Exosome purification method

Abstract

The invention provides an exosome purification method. The exosome purification method comprises the following steps: adding lectin into an exosome-containing to-be-extracted solution, and stirring and mixing; adding a phosphoric acid buffer salt solution, stirring and mixing, washing and diluting to obtain a solution A containing the exosome; introducing the liquid A of the exosome into a first-stage tangential flow ultrafiltration device, and recovering the liquid after ultrafiltration to obtain penetrating fluid; then pumping the penetrating fluid into a second-stage tangential flow ultrafiltration device, and recovering the filtrate to obtain a fluid B containing the exosome; adding a sucrose solution into the solution B containing the exosome, and performing centrifugal filtration in a centrifugal tube to obtain a solution C containing the exosome; carrying out mixed mode chromatography on the liquid C containing the exosome, collecting and flowing through to obtain liquid D containing the exosome; adding hydroxyapatite into a purification column, adding the solution D containing the exosome into the purification column, uniformly stirring and mixing, shaking and incubating, transferring to a affinity chromatography column, and washing and standing to obtain a pure exosome solution. The purification method can improve the extraction efficiency and purity of the exosome.

Description

Technical Field

[0001] The invention belongs to the technical field of potting glue preparation, and specifically relates to a high thermal conductivity potting glue and a preparation method thereof. Background Art

[0002] Exosomes are a type of membrane vesicles that are released into the extracellular matrix after the fusion of intracellular multivesicular bodies with the cell membrane. They are approximately 30-150nm in diameter and contain substances such as proteins, lipids, polysaccharides and RNA. They play an important role in cell communication, drug delivery, immune regulation, tissue repair and cancer treatment. Exosomes provide new strategies and methods for disease diagnosis, treatment and prevention.

[0003] At present, the commonly used methods for extracting exosomes include differential centrifugation, ultrafiltration, centrifugation, etc. The principle of differential centrifugation is to use a high-speed centrifuge to separate cells and extracellular substances. This method has the advantages of simple operation and good separation effect, but it requires a long centrifugation time and may cause the destruction of exosomes. Ultrafiltration is a fast and efficient extraction method. Its principle is to use ultrafiltration membrane to separate cells and extracellular substances. This method has the advantages of simple operation, rapidity and high efficiency, but it requires the selection of a suitable ultrafiltration membrane to avoid membrane contamination and membrane pore size selection problems. Centrifugation is a traditional extraction method. Its principle is to use a centrifuge to separate cells and extracellular substances. This method has the advantages of simple operation and good separation effect, but it requires a long centrifugation time and a high centrifugation speed, and may cause the destruction of exosomes. Summary of the invention

[0004] The purpose of the present invention is to provide a method for purifying exosomes, which improves the purity and extraction efficiency of exosomes while maintaining the biologically active substances in exosomes by combining compound coordination reaction precipitation, secondary tangential flow ultrafiltration, centrifugation, chromatography, freezing and other processes, and promotes their better application.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: A method for purifying exosomes comprises the following steps: S1: adding lectin to the solution to be extracted containing exosomes and stirring to mix; then adding phosphate buffered saline and stirring to mix, washing and diluting to obtain a solution A containing exosomes; S2: passing the exosome liquid A in step S1 into a first-stage tangential flow ultrafiltration device, and recovering the ultrafiltered liquid to obtain a permeate; then pumping the permeate into a second-stage tangential flow ultrafiltration device, recovering the filtrate, and obtaining a liquid B containing exosomes; S3: adding the sucrose solution to the exosome-containing solution B in step S2, and centrifuging and filtering in a centrifuge tube to obtain the exosome-containing solution C; S4: subjecting the exosome-containing solution C in step S3 to mixed-mode chromatography, collecting the flow-through, and obtaining the exosome-containing solution D; S5: Add hydroxyapatite to the purification column, then add the exosome-containing solution D described in step S4 to the purification column, stir to mix, shake and incubate, transfer to the affinity chromatography column, wash and stand to obtain a pure exosome solution.

[0006] Furthermore, step S1 is specifically as follows: by weight, 15-25 parts of lectin are added to 60-100 parts of the liquid to be extracted containing exosomes, and the mixture is stirred at a rate of 300-500 r / min for 5-10 min; then 30-50 parts of phosphate buffered saline are added, and the mixture is stirred at a rate of 400-600 r / min for 10-15 min. After washing and filtering for 3-5 times, a liquid A containing exosomes is obtained.

