Preparation method and application of sheep-derived amniotic fluid exosomes

Abstract

The application relates to the technical field of biomaterials, in particular to a preparation method and application of sheep-derived amniotic fluid exosomes. The preparation method specifically comprises the following steps: amniotic fluid extraction, centrifugal separation, batch ion exchange chromatography and batch hydrophobic chromatography. The batch ion exchange chromatography is used for enriching exosomes and negatively charged proteins and nucleic acids in amniotic fluid by utilizing the negative charge property of the cell membrane structure. The batch hydrophobic chromatography is used for separating hydrophilic substances by utilizing the hydrophobic property of the cell membrane structure. The application aims to collect trace exosomes from a large amount of amniotic fluid, efficiently enrich the exosomes by adopting a batch adsorption method. The method has a mild property, has little damage to the vesicles, does not need to be subjected to ultracentrifugation, and can process several hundred liters of exosome stock solution at a time. The exosomes contain rich growth factors, and the content of placental growth factor is the most.

Description

Technical Field

[0001] This application relates to the technical field of biomaterials, and in particular to a method for preparing and applying sheep-derived amniotic fluid exosomes. Background Technology

[0002] Exosomes, nanoscale membrane vesicles secreted by cells, typically range in diameter from 30 to 150 nm. They contain a rich variety of bioactive molecules, including proteins, nucleic acids, and lipids. These molecules play crucial roles in intercellular communication and effectively regulate cellular physiological functions. Amniotic fluid, the specific environment in which the fetus grows and develops in the uterus, contains a large number of cells and various bioactive molecules. Recent scientific research has also discovered the presence of exosomes in amniotic fluid. These exosomes in amniotic fluid are highly likely to play a vital role in fetal growth and development as well as the mother's health.

[0003] However, currently, the methods for preparing amniotic fluid exosomes and their practical applications remain relatively limited. This urgently requires further in-depth research and vigorous development. Amniotic fluid is widely available, but the content of exosomes in it is negligible, while the volume of amniotic fluid is enormous. Under normal circumstances, exosomes are collected using ultracentrifugation, but this method is extremely time-consuming. Furthermore, during concentration using ultrafiltration, the prolonged shear force can cause vesicle rupture, leading to poor product homogeneity.

[0004] Therefore, it is necessary to find a gentle method to collect exosomes from amniotic fluid. Summary of the Invention

[0005] The purpose of this application is to enable the efficient collection of trace amounts of exosomes from large volumes of amniotic fluid using a batch adsorption method. This method is gentle, causes minimal damage to vesicles, eliminates the need for ultracentrifugation, and allows for high-volume processing, handling hundreds of liters of exosome stock solution at a time. This application provides a method for preparing and applying sheep-derived amniotic fluid exosomes. These exosomes contain abundant growth factors, with placental growth factors being the most abundant.

[0006] In a first aspect, this application provides a method for preparing sheep-derived amniotic fluid exosomes, employing the following technical solution:

[0007] A method for preparing amniotic fluid exosomes derived from sheep, the method specifically includes the following steps: amniotic fluid extraction, centrifugation, batch ion exchange chromatography, and batch hydrophobic chromatography;

[0008] The batch ion exchange chromatography method utilizes the negative charge property of cell membrane structure to enrich exosomes and negatively charged proteins and nucleic acids in amniotic fluid.

[0009] The batch hydrophobic chromatography method utilizes the hydrophobic properties of cell membrane structures to separate hydrophilic substances.

[0010] Optionally, the batch ion exchange chromatography includes the following steps: diluting the supernatant after centrifugation with 20mM phosphate buffer at pH 7.4 to reduce the conductivity to below 4mS / cm; equilibrating DE52 with 20mM phosphate buffer at pH 7.4 and filtering to remove the equilibration buffer; adding the supernatant after centrifugation at a ratio of 1ml DE52 cellulose to 100ml of the supernatant after centrifugation, and placing it on a rotary mixer to slowly mix at 4°C for 4-5 hours;

[0011] After conjugation, the DE52 packing material was introduced into a chromatography column, and the supernatant after centrifugation was filtered by gravity; washing was then performed using washing buffer. The eluent was then mixed with the packing material and slowly incubated on a rotary mixer for 5 minutes, followed by three elutions. The eluents obtained from the three elutions were combined to obtain amniotic fluid exosomes.

