Method for preparing exosome and application

Abstract

The invention relates to the technical field of exosome preparation, and particularly discloses a method for preparing an exosome and application. The method for preparing the exosome comprises the following steps: (1) adjusting the pH value of degreased animal milk to 4.5-4.6 by using an acetic acid-sodium acetate solution, and separating to obtain whey liquid; (2) performing microfiltration on the whey liquid to obtain permeate; (3) washing and filtering the permeate with a NaCl aqueous solution, so that the protein content in the exosome trapped fluid is lower than 2 mu g / mL, and the osmotic pressure is 280-310 mOsm / L; and (4) mixing with an acetic acid-sodium acetate solution to obtain an electrophoresis solution, and carrying out circulating free flow isoelectric focusing electrophoresis. According to the method, the structural integrity of the exosome can be better guaranteed, the overall yield is high, and the purity of the exosome is high.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome preparation, and in particular to a method for preparing exosomes and applications thereof. Background Art

[0002] Exosomes use lipid bilayers as protection to ensure the stability of their contents, and are widely studied as a promising drug delivery system. Milk exosomes have important research value in disease diagnosis, treatment, prevention, and the growth and development of humans and animals. Among them, bovine milk exosomes have attracted much attention due to their rich and low-cost raw materials, good biocompatibility, safety, low immunogenicity, and oral delivery. Milk samples are easy to obtain and do not have a negative impact on dairy cows, which lays the foundation for the study of milk exosomes. Exosomes participate in a variety of biological functions and play an important role. They are considered to be important biomarkers and drug carriers for disease diagnosis, treatment and prognosis. Therefore, the development of simple, efficient and economical exosome separation, purification and enrichment technologies will help early diagnosis and precise treatment of diseases.

[0003] At present, the commonly used methods for isolating exosomes include ultracentrifugation, size screening separation technology (ultrafiltration and size exclusion), immunoaffinity capture, kit method and microfluidics technology. The exosomes obtained by these separation methods have problems such as low purity and / or low concentration. Centrifugation is the most commonly used method, including differential ultracentrifugation and density gradient centrifugation. Differential ultracentrifugation is the most classic and most widely used method for extracting exosomes. It has the advantages of low cost and high yield, but it is dependent on instruments and equipment, time-consuming, easy to be contaminated, low separation purity, and ultra-high-speed centrifugation may destroy the structure of exosomes, which is not suitable for large-scale use. Compared with differential ultracentrifugation, density gradient centrifugation has higher purity of exosomes, but it also has the disadvantages of being time-consuming and destroying the structure of exosomes. Ultrafiltration is also a recognized method for extracting exosomes, with advantages such as rapidity and high efficiency, but there are problems such as the selection of different ultrafiltration membranes, pore blockage caused by vesicles and proteins, protein contamination, and possible loss of a large number of exosomes during the ultrafiltration process. Size exclusion method is based on the difference in the retention of analytes of different molecular sizes in the pores of chemically inert porous stationary phases to achieve separation. During the separation process, exosomes are less damaged and the exosomes obtained are of high purity, but it is not suitable for separating exosomes from large volumes of liquids; immunoaffinity method uses magnetic beads coated with monoclonal antibodies to bind exosomes, which can be used to extract a certain subpopulation of exosomes. It has high specificity, but the operation is time-consuming, the extraction efficiency is low, the cost is high, the output is low, and it is not suitable for a large number of samples. In addition, once the antibody binds to the exosome, it is difficult to separate it under mild elution conditions. Commercial exosome separation and purification kits have received increasing attention due to their simple operation and time saving. Most commercial kits use polymer co-precipitation methods to enrich exosomes, but the purity of exosomes separated by commercial kits is low, and the polymers in them are difficult to remove, which has an adverse effect on subsequent analysis. Microfluidics technology has low yield and low purity, and there are problems such as time-consuming and difficult process standardization and large-scale testing.

[0004] In addition, the research on the use of artificial antibody materials for exosomes has just started. Due to the complex and diverse surface structure of exosomes, the molecular mechanism of artificial antibody recognition between exosomes has not been clearly elucidated. In addition, there are few types of universal ligands for exosomes and the research and development process of specific ligands is highly professional. Therefore, there are few reports on artificial antibodies used for exosome separation and purification.