[0007] Furthermore, the phosphate buffered saline solution is a NaH2PO4-Na2HPO4 mixed solution, and the mass ratio of the two is 1:2-5.

[0008] Furthermore, in step S2: the pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device is ≥0.28μm, and the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is ≤0.02μm; the injection pressure is 3-6PSI, and the molecular weight cutoff of the ultrafiltration membrane is 550-850kD.

[0009] Further, step S3 is specifically as follows: by weight, 60-120 parts of a sucrose aqueous solution with a mass fraction of 40-60% are added to the liquid B containing exosomes, centrifuged at 80000-100000 g for 1-2 hours, and filtered to obtain a liquid C containing exosomes.

[0010] Furthermore, in step S4, the mixed mode chromatography is as follows: the flow rate during the mixed mode chromatography is 3-7 cm / min; when the molecular weight cutoff of the mixed mode chromatography is 400-700 kD, the filler of the mixed mode chromatography is RigoseShell 400 or Capto Core 400.

[0011] Furthermore, in step S5, 35-50 parts by weight of hydroxyapatite purification column are added to the purification column, and then the exosome-containing liquid D described in step S4 is added to the purification column, and stirred and mixed at a rate of 400-600 r / min.

[0012] Furthermore, the shaking incubation time in step S5 is 50-80 min, and the rotation speed is 150-180 rpm.

[0013] Furthermore, the conditions of the chromatography treatment in step S5 are as follows: the balancing solution is 0.1-0.15M Tris-HCl, 0.3-0.5M NaCl, PH7.5-8.1; the eluent is 0.02-0.08M Tris-HCl, 0.4-0.7M NaCl, 0.5-0.8M imidazole, PH7.9; 4 column beds are balanced and 1 column bed is eluted, with a flow rate of 7-10mL / min.

[0014] Furthermore, in step S5, the mixture is washed 2-4 times with a sodium dihydrogen phosphate solution having a mass fraction of 30-40%, and allowed to stand to obtain a pure exosome solution.

[0015] Lectins, as proteins or polypeptides that can recognize and specifically bind to carbohydrate molecules, can bind to specific carbohydrates, and this binding is non-covalent, usually formed through multiple weak interaction forces (such as van der Waals forces, hydrogen bonds, etc.), so they have high specificity and affinity. Phosphate buffered saline is a commonly used buffer solution, which mainly maintains the pH value of the culture medium stable by adjusting the pH value and osmotic pressure. The tangential flow ultrafiltration device is mainly a liquid (usually called the feed liquid) flowing in a direction parallel to the membrane surface, rather than passing through the membrane vertically. This flow mode is called tangential flow. A certain pressure is applied on one side of the membrane, so that small molecules in the liquid (such as water and salts) pass through the membrane pores, while large molecules (such as proteins, virus particles, etc.) are retained and returned to the feed liquid. In this way, the concentration or purification of the target substance can be achieved. Chromatography is a technique used to separate different components in a mixture. It is achieved by introducing the sample between a stationary phase and a mobile phase and utilizing the different distribution coefficients of the components in the mixture between the stationary phase and the mobile phase. When the mixture passes through the chromatographic column, the components have different retention times depending on the strength of their interaction with the stationary phase and the mobile phase, allowing them to be separated. Separation is achieved based on the different distribution behaviors of the components between the two phases.

[0016] Compared with the prior art, the advantages and beneficial effects of the present invention are: The present invention uses processes such as precipitation, centrifugation, ultrafiltration, chromatography, etc., and connects the various steps with each other; in the precipitation stage, the separation and precipitation of relatively pure exosomes and impurities are accelerated; in the ultrafiltration stage, a tangential flow filtration method is adopted and the injection pressure is reduced to avoid damage to the exosomes; subsequently, centrifugation and mixed-mode chromatography are used to compensate for the low impurity protein removal rate brought by ultrafiltration, thereby gradually accelerating the degree of exosome purification, effectively removing the impurity proteins mixed in the exosomes, and ensuring the purity and yield of the exosomes. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] The sources of raw materials of the present invention are as follows: The extract containing exosomes came from Xi'an Ruixi Biotechnology Co., Ltd., item number 153; the lectin came from Shanghai Yuanye Biotechnology Co., Ltd., item number S12055; and the hydroxyapatite came from Shanghai Yuanye Biotechnology Co., Ltd., item number S14126.

