Extraction method of tissue exosome
Exosomes were extracted from tissues through EDTA-2Na chelation and differential centrifugation technology, which solved the problems of difficulty in extracting pure exosomes and cell destruction in the prior art, achieved efficient and stable exosome separation, and improved Purity and activity.
Patent Information
- Application Number
- CN202411599797.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-11
- Publication Date
- 2025-01-07
AI Technical Summary
The prior art is difficult to effectively extract pure exosomes from tissues, and existing methods are prone to destroy cells, resulting in intracellular substance contamination in exosome samples and lack of high-efficiency, specificity and standardized separation methods.
Using EDTA-2Na chelation and differential centrifugation technology, the liver or other organs are cleaved and chelated under ice-cold conditions, followed by multi-stage centrifugation and filter membrane filtration, isolation and extraction of exosomes, reducing cell destruction and improving purity.
It improves the purity and extraction efficiency of exosomes, maintains the activity of exosomes, simplifies the operation process, and EDTA-2Na raw materials are widely available and easy to store.
Smart Images
Figure CN119265107A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of exosome extraction, and specifically to a method for extracting tissue exosomes. Background Art
[0002] At present, the research method of exosomes is mainly to isolate exosomes from cell culture medium or from body fluids, and then conduct the next step of research. However, the study of exosomes in tissues is a supplement to the study of exosomes in vitro, and it can better reflect the composition and functional changes of exosomes in tissues or organs under corresponding physiological or pathological conditions. There are few studies on extracting exosomes from tissues, mainly because tissue exosomes are a mixture of exosomes secreted by multiple cells in the tissue, and it is difficult to determine the function of exosomes secreted by a single cell. Another reason is that the method of isolating tissue exosomes needs to be further developed. The existing method of extracting exosomes from tissues is difficult to avoid destroying cells, which leads to contamination of intracellular substances in exosome samples.
[0003] According to the unique physicochemical properties of exosomes, such as density, size, morphology, marker proteins, hydrophilicity and other characteristics, different methods or a combination of multiple methods can be used to separate and extract exosomes. However, due to the complexity of the biological fluid from which exosomes are derived, which is a mixture of extracellular vesicles and other extracellular substances, and the overlapping physicochemical properties of exosomes and other extracellular vesicles, as well as the heterogeneity between exosomes themselves, these methods cannot completely separate exosomes. At present, there is a lack of standardization in the separation and analysis methods of exosomes, and it is still a challenge to develop a high-yield, high-specificity, repeatable, rapid and effective exosome separation method.
[0004] At present, the main method for extracting tissue exosomes is collagenase hydrolysis. Or directly infiltrating tissue with liquid to release exosomes, among which collagenase hydrolysis is more commonly used. Collagenase hydrolysis uses collagenase to hydrolyze collagen in the intercellular matrix, reducing the adhesion between cells and separating them from each other, thereby releasing exosomes. Collagenase hydrolysis has some disadvantages, such as collagenase needs to work at 37°C, and too high a temperature may be detrimental to the preservation of exosomes. The purchased collagenase may contain other types of enzymes that affect the experiment. In addition, the activity of different batches of collagenase will change due to storage or transportation. It is difficult to grasp the digestion time, which may lead to insufficient or too long digestion time, resulting in less exosome release or excessive digestion and damage to cells. Summary of the invention
[0005] The present invention aims to solve the above technical problems and provides a method for extracting tissue exosomes.
[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:
[0007] A method for extracting tissue exosomes, comprising the following steps:
[0008] S1. Animal Management:
[0009] Rats were selected and the animals were kept in a 12-hour light and 12-hour day and night regime, with free access to food and water;
[0010] S2. Animal anesthesia and pretreatment:
[0011] After the rat is completely anesthetized, fix it on the surgical stand, carefully shave the abdominal villi, disinfect it, and cover the abdomen with a layer of clean plastic wrap;
[0012] S3. Perfusion and liver removal:
[0013] The abdominal cavity was exposed by cross-cutting, and the injection needle was inserted from the hepatic portal vein. After the suture was fixed, ice-cold saline was perfused at a flow rate of 5 ml per minute using a constant flow pump. The thoracic diaphragm was cut immediately after the perfusion began, and the rat was killed. When the liver was flushed until it turned white, the liver was cut and stored on ice for later use.