[0005] The separation and analysis of exosomes is expected to replace the traditional invasive body fluid collection mode (such as blood, urine and cerebrospinal fluid) and be used as a biomarker for early detection of diseases. At the same time, exosomes are not only therapeutic agents themselves, but also carriers for targeted drug delivery and treatment. Therefore, as an important part of clarifying pathophysiological mechanisms and clinical diagnosis and treatment, it is necessary to continue to study the molecular characteristics, biological functions and clinical applications of exosomes in depth, which in turn puts higher requirements on the separation and purification technology of exosomes. However, the body fluid environment is complex, the concentration of exosomes is low, and the size of exosomes is small. In order to meet the needs of scientific research and clinical practice, the rapid, efficient, high-purity and loss-free separation and purification of exosomes has become a major challenge in the field of exosome research. Summary of the invention

[0006] One of the purposes of the present invention is to provide a method for efficiently isolating and obtaining high-purity and high-yield exosomes from milk.

[0007] The present invention provides a method for preparing exosomes, which uses animal milk as a raw material to prepare exosomes, and comprises: (1) Isoelectric precipitation separation: The pH value of the defatted animal milk is adjusted to 4.5-4.6 with an acetic acid-sodium acetate solution, and after sufficient acid precipitation, the precipitate is removed and separated to obtain a whey liquid; the molar ratio of acetic acid to sodium acetate in the acetic acid-sodium acetate solution is 1: (1.4-1.8), and the concentration of acetate ions is 0.055-0.075 mol / L (preferably 0.056-0.071 mol / L); (2) Microfiltration: microfiltration of the whey liquid to remove impurities and obtain a permeate; (3) Ultrafiltration: The permeate is filtered with a NaCl aqueous solution to obtain an exosome retentate; the protein content of the exosome retentate is less than 2 μg / mL and the osmotic pressure is 280-310 mOsm / L; (4) Circulating free-flow isoelectric focusing electrophoresis: the exosome retentate solution is mixed with a carrier ampholyte solution to obtain an electrophoresis solution for electrophoresis, and the components with a pH value of 4-7 are collected; the carrier ampholyte solution is an acetic acid-sodium acetate solution; the concentration of exosomes in the electrophoresis solution is 1.5×10 8 -1×10 10 particles / ml, the molar ratio of acetate ion to sodium ion is 1:(0.3-0.55), and the concentration of acetate ion is 0.02-0.029mol / L.

[0008] Preferably, in step (1) of the method for preparing exosomes of the present invention, the molar ratio of acetic acid to sodium acetate in the acetic acid-sodium acetate solution is 1:(1.6-1.62), more preferably 1:0.617, and the concentration of acetate ions is 0.065 mol / L; And / or, in step (4), the concentration of exosomes in the electrophoresis solution is 2.5×10 9 particles / ml, the molar ratio of acetate ion to sodium ion is 1:(0.42-0.43), preferably 1:0.423, and the concentration of acetate ion is 0.025-0.026 mol / L, preferably 0.02567 mol / L.

[0009] When separating exosomes from animal milk, the present invention generally first removes casein from the animal milk by a specific acid precipitation method, then removes larger particles and impurities (such as bacteria, some milk fat globules and larger suspended particles) by microfiltration, and then further removes components outside the exosomes (such as whey protein) by specific ultrafiltration, and then mixes the obtained ultrafiltration retentate with a specific carrier amphoteric electrolyte solution and performs circulating free-flow isoelectric focusing electrophoresis.

[0010] Specifically, the present invention has found that, when casein acid precipitation is performed, the conventional method in the art can achieve the removal of the target protein, but it is not conducive to ensuring the structural integrity of the exosomes. Therefore, even with the use of circulating free-flow isoelectric focusing electrophoresis, the ideal yield cannot be obtained. When the pH value is adjusted by the specific acetic acid-sodium acetate solution of the present invention, both the effective removal of casein and the improvement of the final recovery rate of exosomes can be achieved.

[0011] In addition, the present invention has also found that when removing impurities, large-sized impurities are first removed by microfiltration, and then ultrafiltration and diafiltration are performed with conventional water. Although small-sized impurities can be removed, it is not conducive to ensuring the integrity of the exosome structure. Instead, the NaCl aqueous solution of the present invention is used for diafiltration, and the osmotic pressure and protein concentration of the retentate are specifically controlled, which can not only ensure that impurities are minimized, but also avoid the introduction of too many ions, taking into account the maintenance of the exosome structure and the subsequent requirements of cyclic free-flow isoelectric focusing electrophoresis.

