A method and device for gentle, low-damage separation, purification, enrichment of EVs and their heterogeneous subpopulations using AF4

Abstract

The present application relates to a method and device for mild, low-damage separation, purification and enrichment of EVs and their heterogeneous subpopulations using AF4. The method for mild, low-damage separation, purification and enrichment of EVs and their heterogeneous subpopulations using AF4 comprises the following steps: separation and purification of EVs samples with a wide particle size distribution according to particle size using AF4; enrichment of the obtained EVs heterogeneous subpopulation samples after separation and purification by changing the AF4 pipeline, connecting a large-volume sample injection pipeline between the original AF4 sample injector and the AF4 system Eclipse DUALTEC instrument to replace the original pipeline, resetting the sample injection program, and switching between aggregation mode and aggregation injection mode to achieve enrichment of EVs samples with different volumes. The method realizes separation and purification of EVs subpopulation mixed groups with a wide particle size distribution, and can realize efficient enrichment of EVs heterogeneous subpopulations, and solves key problems such as deviation of research results caused by excessive protein impurities in EVs samples and changes in biological characteristics of heterogeneous subpopulations during the enrichment process.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of EVs and separation and enrichment of heterogeneous subpopulations thereof, and particularly relates to a method and device for mild and low-damage separation, purification and enrichment of EVs and heterogeneous subpopulations thereof by using AF4. BACKGROUND

[0002] Extracellular vesicles (EVs) refer to double-membrane vesicle-like bodies that are shed from cell membranes or secreted by cells, and are widely present in body fluids. EVs carry rich biological information and are involved in processes such as cell-to-cell communication, cell migration, angiogenesis and immune regulation.

[0003] Studies have shown that EVs are heterogeneous, that is, within an EVs population, each heterogeneous subpopulation is different in biogenesis, physical characteristics (such as size, density, morphology, particle number) and contents (such as proteins, lipids, nucleic acids). The contents carried by EVs are both the response of cells to different physiological and pathological conditions and will produce various biological effects on their recipient cells. Therefore, clearly defining the biophysical characteristics, functions, causes and regulatory mechanisms of EVs heterogeneous subpopulations will not only help to understand their exact role in physiological and pathological processes, but also will help to identify potential diagnostic / prognostic biomarkers in pathological states. This will provide a theoretical basis for accelerating the application of EVs in clinical diagnosis and treatment.

[0004] In 2018, the International Society for Extracellular Vesicles (ISEV) published the latest guidelines: MISEV2018, which suggested using the following operational terms to define EVs: ① physical characteristics: size "small EVs" (sEVs: <100 nm or <200 nm) and "medium / large EVs" (m / lEVs: >200 nm), density; ② biochemical composition (CD63 + / CD81 + -EVs, Annexin A5-stained EVs); ③ using the production conditions or cell sources of EVs for description (such as: tumor cell EXOs, EXOs produced under hypoxia).

[0005] Current studies have analyzed the extracellular RNA (exRNAs) and extracellular protein (exPTNs) of sEVs, mEVs, and lEVs heterogeneous subpopulations of mouse glioma cells by RNA sequencing and quantitative proteomics analysis. It was found that the mRNA of the mEVs subpopulation most closely reflected the transcriptome of the cells, while the exRNAs of the sEVs subpopulation were enriched in small non-coding RNAs. CD9 was mainly a marker of the sEVs subpopulation, while CD81 was always present on the lEVs subpopulation. Nonon protein was mainly present in the lEVs subpopulation.

[0006] The separation of EVs heterogeneous subpopulations is the basis for in-depth research on EVs. The current separation method is mainly centrifugation, such as the commonly used differential ultracentrifugation (DUC), which separates EVs by applying different centrifugal forces to the sample. The separation of EVs heterogeneous subpopulations can be achieved by low-speed (2000g), medium-speed (10000g), and high-speed (100000g) centrifugation. However, the DUC method has the following disadvantages: ① Even at a low centrifugal force, EVs will aggregate, and the absolute separation of EVs subpopulations in the same size range cannot be achieved; ② DUC separation will damage EVs and reduce their activity, which is not conducive to further biological activity research; ③ The separation time is long, the cost is high, and only different EVs subpopulations can be obtained by multiple centrifugations at different speeds; ④ The EVs heterogeneous subpopulations cannot be analyzed and quantified during the separation process; ⑤ It is difficult to separate proteins and cell debris from EVs;