[0014] S4. Chelation:
[0015] Weigh about 1g of liver and place it in a cell culture dish. Add PBS solution with pH 7.4 and 0.5% EDTA-2Na at a mass: volume ratio of 1:10. Cut the liver into small pieces of 2×2×2 mm with a surgical blade on ice. Then place the culture dish on a horizontal shaker at 4°C for chelation for 30 minutes. After the chelation, add an equal volume of DMEM culture medium to supplement the chelated calcium ions and other cations. Then transfer the tissue pieces and supernatant in the culture dish to a 50mL centrifuge tube and store on ice for later use.
[0016] S5. Extraction of exosomes by differential centrifugation:
[0017] The chelated tissue mixture was centrifuged at 3-6°C, 250-350g to remove tissue chunks and cell debris; the supernatant was then transferred to a new centrifuge tube and centrifuged at 3-6°C, 1500-2500g to further remove dead cells and cell debris; the supernatant was again transferred to a new centrifuge tube and centrifuged at 3-6°C, 9000-11000g to remove contamination from large vesicles and mitochondria, the supernatant obtained was filtered through a 0.20-0.24 μm filter membrane, the filtrate was centrifuged at 3-6°C, 99000-103000g for 1.5-2.5h, the precipitate was resuspended with PBS, and then centrifuged again at 3-6°C, 99000-103000g for 1.5-2.5h, the precipitate was resuspended with 90-110 μL PBS and stored at -80°C, which was the prepared exosome sample.
[0018] Preferably, the rat liver is weighed, and a PBS solution containing 0.5% EDTA-2Na at pH 7.4 is added at a ratio of 1 g:10 mL, and the liver is placed on ice and cut into small pieces of 2×2×2 mm.
[0019] Preferably, the supernatant is centrifuged at 300 g, 2000 g, and 10000 g in sequence, and the centrifugation time is 10 min, 20 min, and 60 min, respectively.
[0020] Preferably, the exosome sample is identified by one or a combination of transmission electron microscopy identification, particle size identification or molecular marker identification.
[0021] Preferably, the preparation method of EDTA-2Na is as follows:
[0022] Step 1: Prepare the ingredients:
[0023] The main raw materials for preparing disodium ethylenediaminetetraacetate are ethylenediamine and tetraacetic acid, in which ethylenediamine is solid and tetraacetic acid is liquid. Auxiliary materials are added.
[0024] The second step is to prepare tetraacetic anhydride:
[0025] Add tetraacetic acid into a reaction kettle, heat to 100°C, add hydrogen peroxide as a catalyst, and react for 3-4 hours under an oxygen atmosphere to obtain tetraacetic anhydride;
[0026] The third step is to prepare ethylenediaminetetraacetic acid:
[0027] Tetraacetic anhydride and ethylenediamine are mixed in a ratio of 1:1.3, heated to 180-200°C, and reacted for 2-3 hours. During the heating process, the reactants will undergo a water removal reaction to form ethylenediaminetetraacetic acid. After the reaction is completed, the product is cooled and added to a sodium hydroxide solution for neutralization to obtain a preliminary product of disodium ethylenediaminetetraacetate;
[0028] Step 4: Refining:
[0029] The preliminary product is dissolved in water and filtered to obtain a filtrate. Nitric acid is added to the filtrate to adjust the pH value to about 8.0. Then, sodium carbonate solution is slowly added until a white precipitate is generated. The reaction, precipitation, and filtration steps are then performed to finally obtain EDTA-2Na.
[0030] Preferably, the auxiliary material is one or a combination of hydrogen peroxide, sodium hydroxide and nitric acid.
[0031] Preferably, the liver is replaced by a kidney, spleen or lung.
[0032] After adopting the above method, the present invention has the following advantages:
[0033] The present invention utilizes EDTA-2Na chelation to render most adhesion factors ineffective, so that tissue cells are separated from each other, thereby releasing exosomes in the extracellular matrix, and then using differential ultracentrifugation to separate exosomes from rat liver tissue. Proteomics results show that the EDTA-2Na chelation method extracts more types of proteins, and more exosome proteins can be obtained at the same time. The exosomes of rat liver tissue extracted by the EDTA-2Na chelation method have a higher purity. In addition, the EDTA-2Na chelation method is simple and easy to operate, with stable effects. Compared with the collagenase method at 37°C, the operation at 4°C can better maintain the activity of exosomes. The raw materials of EDTA-2Na are widely available and easy to store.
