CN112899218A

CN112899218A - Dual tangential flow filtration system for exosome extraction, and preparation method and application of exosome

Info

Publication number: CN112899218A
Application number: CN202110138540.6A
Authority: CN
Inventors: 张海心
Original assignee: Ruitai Biotechnology Shenyang Co ltd
Current assignee: Ruitai Biotechnology Shenyang Co ltd
Priority date: 2021-02-01
Filing date: 2021-02-01
Publication date: 2021-06-04

Abstract

The invention discloses a double tangential flow filtration system for exosome extraction, and a preparation method and application of exosomes. The method comprises the following steps: pumping the sample solution containing the exosome into a first storage tank, pumping the sample solution into a first filtering device, circularly concentrating and filtering, and collecting filtrate into a second storage tank; when the volume of the liquid in the first storage tank is concentrated to the minimum operation volume, pumping the liquid in the second storage tank into a second filtering device, and performing circulating concentration and filtering; and collecting the liquid in the second storage tank to obtain the solution of the extracted and purified exosomes. The exosome can be used for exosome medical and beauty products, exosome liquid dressing for promoting wound healing, exosome spray for relieving asthma and acute respiratory distress and preparation of exosome related biological products. The invention extracts and purifies the exosome by two tangential flow filter membranes with different pore sizes, thereby not only removing the cell microvesicles and cell debris with larger particles in a sample, but also extracting and concentrating the exosome to the maximum extent.

Description

Dual tangential flow filtration system for exosome extraction, and preparation method and application of exosome

Technical Field

The invention relates to the technical field of biomedicine, in particular to a dual tangential flow filtration system for exosome extraction and a preparation method and application of exosomes.

Background

Exosomes are vesicles with phospholipid bilayer structure secreted by living cells, have a diameter of 30-150nm and a density of 1.13-1.19g/ml, and can be present in various body fluids, such as serum, plasma, saliva, urine, ascites, spinal fluid, milk, and the like. Exosomes contain a variety of biomolecules, such as mRNA, miRNA, proteins, lipids, etc., that can be delivered to recipient cells, thereby altering the physiological or pathological function of the recipient cells. In recent years, exosomes have attracted considerable attention as an intercellular information transfer tool and biomarkers for various diseases, and have great potential for application in the fields of biomedicine and disease diagnosis.

The high-purity, high-yield and standardized exosome is obtained, and is a precondition for clinical application of the exosome. However, at present, there is no unified standard for the methods for extracting and purifying exosomes, and there are many methods commonly used, such as ultracentrifugation, density gradient centrifugation, ultrafiltration, polymer precipitation, and immunocapture. Although the ultracentrifugation method is a recognized gold standard method for exosome extraction, the operation is time-consuming and labor-consuming, highly depends on manpower, the recovery rate is low, the exosome forms are different in size, and the high-speed centrifugation can damage exosomes to influence downstream experiments. Although the density gradient centrifugation method can obtain very pure exosomes, the method has the disadvantages of complicated operation, poor repeatability, long time consumption and low recovery rate, and is not suitable for extracting exosomes in large batch. The exosome can be conveniently and rapidly extracted by the ultrafiltration method, but the exosome extracted by the method contains large-particle impurity pollution, and downstream application is seriously influenced. The polymer precipitation method is simple and convenient to operate, can be used for extracting exosomes of large-volume samples, but the exosomes extracted by the method are more polluted by impure proteins, are uneven in particle morphology, and influence downstream analysis. Although the immunocapture method can specifically capture exosomes and obtain exosomes with high purity, the method has high cost and low yield, cannot extract all exosomes in a sample, and only can extract exosomes positive for a certain surface antigen. Therefore, the current common exosome extraction method is limited to laboratory operation, and no extraction method suitable for large-scale exosome industrialization exists. Therefore, in order to accelerate clinical transformation of exosomes, a method which is simple to operate, large in sample handling capacity, good in repeatability and suitable for large-scale GMP-level exosome preparation is urgently needed.

Disclosure of Invention

Therefore, the invention provides a dual tangential flow filtration system for exosome extraction, and a preparation method and application of exosomes, so as to solve the problems that the operation is complex, the sample size is small, the repeatability is poor, and the preparation method cannot be used for large-scale GMP-level exosome preparation in the prior art.