[0019] Example 1 This embodiment provides a method for purifying exosomes, comprising the following steps: S1: Add 15 g of lectin to 60 g of the solution containing exosomes to be extracted, stir and mix at a rate of 300 r / min for 5 min; then add 30 g of phosphate buffered saline (NaH 2 PO 4 -Na 2 HPO 4 The mixed solution was stirred at a rate of 400 r / min for 10 min, and washed and filtered three times to obtain liquid A containing exosomes; S2: The liquid A containing exosomes in step S1 is passed into a first-stage tangential flow ultrafiltration device, wherein the pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device is ≥0.28 μm, the injection pressure is controlled to be 3 PSI, the molecular weight cutoff of the ultrafiltration membrane is 550 kD, and the liquid after ultrafiltration is recovered to obtain a permeate; the permeate is then pumped into a second-stage tangential flow ultrafiltration device, wherein the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is ≤0.02 μm, the filtrate is recovered to obtain a liquid B containing exosomes, the injection pressure is controlled to be 4 PSI, and the molecular weight cutoff of the ultrafiltration membrane is 650 kD; S3: Take 60 g of 40% sucrose aqueous solution and add it to liquid B containing exosomes, centrifuge at 80,000 g for 1 h, and filter to obtain liquid C containing exosomes; S4: subjecting the liquid C containing exosomes in step S3 to mixed mode chromatography, collecting the flow-through, and obtaining liquid D containing exosomes; the flow rate during the mixed mode chromatography is 3 cm / min; the molecular weight cut-off of the mixed mode chromatography is 400 kD, and the filler of the mixed mode chromatography is Rigose Shell 400; S5: Add 35g of hydroxyapatite purification column, then add the exosome-containing liquid D described in step S4 to the purification column, stir and mix at a rate of 400r / min, shake and incubate for 50min, and rotate at 150rpm; transfer to the affinity chromatography column, the conditions of the chromatography treatment are: the equilibrium liquid is 0.1mol / L Tris-HCl, 0.3mol / L NaCl, PH7.5; the eluent is 0.02mol / L Tris-HCl, 0.4mol / L NaCl, 0.5mol / L imidazole, PH7.9; balance 4 column beds, elute 1 column bed, the flow rate is 7mL / min; wash twice with 30% mass fraction sodium dihydrogen phosphate solution, and obtain a pure exosome solution after standing.

[0020] Example 2 This embodiment provides a method for purifying exosomes, comprising the following steps: S1: Add 25g of lectin to 100g of the solution containing exosomes to be extracted, stir and mix at a rate of 500r / min for 10min; then add 50g of phosphate buffered saline (NaH 2 PO 4 -Na 2 HPO 4 The mixed solution was stirred at a rate of 600 r / min for 15 min, and washed and filtered 5 times to obtain liquid A containing exosomes; S2: The liquid A containing exosomes in step S1 is passed into a first-stage tangential flow ultrafiltration device, wherein the pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device is ≥0.28 μm, the injection pressure is controlled to be 6 PSI, and the molecular weight cutoff of the ultrafiltration membrane is 850 kD, and the ultrafiltration liquid is recovered to obtain a permeate; then the permeate is pumped into a second-stage tangential flow ultrafiltration device, wherein the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is ≤0.02 μm, and the filtrate is recovered to obtain a liquid B containing exosomes, the injection pressure is controlled to be 6 PSI, and the molecular weight cutoff of the ultrafiltration membrane is 850 kD; S3: 120 g of 60% sucrose aqueous solution was added to liquid B containing exosomes, centrifuged at 100,000 g for 2 h, and filtered to obtain liquid C containing exosomes; S4: subjecting the liquid C containing exosomes in step S3 to mixed mode chromatography, collecting the flow-through, and obtaining liquid D containing exosomes; the flow rate during the mixed mode chromatography is 7 cm / min; the molecular weight cut-off of the mixed mode chromatography is 700 kD, and the filler of the mixed mode chromatography is Capto Core 400; S5: Add 50g of hydroxyapatite purification column, then add the exosome-containing liquid D described in step S4 to the purification column, stir and mix at a rate of 600r / min, shake and incubate for 80min, and rotate at 180rpm; transfer to the affinity chromatography column, the conditions of the chromatography treatment are: the equilibrium liquid is 0.15mol / L Tris-HCl, 0.5mol / L NaCl, PH7.8; the eluent is 0.08mol / L Tris-HCl, 0.7mol / L NaCl, 0.8mol / L imidazole, PH7.9; balance 4 column beds, elute 1 column bed, the flow rate is 10mL / min; wash 4 times with 40% mass fraction sodium dihydrogen phosphate solution, and obtain a pure exosome solution after standing.