[0007] In actual research, protein impurities or cell debris in the biological matrix can seriously affect the reliability of EVs research. Protein impurities or cell debris can make it difficult to accurately quantify the protein concentration of EVs samples, which can significantly affect the downstream analysis results, making it difficult to compare, reproduce, and analyze the results between groups, and cannot exclude the effects of protein impurities and cell debris, ultimately affecting the authenticity of EVs biological activity research. However, current research is mostly based on the extraction of EVs and direct research, and there is a lack of suitable purification methods. Therefore, developing an effective purification scheme for in-depth research on the biological activity of EVs is an urgent problem in the EVs field. Therefore, developing a low-cost, mild, and preserving the biological characteristics of EVs sample separation and purification enrichment method has become an urgent problem in the field of EVs heterogeneous subpopulation research.

[0008] Asymmetric field flow fractionation (AF4) is a separation method based on the molecular diffusion of the sample and the cross-flow in the opposite direction of the sample molecular diffusion. AF4 has no stationary phase and filler, and the separation process is completed in a fluid environment, so it has the characteristics of low shear force, mild separation conditions, and suitability for the separation and characterization of biological macromolecules. During the separation process, small particle size samples are closer to the center of the parabolic laminar flow in the pool channel due to their small diffusion coefficient, and are eluted first. Large particle size samples are far away from the center of the parabolic laminar flow, and are eluted later, thus achieving sample separation. In addition, AF4 can be coupled with a multi-angle laser detector (MALS) to provide information such as particle size distribution and particle number of the sample. By optimizing the AF4 separation conditions, protein impurities and EVs samples can be separated. Protein impurities are eluted first due to their small particle size, and vesicles are eluted later, thus achieving the purification of EVs samples and the separation of EVs heterogeneous subpopulations.

[0009] When AF4 and other chromatography techniques (such as size exclusion chromatography) are used to separate EVs heterogeneous subpopulations, the mobile phase will dilute the EVs, resulting in a decrease in sample concentration. In the past, ultrafiltration was used to enrich EVs heterogeneous subpopulation samples. Ultrafiltration, like centrifugation, applies centrifugal force and also encounters problems such as EVs sample aggregation and biological damage caused by centrifugation. A mild and low-damage method for enriching EVs heterogeneous subpopulation samples is needed. AF4 technology can be used to solve this problem.

[0010] However, the obtained EVs heterogeneous subpopulation samples are usually low in concentration and large in volume, so a large volume sample needs to be injected. The maximum injection volume of the AF4 injection ring is 100 μL, which cannot directly achieve the purpose of enrichment. Moreover, there is no commercial large-volume injection ring, and it is difficult to realize the customization of large-volume injection rings due to high cost and time-consuming. In addition, due to the limitation of the AF4 mobile phase unidirectional filter (<100 nm), samples larger than 100 nm cannot be pumped into the pool channel by adding to the mobile phase. Therefore, by modifying the AF4 pipeline, large-volume sample injection can be achieved. At present, there is no low-cost and low-damage enrichment method for EVs biological activity. Therefore, based on the mild and low-damage technical characteristics of AF4, a low-cost and low-damage method for enriching EVs heterogeneous subpopulations can be developed by modifying the pipeline based on the original AF4 instrument. This method not only helps to efficiently enrich EVs heterogeneous subpopulation samples, but also can be connected to a detector for online enrichment and detection according to the needs, which is of great significance for the study of EVs function. Moreover, it can be further applied to the enrichment of trace amounts of nano-sized particulate matter in the environment, providing a new method for the study of trace amounts of nano-sized particulate matter in the environment. SUMMARY

[0011] The purpose of the present application is to provide a method for mild, low-damage separation, purification and enrichment of EVs and their heterogeneous subpopulations using AF4, which realizes the separation and characterization of the mixed population of platelet-derived extracellular vesicles (PEVs) with a wide size distribution of 30-400 nm in size distribution in the experiment, and can realize the efficient enrichment of EVs heterogeneous subpopulations, and solve the problems of result deviation caused by protein impurities and biological property changes caused by heterogeneous subpopulation enrichment in current EVs research.