In order to achieve the above purpose, the invention provides the following technical scheme:

according to an aspect of the present invention, there is provided a method for preparing exosomes using a dual tangential flow filtration system for exosome extraction, the method comprising:

step one, pretreating a sample solution to obtain a sample solution containing exosomes;

placing the sample solution containing the exosomes in a first storage tank, opening a first peristaltic pump, pumping the sample solution containing the exosomes into a first filtering device, circularly concentrating and filtering, and collecting the filtered solution to a second storage tank;

step three, when the volume of the liquid in the first storage tank is concentrated to the minimum operation volume, adding sterile PBS buffer solution into the first storage tank, continuously pumping the liquid in the first storage tank into the first filtering device, continuously circularly concentrating and filtering, and collecting the filtrate to the second storage tank;

step four, when the volume of the liquid in the first storage tank is concentrated to the minimum operation volume, a second peristaltic pump is started, and the liquid in the second storage tank is pumped into a second filtering device for cyclic concentration and filtration;

step five, when the volume of the liquid in the second storage tank is concentrated to the minimum operation volume, adding sterile PBS buffer solution into the second storage tank, continuously pumping the liquid in the second storage tank into a second filtering device, circularly concentrating and filtering, when the volume of the liquid in the second storage tank is concentrated to a certain volume, collecting the liquid in the second storage tank, and obtaining the solution for extracting and purifying the exosomes;

adding a protective solution into the exosome solution, and freezing and storing at-80 ℃ or preparing the exosome solution into exosome freeze-dried powder;

the first filtering device is a filtering system provided with a tangential flow filter membrane F-1, and the interception pore size of the tangential flow filter membrane F-1 is 0.15-0.45 mu m; the second filtration device is a filtration system provided with a tangential flow through filter membrane F-2, the tangential flow through filter membrane F-2 having a molecular weight cut-off of 50-750 kD.

Further, the sample solution is a biological fluid or a cell culture supernatant; wherein the biological fluid comprises pleural effusion, urine, and milk; the cell culture supernatant comprises umbilical cord mesenchymal stem cells, placenta mesenchymal stem cells, dental pulp stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, nerve stem cells, hair follicle stem cells, skin stem cells, hematopoietic stem cells, induced pluripotent stem cells, embryonic stem cells or cell culture supernatant of differentiated cells induced by the stem cells.

Furthermore, the sample is pretreated by adding the sample into a centrifuge tube, centrifuging for 5-50min at 12000g of 5000-.

Further, the minimum operation volume of the first filtering device is 50-500 mL; the minimum operation volume of the second filtering device is 50-500 mL.

Further, the revolution numbers of the peristaltic pumps of the tangential flow filtration system are all 50-500 rpm/min.

Further, in the sixth step, the protective solution is a trehalose solution with a concentration of 0-100 mmol/L.

Further, in the sixth step, the process for preparing the exosome freeze-dried powder comprises the following steps:

freezing at-35 deg.C to-45 deg.C for 2-5h under normal pressure;

then freezing for 2-5h at-35 deg.C to-45 deg.C under the vacuum condition with vacuum degree value of 0.04mbar, then heating to-20 deg.C to-30 deg.C, freeze drying for 7-14h, continuing heating to-10 deg.C to 5 deg.C, freeze drying for 1-5h, and finally heating to 25 deg.C to 37 deg.C, and drying for 2-5h to obtain exosome freeze-dried powder.

Further, in the sixth step, the prepared exosome freeze-dried powder is stored at 4 ℃.

A second aspect of the invention provides a dual tangential flow filtration system for preparation of exosome extraction, comprising a first storage tank, a second storage tank, a first filtration device, a second filtration device, a first peristaltic pump, a second peristaltic pump;

wherein, the outlet of the first storage tank is connected with the inlet of the first filtering device through a first peristaltic pump, the outlet of the first filtering device is connected with the inlet of the first storage tank, and the filtered liquid outlet is connected with the first inlet of the second storage tank; an outlet of the second storage tank is connected with an inlet of a second filtering device through a second peristaltic pump, an outlet of the second filtering device is connected with a second inlet of the second storage tank, and liquid filtered by the second filtering device directly flows out of the system;

The exosome prepared by the method provided by the third aspect of the invention is applied to the preparation of medical and American products, exosome fluid dressing for promoting wound healing, exosome spray for relieving asthma and acute respiratory distress, and exosome-related biological products.