[0012] Optionally, the washing solution is 20 mM phosphate and 10 mM sodium chloride at pH 7.4.

[0013] Optionally, the eluent is 20 mM phosphate and 50 mM sodium chloride at pH 7.4.

[0014] Optionally, the batch hydrophobic chromatography includes the following steps: adding 2M ammonium sulfate to the supernatant after centrifugation; equilibrating the phenyl hydrophobic packing material with a 20mM phosphate and 2M ammonium sulfate equilibration buffer solution at pH 7.4, and filtering to remove the equilibration buffer solution; adding the supernatant after centrifugation at a ratio of 1ml phenyl hydrophobic packing material to 100ml supernatant after centrifugation, and placing it on a rotary mixer for slow mixing at 4°C for 4-5 hours;

[0015] After binding, the phenyl hydrophobic packing material was introduced into a chromatography column, and the supernatant after centrifugation was filtered by gravity. Washing was performed using washing buffer. Eluent 1 and eluent 2 were mixed with the packing material sequentially, and then slowly mixed and incubated on a rotary mixer for 5 minutes, eluting three times each. The eluents obtained from the three elutions with eluent 2 were combined to obtain amniotic fluid exosomes.

[0016] Optionally, the washing solution is 20 mM phosphate and 1 M sodium chloride at pH 7.4.

[0017] Optionally, the eluent 1 is 20 mM phosphate and 500 mM sodium chloride at pH 7.4.

[0018] Optionally, the eluent 2 is 20 mM phosphate and 150 mM sodium chloride at pH 7.4.

[0019] Optionally, the source of the sheep is sheep.

[0020] Secondly, this application provides the use of the sheep-derived amniotic fluid exosomes prepared by the above-described method in the preparation of compositions for tissue repair, regeneration, and / or disease treatment.

[0021] Optionally, the composition is a cosmetic.

[0022] In summary, this application includes at least one of the following beneficial technical effects:

[0023] 1. This application provides, for the first time, a method for preparing sheep-derived amniotic fluid exosomes. This method is simple, efficient, and low-cost, does not harm ewes or lambs, and allows for large-scale preparation of sheep-derived amniotic fluid exosomes.

[0024] 2. This application found that sheep-derived amniotic fluid exosomes contain abundant growth factors, with placental growth factor having the highest content, providing a new direction and basis for the application of sheep amniotic fluid exosomes.

[0025] 3. The preparation method of this application can prepare sheep-derived amniotic fluid exosomes on a large scale, providing sufficient raw material guarantee for their application.

[0026] 4. Sheep-derived amniotic fluid exosomes have broad application prospects and can provide new tools and raw materials for tissue repair and regeneration, disease treatment, cosmetics and other fields. Attached Figure Description

[0027] Figure 1 The TEM results are for the amniotic fluid exosomes obtained in Example 4 of this application. Detailed Implementation

[0028] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0030] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0032] This application provides a method for preparing sheep-derived amniotic fluid exosomes. The preparation method specifically includes the following steps:

[0033] (1) Collection of amniotic fluid from sheep

[0034] Healthy sheep in late pregnancy were selected, and amniotic fluid was collected during farrowing. The collection process strictly followed clean operating procedures to ensure the quality and safety of the amniotic fluid.

[0035] The collected amniotic fluid was immediately placed in a sterile centrifuge tube and stored at 4°C for further processing as soon as possible.

[0036] (2) Centrifugal separation

[0037] The collected amniotic fluid was centrifuged at 3000 rpm for 15 minutes at 4°C to remove cells and debris.

[0038] Take the supernatant and centrifuge it at 10,000 rpm for 30 min at 4°C to further remove small particles and impurities.

[0039] Further filtration and sterilization were performed using a 0.22µm polyethersulfone filter membrane to obtain the supernatant after centrifugation.

[0040] (3) Batch processing ion exchange chromatography

[0041] By utilizing the negative charge of cell membrane structures, exosomes and negatively charged proteins and nucleic acids in amniotic fluid can be enriched, thereby removing other impurities in the amniotic fluid and greatly reducing its volume.