[0021] Example 3 This embodiment provides a method for purifying exosomes, comprising the following steps: S1: Add 20g of lectin to 80g of the solution containing exosomes to be extracted, stir and mix at a rate of 450r / min for 7min; then add 40g of phosphate buffered saline (NaH 2 PO 4 -Na 2 HPO 4 The mixed solution was stirred at a rate of 550 r / min for 12 min, and washed and filtered four times to obtain liquid A containing exosomes; S2: The liquid A containing exosomes in step S1 is passed into a first-stage tangential flow ultrafiltration device, wherein the pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device is ≥0.28 μm, the injection pressure is controlled to be 5 PSI, the molecular weight cutoff of the ultrafiltration membrane is 700 kD, and the liquid after ultrafiltration is recovered to obtain a permeate; then the permeate is pumped into a second-stage tangential flow ultrafiltration device, wherein the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is ≤0.02 μm, the filtrate is recovered to obtain a liquid B containing exosomes, the injection pressure is controlled to be 4 PSI, and the molecular weight cutoff of the ultrafiltration membrane is 650 kD; S3: Take 100 g of 55% sucrose aqueous solution and add it to liquid B containing exosomes, centrifuge at 90,000 g for 1.5 h, and filter to obtain liquid C containing exosomes; S4: subjecting the liquid C containing exosomes in step S3 to mixed mode chromatography, collecting the flow-through, and obtaining a liquid D containing exosomes; the flow rate during the mixed mode chromatography is 5 cm / min; the molecular weight cut-off of the mixed mode chromatography is 600 kD, and the filler of the mixed mode chromatography is Capto Core 400; S5: Add 45g of hydroxyapatite purification column, then add the exosome-containing liquid D described in step S4 to the purification column, stir and mix at a rate of 500r / min, shake and incubate for 70min, and rotate at a speed of 175rpm; transfer to an affinity chromatography column, and the conditions of the chromatography treatment are as follows: the equilibrium liquid is 0.13mol / L Tris-HCl, 0.4mol / L NaCl, PH7.9; the eluent is 0.06mol / L Tris-HCl, 0.55mol / L NaCl, 0.7mol / L imidazole, PH7.9; balance 4 column beds, elute 1 column bed, and the flow rate is 8mL / min; wash 3 times with 35% mass fraction sodium dihydrogen phosphate solution, and obtain a pure exosome solution after standing.

[0022] Comparative Example 1 The difference between this comparative example and Example 1 is that in step S1, lectin and phosphate buffered saline solution treatment are not performed, and the other steps are the same.

[0023] Comparative Example 2 The difference between this comparative example and Example 1 is that the tangential flow ultrafiltration device is not used in step S2, and the other steps are the same.

[0024] Comparative Example 3 The difference between this comparative example and Example 1 is that the sucrose solution treatment in step S3 is not performed, and the other steps are the same.

[0025] Comparative Example 4 The difference between this comparative example and Example 1 is that the chromatography treatment in step S4 is not performed, and the other steps are the same.

[0026] Comparative Example 5 The difference between this comparative example and Example 1 is that hydroxyapatite treatment is not performed in step S5, and the other steps are the same.

[0027] Comparative Example 6 The difference between this comparative example and Example 1 is that no affinity chromatography column treatment is performed in step S5, and the other steps are the same.

[0028] Performance Test: TEM characterization of exosomes: Take the exosomes extracted from each embodiment and comparative example, drop them onto the copper grid, negatively stain with uranyl acetate, wash the copper grid twice with deionized water, dry it, and perform electron microscopy detection and imaging at 100kv to obtain the morphology and size of the exosomes. Exosome particle size measurement: Take out 10μL of the obtained exosomes and dilute them to 30μL; first use the standard sample to perform the instrument performance test and then load the exosome sample. Quantitative detection of total protein concentration: Rapidly melt the exosomes at 37°C and quickly add 5× RIPA lysis buffer. After mixing, lyse on ice for 30min, mixing during the period. Prepare the standard sample for measuring protein concentration by BCA method, take 5μL of sample and add it to the BCA mixed solution, and mix. Incubate at 37°C for 30min, and detect the absorbance value at OD562 nm on the microplate reader. Exosome yield = exosome weight / mass of the liquid to be extracted containing exosomes × 100%. The results are shown in the table below: Table 1 Performance test results

[0029] From the above performance test results, it can be seen that the purity, average particle size, protein concentration and exosome yield of Examples 1-3 are all good. In particular, the comprehensive performance of Implementation 3 is the most outstanding. However, because the comparative example does not adopt the necessary technical solution, it is significantly worse than the embodiment in the corresponding performance test. The above experimental results further prove the importance of the technical solution defined in the present invention for its technical effect.