[0034] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or technical descriptions will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0036] Figure 1 This is a flow chart of the EDTA-2Na chelation method for extracting exosomes from rat liver tissue;
[0037] Figure 2 This is a result diagram of the immunoblotting of the present invention comparing three methods for extracting exosomes from liver tissue;
[0038] Figure 3 Schematic diagram of proteome analysis using three methods of the present invention. DETAILED DESCRIPTION
[0039] Specific embodiments of the present invention will now be mentioned in detail. Although the present invention is described in conjunction with these specific embodiments, it should be appreciated that it is not intended to limit the present invention to these specific embodiments. On the contrary, these embodiments are intended to cover substitutions, changes or equivalent embodiments that may be included in the spirit and scope of the invention defined by the claims. In the following description, a large number of specific details are set forth in order to provide a comprehensive understanding of the present invention. The present invention can be implemented without some or all of these specific details. In other cases, in order not to make the present invention unnecessarily obscure, well-known process operations are not described in detail.
[0040] When used in conjunction with "including," "methods comprising," or similar language in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
[0041] The present invention is further described in detail below in conjunction with the full text.
[0042] Combined with Figure 1-Figure 3 , a method for extracting tissue exosomes, comprising the following steps:
[0043] S1. Animal Management:
[0044] Rats were selected and kept under a 12-hour light and 12-hour day and night regime, with free access to food and water.
[0045] Specifically, 6-8 weeks old, 200-240 g SPF-grade Sprague Dawley rats were purchased from Shrek Jingda Company. The animals were raised under a 12-hour light and 12-hour day and night regime, with free access to food and water.
[0046] S2. Animal anesthesia and pretreatment:
[0047] After the rat is completely anesthetized, fix it on the surgical stand, carefully shave the abdominal villi, disinfect it, and cover the abdomen with a layer of clean plastic wrap;
[0048] Specifically,
[0049] Inject 0.2-0.4 ml of 3% sodium pentobarbital for every 200g rat, fix the rat on the operating frame after it is completely anesthetized, carefully shave the abdominal villi, disinfect the abdomen with 75% alcohol and iodine, and cover the abdomen with a layer of clean plastic wrap.
[0050] S3. Perfusion and liver removal:
[0051] The abdominal cavity was exposed by cross-cutting, and the injection needle was inserted from the hepatic portal vein. After the suture was fixed, ice-cold saline was perfused at a flow rate of 5 ml per minute using a constant flow pump. The thoracic diaphragm was cut immediately after the perfusion began, and the rat was killed. When the liver was flushed until it turned white, the liver was cut and stored on ice for later use.
[0052] S4. Chelation:
[0053] Weigh about 1g of liver and place it in a cell culture dish. Add PBS solution with pH 7.4 and 0.5% EDTA-2Na at a mass (g): volume (mL) ratio of 1:10. Cut the liver into 2×2×2 mm pieces with a surgical blade on ice. Then place the culture dish on a horizontal shaker at 4°C for chelation for 30 minutes. After the chelation is completed, add an equal volume of DMEM culture medium to supplement the chelated calcium ions and other cations. Then transfer the tissue pieces and supernatant in the culture dish to a 50mL centrifuge tube and store on ice for later use.
[0054] S5. Extraction of exosomes by differential centrifugation:
[0055] The chelated tissue mixture was centrifuged at 3-6°C, 250-350g to remove tissue chunks and cell debris; the supernatant was then transferred to a new centrifuge tube and centrifuged at 3-6°C, 1500-2500g to further remove dead cells and cell debris; the supernatant was again transferred to a new centrifuge tube and centrifuged at 3-6°C, 9000-11000g to remove contamination from large vesicles and mitochondria, the supernatant obtained was filtered through a 0.20-0.24 μm filter membrane, the filtrate was centrifuged at 3-6°C, 99000-103000g for 1.5-2.5h, the precipitate was resuspended with PBS, and then centrifuged again at 3-6°C, 99000-103000g for 1.5-2.5h, the precipitate was resuspended with 90-110 μL PBS and stored at -80°C, which was the prepared exosome sample.
[0056] The rat liver was weighed, and a PBS solution containing 0.5% EDTA-2Na at pH 7.4 was added at a ratio of 1 (g): 10 (mL), and the liver was cut into small pieces of 2×2×2 mm on ice.