The invention has the following advantages:

the invention provides a double tangential flow filtration system for extracting and purifying exosomes from a large-volume sample, which extracts and purifies exosomes through two tangential flow filtration membranes with different pore sizes, can remove cell microvesicles and cell fragments with larger particles in the sample, can extract and concentrate exosomes to the maximum extent, and is very suitable for large-scale production and preparation of exosomes.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

FIG. 1 is a schematic diagram of the principle of purifying an exosome by a tangential flow filtration membrane provided by the invention, wherein (1) is a schematic diagram of a tangential flow filtration membrane F-1 and (2) is a schematic diagram of a tangential flow filtration membrane F-2;

FIG. 2 is a schematic diagram of a dual tangential flow filtration system of the present invention, wherein 1 is a first storage tank, 2 is a first peristaltic pump, 3 is a first filtration device, 4 is a second storage tank, 5 is a second filtration device, and 6 is a second peristaltic pump;

FIG. 3 is a microscope image of an umbilical cord mesenchymal stem cell provided by the present invention;

FIG. 4 is a diagram of the flow detection result of the umbilical cord mesenchymal stem cell specific protein CD34 provided by the present invention;

FIG. 5 is a diagram of the flow detection result of the umbilical cord mesenchymal stem cell specific protein CD45 provided by the present invention;

FIG. 6 is a diagram of the flow detection result of the umbilical cord mesenchymal stem cell specific protein CD73 provided by the present invention;

FIG. 7 is a diagram of the flow detection result of the umbilical cord mesenchymal stem cell specific protein CD90 provided by the present invention;

FIG. 8 is a diagram of the flow detection result of the umbilical cord mesenchymal stem cell specific protein CD105 provided by the present invention;

FIG. 9 is an electron microscope detection result diagram of exosomes extracted in comparative example provided by the present invention;

FIG. 10 is an electron microscope examination result diagram of exosomes extracted in the example provided by the present invention;

FIG. 11 is a graph showing the results of detecting the concentration of exosome proteins obtained by different extraction methods of quantitative detection of BSA proteins in the present invention, wherein A is the result of detecting the concentration of exosome proteins obtained from the control group, and B is the result of detecting the concentration of exosome proteins obtained from the examples;

FIG. 12 is a graph showing the results of detecting the number of exosomes obtained by NTA detection with different extraction methods in the present invention, wherein A is the result of detecting the number of exosomes obtained from the control group, and B is the result of detecting the number of exosomes obtained from the examples;

FIG. 13 is a graph of the relative purity assay results for exosomes from different extraction methods provided by the present invention;

FIG. 14 is a graph showing the detection results of the marker proteins, CD9, CD63 and ALIX, of exosomes prepared by Western Blot detection according to the present invention, (1) shows the detection results of the exosomes obtained in the examples, and (2) shows the detection results of the exosomes obtained in the control examples;

FIG. 15 is a graph showing the results of the cell proliferation assay using exosomes prepared by different extraction methods provided in the present invention, wherein A is an exosome group and B is a blank control group;

FIG. 16 is a graph showing the results of different concentrations of protectant provided by the present invention on exosome particle concentration;

FIG. 17 is a graph showing the effect of various concentrations of protectant provided by the present invention on the promotion of cell proliferation by exosomes.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1A method for the preparation of exosomes using a dual tangential flow filtration system for exosome extraction provided by the present invention

Umbilical cord mesenchymal stem cell culture

1. Extraction and preparation of umbilical cord mesenchymal stem cells

Collecting umbilical cord of newborn from operating room, washing umbilical cord with sterile PBS buffer solution, removing blood vessel with scissors and forceps, stripping Fahrenheit gelatin tissue, and sufficiently cutting tissue to 1mm³Tissue mass of size. The tissue blocks were spread evenly on a petri dish and placed in an incubator for 1h (culture conditions of 5% CO)₂At 37 ℃ C. When the tissue block adheres to the wall firmly, adding a proper amount of stem cell culture medium, and culturing in an incubator (the culture condition is 5% CO)₂At 37 ℃ C. After the cells adhere well, the culture medium is slightly sucked off, the cells are gently rinsed once by the stem cell culture medium, 10mL of fresh stem cell culture medium is added and the cells are cultured in an incubator (the culture condition is 5% CO)₂At 37 ℃ C. Digesting with digestive juice (0.25% trypsin) when the adherent cells in the culture dish are 70% -85% full, adding stop solution to stop digestion when the cells begin to fall off and float, slightly blowing the cells into single cell suspension by a pipette, centrifuging at 1000rpm for 10min, then discarding the supernatant, and finally re-suspending with 10mL of fresh stem cell culture medium to obtain the primary umbilical cord mesenchymal stem cells (P)₀) As shown in fig. 3, the microscope observation picture of the primary umbilical cord mesenchymal stem cells.