[0012] Another purpose of the present application is to provide a device for a method for mild, low-damage separation, purification and enrichment of EVs and their heterogeneous subpopulations using AF4.

[0013] In order to achieve the above purposes, the technical scheme adopted by the present application is:

[0014] A method for mild, low-damage separation, purification and enrichment of EVs and their heterogeneous subpopulations using AF4, comprising the following steps:

[0015] 1) Separation and purification: using AF4 to separate EVs samples with a wide size distribution according to particle size, protein impurities are separated from EVs, and at the same time EVs are eluted out from small to large, realizing separation and purification of EVs;

[0016] 2) Enrichment: different EVs heterogeneous subpopulation samples obtained after separation are enriched by AF4, a large volume sampling pipeline is connected between the original AF4 sampler and the AF4 system Eclipse DUALTEC instrument to replace the original pipeline, and the sampling program is reset to switch between aggregation mode and aggregation sampling mode to realize enrichment of different volumes of EVs samples.

[0017] Further, the separation and purification in step 1) is gradient elution, which is cross-flow flow rate of 2 mL / min to 0.25 mL / min within 5 min, and then 0.25 mL / min to 0.09 mL / min within 30 min.

[0018] Further, the separation and purification in step 1) is gradient elution, which is cross-flow flow rate of 2 mL / min to 0.25 mL / min within 5 min, and then 0.25 mL / min to 0.09 mL / min within 30 min.

[0019] Further, the separation and purification in step 1) is gradient elution, which is cross-flow flow rate of 2 mL / min to 0.25 mL / min within 5 min, and then 0.25 mL / min to 0.09 mL / min within 30 min.

[0020] Further, the enrichment in step 2) is achieved by connecting a large volume injection line between the injector and the AF4 system Eclipse DUALTEC instrument to replace the original line.

[0021] Further, the enrichment in step 2) is achieved by changing the injection program setting during enrichment, first performing the concentration mode, then performing the concentration injection mode, and then switching between the concentration mode and the concentration injection mode according to the sample amount to be enriched.

[0022] Further, the enrichment in step 2) is achieved by changing the injection program setting during enrichment, first performing the concentration mode, then performing the concentration injection mode, and then switching between the concentration mode and the concentration injection mode according to the sample amount to be enriched.

[0023] Further, in the concentration mode, the mobile phase does not pass through the injector, but enters the AF4 system Eclipse DUALTEC instrument from the mobile phase bottle through the liquid phase pump, and then enters the cell channel through the inlet and outlet of the cell channel, forming a convection near the cell channel injection port.

[0024] Further, in the concentration injection mode, the mobile phase passes through the injector, and the sample in the large volume line is brought into the cell channel through the cell channel injection port, at which time in the cell channel, the solvent and any substance smaller than the pore size of the semi-permeable membrane can pass through the semi-permeable membrane into the waste pipe, and the vesicles larger than the pore size of the semi-permeable membrane can be retained in the cell channel.

[0025] The device for the method of gentle, low-damage separation, purification and enrichment of EVs and their heterogeneous subpopulations using AF4 includes an AF4 separation and purification module and an AF4 enrichment module, wherein the AF4 enrichment module is achieved by connecting a large volume injection line between the injector and the AF4 system Eclipse DUALTEC instrument to replace the original line.

[0026] The beneficial effects of the present application are as follows:

[0027] The present application uses AF4 to achieve separation, purification and characterization of a mixed population of EVs subpopulations with a wide particle size distribution of 30-400 nm actually extracted in experiments. Through AF4 separation, the removal of protein impurities in EVs samples and the separation of EVs heterogeneous subpopulation samples can be achieved at the same time, solving the problem that the accuracy of EVs function research is questioned due to the presence of protein impurities in current EVs research.