[0042] The specific operating procedure is as follows: Dilute the supernatant after centrifugation with 20mM phosphate buffer (pH 7.4) to reduce the conductivity to below 4 mS / cm. Equilibrate DEAE cellulose (DE52) with 20mM phosphate buffer (pH 7.4), and filter to remove the equilibration buffer. Add the supernatant after centrifugation at a ratio of 1 ml DE52 cellulose to 100 ml of supernatant, and place the mixture on a rotary mixer. Mix slowly at 4°C for 4-5 hours. Avoid generating excessive foam during mixing.

[0043] After binding, the DE52 packing material was introduced into the chromatography column, and the supernatant after centrifugation was filtered by gravity. The column was washed with washing buffer (20 mM phosphate, 10 mM sodium chloride, pH 7.4). The eluent (20 mM phosphate, 50 mM sodium chloride, pH 7.4) was then mixed with the packing material and slowly incubated on a rotary mixer for 5 min, followed by three elutions. The three eluents were then combined to obtain the eluent for ion exchange chromatography.

[0044] (4) Batch hydrophobic chromatography

[0045] By utilizing the hydrophobic properties of cell membrane structure, hydrophobic chromatography can be used to separate some hydrophilic substances in one step.

[0046] The specific operating procedure is as follows: Add 2M ammonium sulfate to the eluent of ion exchange chromatography. Equilibrate the phenyl hydrophobic packing material with equilibration buffer (20mM phosphate and 2M ammonium sulfate at pH 7.4), and filter to remove the equilibration buffer. Add the ion exchange chromatography eluent at a ratio of 1 ml phenyl hydrophobic packing material to 100 ml ion exchange chromatography eluent, and place the mixture on a rotary mixer. Mix slowly at 4°C for 4-5 hours. Avoid generating excessive foam during mixing.

[0047] After conjugation, the phenyl hydrophobic packing material was introduced into a chromatography column, and the eluent from the ion exchange chromatography was filtered by gravity. Washing was performed using washing buffer (20 mM phosphate, 1 M sodium chloride, pH 7.4). The packing material was then mixed sequentially with eluent 1 (20 mM phosphate, 500 mM sodium chloride, pH 7.4) and eluent 2 (20 mM phosphate, 150 mM sodium chloride, pH 7.4), and incubated slowly on a rotary mixer for 5 min, eluting three times for each eluent. The three eluents obtained from eluent 2 were combined to obtain amniotic fluid exosomes.

[0048] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0049] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0050] The present application will be further described in detail below with reference to the embodiments and test results.

[0051] Example 1

[0052] This embodiment provides a method for preparing amniotic fluid exosomes. The subject is a healthy sheep in late pregnancy.

[0053] The above preparation method specifically includes the following steps:

[0054] (1) Collection of amniotic fluid from sheep

[0055] Healthy sheep in late pregnancy were selected, and amniotic fluid was collected during farrowing. The collection process strictly followed clean operating procedures to ensure the quality and safety of the amniotic fluid.

[0056] The collected amniotic fluid was immediately placed in a sterile centrifuge tube and stored at 4°C for further processing as soon as possible.

[0057] (2) Centrifugal separation

[0058] The collected amniotic fluid was centrifuged at 3000 rpm for 15 minutes at 4°C to remove cells and debris.

[0059] Take the supernatant and centrifuge it at 10,000 rpm for 30 min at 4°C to further remove small particles and impurities.

[0060] Further filtration and sterilization were performed using a 0.22µm polyethersulfone filter membrane to obtain the supernatant after centrifugation.

[0061] (3) Ultrafiltration Concentration

[0062] The supernatant after centrifugation was concentrated by ultrafiltration using an external pressure ultrafiltration membrane (low shear force). The ultrafiltration membrane had a molecular weight cutoff of 100 kDa. The ultrafiltration process was carried out at 4°C, a pressure of 0.1 MPa, and a flow rate of 0.5 L / min. The volume of the concentrated liquid was reduced to 1 / 20 of its original volume.

