Exosome separation and extraction method

Abstract

The invention relates to an exosome separation and extraction method, in particular to a human peripheral blood macrophage exosome separation and extraction method. The invention discloses a method for separating and extracting human peripheral blood macrophage exosomes, which has the characteristics of convenience in collection, safety and reliability compared with a common separation and extraction method, avoids the problems of ethical heredity and rejection anaphylaxis of exosomes of heterologous tissue cells or exosomes of allogeneic tissue cells such as umbilical cord, abdominal cavity and the like, and has the advantages of simple operation and high efficiency. The exosome is easy to apply clinically and is an ideal source exosome.

Description

Technical Field

[0001] The present invention relates to a method for separating and extracting exosomes, and in particular to a method for separating and extracting exosomes from human peripheral blood macrophages. Background Art

[0002] Exosomes are nanoscale vesicles secreted by cells and are widely present in various biological samples. They contain a variety of bioactive molecules, such as proteins, nucleic acids, lipids, etc., and play an important role in intercellular communication and material transport. The separation and extraction of exosomes is of great significance for studying their biological characteristics, developing drug carriers, and disease diagnosis. However, due to the small size of exosomes, their low concentration in biological samples, and the presence of a large number of interfering substances, their separation and extraction are difficult.

[0003] Exosomes can be isolated and extracted from almost all tissue cells. However, considering the feasibility of future clinical applications, there are ethical and genetic issues in the use of exosomes from xenogeneic tissue cells in humans. Exosomes from allogeneic tissue cells such as umbilical cord and peritoneal cavity have the problem of rejection allergic reactions and are relatively difficult to collect. However, exosomes from autologous peripheral blood macrophages of the human body are easy to collect, safe and reliable, and do not have ethical genetic and rejection allergic reaction issues. They are easier to use in clinical practice and are an ideal source of exosomes.

[0004] At present, the commonly used exosome separation and extraction methods include ultracentrifugation, filtration centrifugation, density gradient centrifugation, immunomagnetic beads, PS affinity method, chromatography, microfluidic separation and immunological separation. These separation and extraction methods have their own advantages and disadvantages. The common problems are complex operation, low extraction efficiency, and insufficient separation purity. Therefore, we proposed a method for the separation and extraction of exosomes.

[0005] In addition, during the separation and extraction of exosomes, we used PVDF membranes for dynamic filtration. However, the existing filtration membranes have low filtration efficiency and strong protein adsorption, which affects the subsequent separation of exosomes. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention discloses a method for separating and extracting exosomes, comprising the following steps: Step 1: Collect fresh anticoagulated peripheral blood from normal subjects, centrifuge, discard the upper plasma, obtain the lower precipitated cells, add PBS buffer to make a cell suspension, slowly add the cell suspension to the upper layer of lymphocyte stratification fluid, centrifuge, and obtain mononuclear cells (PBMC); Step 2: Add the prepared centrifugal media of various concentrations to the centrifuge tube in order of thick to thin, and finally add the mononuclear cell suspension to perform discontinuous density gradient centrifugation, and aspirate the macrophage layer to obtain purified macrophages; Step 3: Mix the macrophages with PBS buffer in a certain ratio and mix by hand or with a blender to make a cell suspension; Step 4: Separate the cells of the cell suspension from the supernatant, transfer the supernatant to a centrifuge tube using a pipette, and centrifuge at a centrifugal force of 3000xg-5000xg for 8-12 minutes to separate the precipitate and the supernatant; Step 5: Add the supernatant to the first dynamic filter membrane device for filtration, and rotate the dynamic filter membrane device at a speed of 100 rpm at room temperature to allow the exosomes in the macrophages to pass through the filter membrane into the collection vessel; Step 6: Wash the collected filtrate with 0.9% PBS buffer, centrifuge at 1000 rpm for 15 minutes at room temperature, and collect the supernatant; Step 7: Repeat steps 4 and 6 at least twice, centrifuge the collected supernatant using an ultracentrifuge, and pass it through a second dynamic filtration membrane device to separate and collect exosomes.

[0007] Preferably, the centrifugation in step 1 is carried out at room temperature and 2000 rpm for 20 minutes.

[0008] Preferably, the discontinuous density gradient centrifugation medium in step 2 is Fcoll, and the centrifugation is carried out at 4°C and 2000 g / min.

[0009] Preferably, the macrophages to be separated in step three are mixed with PBS buffer at a volume ratio of 10:1.

[0010] Preferably, the cells are peripheral blood macrophages.