2. Identification of umbilical cord mesenchymal stem cells

Obtaining primary stem cell P₀Then, sterile PBS buffer was added to adjust the cell concentration to 1X 10⁶and/mL, taking five 200 mu L cell suspensions, respectively adding 5 mu L of fluorescently-labeled monoclonal antibody CD34, 5 mu L of fluorescently-labeled monoclonal antibody CD45, 5 mu L of fluorescently-labeled monoclonal antibody CD73, 5 mu L of fluorescently-labeled monoclonal antibody CD90 and 5 mu L of fluorescently-labeled monoclonal antibody CD105, mixing uniformly, incubating at room temperature in the dark for 20min, and detecting in a flow cytometer, wherein the flow detection result graphs of umbilical cord mesenchymal stem cell specific proteins CD34, CD45, CD73, CD90 and CD105 are respectively shown in figures 4, 5, 6, 7 and 8.

3. Culture of umbilical cord mesenchymal stem cells

Culturing the primary umbilical cord mesenchymal stem cells in a stem cell culture medium (the culture condition is 5% CO)₂And at 37 ℃), when the cells are fully spread by 90 percent at the adherent temperature, centrifuging at 5000g for 10min, and collecting cell culture supernatant to obtain the umbilical cord mesenchymal stem cell solution.

Two, dual tangential flow filtration system for extracting and purifying exosome

1. Pretreatment of umbilical cord mesenchymal stem cell solution

Adding the umbilical cord mesenchymal stem cell solution into a centrifuge tube, centrifuging for 5-50min at 12000g of 5000-;

2. extraction and purification of exosome by dual tangential flow filtration system

(1) Adding sterile water into the first storage tank 1 and the second storage tank 4, respectively opening the peristaltic pumps (the first peristaltic pump 2 and the second peristaltic pump 6) and flushing the system;

(2) adding 4L of pretreated sample solution into a first storage tank 1, opening a first peristaltic pump 2 to circulate the system, concentrating and filtering the pretreated sample solution by a first filtering device 3 provided with a tangential flow filter membrane F-1, wherein the principle of the tangential flow filter membrane F-1 is shown in a (1) in a figure 1, and enabling the solution containing exosomes to enter a second storage tank 4; when the liquid volume in the first storage tank 1 is the minimum operation volume, 2L of sterile PBS buffer solution is added to continue concentration and filtration to the minimum operation volume;

(3) when the liquid volume in the second storage tank 4 exceeds 400mL, a second peristaltic pump 6 is opened to circulate the system, and the solution containing the exosome passes through a second filtering device 5 provided with a tangential flow filter membrane F-2 to be concentrated and filtered, wherein the principle of the tangential flow filter membrane F-2 is as shown in a (2) in a figure 1, and small molecular substances are removed;

(4) and when the liquid volume in the second storage tank 4 is the minimum operation volume, adding 2000mL of PBS buffer solution into the second storage tank 4, continuing to concentrate and filter until the solution in the second storage tank 4 is 200mL, and collecting the solution to obtain the exosome extracted and purified by the dual tangential flow filtration system.

Example 2A Dual tangential flow filtration System for exosome extraction

The dual tangential flow filtration system comprises a first storage tank 1, a second storage tank 4, a first filtration device 3, a second filtration device 5, a first peristaltic pump 2, a second peristaltic pump 6;

wherein, the outlet of the first storage tank is connected with the inlet of the first filtering device through a first peristaltic pump, the outlet of the first filtering device is connected with the inlet of the first storage tank, and the filtered liquid outlet is connected with the first inlet of the second storage tank; the outlet of the second storage tank is connected with the inlet of the second filtering device through a second peristaltic pump, the outlet of the second filtering device is connected with the second inlet of the second storage tank, and the liquid filtered by the second filtering device directly flows out of the system.