[0063] (4) Exosome extraction

[0064] Amniotic fluid exosomes were extracted from sheep using ultracentrifugation. The concentrated ultrafiltration liquid was ultracentrifuged at 100,000 rpm for 2 hours at 4°C, and the resulting precipitate was the amniotic fluid exosomes. The precipitate was washed three times with PBS buffer to remove residual impurities.

[0065] Example 2

[0066] This embodiment provides a method for preparing amniotic fluid exosomes. The subject is a healthy sheep in late pregnancy.

[0067] The above preparation method specifically includes the following steps:

[0068] (1) Collection of amniotic fluid from sheep: The procedure was carried out according to the corresponding steps in Example 1.

[0069] (2) Centrifugation: Performed according to the corresponding steps in Example 1.

[0070] (3) Batch processing ion exchange chromatography

[0071] By utilizing the negative charge of cell membrane structures, exosomes and negatively charged proteins and nucleic acids in amniotic fluid can be enriched, thereby removing other impurities in the amniotic fluid and greatly reducing its volume.

[0072] The specific operating procedure is as follows: Dilute the supernatant after centrifugation with 20mM phosphate buffer (pH 7.4) to reduce the conductivity to below 4 mS / cm. Equilibrate DEAE cellulose (DE52) with 20mM phosphate buffer (pH 7.4), and filter to remove the equilibration buffer. Add the supernatant after centrifugation at a ratio of 1 ml DE52 cellulose to 100 ml of supernatant, and place the mixture on a rotary mixer. Mix slowly at 4°C for 4-5 hours. Avoid generating excessive foam during mixing.

[0073] After conjugation, the DE52 packing material was introduced into a chromatography column, and the supernatant after centrifugation was filtered by gravity. The column was washed with washing buffer (20 mM phosphate, 10 mM sodium chloride, pH 7.4). The eluent (20 mM phosphate, 50 mM sodium chloride, pH 7.4) was then mixed with the packing material and slowly incubated on a rotary mixer for 5 minutes, eluting three times. The three eluents were combined to obtain amniotic fluid exosomes.

[0074] Example 3

[0075] This embodiment provides a method for preparing amniotic fluid exosomes. The subject is a healthy sheep in late pregnancy.

[0076] The above preparation method specifically includes the following steps:

[0077] (1) Collection of amniotic fluid from sheep: The procedure was carried out according to the corresponding steps in Example 1.

[0078] (2) Centrifugation: Performed according to the corresponding steps in Example 1.

[0079] (3) Batch hydrophobic chromatography

[0080] By utilizing the hydrophobic properties of cell membrane structure, hydrophobic chromatography can be used to separate some hydrophilic substances in one step.

[0081] The specific operating procedure is as follows: Add 2M ammonium sulfate to the supernatant after centrifugation. Equilibrate the phenyl hydrophobic filler with equilibration buffer (20mM phosphate, 2M ammonium sulfate, pH 7.4), and filter to remove the equilibration buffer. Add the supernatant after centrifugation at a ratio of 1ml phenyl hydrophobic filler to 100ml supernatant, and place the mixture on a rotary mixer. Mix slowly at 4℃ for 4-5 hours. Avoid generating excessive foam during mixing.

[0082] After conjugation, the phenyl hydrophobic packing material was introduced into a chromatography column, and the supernatant after centrifugation was filtered by gravity. Washing was performed using washing buffer (20 mM phosphate, 1 M sodium chloride, pH 7.4). The packing material was then mixed sequentially with eluent 1 (20 mM phosphate, 500 mM sodium chloride, pH 7.4) and eluent 2 (20 mM phosphate, 150 mM sodium chloride, pH 7.4), and incubated slowly on a rotary mixer for 5 min, eluting three times each. The eluents obtained from the three elutions with eluent 2 were combined to obtain amniotic fluid exosomes.

[0083] Example 4

[0084] This embodiment provides a method for preparing amniotic fluid exosomes. The subject is a healthy sheep in late pregnancy.

[0085] The above preparation method specifically includes the following steps:

[0086] (1) Collection of amniotic fluid from sheep: The procedure was carried out according to the corresponding steps in Example 1.

[0087] (2) Centrifugation: Performed according to the corresponding steps in Example 1.

[0088] (3) Batch ion exchange chromatography: Performed according to the corresponding steps in Example 2.