[0028] And by modifying the AF4 pipeline and resetting the sample injection program, the efficient enrichment of EVs heterogeneous subpopulation samples can be realized, and the problem of damaging the biological characteristics of EVs heterogeneous subpopulation in the separation and enrichment process is solved. The method has the characteristics of low cost, high flexibility, easy operation, etc. Compared with the commonly used ultrafiltration method for enriching extracellular vesicles, the enrichment multiple of the ultrafiltration method is 80 to 100 times; the enrichment multiple of this method can be as high as 160 times or even higher, and it has higher advantages in enrichment effect. Using AF4 method for enrichment, more EVs are observed under electron microscope and the morphology is better, which is more conducive to the further research on EVs heterogeneous subpopulation. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Figure for AF4 separation and purification results of platelet-derived extracellular vesicles (PEVs) in Example 1;

[0030] Figure 2 Figure for AF4 separation and enrichment principle diagram of PEVs in Example 1;

[0031] Figure 3 Figure for AF4 pipeline modification and enrichment principle diagram of EVs in Example 1;

[0032] Figure 4 Figure for TEM imaging analysis of sPEVs, mPEVs and lPEVs subpopulations after AF4 concentration and ultrafiltration concentration in Example 1. DETAILED DESCRIPTION

[0033] The application will be further described below in conjunction with the embodiments of the application and the drawings.

[0034] Example 1

[0035] The method for mild and low-damage separation, purification and enrichment of EVs and their heterogeneous subpopulations by AF4 in this embodiment includes the following steps:

[0036] 1) Separation and purification of EVs heterogeneous subpopulations:

[0037] The following method is used to prepare PEVs sample: take 10 mL of rat whole blood in a sodium citrate anticoagulant tube, then use a low-speed centrifuge at 22°C, 800 rpm / min for 10 min, take the supernatant (plasma) in a 15 ml centrifuge tube, centrifuge at 22°C, 3000 rpm / min for 10 min, discard 1 / 2 of the supernatant, resuspend the remaining supernatant and sediment to obtain platelet-rich plasma. Then couple biotinylated TIM4-Fc protein with streptavidin magnetic beads, extract PEVs from platelet-rich plasma, filter with 0.8 μm water phase filter membrane, and place in a liquid phase sample injection bottle (since the sample volume is small, an auxiliary inner cannula is needed in the sample injection bottle).

[0038] PEVs were separated, purified and characterized by AF4. The separation and purification conditions were as follows: the gradient elution was decreased from 2 mL / min to 0.25 mL / min within 5 min of cross-flow flow rate; the flow rate was decreased from 0.25 mL / min to 0.09 mL / min in the last 30 min; the injection volume was 100 μL; the detector flow rate was 1 mL / min; the type of membrane was 10 kDa RC membrane, the mobile phase was 10 mM PBS solution, and the separation program was shown in Table 1. The results are shown in Figure 1 PEVs samples were separated, purified and characterized by AF4-MALS, the particle size distribution of PEVs was 30 nm-400 nm, the protein impurity peak time was 8-11 min, the PEVs sample peak time was 11-40 min, and the established PEVs analysis method had good reproducibility.

[0039] Table 1: AF4 separation PEVs program

[0040]

[0041] According to the MISEV2018, the PEVs are divided into three subgroups according to the size, i.e. small (<100 nm), medium (100-200 nm), and large (>200 nm), namely sPEVs, mPEVs, and lPEVs. The AF4 separation and characterization results of the heterogeneous subgroups are shown in Table 2. As shown in Table 2, the AF4-MALS detection of the s subgroup of PEVs heterogeneous subgroups has an average radius of 38.17 nm; the m subgroup has an average radius of 63.6 nm; and the l subgroup has an average radius of 123.1 nm, which has good reproducibility. At the same time, the particle concentration of the PEVs heterogeneous subgroups can be determined.

[0042] Table 2: AF4-MALS separation and detection of PEVs heterogeneous subgroups particle size and particle number results

[0043]

[0044] AF4-MALS separation and detection of EVs heterogeneous subgroups, one injection, separation, purification and detection are carried out at the same time, and the separated fractions can be collected for further study of the heterogeneous subgroups. As shown in Table 3. The AF4 separation and characterization takes 1 h, which is shorter than the centrifugal separation which takes 6-7 h, and AF4 has the advantage of short time consumption; and the separation uses PBS as the mobile phase, which is low in cost; at the same time, it can provide the particle size distribution and average radius information of the heterogeneous subgroups, and can minimize the aggregation of EVs samples and maximize the preservation of the biological characteristics of the samples, which is more conducive to the further study of the heterogeneous subgroups.