[0012] Furthermore, the present invention specifically selects acetic acid-sodium acetate solution as the carrier ampholyte solution when performing circulating free-flow isoelectric focusing electrophoresis, and controls the concentration of the main substances in the electrophoresis solution, so that no additional ions are introduced, and the effective separation of exosomes during electrophoresis is ensured, thereby obtaining the effect of high purity and good yield. If the concentrations of the main substances are not well matched, an effective pH gradient cannot be formed in the electrophoresis medium or the structure of the exosomes is damaged. This will result in the inability to obtain effective purification results.

[0013] In the present invention, animal milk refers to the milk of mammals such as cow's milk, goat's milk, camel's milk, deer's milk, horse's milk, etc.

[0014] The animal milk of the present invention can be defatted by conventional methods in the art, such as centrifuging at a temperature of 45-50° C. (preferably 45° C.) and a rotation speed of 3500-5500 rpm (preferably 4500 rpm).

[0015] The animal milk of the present invention is preferably subjected to conventional milk purification operations before defatting, for example, centrifuging in a centrifugal milk purifier at a temperature of 1-10° C. (preferably 4-7° C.) and a rotation speed of 3500-5500 rpm for 10-20 minutes.

[0016] In step (2) of the method for preparing exosomes of the present invention, during the microfiltration, the pore size of the microfiltration membrane is 0.6-1.0 μm, the temperature is 4-7° C., and the pressure is 2-6 bar.

[0017] In step (3) of the method for preparing exosomes of the present invention, during the ultrafiltration, the pore size of the ultrafiltration membrane is 200-400 kDa, the temperature is 4-7°C, and the pressure is 0.05-0.2 MPa; The treatment under the microfiltration and ultrafiltration conditions of the present invention is conducive to ensuring the purity and recovery rate of the final exosomes (reducing the impact on the exosome structure). If the temperature during microfiltration and ultrafiltration is too high, or the microfiltration and ultrafiltration pressures and pore sizes are not well matched, the purity and / or recovery rate of the exosomes will be reduced. Therefore, it is preferred to use the above-mentioned treatment conditions of the present invention.

[0018] In step (3) of the method for preparing exosomes of the present invention, the concentration of NaCl in the NaCl aqueous solution is 0.8%-1%.

[0019] In the present invention, when diafiltration is performed with a NaCl aqueous solution, a person skilled in the art can adjust the concentration and dosage of the NaCl aqueous solution according to common sense, so as to simultaneously control the osmotic pressure and protein concentration of the retentate to the limits of the present invention. For example, if the concentration of the NaCl aqueous solution is high, the dosage of the NaCl aqueous solution can be appropriately reduced to avoid excessive osmotic pressure while controlling the protein concentration; if the concentration of the NaCl aqueous solution is low, the dosage of the NaCl aqueous solution can be appropriately increased to more fully wash away impurities under the premise of appropriate osmotic pressure.

[0020] More preferably, in step (2) of the method for preparing exosomes of the present invention, during the microfiltration, the pore size of the microfiltration membrane is 0.8 μm, the temperature is 6° C., and the pressure is 4 bar; And / or, in step (3), during the ultrafiltration, the pore size of the ultrafiltration membrane is 300 kDa, the temperature is 6° C., and the pressure is 0.1 MPa; and the concentration of NaCl in the NaCl aqueous solution is 0.9%.

[0021] In step (4) of the method for preparing exosomes of the present invention, the pressure of the circulating free-flow isoelectric focusing electrophoresis is 300-500 V, and the temperature of the separation chamber during focusing is 2-8° C.

[0022] Preferably, in step (4) of the method for preparing exosomes of the present invention, the pressure of circulating free-flow isoelectric focusing electrophoresis is 400 V and the temperature is 4° C.

[0023] Temperature has an impact on the structure of exosomes, and also affects the degree of dissociation and buffering capacity of amphoteric electrolytes. Those skilled in the art can select according to common sense, preferably using the circulating free-flow isoelectric focusing electrophoresis conditions of the present invention, which can be well matched with a specific electrophoresis solution to achieve an ideal exosome separation effect.