[0089] (4) Batch hydrophobic chromatography: The amniotic fluid exosomes obtained by batch ion exchange chromatography were subjected to batch hydrophobic chromatography according to the corresponding steps in Example 3 to obtain an amniotic fluid exosome solution.

[0090] Example 5

[0091] This embodiment provides a method for preparing amniotic fluid exosomes. The subject is a healthy sheep in late pregnancy.

[0092] The above preparation method specifically includes the following steps:

[0093] (1) Collection of amniotic fluid from sheep: The procedure was carried out according to the corresponding steps in Example 1.

[0094] (2) Centrifugation: Performed according to the corresponding steps in Example 1.

[0095] (3) Flow-through ion exchange chromatography

[0096] The supernatant after centrifugation was diluted with 20 mM phosphate buffer (pH 7.4) to reduce its conductivity to below 4 mS / cm. DEAE cellulose (DE52) was loaded into a 1 ml chromatography column and connected to an AKTA medium-pressure chromatography system. The column was equilibrated with 20 mM phosphate buffer (pH 7.4) at a flow rate of 1 ml / min. 100 ml of the centrifuged supernatant was then flowed through the column, and loading was stopped when the column pressure reached 0.3 MPa.

[0097] After binding, the mixture is washed with a washing buffer (20 mM phosphate and 10 mM sodium chloride at pH 7.4). Then, it is eluted with an elution buffer (20 mM phosphate and 50 mM sodium chloride at pH 7.4). The resulting solution is the amniotic fluid exosome solution.

[0098] Performance test results

[0099] The performance of the amniotic fluid exosomes prepared in Examples 1-5 above was tested.

[0100] (I) Processing Capacity Analysis

[0101] The results of the processing capability analysis for Examples 1-5 are shown in Table 1.

[0102] Table 1 Processing capacity of Examples 1-5

[0103] 1 50 10 Ultracentrifuges have small capacities and long centrifugation times. 2 200-300 0.5 - 3 200-300 0.5 - 4 200-300 0.5 - 5 50 0.5 Column clogging

[0104] As shown in Table 1, Example 1 uses traditional centrifugation to extract exosomes. Since ultracentrifuges are typically angle rotor benchtop centrifuges with a rotor capacity of 1-5 ml, the maximum batch size is 10 ml; and the processing time for each batch is at least 2 hours. Therefore, processing 50 ml would require 10 hours, making industrial-scale production difficult.

[0105] In addition, Example 5 uses the flow-through adsorption method. Since enrichment is easily caused at the column head of the chromatography column, the chromatography column will become blocked. Therefore, when 1 ml of packing material is used to process 50 ml of amniotic fluid, the column force increases significantly and cannot continue to process. Therefore, compared with the batch processing method, the flow-through adsorption method has certain limitations in industrial scale-up.

[0106] Based on the above, Examples 2-4 use batch adsorption method for extraction and purification, which has a large processing capacity, short processing time, and is easy to scale up industrially.

[0107] (II) Particle Size Analysis

[0108] The particle size of the amniotic fluid exosomes obtained in each example was analyzed using dynamic light scattering (DLS). The analysis results are shown in Table 2.

[0109] Table 2. Results of particle size analysis and total protein analysis

[0110]

[0111] As shown in Table 2, the particle size of sheep amniotic fluid exosomes is mainly distributed between 30-150 nm, with an average particle size of 80 nm.

[0112] (III) Total Protein Analysis of Exosomal

[0113] Exosomes were extracted using ultracentrifugation, and the total protein content of the amniotic fluid exosomes obtained in each example was analyzed.

[0114] Take 1 ml of amniotic fluid exosomes obtained in each example and centrifuge at 100,000 rpm for 2 hours at 4°C. Collect the supernatant and precipitate after centrifugation. Resuspend the precipitate in 100 μL of cell lysis buffer. Determine the protein content released by the exosomes and the protein content in the supernatant using the BCA method. The purity is calculated as follows: protein content in exosomes / (protein content in exosomes + protein content in supernatant).

[0115] The test results are shown in Table 2.