[0011] Preferably, the filter membrane of the first dynamic filter membrane device is one or more of polyvinylidene fluoride filter membrane, polytetrafluoroethylene filter membrane, nitrocellulose filter membrane or nylon filter membrane, and its pore size is 0.8-1.5 microns; in the step 5, negative pressure is applied above the filter membrane of the dynamic filter membrane device, and the negative pressure is 0.05-0.2MPa Preferably, the pore size of the filter membrane of the second dynamic filter membrane device in step seven is 0.2-0.4 microns, and it is made of polyvinylidene fluoride; the ultracentrifuge in step seven is centrifuged at 10°C-15°C and 12000-18000rpm for 20-30 minutes.

[0012] Preferably, the buffer in step six is ​​a phosphate buffer with a pH of 7.0-7.5, and the amount of the buffer added is 0.5%-1.5% of the total volume of the supernatant.

[0013] Preferably, the preparation method of the second dynamic filter membrane is as follows: 1) Mix polyvinylidene fluoride, N, N-dimethylformamide and petroleum ether at a temperature of 60-90°C, stir and disperse for 5-9 minutes, add triacetin and polyurethane, ultrasonically disperse for 10-20 minutes, continue stirring for 2-4 hours, add polymethyl methacrylate, stir and mix for 8-12 minutes to obtain PVDF spinning solution; 2) The PVDF spinning solution prepared in step 1) is subjected to electrospinning to obtain a nanofiber membrane to obtain a PVDF nanofiber membrane; the PVDF nanofiber membrane is placed in a supercritical fluid reactor, a supercritical fluid is introduced, and the temperature is heated at 150-200°C and a pressure of 10-14 MPa for 6-10 minutes, and the temperature is reduced at a rate of 0.4-0.8°C per minute to 60-80°C, and then cooled to room temperature to obtain a PVDF dynamic filter membrane.

[0014] Preferably, the weight ratio of polyvinylidene fluoride to triacetin is (0.2-0.4):1; the weight ratio of polyvinylidene fluoride to triacetin is 0.3:1; the weight ratio of polyurethane to polymethyl methacrylate is (1.5-2):1; the weight ratio of polyurethane to polymethyl methacrylate is 1.7:1; the viscosity of polyvinylidene fluoride is 9000-15000mPa·s; PVDF nanofiber membrane, the thickness of the PVDF nanofiber membrane is 110-130μm.

[0015] Compared with the prior art, the present invention has the following advantages: First, the present invention separates and extracts human peripheral blood macrophage exosomes. Compared with the commonly used separation and extraction of exosomes from xenogeneic tissue cells or allogeneic tissue cells such as umbilical cord and peritoneal cavity, the present invention has the characteristics of convenient collection, safety and reliability, and does not have ethical inheritance and rejection allergic reaction problems. It is easier to apply clinically and is an ideal source of exosomes. Peripheral blood mononuclear cells are obtained by centrifugation, and more purified macrophages are obtained by discontinuous density gradient centrifugation, which will increase the reliability of subsequent experiments.

[0016] Secondly, the present invention can better protect the physicochemical structure and biological activity of exosomes by mixing peripheral blood with PBS buffer, and the supernatant is filtered through a dynamic filter membrane device and repeated at least twice, which can increase the exosome yield.

[0017] In addition, the PVDF dynamic filter membrane prepared by the present invention has a high porosity of more than 90%, which improves the separation purity of exosomes and separates purer exosomes. Its porosity reaches 90%, which improves the separation purity of exosomes and reduces the adhesion of proteins. It can further separate purer exosomes, and the extraction purity of exosomes can reach more than 90%; the present application adds polyurethane and polymethyl methacrylate to the PVDF spinning solution, which can enhance the interaction between fibers and improve the mechanical strength of the PVDF fiber membrane. The ratio of PVDF to organic solvent also has a certain effect on the mechanical strength of the PVDF fiber membrane, and the added polyurethane can improve the tensile strength and tensile strength of the PVDF nanofiber membrane, which can enhance The contact area between the large fibers is increased to improve the mechanical strength of the PVDF nanofiber membrane; the present application also adds triacetin and petroleum ether, triacetin can change the internal structure of the polyvinylidene fluoride nanofiber membrane, and petroleum ether, as a porogen, can affect the morphology of the fiber and promote the diffusion of solutes inside the membrane, thereby affecting its structure. The combination of thermodynamics and fluid dynamics of the supercritical fluid of the present application has a very obvious effect on the preparation of high-porosity fibers; the interaction between the supercritical fluid and the PVDF nanofiber membrane of the present application also makes its porosity reach more than 90%, and the fiber has good morphology control, its shape is cylindrical, and the pore size is 500-800nm, which can improve the porosity of the nanofiber membrane and improve the separation purity of exosomes. DETAILED DESCRIPTION