[0024] In the electrophoresis of the present invention, a stable electrophoresis voltage is provided and maintained by the continuous circulation of the anolyte and the catholyte. Those skilled in the art can select the anolyte and the catholyte, as well as the circulation pump speeds of the electrophoresis liquid and the anolyte and the catholyte according to common sense, so as to ensure that the electrophoresis can be realized (for example, the circulation pump speed of the electrophoresis liquid is set to 1-3 ml / min, and the circulation pump speeds of the anolyte and the catholyte are set to 2-4 ml / min, and the circulation pump speed of the electrophoresis liquid is set to be lower than the circulation pump speeds of the anolyte and the catholyte).

[0025] The method for determining the end point of the circulating free-flow isoelectric focusing electrophoresis of the present invention is common knowledge in the art, that is, when the current drops to 1-5 mA and varies within ±1 mA within 30 minutes, it means that the isoelectric focusing is completed and the target can be collected.

[0026] In step (1) of the method for preparing exosomes of the present invention, the fat content in the defatted animal milk is ≤ 0.05%, which is conducive to the subsequent electrophoresis.

[0027] In step (1) of the method for preparing exosomes of the present invention, the whey liquid is obtained by removing the precipitate and separating by a conventional centrifugation method in the art; preferably, the centrifugation temperature is 0-6°C, more preferably 4°C, and the rotation speed is 8000-15000 rpm, more preferably 10000 rpm.

[0028] The present invention also provides the use of the above method for preparing exosomes in improving the purity of exosomes when exosomes are isolated from milk.

[0029] The present invention also provides an application of the above method for preparing exosomes in improving the recovery rate of exosomes when isolating exosomes from milk.

[0030] The beneficial effects of the present invention are at least: The method of the present invention can process raw materials on a large scale and is suitable for the industrial enrichment of exosomes in animal milk. The entire method has a high recovery rate (above 70%), and the exosomes obtained are of high purity (80-99%), which can reduce the production cost of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a representative transmission electron micrograph of the fractions with a pH value of 4-7 collected after circulating free-flow isoelectric focusing electrophoresis in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0033] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0034] Example 1 This embodiment provides a method for preparing exosomes, which is as follows: 1. Cleansing milk 10L of raw milk was processed by a centrifugal milk purifier at a temperature of 4°C, a rotation speed of 4500 rpm, and a time of 10 min.

[0035] 2. Centrifugal separation The skimmed milk with a fat content of 0.05% was obtained by centrifugation at a centrifugal separator with a centrifugal temperature of 45°C and a rotation speed of 4500 rpm.

[0036] 3. Isoelectric point precipitation separation In the skimmed milk of step 2, acetic acid-sodium acetate solution (the concentration of acetate ion is 0.056 mol / L, and the molar ratio of acetic acid to sodium acetate is 1:1.408) is added dropwise, the pH is adjusted to 4.5 (the osmotic pressure of the solution is 293 mOsm / L), and after acid precipitation for 1 hour, centrifugation is performed using a centrifuge at a centrifugal temperature of 6°C and a rotation speed of 15,000 rpm for 2 hours to obtain a whey solution.

[0037] 4. Microfiltration The whey liquid is microfiltered to obtain a clear permeate. The pore size of the microfiltration membrane is 0.6 μm, the microfiltration temperature is 4°C, and the pressure is 2 bar.

[0038] 5. Ultrafiltration The permeate was diafiltered with an aqueous NaCl solution (0.8%) using an ultrafiltration membrane until the protein content in the retentate was less than 2 μg / mL and the osmotic pressure was 289 mOsm / L.

[0039] The pore size of the ultrafiltration membrane was 400 kDa, the ultrafiltration temperature was controlled at 4 °C, and the pressure was 0.2 MPa; 6. Free-flow isoelectric focusing (FFIEF) A. The retentate solution was mixed with the carrier ampholyte solution (acetic acid-sodium acetate solution) to obtain the electrophoresis solution. The exosome concentration in the electrophoresis solution was 1.5×10 8 particles / ml, the molar ratio of acetate ion to sodium ion is 1:0.323; the concentration of acetate ion is 0.02065mol / L.