[0116] Table 2 shows that sheep amniotic fluid exosomes are rich in protein, with an average of 500 μg of protein per milliliter. In Example 1, ultracentrifugation effectively removed free protein, resulting in high purity. Examples 2, 3, and 5 contained more impurities, mainly due to the strong adsorption capacity of the chromatography columns for some free protein impurities, leading to insufficient removal in a single step. Example 4, through a two-step purification process, achieved a protein purity in the amniotic fluid exosomes close to that of Example 1.

[0117] (iv) Nucleic acid content determination

[0118] The nucleic acid content in the amniotic fluid exosomes obtained in each example was determined by real-time quantitative PCR. The results are shown in Table 3.

[0119] Table 3. Analysis of nucleic acid and cytokine content

[0120] 1 12 8 140 20 2 15 10 190 35 3 13 9 184 38 4 14 9.5 180 35 5 11 9.5 195 40

[0121] As shown in Table 3, the sheep amniotic fluid exosomes obtained in each embodiment contain a variety of nucleic acid molecules, such as miRNA and mRNA.

[0122] (V) Cytokine Content Analysis

[0123] The levels of placental growth factor and insulin-like growth factor in the amniotic fluid exosomes obtained in each example were determined using enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 3.

[0124] As shown in Table 3, the sheep amniotic fluid exosomes obtained in each example contain abundant growth factors. The cytokine content in the sheep amniotic fluid exosomes obtained in Examples 2-5 is basically the same, but significantly higher than that in Example 1. This may be because a small amount of free cytokines were also extracted from the amniotic fluid during the purification of the amniotic fluid exosomes in Examples 2-5.

[0125] (vi) Morphological observation

[0126] The morphology of the amniotic fluid exosomes obtained in Example 4 was observed using transmission electron microscopy (TEM). The results are as follows: Figure 1 As shown.

[0127] Depend on Figure 1 It is known that sheep amniotic fluid exosomes are round or elliptical membrane vesicles with complete membrane structure and clear internal structure.

[0128] Example 6

[0129] This embodiment provides the application of the amniotic fluid exosomes obtained in Example 4 in tissue repair and regeneration.

[0130] The specific method is as follows: Twenty mice were selected and randomly divided into four groups of five each. Superficial and deep second-degree skin wound models were created on the backs of the mice. The experimental group mice had sheep amniotic fluid exosomes prepared in Example 4 applied to the wound site, while the control group mice had physiological saline applied. The healing of the wounds was observed daily, and the wound area was measured. The healing rate and healing time at 7 and 14 days were calculated.

[0131] The results are shown in Table 4.

[0132] Table 4. Application of amniotic fluid exosomes obtained in Example 4 in tissue repair and regeneration.

[0133]

[0134] As shown in Table 4, the skin wound healing speed of mice in the experimental group was significantly accelerated.

[0135] Example 7

[0136] This embodiment provides the application of the amniotic fluid exosomes obtained in Example 4 in the field of cosmetics.

[0137] The specific method is as follows: Prepare a cosmetic emulsion containing the amniotic fluid exosomes obtained in Example 4.

[0138] Formula 1 is as follows: 0.01% amniotic fluid exosomes, 0.1% hyaluronic acid, 0.5% vitamin E, 3% glycerin, 0.1% emulsifier, 0.5% preservative, and the balance being deionized water.

[0139] Formula 2 is as follows: Collagen (Type I, Zhejiang Chongshan Biological Products Co., Ltd.) 0.01%, Hyaluronic Acid 0.1%, Vitamin E 0.5%, Glycerin 3%, Emulsifier 0.1%, Preservative 0.5%, Deionized Water balance.

[0140] Thirty volunteers were recruited for a trial experiment and randomly divided into three groups of 10 each. The experimental group used formula 1 of the cosmetic lotion, while the control group used formula 2. The lotion was applied twice daily, morning and evening, for eight weeks. Before use, and after four and eight weeks of use, the volunteers' skin was assessed, including the degree of wrinkles, skin texture, pore size, skin moisture content, and skin elasticity recovery rate. The degree of wrinkles, skin texture, and pore size were assessed using the Visia skin analyzer, while skin moisture content and skin elasticity recovery rate were assessed using the CK skin analyzer.

[0141] The test results after 8 weeks of use are shown in Table 5.