[0057] The supernatant was centrifuged at 300g, 2000g and 10000g in sequence, and the centrifugation time was 10min, 20min and 60min respectively.
[0058] The exosome sample is identified by one or a combination of transmission electron microscopy identification, particle size identification or molecular marker identification.
[0059] The preparation method of the EDTA-2Na is as follows:
[0060] Step 1: Prepare the ingredients:
[0061] The main raw materials for preparing disodium ethylenediaminetetraacetate are ethylenediamine and tetraacetic acid, in which ethylenediamine is solid and tetraacetic acid is liquid. Auxiliary materials are added.
[0062] The second step is to prepare tetraacetic anhydride:
[0063] Add tetraacetic acid into a reaction kettle, heat to 100°C, add hydrogen peroxide as a catalyst, and react for 3-4 hours under an oxygen atmosphere to obtain tetraacetic anhydride;
[0064] The third step is to prepare ethylenediaminetetraacetic acid:
[0065] Tetraacetic anhydride and ethylenediamine are mixed in a ratio of 1:1.3, heated to 180-200°C, and reacted for 2-3 hours. During the heating process, the reactants will undergo a water removal reaction to form ethylenediaminetetraacetic acid. After the reaction is completed, the product is cooled and added to a sodium hydroxide solution for neutralization to obtain a preliminary product of disodium ethylenediaminetetraacetate;
[0066] Step 4: Refining:
[0067] The preliminary product is dissolved in water and filtered to obtain a filtrate. Nitric acid is added to the filtrate to adjust the pH value to about 8.0. Then, sodium carbonate solution is slowly added until a white precipitate is generated. The reaction, precipitation, and filtration steps are then performed to finally obtain EDTA-2Na.
[0068] The auxiliary material is one or a combination of hydrogen peroxide, sodium hydroxide and nitric acid.
[0069] Embodiment 1:
[0070] A method for extracting tissue exosomes, characterized in that it comprises the following steps:
[0071] S1. Animal Management:
[0072] 6-week-old, 200g SPF-grade Sprague Dawley rats were purchased from Shrek Jingda Co., Ltd. Animals were housed under a 12-hour light and 12-hour day and night regime, with free access to food and water.
[0073] S2. Animal anesthesia and pretreatment:
[0074] Each 200g rat was injected with 0.2 ml of 3% sodium pentobarbital, and after the rat was completely anesthetized, it was fixed on the surgical frame, and the abdominal villi were carefully shaved. The abdomen was disinfected with 75% alcohol and iodine solution, and then covered with a layer of clean plastic wrap.
[0075] S3. Perfusion and liver removal:
[0076] The abdominal cavity was exposed by cross-cutting, and the injection needle was inserted from the hepatic portal vein. After the suture was fixed, ice-cold saline was perfused at a flow rate of 5 ml per minute using a constant flow pump. The thoracic diaphragm was cut immediately after the perfusion began, and the rat was killed. When the liver was flushed until it turned white, the liver was cut and stored on ice for later use.
[0077] S4. Chelation:
[0078] Weigh about 1g of liver and place it in a cell culture dish. Add PBS solution with pH 7.4 and 0.5% EDTA-2Na at a mass (g): volume (mL) ratio of 1:10. Cut the liver into 2×2×2 mm pieces with a surgical blade on ice. Then place the culture dish on a horizontal shaker at 4°C for chelation for 30 minutes. After the chelation is completed, add an equal volume of DMEM culture medium to supplement the chelated calcium ions and other cations. Then transfer the tissue pieces and supernatant in the culture dish to a 50mL centrifuge tube and store on ice for later use.
[0079] S5. Extraction of exosomes by differential centrifugation:
[0080] The chelated tissue mixture was centrifuged at 4°C, 300g for 10 minutes to remove tissue blocks and cell debris; the supernatant was then transferred to a new centrifuge tube and centrifuged at 4°C, 2000g for 20 minutes to further remove dead cells and cell debris; the supernatant was again transferred to a new centrifuge tube and centrifuged at 4°C, 10000g for 60 minutes to remove contamination from large vesicles and mitochondria. The obtained supernatant was filtered through a 0.22μm filter membrane, and the filtrate was centrifuged at 4°C, 100000g for 2h. The obtained precipitate was resuspended with PBS, and then centrifuged again at 4°C, 100000g for 2h. The obtained precipitate was resuspended with 100μL PBS and stored at -80°C, which was the prepared exosome sample.