Wherein the first filtering device 3 is a filtering device provided with a tangential flow filter membrane F-1; the second filter device 5 is a filter device provided with a tangential flow through filter membrane F-2.

The filtering system is characterized in that liquid in a first storage tank 1 is pumped into a first filtering device 3 through a first peristaltic pump 2, the liquid purified by the first filtering device flows into a second storage tank 4, and the liquid reserved in the first filtering device 3 flows back into the first storage tank 1; the second storage tank 4 is pumped into the second filtering device 5 through the second peristaltic pump 6, the liquid without the exosomes purified by the second filtering device directly flows out of the system, the liquid retained in the second filtering device 5 flows back into the second storage tank 4 again, and the liquid flowing back into the second storage tank 4 again is the exosomes obtained through purification.

Comparative example ultracentrifugation method for extracting exosome

And adding 20mL of the pretreated sample solution into an ultracentrifuge tube, centrifuging for 2h at 100000g at 4 ℃, and carrying out heavy suspension by using 1mL of sterile PBS buffer solution to obtain the exosome extracted and purified by the ultracentrifuge method.

Experimental example 1 comparison of different extraction methods

1. Exosome electron microscope contrast

The exosomes obtained in the example and the comparative example are respectively fixed on a sample-carrying copper net by using glutaraldehyde solution, reacted for 5min, and then ddH is used₂And O, washing the copper mesh, putting the copper mesh in uranyl oxalate solution for incubation for 5min, finally, absorbing excess liquid on filter paper, putting the copper mesh in a sample box after the copper mesh is dried, and taking an electron microscope picture at 80 kV. As shown in fig. 10, electron microscope examination results of the purified exosomes extracted in the example are shown, and electron microscope examination results of the exosomes extracted in the comparative example are shown in fig. 9. The result shows that the structure of the saucer-shaped double-layer vesicle can be seen under an electron microscope, the particle size is within the range of 30-150nm, the structure is clear, and no obvious difference exists.

2. Comparison of exosome protein concentrations

The protein concentration of the exosomes obtained in the example and the comparative example is detected by adopting a BCA method, equal volume of Ripa lysate (50mM Tris, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, pH 7.4) is respectively added into the exosome solution obtained in the collected example and the comparative example, the mixture is fully mixed, the mixture is kept stand at 4 ℃ for 30min, and the exosome protein concentration of the exosome protein solution after the lysis treatment is determined according to the operation requirement of the BCA detection kit instruction. The experimental results are shown in fig. 11, and the exosome protein concentration detection result graph is prepared by adopting different extraction methods for quantitative detection of BSA protein. The concentration of the exosome protein extracted by the comparative example is obviously higher than that of the example, and the concentration of the exosome protein extracted by the comparative example is about 3 times that of the example.

3. Comparison of exosome particle range and concentration

The exosome particle concentrations obtained in the examples and comparative examples were diluted to 1 × 10⁷Perml and 1X 10⁹In the/mL range. And (3) detecting the exosome concentrations obtained in the examples and the comparative examples respectively by adopting a nano-particle size tracing analyzer. As can be seen from fig. 12, the particle concentration of the exosomes obtained in the example was about 3 times that of the exosomes obtained in the comparative example, and the example could obtain more exosomes with higher recovery rate.

The relative purities of exosomes can be simply compared according to the ratio of particle concentration to protein concentration of exosomes (the greater the ratio of particle concentration to protein concentration, the higher the purity of exosomes). As shown in FIG. 13, the exosomes obtained in the example have a particle concentration of protein 9 times higher than that of the exosomes obtained by the ultracentrifugation method, the exosomes obtained in the example have higher purity, and the exosomes obtained in the comparative example may contain a large amount of contaminating protein.