[0142] Table 5. Application of amniotic fluid exosomes obtained in Example 4 in the cosmetics field.

[0143] Visia Improvement rate of skin wrinkles (%) 25±3 13±2 Visia Improvement in skin texture (%) 20±2 16±4 Visia Pore ​​refinement (%) 17±2 9±5 CK Skin moisture content (%) 32±5 21±3 CK Skin elasticity recovery rate (%) 28±5 16±4

[0144] As shown in Table 5, using cosmetic lotions containing amniotic fluid exosomes can significantly improve skin problems such as wrinkles, sagging, and dryness, improve skin elasticity and firmness, and make the skin smoother and more delicate.

[0145] In summary, the sheep amniotic fluid exosomes provided in this application contain abundant growth factors, with placental growth factor being the most abundant. Furthermore, the preparation method described in this application is simple, efficient, and low-cost, enabling large-scale preparation of sheep amniotic fluid exosomes. Sheep amniotic fluid exosomes have broad application prospects, providing new tools and raw materials for tissue repair and regeneration, disease treatment, cosmetics, and other fields.

[0146] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. An industrial method for preparing sheep-derived amniotic fluid exosomes, characterized in that, The preparation method specifically includes the following steps: amniotic fluid extraction, centrifugation, batch ion exchange chromatography, and batch hydrophobic chromatography. The centrifugal separation includes the following steps: The collected amniotic fluid was centrifuged at 3000 rpm for 15 minutes at 4°C to remove cells and debris. Take the supernatant and centrifuge it at 10,000 rpm for 30 min at 4℃ to further remove small particles and impurities; Further filtration and sterilization were performed using a 0.22µm polyethersulfone filter membrane to obtain the supernatant after centrifugation. The batch ion exchange chromatography includes the following steps: Dilute the supernatant after centrifugation with 20mM phosphate buffer at pH 7.4 to reduce the conductivity to below 4mS / cm; equilibrate DE52 with 20mM phosphate buffer at pH 7.4 and filter to remove the equilibration buffer; add the supernatant after centrifugation at a ratio of 1ml DE52 cellulose to 100ml supernatant, and place on a rotary mixer to mix slowly at 4°C for 4-5 hours. After the combination is complete, the DE52 packing material is introduced into the chromatography column, and the supernatant after centrifugation is filtered by gravity; the eluent is washed with washing solution; the eluent is then mixed with the packing material and slowly mixed and incubated on a rotary mixer for 5 minutes, and eluted three times; the eluents obtained from the three times are mixed together to obtain the supernatant after ion exchange chromatography. In the batch ion exchange chromatography, the washing buffer is 20 mM phosphate and 10 mM sodium chloride at pH 7.4; the eluent is 20 mM phosphate and 50 mM sodium chloride at pH 7.

4. The batch hydrophobic chromatography method includes the following steps: Add 2M ammonium sulfate to the supernatant after ion exchange chromatography; equilibrate the phenyl hydrophobic packing material with a 20mM phosphate and 2M ammonium sulfate buffer solution at pH 7.4, and filter to remove the equilibration buffer solution; add the phenyl hydrophobic packing material to the supernatant after ion exchange chromatography at a ratio of 1ml of phenyl hydrophobic packing material to 100ml of supernatant after ion exchange chromatography, and place the mixture on a rotary mixer to mix slowly at 4℃ for 4-5h; After the combination was completed, the phenyl hydrophobic packing material was introduced into the chromatography column, and the supernatant after ion exchange chromatography was filtered by gravity. The packing material was washed with washing solution. The packing material was mixed with elution 1 and elution 2 respectively, and then slowly mixed and incubated on a rotary mixer for 5 min. Each was eluted three times. The eluents obtained from the three elutions with elution 2 were mixed together to obtain amniotic fluid exosomes. In the batch hydrophobic chromatography, the washing solution is 20 mM phosphate and 1 M sodium chloride at pH 7.4; the eluent 1 is 20 mM phosphate and 500 mM sodium chloride at pH 7.4; and the eluent 2 is 20 mM phosphate and 150 mM sodium chloride at pH 7.

4.

2. The industrial preparation method according to claim 1, characterized in that, The sheep source is sheep.

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