[0030] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for purifying exosomes, characterized in that: The steps include: S1: adding lectin to the solution to be extracted containing exosomes and stirring to mix; then adding phosphate buffered saline and stirring to mix, washing and diluting to obtain a solution A containing exosomes; S2: passing the exosome liquid A in step S1 into a first-stage tangential flow ultrafiltration device, and recovering the ultrafiltered liquid to obtain a permeate; then pumping the permeate into a second-stage tangential flow ultrafiltration device, recovering the filtrate, and obtaining a liquid B containing exosomes; S3: adding the sucrose solution to the exosome-containing solution B in step S2, and centrifuging and filtering in a centrifuge tube to obtain the exosome-containing solution C; S4: subjecting the exosome-containing solution C in step S3 to mixed-mode chromatography, collecting the flow-through, and obtaining the exosome-containing solution D; S5: Add hydroxyapatite to the purification column, then add the exosome-containing solution D described in step S4 to the purification column, stir to mix, shake and incubate, transfer to the affinity chromatography column, wash and stand to obtain a pure exosome solution.

2. The method for purifying exosomes according to claim 1, characterized in that: Step S1 is specifically as follows: by weight, add 15-25 parts of lectin to 60-100 parts of the liquid to be extracted containing exosomes, stir and mix at a rate of 300-500 r / min for 5-10 minutes; then add 30-50 parts of phosphate buffered saline, stir and mix at a rate of 400-600 r / min for 10-15 minutes, wash and filter 3-5 times to obtain liquid A containing exosomes.

3. The method for purifying exosomes according to claim 2, characterized in that: The phosphate buffered saline solution is a NaH2PO4-Na2HPO4 mixed solution, and the mass ratio of the two is 1:2-5.

4. The method for purifying exosomes according to claim 1, characterized in that: In step S2: the pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device is ≥0.28μm, and the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is ≤0.02μm; the injection pressure is 3-6PSI, and the molecular weight cutoff of the ultrafiltration membrane is 550-850kD.

5. The method for purifying exosomes according to claim 1, characterized in that: Step S3 is specifically as follows: by weight, 60-120 parts of a sucrose aqueous solution with a mass fraction of 40-60% are added to the liquid B containing exosomes, centrifuged at 80000-100000 g for 1-2 hours, and filtered to obtain the liquid C containing exosomes.

6. The method for purifying exosomes according to claim 1, characterized in that: The mixed mode chromatography in step S4 is as follows: the flow rate during the mixed mode chromatography is 3-7 cm / min; when the molecular weight cutoff of the mixed mode chromatography is 400-700 kD, the filler of the mixed mode chromatography is Rigose Shell 400 or Capto Core 400.

7. The method for purifying exosomes according to claim 1, characterized in that: In step S5, 35-50 parts by weight of hydroxyapatite purification column are added to the purification column, and then the exosome-containing liquid D described in step S4 is added to the purification column, and stirred at a rate of 400-600 r / min.

8. The method for purifying exosomes according to claim 1, characterized in that: The shaking incubation time in step S5 is 50-80 min, and the rotation speed is 150-180 rpm.

9. The method for purifying exosomes according to claim 1, characterized in that: The conditions of the chromatography treatment in step S5 are as follows: the balancing solution is 0.1-0.15M Tris-HCl, 0.3-0.5M NaCl, pH 7.5-8.1; the eluent is 0.02-0.08M Tris-HCl, 0.4-0.7M NaCl, 0.5-0.8M imidazole, pH 7.9; 4 column beds are balanced and 1 column bed is eluted, with a flow rate of 7-10mL / min.

10. The method for purifying exosomes according to claim 1, characterized in that: In step S5, the mixture is washed 2-4 times with a sodium dihydrogen phosphate solution having a mass fraction of 30-40%, and then allowed to stand to obtain a pure exosome solution.

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