4. Exosome Western Blot detection

The exosomes obtained in the example and the comparative example are respectively added with a proper amount of loading buffer, and the mixture is heated in a boiling water bath to fully denature the protein. And (3) configuring SDS-PAGE gel, loading the protein into a loading hole of the SDS-PAGE gel, and electrophoretically separating a protein sample and transferring the protein sample to a membrane. After the film transfer is finished, the protein film is placed in 5% of sealing liquid for sealing. After blocking was complete, the primary antibody (CD9, CD63, ALIX) was incubated overnight at 4 ℃ and then with the enzyme-labeled secondary antibody at room temperature for 1 h. And finally, placing the protein film in ECL luminous liquid, reacting for 2min, and then placing the protein film into a chemiluminescence imaging system for color development imaging. The experimental results of CD9, CD63 and ALIX protein Western Blot of the exosomes obtained in the examples and the comparative examples are shown in FIG. 14, the bands of the exosomes extracted and purified in the examples are clear and bright, while the bands of the exosomes extracted in the comparative examples are lower in definition due to the fact that the exosomes obtained by the purification method of the comparative examples are mixed with the foreign proteins, and the fact that the exosomes extracted in the comparative examples are lower in purity than the exosomes extracted in the examples can also be shown.

5. Exosome bioactivity comparison

Detection implementation by MTT methodProliferation potency of exosomes obtained in examples and comparative examples. Taking umbilical cord mesenchymal stem cells at a rate of 5 × 10 per well³Each cell was seeded in a 96-well plate, and the exosomes obtained in example and comparative example were diluted to the same concentration, 10. mu.L each was added to each cultured cell, and 10. mu.L of the medium without exosomes was used as a control. After 3 days of incubation, 20. mu.L of MTT solution (5mg/mL) was added to each well. Incubation was continued for 4h, the culture was terminated and the culture supernatant from the wells was carefully aspirated. Add 150. mu.L of LDMSO to each well and shake for 10 min. OD values were measured at 450nm with a microplate reader, 6 replicates per group, and the average was taken. The result is shown in fig. 15, the umbilical cord mesenchymal stem cell exosome plays an obvious role in promoting cell proliferation, the effect of the exosome obtained in the example on cell proliferation is obviously higher than that of the comparative example, the result is caused by that the membrane structure of the exosome is damaged by the strong centrifugal force of the ultracentrifugation method, and the mode for extracting the exosome in the example is milder, so that the biological activity of the exosome is better ensured.

Experimental example 2 storage of exosomes

1. Effect of different storage conditions on exosome concentration

1.1 preparation of exosome freeze-dried powder

Preparing 500mM of trehalose mother liquor, adding the trehalose mother liquor into the exosome solution obtained in the embodiment in proportion to obtain exosome solutions with trehalose final concentrations of 0mM, 20mM, 40mM, 60mM, 80mM and 100mM respectively, sucking 2mL of the prepared exosome solutions respectively and placing the solutions into 5mL glass bottles, and sucking 2mL of the prepared exosome solutions respectively and placing the solutions into 5mL centrifuge tubes. The 5mL centrifuge tubes were stored at 4 ℃ and-80 ℃. Covering a 5mL glass bottle with a rubber plug, placing the glass bottle in a vacuum freeze dryer, and starting a freeze-drying program:

(1) freezing at-35 deg.C to-45 deg.C for 2-5h under normal pressure;

(2) starting a vacuum pump to ensure that the vacuum value is 0.04mbar, and freezing for 2-5h at the temperature of-35 to-45 ℃;

(3) keeping the vacuum degree unchanged, heating to-20 to-30 ℃, and freeze-drying for 7-14 h;

(4) keeping the vacuum degree unchanged, heating to the temperature of-10 to 5 ℃, and freeze-drying for 1 to 5 hours;

(5) heating to 25-37 ℃ and drying for 2-5h without changing the vacuum degree;

(6) and (5) obtaining the exosome freeze-dried powder after the program is finished.

1.2 Effect of different storage conditions on exosome concentration

Standing the exosome freeze-dried powder stored at 4 ℃, the exosome solution stored at 4 ℃ and the exosome solution stored at-80 ℃ for 3 months, carrying out nano-particle size tracing analysis (the exosome freeze-dried powder is redissolved to 2mL), and comparing the particle concentrations of exosomes respectively. As can be seen in FIG. 16, the particle concentration of the exosome solution stored at 4 ℃ is reduced by about 10 times, which indicates that the exosome stored at 4 ℃ for 3 months is broken or aggregated, and the addition of trehalose does not play a role in protecting the exosome under the condition. Exosomes stored at minus 80 ℃ and lyophilized powder stored at 4 ℃ have particle concentration close to that of fresh exosomes, but the particle number of exosomes is larger along with the increase of trehalose concentration, which indicates that trehalose can enable exosomes resuscitated after refrigeration or lyophilization to have better dispersibility, when the trehalose concentration is 60mM, the exosome concentration is closest to that of the fresh exosomes, and the particle concentration of exosomes stored at 4 ℃ of lyophilized powder is slightly higher than that of the exosomes stored at minus 80 ℃, so the optimal exosome storage condition is that the final concentration of trehalose is 60mM and the exosomes are stored at 4 ℃.