[0018] A method for separating and extracting exosomes comprises the following steps: Step 1: Collect fresh anticoagulated peripheral blood from normal subjects, centrifuge at room temperature and 2000 rpm for 20 minutes, discard the upper plasma, obtain the lower precipitated cells, add PBS buffer to make a cell suspension, slowly add the cell suspension to the upper layer of lymphocyte stratification solution, centrifuge, and obtain mononuclear cells (PBMC); Step 2: Add the prepared centrifugal media of various concentrations to the centrifuge tube in order of thick to thin. The discontinuous density gradient centrifugation medium is Fcoll. Finally, add the mononuclear cell suspension and centrifuge at 4°C and 2000 speed for 20 minutes for discontinuous density gradient centrifugation. Aspirate the macrophage layer to obtain purified macrophages. Step 3: Mix the macrophages with PBS buffer at a volume ratio of 10:1 and mix by hand or with a blender to make a cell suspension; Step 4: Separate the cells of the cell suspension from the supernatant, transfer the supernatant to a centrifuge tube using a pipette, and centrifuge at 3000 x g for 12 minutes to separate the precipitate and the supernatant; Step 5: Add the supernatant to the first dynamic membrane filter device for filtration. The filter membrane uses a common commercial filter membrane with a pore size of 1 micron. The dynamic membrane filter device is rotated at a speed of 100 rpm at room temperature. A negative pressure of 0.1 MPa is applied above the filter membrane of the dynamic membrane filter device to allow the exosomes in the macrophages to pass through the filter membrane into the collection vessel. Step 6: Wash the collected filtrate with 0.9% PBS buffer, the buffer is a phosphate buffer of pH 7.2, the amount of the buffer added is 1% of the total volume of the supernatant, centrifuge at room temperature at 1000 rpm for 15 minutes, and collect the supernatant; Step 7: Repeat steps 4 and 6 twice, and centrifuge the collected supernatant with an ultracentrifuge at a speed of 12,000 rpm and 15°C for 20 minutes, and pass it through a second dynamic filtration membrane device. The filter membrane of the second dynamic filtration membrane device also uses an ordinary commercial filter membrane with a pore size of 0.3 microns to separate and collect exosomes.

[0019] The obtained exosomes were characterized, the exosome protein concentration was determined by the BCA method, the exosome particle size was determined by the NTA method, the exosome morphology was observed by transmission electron microscopy, and the purity of the exosome extraction was calculated. The results showed that the exosome protein concentration was 0.8 mg / mL, the particle size was mainly distributed in the range of 50-150 nm, and the purity of the exosome extraction was 81%.

[0020] Example 2: The exosome separation and extraction method of this example is basically the same as that of Example 1, except that: the preparation method of the second dynamic filter membrane in step 7 of Example 2 is as follows: 1) Mix polyvinylidene fluoride, N, N-dimethylformamide and petroleum ether at a temperature of 75°C, stir and disperse for 7 minutes, add triacetin according to the weight ratio of polyvinylidene fluoride to triacetin of 0.3:1, then add polyurethane and polymethyl methacrylate according to the weight ratio of polyurethane to polymethyl methacrylate of 1.7:1, ultrasonically disperse for 15 minutes, continue stirring for 3 hours, add polymethyl methacrylate, stir and mix for 10 minutes to obtain PVDF spinning solution; the viscosity of polyvinylidene fluoride is 12000mPa·s; 2) The PVDF spinning solution prepared in step 1) is subjected to electrospinning to obtain a nanofiber membrane to obtain a PVDF nanofiber membrane, wherein the thickness of the PVDF nanofiber membrane is 120 μm; the PVDF nanofiber membrane is placed in a supercritical fluid reactor, a supercritical fluid is introduced, and the temperature is heated at 175°C and a pressure of 12 MPa for 8 minutes, and the temperature is lowered at a rate of 0.6°C per minute to 70°C, and cooled to room temperature to obtain a PVDF dynamic filter membrane; the pore size of the filter membrane of the first dynamic filter membrane device is 1 micron; a negative pressure is applied above the filter membrane of the dynamic filter membrane device, and the negative pressure is 0.1 MPa.

[0021] In step 6 and step 7, the filter membrane of the second dynamic filter membrane device also adopts the PVDF filter membrane defined in the present invention, and its pore size is 0.3 micron; in step 7, the ultracentrifuge is centrifuged at a speed of 15000 rpm and 10°C for 30 minutes.

[0022] The obtained exosomes were characterized under the same conditions and methods as in Example 1. The results showed that the exosome protein concentration was 1.2 mg / mL, the particle size was mainly distributed in the range of 75-100 nm, and the exosome extraction purity was as high as 94%. Compared with Example 1, after using the PVDF filter membrane defined in the present invention, the extraction purity of the exosomes was significantly improved, and the protein concentration was also increased, indicating that the PVDF filter membrane of the present invention has a better effect in the separation and extraction of exosomes.

[0023] Although the specific embodiments of the present invention are described in detail above, the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in this field without departing from the purpose of the present invention, and modifications or deformations without creative labor are still within the protection scope of the present invention.