[0040] B. Set the constant voltage to 300V, the electrophoresis liquid pump speed to 3ml / min, the pump speed of the anode buffer solution (100mM H3PO4) and the cathode buffer solution (100mM NaOH) to 4ml / min, and start RFFIEF separation and focusing of the electrophoresis liquid. The separation chamber temperature is set to 8℃ during focusing. RFFIEF is cycled back and forth until the current drops to 3mA and the change is within ±1mA within 30min, and the components with pH values ​​of 4-7 are collected.

[0041] Example 2 This embodiment provides a method for preparing exosomes, which is as follows: 1. Cleansing milk 10L of raw milk was processed by a centrifugal milk purifier at a temperature of 4°C, a rotation speed of 4500 rpm, and a time of 10 min.

[0042] 2. Centrifugal separation The skimmed milk with a fat content of 0.05% was obtained by centrifugation at a centrifugal separator with a centrifugal temperature of 45°C and a rotation speed of 4500 rpm.

[0043] 3. Isoelectric point precipitation separation In the skimmed milk of step 2, acetic acid-sodium acetate solution (the concentration of acetate ion is 0.071 mol / L, and the molar ratio of acetic acid to sodium acetate is 1:1.786) is added dropwise, the pH is adjusted to 4.6 (the osmotic pressure of the solution is 308 mOsm / L), and after acid precipitation for 1 hour, centrifugation is performed using a centrifuge at a centrifugal temperature of 0°C and a rotation speed of 8000 rpm for 2 hours to obtain a whey solution.

[0044] 4. Microfiltration The whey liquid is microfiltered to obtain a clear permeate. The pore size of the microfiltration membrane is 1 μm, the microfiltration temperature is 7°C, and the pressure is 6 bar.

[0045] 5. Ultrafiltration The permeate was diafiltered with an aqueous NaCl solution (1%) using an ultrafiltration membrane until the protein content in the retentate was less than 2 μg / mL and the osmotic pressure was 281 mOsm / L.

[0046] The pore size of the ultrafiltration membrane was 200 kDa, the ultrafiltration temperature was controlled at 7 °C, and the pressure was 0.05 MPa; 6. Free-flow isoelectric focusing (FFIEF) A. The retentate solution was mixed with the carrier ampholyte solution (acetic acid-sodium acetate solution) to obtain the electrophoresis solution. The exosome concentration in the electrophoresis solution was 1×10 10 particles / ml, the molar ratio of acetate ion to sodium ion is 1:0.523; the concentration of acetate ion is 0.02875mol / L.

[0047] B. Set the constant voltage to 500V, the electrophoresis liquid pump speed to 1ml / min, the pump speed of the anode buffer solution (100mM H3PO4) and the cathode buffer solution (100mM NaOH) to 3ml / min, and start RFFIEF separation and focusing of the electrophoresis liquid. The separation chamber temperature is set to 2℃ during focusing. RFFIEF is repeated until the current drops to 3mA and ends when the change is ±1mA within 30min. Collect the components with pH values ​​of 4-7.

[0048] Example 3 This embodiment provides a method for preparing exosomes, which is as follows: 1. Cleansing milk 10L of raw milk was processed by a centrifugal milk purifier at a temperature of 5°C, a rotation speed of 4500 rpm, and a time of 10 min.

[0049] 2. Centrifugal separation The skimmed milk with a fat content of 0.05% was obtained by centrifugation at a centrifugal separator with a centrifugal temperature of 45°C and a rotation speed of 4500 rpm.

[0050] 3. Isoelectric point precipitation separation In the skimmed milk of step 2, acetic acid-sodium acetate solution (the concentration of acetate ion is 0.065 mol / L, and the molar ratio of acetic acid to sodium acetate is 1:1.617) is added dropwise, the pH is adjusted to 4.6 (the osmotic pressure of the solution is 301 mOsm / L), and after acid precipitation for 1 hour, centrifugation is performed using a centrifuge at a centrifugal temperature of 4°C and a rotation speed of 10,000 rpm for 2 hours to obtain a whey solution.

[0051] 4. Microfiltration The whey liquid is microfiltered to obtain a clear permeate. The pore size of the microfiltration membrane is 0.8 μm, the microfiltration temperature is 6°C, and the pressure is 4 bar.

[0052] 5. Ultrafiltration The permeate was diafiltered with an aqueous NaCl solution (0.9%) using an ultrafiltration membrane until the protein content in the retentate was less than 2 μg / mL and the osmotic pressure was 283 mOsm / L.