2. Effect of different storage conditions on the biological Activity of exosomes

The exosome freeze-dried powder stored at 4 ℃, the exosome solution stored at 4 ℃ and the exosome solution stored at-80 ℃ are respectively used for detecting the cell proliferation capacity of exosomes with different trehalose concentrations and different storage methods by adopting an MTT method, as can be seen in figure 17, the cell proliferation promoting capacity of the exosome solution stored at 4 ℃ is greatly reduced, which indicates that the exosomes stored under the condition lose biological activity. Exosomes stored at-80 ℃ and lyophilized powder stored at 4 ℃ have better cell proliferation promoting capacity, the capacity is increased along with the increase of trehalose concentration, the optimal trehalose concentration is 60mM, the bioactivity of exosomes stored at the lyophilized powder at 4 ℃ is slightly higher than that of exosomes stored at-80 ℃, and the result is matched with the result of exosome concentration.

The invention provides a method for extracting and purifying exosomes by a double tangential flow filtration system, which can be used for extracting and purifying exosomes from various large-volume samples, is simple to operate, can obtain exosomes with high purity and high yield, can realize automatic batch production of the exosomes, can prepare GMP-grade exosomes, and greatly accelerates the technical development of an exosome-related biological product and the process of clinical transformation application.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for producing exosomes for a dual tangential flow filtration system for exosome extraction, the method comprising:

step two, placing the sample solution containing the exosomes in a first storage tank, opening a first peristaltic pump, pumping the sample solution containing the exosomes into a first filtering device, circularly concentrating and filtering, and collecting filtrate to a second storage tank;

2. A method for the preparation of exosomes according to claim 1, characterised in that the sample solution is a biological fluid or a cell culture supernatant; wherein the biological fluid comprises pleural effusion, urine, and milk; the cell culture supernatant comprises umbilical cord mesenchymal stem cells, placenta mesenchymal stem cells, dental pulp stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, nerve stem cells, hair follicle stem cells, skin stem cells, hematopoietic stem cells, induced pluripotent stem cells, embryonic stem cells or cell culture supernatant of differentiated cells induced by the stem cells.

3. The method for preparing exosomes through the dual tangential flow filtration system for exosome extraction as claimed in claim 1, wherein the sample is pretreated by adding the sample into a centrifuge tube, centrifuging for 5-50min at 5000-12000g, taking the supernatant, and filtering and sterilizing the supernatant through a 0.22-0.45 μm filter membrane.

4. A method for the preparation of exosomes according to claim 1, characterised in that the minimum running volume of the first filtration means is 50-500 mL; the minimum operation volume of the second filtering device is 50-500 mL.

5. A method for the preparation of exosomes according to claim 1 with a dual tangential flow filtration system for exosome extraction, characterized in that the first and second peristaltic pumps of the tangential flow filtration system are each in revolutions of 50-500 rpm/min.

6. The method for preparing exosomes according to claim 1, wherein in the sixth step, the protective solution is a trehalose solution with a concentration of 0-100 mmol/L.

7. The method for preparing exosomes according to claim 1, wherein in the step six, the process for preparing exosome lyophilized powder comprises:

freezing at-35 deg.C to-45 deg.C for 2-5h under normal pressure;

8. The method for preparing exosomes according to claim 1, wherein in step six, the prepared exosome lyophilized powder is stored at 4 ℃.

9. A dual tangential flow filtration system for use in the preparation of exosome extracts of claims 1-8, comprising a first storage tank, a second storage tank, a first filtration device, a second filtration device, a first peristaltic pump, a second peristaltic pump;

10. Use of exosomes prepared by the process of claims 1-8 in the preparation of an ophthalmic product, an exosome fluid dressing for promoting wound healing, an exosome spray for relieving asthma and acute respiratory distress, and an exosome-related biologic.