CN115094030A

CN115094030A - Clinical-grade high-purity exosome purification method

Info

Publication number: CN115094030A
Application number: CN202210789295.XA
Authority: CN
Inventors: 金倞; 雷欣华; 梁磊; 高雷
Original assignee: Beijing Jizhongke Biotechnology Co ltd
Current assignee: Beijing Jizhongke Biotechnology Co ltd
Priority date: 2022-07-05
Filing date: 2022-07-05
Publication date: 2022-09-23
Anticipated expiration: 2042-07-05
Also published as: CN115094030B

Abstract

The invention provides a purification method of clinical-grade high-purity exosomes, and relates to the technical field of exosome purification. The invention carries out exosome purification based on a method combining tangential flow filtration and size exclusion chromatography, and can be used for preparing high-purity exosome meeting clinical requirements. The purification method provided by the invention realizes concentration and purification of the natural exosome by virtue of a physical principle, no exogenous substance is introduced during the process, and finally the natural exosome product exists in the physiological saline for injection, thereby meeting the clinical application requirement.

Description

Clinical-grade high-purity exosome purification method

Technical Field

The invention belongs to the technical field of exosome purification, and particularly relates to a purification method of clinical-grade high-purity exosomes.

Background

Exosomes (exosomes) are Extracellular Vesicles (EVs) of nanometer size 30-150nm, which play an important role in intercellular communication and are able to modulate the behavior of receptor cells through autocrine, paracrine, endocrine and/or near-secretory cell signaling patterns. Exosomes are naturally produced by a variety of cell types and are a natural vector for transferring proteins, lipids, DNA, microRNA and mRNA to adjacent or more distant cells. Exosomes play an important role in cancer progression, angiogenesis, metastatic niche formation, organ-specific metastasis, tumor microenvironment remodeling, immunosuppression, and the like, and have received considerable attention.

On the one hand, natural and engineered exosomes have been identified to play important roles in many different disease models, such as neurodegenerative diseases, cancer and wound healing. Meanwhile, the exosome is used for treating COVID-19 by virtue of the anti-inflammatory activity of the exosome, and has great potential. Furthermore, as a natural carrier for biomolecules, the use of exosomes as delivery systems for stable and targeted drugs is another important direction of development.

On the other hand, exosomes represent tissue-specific molecules with higher stability, which can be used as disease-specific biomarkers. Exosomes can be isolated from small amounts of biological fluids and clinical samples, and exosomes in patient fluids have become a promising source of biomarker development. Also, since exosomes can be regulated in their release and composition by environmental factors, they can also serve as markers of disease state and therapeutic outcome.

Exosomes are of great interest because of their role in pathobiological processes and are being explored as tools for disease diagnosis and treatment. With the increasing potential for their clinical use, it has become imperative to optimize their isolation methods to achieve maximum yield, purity and assay reproducibility. Therefore, various separation methods based on different principles have been developed for exosome separation.

One of the major challenges in exosome isolation is the elimination of nanoscale contaminants, including cell-free nucleic acids and lipoproteins, which interfere with the accuracy of exosome biomarkers. Ultracentrifugation is currently the major technique for exosome isolation, and is a gold standard method for exosome isolation and identification recognized by industry agencies and personnel. Nevertheless, the exosomes obtained by ultracentrifugation techniques still do not meet the standards required for clinical applications, with the main limitations being their achievable exosome purity, yield, integrity and long processing time. Therefore, innovative technologies need to be developed to improve exosome purification in terms of efficiency, purity, yield, speed and robustness.

Disclosure of Invention

In view of the above, the present invention provides a clinical-grade high-purity exosome purification method, which is an exosome purification method based on a combination of a tangential flow filtration method and a size exclusion chromatography method, so as to prepare high-purity exosomes meeting clinical requirements.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for purifying high-purity exosomes, which comprises the following steps: sequentially subjecting the supernatant of the cultured exosome-secreting cells to TFF tangential flow purification and size exclusion chromatography purification;

the supernatant comprises a mixture of the compounds based on Rooster Nourich ^TM -MSC-XF mesenchymal stem cell expansion medium and Rooster Collection ^TM -cell supernatant collected under 2D culture conditions or 3D bioreactor culture system in EV exosome-rich medium.

Preferably, the method further comprises centrifuging at 4 ℃ for 1h at 3000g to remove impurities before purifying the supernatant.