[0045] Table 3 Comparison of centrifugation and AF4 separation of EVs methods

[0046]

[0047] 2) Enrichment of PEVs heterogeneous subpopulations

[0048] As Figure 3 The principle of AF4 enrichment of EVs is shown in Figure 1. A 10 mL large volume injection line is connected between the injector and the AF4 system Eclipse DUALTEC instrument, replacing the original line. Enrichment of EVs is achieved by Focus mode and Focus inject mode. By switching between the two modes, different volumes of EVs samples can be enriched according to the actual EVs sample volume, which is flexible and easy to operate.

[0049] The specific operation is as follows:

[0050] First, change the AF4 program settings. Set AF4 to Focus mode for 1 min, and Cross Flow to constant 2 mL / min. In this Focus mode, the mobile phase enters the AF4 system Eclipse DUALTEC instrument from the mobile phase bottle through the liquid phase pump, then enters the pool channel through the AF4 system Eclipse DUALTEC instrument, and finally enters the Input and Output ports of the pool channel near the sample inlet. At this time, the flow phase does not pass through the injector.

[0051] The sPEVs subpopulation sample in the PEVs heterogeneous subpopulation sample collected in step 1) is vortexed and mixed. A PEEK Pruril joint is connected to one end of a 10 mL syringe. A 10 mL ETEF tube is prepared, with PEEK integral high-pressure joints connected to both ends. One end of the 10 mL ETEF tube is connected to the Pruril joint of the 10 mL syringe, and one end is placed in the collected sPEVs subpopulation sample tube. The sPEVs subpopulation sample is sucked into the 10 mL ETEF tube using the suction force of the syringe. This process can minimize the presence of air bubbles in the ETEF tube to prevent air bubbles from being pumped into the AF4 pool channel, which can reduce the recovery rate of the sPEVs subpopulation sample.

[0052] The AF4 system Eclipse DUALTEC instrument As Out port and injector outlet position tubing were disconnected, and the ETEF tube containing the sPEVs subpopulation sample was connected to the AF4 system Eclipse DUALTEC instrument As Out port and injector outlet position. The flow phase PBS was filled in the liquid phase injection bottle and placed in the injector set position. The time required for the entire volume of sPEVs subpopulation sample in the ETEF tube to enter the channel and reach equilibrium was calculated by calculating the actual flow rate of the AF4 tubing and the time of the sample path tubing. When the focusing flow rate was 2.0 mL / min, the actual injection rate at the injection port end was about 0.4 mL / min, and the sample focusing relaxation equilibrium time required 3-5 min. Therefore, the online enrichment time of 10 mL sample was 25 min, and 5 min of sample equilibrium time was added to ensure that the sample was fully balanced in the channel. The AF4 was set to the aggregation mode for 1 min, and the cross flow was constant at 2 mL / min; the aggregation injection mode was set to last for 30 min, and the cross flow was constant at 2 mL / min.

[0053] In the concentration mode, the mobile phase from the mobile phase bottle enters the AF4 system Eclipse DUALTEC instrument through the liquid phase pump, and then passes through the sample injector. The sample in the ETEF pipeline can be taken into the cell channel through the In Let port of the cell channel, and at the same time, the mobile phase enters the cell channel through the Outlet port, and reaches equilibrium at the sample injection port of the cell channel. At this time, in the cell channel, the sPEVs subpopulation sample is subjected to the action of the downward cross-flow and the action of the upward diffusion force of the sample itself, and the directions of the two forces are opposite. The sample gradually reaches equilibrium under the action of the two opposite forces, and the equilibrium position is a certain distance away from the cell membrane, which can minimize the adsorption of the sample. Solvents and any substances smaller than the pore size of the semi-permeable membrane can pass through the semi-permeable membrane into the waste pipe, and vesicles larger than the pore size of the semi-permeable membrane can be retained in the cell channel. The large volume sPEVs subpopulation sample can be enriched near the sample injection port, and PBS as the mobile phase can maximize the protection of the biological properties of the sample. The PEVs heterogeneous subpopulation fractions collected by repeated injection of 5 times are 30 mL of sPEVs sample, 45 mL of mPEVs sample, and 60 mL of lPEVs sample. When the volume of the sample to be enriched exceeds the volume of the ETEF pipeline, a larger volume pipeline (20 mL / 30 mL) can be replaced or the Focus, Focus inject program can be repeatedly increased when setting the AF4 program. In the Focus mode, the ETEF pipeline is replaced, the sample is reloaded, and the enrichment of samples of different volumes is realized. Because the AF4 cavity volume is in the order of microliters, when a large volume of sample solution (a few milliliters or even a few hundred milliliters) is pumped in, the solution volume of the particles retained in the separation chamber can be changed from a large volume to the AF4 cell channel capacity volume (about 380 μL), thereby achieving the purpose of enrichment. Through the switching of the two modes, the enrichment of EVs samples of different volumes is realized. The enrichment of EVs samples of different volumes can also be achieved by replacing the ETEF pipeline according to the actual EVs sample or other trace particle samples in the environment. The cost is low, and the operation is flexible and easy to operate.

