcg 20 <210> 9 <211> 20 <212> DNA <213> Artificial sequences <220> <223> Forward primer IL-6-F for qRT-PCR of the IL-6 gene <400> 9 acccccagga gaagattcca 20 <210> 10 <211> 20 <212> DNA <213> Artificial sequences <220> <223> The reverse primer for the IL-6 gene used in qRT-PCR: IL-6-R <400> 10 gatgccgtcg aggatgtacc 20 <210> 11 <211> 20 <212> DNA <213> Artificial sequences <220> <223> Forward primer IL-1β-F for qRT-PCR of the IL-1β gene <400> 11 gcagccatgg cagaagtacc 20 <210> 12 <211> twenty four <212> DNA <213> Artificial sequences <220> <223> The reverse primer for the IL-1β gene used in qRT-PCR: IL-1β-R <400> 12 agtcatcctc attgccactg taat 24 <210> 13 <211> twenty two <212> DNA <213> Artificial sequences <220> <223> Forward primer TNF-α-F for qRT-PCR of the TNF-α gene <400> 13 tagcccatgt tgtagcaaac cc 22 <210> 14 <211> twenty one <212> DNA <213> Artificial sequences <220> <223> The reverse primer TNF-α-R for the TNF-α gene used in qRT-PCR <400> 14 ttatctctca gctccacgcc a 21

Claims

1. A method for producing exosomes, characterized in that: The method includes: Avian embryonic mesenchymal stem cells (AMSCs) were cultured in a medium containing 0.1 to 50 µM of 2,3,4′,5-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) to obtain AMSC cell cultures; and Exosomes were collected from the cell culture.

2. The method according to claim 1, characterized in that: The culture lasted 24 to 96 hours.

3. The method according to claim 1, characterized in that: The exosomes were collected through size-selective or density-selective separation processes.

4. The method according to claim 3, characterized in that: The separation process is a combination of tangential flow filtration and size-exclusion chromatography.

5. An exosome, characterized in that: The exosomes are prepared by the method described in any one of claims 1 to 4.

6. The exosomes according to claim 5, characterized in that: The exosomes have a particle size ranging from 50 to 200 nm.

7. The exosome according to claim 5, characterized in that: The exosomes have an average particle size ranging from 50 to 100 nm.

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