[0053] The pore size of the ultrafiltration membrane was 300 kDa, the ultrafiltration temperature was controlled at 6 °C, and the pressure was 0.1 MPa; 6. Free-flow isoelectric focusing (FFIEF) A. The retentate solution was mixed with the carrier ampholyte solution (acetic acid-sodium acetate solution) to obtain the electrophoresis solution. The exosome concentration in the electrophoresis solution was 2.5×10 9 particles / ml, the molar ratio of acetate ion to sodium ion is 1:0.423; the concentration of acetate ion is 0.02567mol / L.

[0054] B. Set the constant voltage to 400V, the electrophoresis liquid pump speed to 2ml / min, the pump speed of the anode buffer solution (100mM H3PO4) and the cathode buffer solution (100mM NaOH) to 3ml / min, and start RFFIEF separation and focusing of the electrophoresis liquid. The separation chamber temperature is set to 4℃ during focusing. RFFIEF is cycled back and forth until the current drops to 3mA and the change is within ±1mA within 30min, and the components with pH values ​​of 4-7 are collected.

[0055] Comparative Example 1 This comparative example provides a method for preparing exosomes, which is basically the same as Example 1, except that in step (1), acetic acid solution is used to adjust the pH value, and NaCl is added at the same time as acetic acid to maintain the osmotic pressure of the acid precipitation system between 293 mOsm / L.

[0056] Comparative Example 2 This comparative example provides a method for preparing exosomes, which is substantially the same as that of Example 1, except that in step (4), the exosome retentate is mixed with an acetic acid-sodium acetate solution to make the concentration of exosomes in the electrophoresis solution 2.5×10 11 The molar ratio of acetate ions to sodium ions and the concentration of acetate ions are the same as those in Example 1.

[0057] Comparative Example 3 This comparative example provides a method for preparing exosomes, which is basically the same as Example 1, except that in step (4), the exosome retentate is mixed with the acetic acid-sodium acetate solution so that the concentration of exosomes and the concentration of acetate ions in the electrophoresis solution are the same as those in Example 1, and the molar ratio of acetate ions to sodium ions is changed to 1:0.21.

[0058] Comparative Example 4 This comparative example provides a method for preparing exosomes, which is basically the same as Example 1, except that in step (4), a sodium hydrogen phosphate-citric acid solution is used as a carrier ampholyte solution, and the concentration of exosomes in the electrophoresis solution is the same as that in Example 1, the molar ratio of sodium hydrogen phosphate to citric acid is 1:1.335, and the concentration of sodium ions is 0.671 mol / L.

[0059] Comparative Example 5 This comparative example provides a method for preparing exosomes, which is basically the same as Example 1, except that in step (4), Tris-glycine solution is used as the carrier ampholyte solution, and the concentration of exosomes in the electrophoresis solution is the same as that in Example 1, with the concentration of tris(hydroxymethyl)methylglycine (TRIS) being 0.0088 mol / L and the concentration of glycine being 0.0112 mol / L.

[0060] Experimental example This experimental example evaluates the exosome preparation effect of the above examples and comparative examples. Specifically, based on the TEM identification and analysis of the component particle morphology technology, the TEM imaging analysis of the samples to be tested in each example was performed. It was observed that the particles of 30-150nm under the transmission electron microscope had the typical morphological characteristics of exosomes - mostly cup-shaped round or quasi-round membrane vesicles, and the double membrane structure of the vesicles was visible, with low electron density components in the center, concentrated distribution and clear boundaries. Typical observation results are as follows Figure 1 As shown, the purity and recovery rate of exosomes were then tested according to the following method. The results are shown in Table 1: 1. The exosome concentration (total concentration of particles with a particle size between 30 and 150 nm) in the exosome retentate obtained in step 5 of each case was detected by the NTA method and calculated as A1 (particles / mL).

[0061] 2. The particle size distribution and concentration of particles of each size contained in the exosome product obtained in step 6 of each example were detected by the NTA method. According to the particle size distribution diagram, particles with a size of 30-150 nm were selected, and their particle concentrations were added up to be calculated as A2 (particles / mL); the concentrations of particles of all particle sizes were added up to obtain the total particle concentration, which was calculated as A3 (particles / mL).