[0054] After the sample enrichment is completed, stop the liquid phase pump. Connect the PEEK Pruril joint at the 5 mL syringe port, connect the PEEK integral high-pressure joint at the other end of the PEEK flange pipe joint containing a 15 cm HPLC 1 / 16 pipeline connection line, and connect it with the PEEK Pruril joint connected to the syringe. Next, connect the PEEK flange pipe joint at the cell channel outlet port; connect another PEEK flange pipe joint containing a 15 cm HPLC 1 / 16 pipeline connection line at the cell channel inlet port, and connect the other end of the pipeline to a 1.5 mL EP tube. To minimize dilution of the sample, pump 1 mL of air into the syringe. At this time, the air pressure in the syringe pushes the enriched sPEVs sample out, and about 380 μL of the sample is collected in the EP tube, which is stored at -80°C.

[0055] After AF4 concentration, the volume of each subpopulation sample can be reduced to the volume of the chamber.

[0056] The EVs heterogeneous subpopulation enrichment procedure is shown in Table 4.

[0057] Table 4 EVs heterogeneous subpopulation enrichment procedure

[0058]

[0059] The enrichment fold of sPEVs, mPEVs, and lPEVs heterogeneous subpopulation samples after AF4 concentration after pipeline modification is shown in Table 5.

[0060] Table 5 Enrichment fold of sPEVs, mPEVs, and lPEVs heterogeneous subpopulation samples after AF4 concentration after pipeline modification after 5 times of separation and collection

[0061]

[0062] To verify the accuracy and feasibility of AF4 concentration, the experiment also compared the commonly used ultrafiltration method for concentrating EVs heterogeneous subpopulations. The sPEVs, mPEVs, and lPEVs subpopulation samples concentrated by ultrafiltration and AF4 were negatively stained with uranyl acetate dye, and TEM imaging analysis was performed. The results are shown in Figure 4 It can be seen under the electron microscope that the number of EVs heterogeneous subpopulations after ultrafiltration concentration is less than that after AF4 concentration, and the observed EVs subpopulations all have damaged membrane structures. This may be due to the large centrifugal force applied by the ultrafiltration method, which causes the sample to precipitate or adsorb on the ultrafiltration column. AF4 is enriched in a fluid environment without applying high-speed external force, and the diffusion force of the sample particles will cause the sample and the membrane to have a distance, which will minimize the adsorption of the sample. The EVs sample after enrichment has a good shape and an intact membrane structure, which retains the biological characteristics of the sample and is more conducive to further research on EVs heterogeneous subpopulations. It is proved that AF4 can have a better enrichment effect on EVs than ultrafiltration.

[0063] The device for the method of using AF4 to gently and low-damage separate, purify, and enrich EVs and their heterogeneous subpopulations of the present embodiment includes an AF4 separation module and an AF4 enrichment module. The AF4 enrichment module is a 10 mL large-volume sample inlet pipeline connected between the sample inlet and the AF4 system Eclipse DUALTEC, replacing the original pipeline. In specific use, different volumes of large-volume sample inlet pipelines can be selected according to the amount of EVs and their heterogeneous subpopulations that need to be enriched.