[0081] Embodiment 2:
[0082] A method for extracting tissue exosomes, characterized in that it comprises the following steps:
[0083] S1. Animal Management:
[0084] 8-week-old, 240 g SPF-grade Sprague Dawley rats were purchased from Shrek Jingda Co., Ltd. Animals were housed under a 12-hour light and 12-hour day and night regime, with free access to food and water.
[0085] S2. Animal anesthesia and pretreatment:
[0086] Each 200g rat was injected with 0.3 ml of 3% sodium pentobarbital, and after the rat was completely anesthetized, it was fixed on the surgical frame, and the abdominal hair was carefully shaved. The abdomen was disinfected with 75% alcohol and iodine, and then covered with a layer of clean plastic wrap.
[0087] S3. Perfusion and liver removal:
[0088] The abdominal cavity was exposed by cross-cutting, and the injection needle was inserted into the vein. After the suture was fixed, ice-cold saline was perfused at a flow rate of 5 ml per minute using a constant flow pump. The thoracic diaphragm was cut immediately after the perfusion began, and the rat was killed. When the kidney was flushed to white, the kidney was cut and stored on ice for later use.
[0089] S4. Chelation:
[0090] Weigh about 1g of kidney and place it in a cell culture dish. Add PBS solution with pH 7.4 and 0.5% EDTA-2Na at a mass (g): volume (mL) ratio of 1:10. Cut the kidney into 2×2×2 mm small pieces with a surgical blade on ice. Then place the culture dish on a horizontal shaker at 4°C for chelation for 30 minutes. After the chelation is completed, add an equal volume of DMEM culture medium to supplement the chelated calcium ions and other cations. Then transfer the tissue pieces and supernatant in the culture dish to a 50mL centrifuge tube and store on ice for later use.
[0091] S5. Extraction of exosomes by differential centrifugation:
[0092] The chelated tissue mixture was centrifuged at 4°C, 300g for 10 minutes to remove tissue blocks and cell debris; the supernatant was then transferred to a new centrifuge tube and centrifuged at 4°C, 2000g for 20 minutes to further remove dead cells and cell debris; the supernatant was again transferred to a new centrifuge tube and centrifuged at 4°C, 10000g for 60 minutes to remove contamination from large vesicles and mitochondria. The obtained supernatant was filtered through a 0.22μm filter membrane, and the filtrate was centrifuged at 4°C, 100000g for 2h. The obtained precipitate was resuspended with PBS, and then centrifuged again at 4°C, 100000g for 2h. The obtained precipitate was resuspended with 100μL PBS and stored at -80°C, which was the prepared exosome sample.
[0093] Comparative Example:
[0094] Exosomes were extracted from rat livers of equal mass using the infiltration method and collagenase enzymatic hydrolysis method.
[0095] like Figure 2 As shown, EDTA-2Na-1 and EDTA-2Na-2 are EDTA-2Na chelation methods for exosome extraction, and the infiltration method (JR) and collagenase enzymatic method (collagenaseII) are comparative examples for exosome extraction. Compared with the other two methods, the results of the method of the present invention show that the EDTA-2Na chelation method enriches more exosome marker proteins HSP70, TSG101, CD63, CD81, CD9, and less endoplasmic reticulum pollutant protein Calnexin and Golgi pollutant protein GM130. At the same time, the proteomics results show that the EDTA-2Na chelation method extracts more types of proteins, and more exosome proteins can be obtained, such as Figure 3 shown.
[0096] Therefore, the purity of rat tissue exosomes extracted by EDTA-2Na chelation method is higher. In addition, EDTA-2Na chelation method is simple and easy to use, with stable effect. Compared with collagenase method at 37℃, operation at 4℃ can better maintain the activity of exosomes. EDTA-2Na raw materials are widely available and easy to store. EDTA-2Na chelation method can extract more and purer tissue exosomes.
[0097] The present invention and its embodiments are described above, and such description is not restrictive. What is shown in the full text is only one of the embodiments of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.