[0062] For specific detection methods, please refer to: Human Mesenchymal Stem Cell Exosome Preparation and Inspection Specifications, T / FDSA 0049-2024, Group Standard.

[0063] 3. Calculate the purity and recovery rate of exosomes using the following formula: Exosome purity = A2 / A3 × 100%; exosome recovery rate = A2 / A1 × 100%.

[0064] Table 1

[0065] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing exosomes, using animal milk as a raw material to prepare exosomes, characterized in that: include: (1) Isoelectric precipitation separation: The pH value of the defatted animal milk is adjusted to 4.5-4.6 with an acetic acid-sodium acetate solution, and after sufficient acid precipitation, the precipitate is removed and separated to obtain a whey liquid; the molar ratio of acetic acid to sodium acetate in the acetic acid-sodium acetate solution is 1: (1.4-1.8), and the concentration of acetate ions is 0.055-0.075 mol / L; (2) Microfiltration: microfiltration of the whey liquid to remove impurities and obtain a permeate; (3) Ultrafiltration: The permeate is filtered with a NaCl aqueous solution to obtain an exosome retentate; the protein content of the exosome retentate is less than 2 μg / mL and the osmotic pressure is 280-310 mOsm / L; (4) Circulating free-flow isoelectric focusing electrophoresis: the exosome retentate solution is mixed with a carrier ampholyte solution to obtain an electrophoresis solution for electrophoresis, and the components with a pH value of 4-7 are collected; the carrier ampholyte solution is an acetic acid-sodium acetate solution; the concentration of exosomes in the electrophoresis solution is 1.5×10 8 -1×10 10 particles / ml, the molar ratio of acetate ion to sodium ion is 1:(0.3-0.55), and the concentration of acetate ion is 0.02-0.029 mol / L.

2. The method for preparing exosomes according to claim 1, characterized in that: In step (1), the molar ratio of acetic acid to sodium acetate in the acetic acid-sodium acetate solution is 1:(1.6-1.62), and the concentration of acetate ions is 0.065 mol / L; And / or, in step (4), the concentration of exosomes in the electrophoresis solution is 2.5×10 9 particles / ml, the molar ratio of acetate ion to sodium ion is 1:(0.42-0.43), and the concentration of acetate ion is 0.025-0.026mol / L.

3. The method for preparing exosomes according to claim 1 or 2, characterized in that: In step (2), during the microfiltration, the pore size of the microfiltration membrane is 0.6-1.0 μm, the temperature is 4-7° C., and the pressure is 2-6 bar.

4. The method for preparing exosomes according to claim 1 or 2, characterized in that: In step (3), during the ultrafiltration, the pore size of the ultrafiltration membrane is 200-400 kDa, the temperature is 4-7°C, and the pressure is 0.05-0.2 MPa; And / or, the concentration of NaCl in the NaCl aqueous solution is 0.8%-1%.

5. The method for preparing exosomes according to claim 1 or 2, characterized in that: In step (2), during the microfiltration, the pore size of the microfiltration membrane is 0.8 μm, the temperature is 6° C., and the pressure is 4 bar; And / or, in step (3), during the ultrafiltration, the pore size of the ultrafiltration membrane is 300 kDa, the temperature is 6° C., and the pressure is 0.1 MPa; and the concentration of NaCl in the NaCl aqueous solution is 0.9%.

6. The method for preparing exosomes according to claim 1 or 2, characterized in that: In step (4), the pressure of the circulating free-flow isoelectric focusing electrophoresis is 300-500 V and the temperature is 2-8°C.

7. The method for preparing exosomes according to claim 6, characterized in that: In step (4), the pressure of the circulating free-flow isoelectric focusing electrophoresis is 400 V and the temperature is 4°C.

8. The method for preparing exosomes according to claim 1 or 2, characterized in that: In step (1), the fat content in the defatted animal milk is ≤ 0.05%; And / or, removing the precipitate by centrifugation to obtain whey liquid; the centrifugation temperature is 0-6°C and the rotation speed is 8000-15000 rpm.

9. Use of the method for preparing exosomes according to any one of claims 1 to 8 in improving the purity of exosomes when isolating exosomes from milk.

10. Use of the method for preparing exosomes according to any one of claims 1 to 8 in improving the recovery rate of exosomes when isolating exosomes from milk.

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