Claims

1. A method for gentle, low-damage separation, purification, enrichment of EVs and their heterogeneous subpopulations using AF4, characterized in that, The method comprises the following steps: Separation and purification: AF4 is used to separate protein impurities from EVs and separate EVs and their heterogeneous subpopulations according to particle size. Enrichment: the EVs heterogeneous subpopulation samples of different particle sizes obtained after separation are enriched by AF4, a large-volume sampling pipeline is connected between the original AF4 sampler and the AF4 system Eclipse DUALTEC instrument to replace the original pipeline, the large-volume sampling pipeline is connected to the As Out port of the AF4 system Eclipse DUALTEC instrument and the outlet position of the sampler, and the enrichment of EVs samples of different volumes is realized by switching the original aggregation, sampling and elution mode to the aggregation mode and the aggregation sampling mode; the volume of the large-volume sampling pipeline is 10 mL-30 mL. In step 1), the separation and purification, the gradient elution is cross-flow flow rate of 2 mL / min to 0.25 mL / min within 5 min, and then 0.25 mL / min to 0.09 mL / min within 30 min.

2. The method for gentle, low-damage separation, purification, enrichment of EVs and their heterogeneous subpopulations by AF4 according to claim 1, characterized in that, In step 1), the separation and purification, the sampling amount is 100 μL, the detector flow rate is 1 mL / min, the type of membrane is 10 kDa RC membrane, and the mobile phase is 10 mM PBS solution.

3. The method for mild, low-damage separation, purification, enrichment of EVs and their heterogeneous subpopulations by AF4 according to claim 1, characterized in that, In step 1), the purification, according to the different retention times, the protein impurities and EVs samples are separated to achieve the purpose of EVs sample purification.

4. The method for mild, low-damage separation, purification, enrichment of EVs and their heterogeneous subpopulations by AF4 according to claim 1, characterized in that, In step 2), the enrichment, the sampling program is changed, the aggregation mode is performed first, then the aggregation sampling mode is performed, and then the sample amount is enriched according to the need.

5. The method for mild, low-damage separation, purification, enrichment of EVs and their heterogeneous subpopulations by AF4 according to claim 1, characterized in that, In step 2), the enrichment, the cross-flow flow rate is constant at 2 mL / min, the type of membrane is 10 kDa RC membrane, and the mobile phase is 10 mM PBS solution.

6. The method for mild, low-damage separation, purification, enrichment of EVs and their heterogeneous subpopulations by AF4 according to claim 1, characterized in that, The elution mode is the aggregation mode and the aggregation sampling mode. In the aggregation mode, by setting the sampling program, the mobile phase does not pass through the sampler, enters the AF4 system Eclipse DUALTEC instrument from the mobile phase bottle through the liquid phase pump, and then enters the cell channel through the inlet and outlet ends of the cell channel, and a convection is formed near the cell sampling port.

7. The method for gentle, low-damage separation, purification, enrichment of EVs and their heterogeneous subpopulations by AF4 according to claim 1, characterized in that, In the aggregation sampling mode, the mobile phase passes through the sampler, and the sample in the large-volume pipeline can be brought into the cell channel through the cell sampling port, at this time, in the cell channel, the solvent and any substance smaller than the pore size of the semi-permeable membrane can pass through the semi-permeable membrane into the waste liquid pipe, and the vesicles larger than the pore size of the semi-permeable membrane can be retained in the cell channel.

8. The method for gentle, low-damage separation, purification, enrichment of EVs and their heterogeneous subpopulations by AF4 according to claim 1, characterized in that, ​ 9. A device for the gentle, low-damage separation, purification, enrichment of EVs and their heterogeneous subpopulations using AF4, characterized in that, The AF4 separation module, the AF4 enrichment module, the AF4 enrichment module is connected with a large volume injection pipeline between a sample injector and an AF4 system Eclipse DUALTEC instrument to replace the original pipeline; the large volume injection pipeline is connected between an As Out port of the AF4 system Eclipse DUALTEC instrument and an outlet position of the sample injector, and the enrichment of EVs samples with different volumes is realized by switching the original aggregation, injection and elution mode to the aggregation mode and the aggregation injection mode; the volume of the large volume injection pipeline is 10 mL-30 mL.

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