Claims
1. A method for extracting tissue exosomes, characterized in that: The following steps are involved: S1. Animal Management: Rats were selected and the animals were kept in a 12-hour light and 12-hour day and night regime, with free access to food and water; S2. Animal anesthesia and pretreatment: After the rat is completely anesthetized, fix it on the surgical stand, carefully shave the abdominal villi, disinfect it, and cover the abdomen with a layer of clean plastic wrap; S3. Perfusion and liver removal: The abdominal cavity was exposed by cross-cutting, and the injection needle was inserted from the hepatic portal vein. After the suture was fixed, ice-cold saline was perfused at a flow rate of 5 ml per minute using a constant flow pump. The thoracic diaphragm was cut immediately after the perfusion began, and the rat was killed. When the liver was flushed until it turned white, the liver was cut and stored on ice for later use. S4. Chelation: Weigh about 1g of liver and place it in a cell culture dish. Add PBS solution with pH 7.4 and 0.5% EDTA-2Na at a mass: volume ratio of 1:
10. Cut the liver into small pieces of 2×2×2 mm with a surgical blade on ice. Then place the culture dish on a horizontal shaker at 4°C for chelation for 30 minutes. After the chelation, add an equal volume of DMEM culture medium to supplement the chelated calcium ions and other cations. Then transfer the tissue pieces and supernatant in the culture dish to a 50mL centrifuge tube and store on ice for later use. S5. Extraction of exosomes by differential centrifugation: The chelated tissue mixture was centrifuged at 3-6°C, 250-350 g to remove tissue chunks and cell debris; The supernatant was then transferred to a new centrifuge tube and centrifuged at 1500-2500 g at 3-6 °C to further remove dead cells and cell debris; The supernatant was transferred to a new centrifuge tube again and centrifuged at 3-6°C, 9000-11000g to remove contamination from large vesicles and mitochondria. The supernatant was filtered through a 0.20-0.24 μm filter membrane, and the filtrate was centrifuged at 3-6°C, 99000-103000g for 1.5-2.5 h. The precipitate was resuspended in PBS and then centrifuged again at 3-6°C, 99000-103000g for 1.5-2.5 h. The precipitate was resuspended in 90-110 μL PBS and stored at -80°C to prepare the exosome sample.
2. The method for extracting tissue exosomes according to claim 1, characterized in that: The rat liver was weighed, and a PBS solution containing 0.5% EDTA-2Na at pH 7.4 was added at a ratio of 1 g:10 mL. The liver was placed on ice and cut into small pieces of 2×2×2 mm.
3. The method for extracting tissue exosomes according to claim 1, characterized in that: The supernatant was centrifuged at 300g, 2000g and 10000g in sequence, and the centrifugation time was 10min, 20min and 60min respectively.
4. The method for extracting tissue exosomes according to claim 1, characterized in that: The exosome sample is identified by one or a combination of transmission electron microscopy identification, particle size identification or molecular marker identification.
5. The method for extracting tissue exosomes according to claim 1, characterized in that: The preparation method of the EDTA-2Na is as follows: Step 1: Prepare the ingredients: The main raw materials for preparing disodium ethylenediaminetetraacetate are ethylenediamine and tetraacetic acid, in which ethylenediamine is solid and tetraacetic acid is liquid. Auxiliary materials are added. The second step is to prepare tetraacetic anhydride: Add tetraacetic acid into a reaction kettle, heat to 100°C, add hydrogen peroxide as a catalyst, and react for 3-4 hours under an oxygen atmosphere to obtain tetraacetic anhydride; The third step is to prepare ethylenediaminetetraacetic acid: Tetraacetic anhydride and ethylenediamine are mixed in a ratio of 1:1.3, heated to 180-200°C, and reacted for 2-3 hours. During the heating process, the reactants will undergo a water removal reaction to form ethylenediaminetetraacetic acid. After the reaction is completed, the product is cooled and added to a sodium hydroxide solution for neutralization to obtain a preliminary product of disodium ethylenediaminetetraacetate; Step 4: Refining: The preliminary product is dissolved in water and filtered to obtain a filtrate. Nitric acid is added to the filtrate to adjust the pH value to 8.0, and then a sodium carbonate solution is slowly added until a white precipitate is generated. The reaction, precipitation, and filtration steps are then performed to finally obtain EDTA-2Na.
6. The method for extracting tissue exosomes according to claim 5, characterized in that: The auxiliary material is one or a combination of hydrogen peroxide, sodium hydroxide and nitric acid.
7. The method for extracting tissue exosomes according to claim 1, characterized in that: The liver may be replaced by a kidney, spleen or lung.