Claims

1. A method for separating and extracting exosomes, characterized in that: The following steps are involved: Step 1: Collect fresh anticoagulated peripheral blood from normal subjects, centrifuge, discard the upper plasma, obtain the lower precipitated cells, add PBS buffer to make a cell suspension, slowly add the cell suspension to the upper layer of lymphocyte stratification fluid, centrifuge, and obtain mononuclear cells (PBMC); Step 2: Add the prepared centrifugal media of various concentrations to the centrifuge tube in order of thick to thin, and finally add the mononuclear cell suspension to perform discontinuous density gradient centrifugation, and aspirate the macrophage layer to obtain purified macrophages; Step 3: Mix the macrophages with PBS buffer in a certain proportion and mix by hand or with a blender to make a cell suspension; Step 4: Separate the cells of the cell suspension from the supernatant, transfer the supernatant to a centrifuge tube using a pipette, and centrifuge at a centrifugal force of 3000xg-5000xg for 8-12 minutes to separate the precipitate and the supernatant; Step 5: Add the supernatant to the first dynamic filter membrane device for filtration, and rotate the dynamic filter membrane device at a speed of 100 rpm at room temperature to allow the exosomes in the macrophages to pass through the filter membrane into the collection vessel; Step 6: Wash the collected filtrate with 0.9% PBS buffer, centrifuge at 1000 rpm for 15 minutes at room temperature, and collect the supernatant; Step 7: Repeat steps 4 and 6 at least twice, centrifuge the collected supernatant using an ultracentrifuge, and pass it through a second dynamic filtration membrane device to separate and collect exosomes.

2. The method for separating and extracting exosomes according to claim 1, characterized in that: In step 1, the centrifugation was carried out at room temperature and 2000 rpm for 20 minutes.

3. The method for separating and extracting exosomes according to claim 1, characterized in that: In the step 2, the discontinuous density gradient centrifugation medium is Fcoll, and the centrifugation is carried out at 4° C. and 2000 g / min.

4. The method for separating and extracting exosomes according to claim 1, characterized in that: The macrophages to be separated in step 3 are mixed with PBS buffer at a volume ratio of 10:

1.

5. The method for separating and extracting exosomes according to claim 1, characterized in that: The filter membrane of the first dynamic filter membrane device is one or more of polyvinylidene fluoride filter membrane, polytetrafluoroethylene filter membrane, nitrocellulose filter membrane or nylon filter membrane, and its pore size is 0.8-1.5 microns; in the step five, negative pressure is applied above the filter membrane of the dynamic filter membrane device, and the negative pressure is 0.05-0.2MPa.

6. The method for isolating and extracting exosomes according to claim 1, characterized in that: The pore size of the filter membrane of the second dynamic filter membrane device in step seven is 0.2-0.4 microns, and it is made of polyvinylidene fluoride; the ultracentrifuge in step seven is centrifuged at 10°C-15°C and 12000-18000rpm for 20-30 minutes.

7. The method for separating and extracting exosomes according to claim 1, characterized in that: The buffer in step six is ​​a phosphate buffer with a pH of 7.0-7.5, and the amount of the buffer added is 0.5%-1.5% of the total volume of the supernatant.

8. The method for separating and extracting exosomes according to claim 5, characterized in that: The preparation method of the second dynamic filter membrane is as follows: 1) Mix polyvinylidene fluoride, N, N-dimethylformamide and petroleum ether at a temperature of 60-90°C, stir and disperse for 5-9 minutes, add triacetin and polyurethane, ultrasonically disperse for 10-20 minutes, continue stirring for 2-4 hours, add polymethyl methacrylate, stir and mix for 8-12 minutes to obtain PVDF spinning solution; 2) The PVDF spinning solution prepared in step 1) is subjected to electrospinning to obtain a nanofiber membrane to obtain a PVDF nanofiber membrane; the PVDF nanofiber membrane is placed in a supercritical fluid reactor, a supercritical fluid is introduced, and the temperature is heated at 150-200°C and a pressure of 10-14 MPa for 6-10 minutes, and the temperature is reduced at a rate of 0.4-0.8°C per minute to 60-80°C, and then cooled to room temperature to obtain a PVDF dynamic filter membrane.

9. The method for separating and extracting exosomes according to claim 7, characterized in that: The weight ratio of polyvinylidene fluoride to triacetin is (0.2-0.4):1; the weight ratio of polyvinylidene fluoride to triacetin is 0.3:1; the weight ratio of polyurethane to polymethyl methacrylate is (1.5-2):1; the weight ratio of polyurethane to polymethyl methacrylate is 1.7:1; the viscosity of polyvinylidene fluoride is 9000-15000mPa·s; PVDF nanofiber membrane, the thickness of the PVDF nanofiber membrane is 110-130μm.