Preferably, the apparatus for tangential flow purification of TFF comprises a hollow fiber module.

Preferably, the hollow fibers in the hollow fiber module have a pore size of 200 nm.

Preferably, prior to performing said size exclusion chromatography purification, further comprising concentration of exosomes.

Preferably, the concentration comprises using a filter element comprising polysulfone hollow fibers having a pore size of 20 nm.

Preferably, the size exclusion chromatography purification comprises using a SEC chromatography column packed with porous gel particles.

Preferably, the eluent used for size exclusion chromatography purification is PBS.

The invention also provides application of the exosome prepared by the purification method in preparation of a clinical exosome reagent.

Preferably, before preparing the clinical exosome reagent, the method further comprises replacing the solvent of the prepared exosome with physiological saline.

Has the advantages that: the invention provides a clinical-grade high-purity exosome purification method, which is an exosome purification method based on the combination of a tangential flow filtration method and a size exclusion chromatography method and can be used for preparing high-purity exosomes meeting clinical requirements. In the upstream TFF tangential flow purification process, the biomembranes with different porosities can separate particles with different molecular weights, further remove residual particle pollutants, replace an exosome solvent culture medium with NaCl Buffer, and simultaneously stably and efficiently separate and concentrate exosomes with specific particle size ranges. The initial high purity and high concentration of the exosome sample solves the problem that the exosome sample is easily polluted by non-exosome particles in an SEC process flow, reduces the pollution of high-abundance protein, realizes the fine purification of exosome, and ensures the purity and yield of the exosome at the end of the process flow. The purification method provided by the invention realizes concentration and purification of the natural exosome by virtue of a physical principle, no exogenous substance is introduced during the process, and finally the natural exosome product exists in the physiological saline for injection, thereby meeting the clinical application requirement.

Drawings

FIG. 1 is a schematic diagram of the structure of TFF-MV;

FIG. 2 is a schematic diagram of the loading process of exosomes during purification of TFF-MV;

FIG. 3 is a schematic diagram of TFF-MV tangential flow filtration;

FIG. 4 is a schematic representation of TFF-MV wash residue particles;

FIG. 5 is a schematic structural diagram of TFF-Easy;

FIG. 6 is a schematic diagram of exosome loading process at EV concentration;

FIG. 7 is a schematic representation of TFF-Easy tangential flow concentration;

FIG. 8 is a schematic view of the operation before recovery of the concentrated sample;

FIG. 9 is a schematic of the recovery of concentrated exosomes;

FIG. 10 shows the result of NTA detection of exosomes before and after 2D environmental purification;

FIG. 11 shows the result of NTA detection of exosomes before and after purification in a 3D environment;

FIG. 12 shows electron microscope detection results of exosomes before and after 2D/3D environmental purification;

FIG. 13 shows the detection results of Western Blot on exosomes before and after 2D/3D environmental purification; in the figure, 1.3D samples before purification; 2.3D-TFF-MV purified sample; 3.3D-TFF-Easy purified sample; 4.3D-SEC purified sample; 5.2D-SEC purified sample; 2D-TFF-Easy purified sample; 7.2D-samples before purification;

FIG. 14 is the composition of exosome NanoView detection subpopulations before and after 2D/3D environmental purification;

FIG. 15 shows the particle size distribution of specific subsets of exosomes before and after 2D environmental purification;

FIG. 16 is the particle size distribution of specific subpopulations of exosomes before and after purification in a 3D environment;

FIG. 17 is a comparison of the fluorescence intensities of specific subsets of exosomes before and after purification in a 2D environment;

FIG. 18 is a comparison of exosome-specific subset fluorescence intensity before and after 3D environmental purification;

FIG. 19 is a schematic diagram of the low sample adsorption modified polyethersulfone membrane hollow fiber tangential flow filtration principle;

FIG. 20 shows Exo-spin ^TM -EX04 size exclusion chromatography purification of exosomes schematic, 1. column temperature equilibration; 2. absorbing and discarding the protective agent; 3. balancing a column bed; 4. concentrating the sample for addition; 5. elution of purified exosomes

Detailed Description

The invention provides a purification method of clinical-grade high-purity exosomes, which comprises the following steps: sequentially subjecting the supernatant of cultured exosome receptor cells to TFF tangential flow purification and size exclusion chromatography purification;

said supernatant comprises a mixture of a Rooster Noursh-based supernatant ^TM -MSC-XF mesenchymal stem cell expansion medium and RossterCollect ^TM -cell supernatant collected under 2D culture conditions or 3D bioreactor culture system in EV exosome-rich medium.

The TFF of the present invention is a filtration process using tangential flow across the surface to avoid the formation of a filter cake. The filter has an optimal pore size, can remove small-sized protein impurities, and can effectively separate and concentrate exosomes. Clogging is less likely than in conventional filtration due to the lower likelihood of cake formation.

The Size Exclusion Chromatography (SEC) according to the present invention preferably allows the separation of components in a sample according to hydrodynamic volume; SEC is usually carried out using Sepharose CL-2B or similar fixed columns, where the fractions are eluted with PBS; SEC is simple, robust and scalable, does not require expensive equipment, and can be used under mild elution conditions, applicable to a variety of application scenarios. In addition, SEC is an efficient exosome isolation method for downstream proteomic analysis.

The invention has limitations on the source of exosomes for purification, based on rooster nourish ^TM -MSC-XF mesenchymal stem cell expansion medium and RossterCollect ^TM Cell supernatants collected from EV exosome-rich medium under T-1752D culture conditions and FiberCell 3D bioreactor culture system, and the method of the invention further comprises centrifugation for 1h at 4 ℃ and 3000g before exosome purification, impurity removal and pretreatment before fine purification. In the invention, the exosome obtained under a FiberCell 3D culture system is preferably adopted for subsequent purification, for example, in the embodiment, Mesenchymal Stem Cells (MSC) are used as exosome donor cells, the MSC is used for obtaining the exosome in the FiberCell low-particle culture medium system, the FiberCell material per se is a vacuum fiber column with a certain porosity, a sample in an internal channel is not polluted by macromolecules of an external culture medium circulation system, and the purity of an initial sample is ensured.

The present invention, after the above pre-treatment, performs a tangential flow purification of TFF, preferably comprising a hollow fiber (Hollowfiber) module in the apparatus. Sources of the HollowFiber of the present invention preferably include TFF-MV and TFF-Easy from Hansa BioMed, or the GREEN, Steamer, Reuse series of different pore sizes and membrane retention capacity products of hollow fiber modules from Saedolis, Germany, where the hollow fiber Tangential Flow Filtration (TFF) modules provide high performance separations including cell collection, clarification, perfusion, washing and concentration in various upstream and downstream process unit operations. They are characterized by being made of modified polyethersulfone membrane (m-PES) with low sample adsorption, stably providing high sample processing throughput and high sample recovery, and the separation principle is based on the interception of different sized molecules of sample solution passing through the pore size of modified polyethersulfone membrane (FIG. 19), and TFF-MV and TFF-Easy are exemplified in the examples, but they cannot be regarded as the full protection scope of the present invention.

The structure of the TFF-MV is shown in figure 1, the TFF-MV is a filter element (200nm hole) containing polysulfone hollow fibers, and can separate medium/large extracellular vesicles from small EV or other fluids (cell culture medium, urine, blood plasma, blood serum) from different sources. Small EVs, proteins and other soluble molecules pass through the hollow fiber pores (permeate), while medium/large EVs are concentrated in the retentate. Large EVs can be easily collected by a syringe in a cartridge filter. TFF-MV is sterile. Once the filter was used and can be sterilized by Beta irradiation, TFF-MV could not be autoclaved and stored at room temperature. In the present invention, filtration of EV using said TFF-MV preferably comprises filtration feeding, tangential flow filtration, washing of residual small particle extracellular exosomes and enrichment of small EV.

In the embodiment of the present invention, the filtered sample injection preferably includes: removing the screw from the sample injection nozzle; a syringe filter is inserted in the nozzle, the device is compatible with a variety of filter types, the size of the filter to be used depends on the user, 0.22 μm is selected in the embodiment; taking a syringe A with a sample and inserting the syringe A into the top of the filter; inserting a clean empty syringe into the tangential flow filtration nozzle (position 2 in FIG. 1); rotating the valve 3 to the position indicated by the arrow in fig. 2; the sample was injected into the filter by pushing the plunger of syringe a, the sample passed through the filter, filling syringe B.

In an embodiment of the present invention, the tangential flow filtration preferably comprises: rotating the valve 3 to the position shown in figure 3; the lid is removed and the permeation nozzle 4b is opened; a permeate collection tube was placed below the nozzle at position 4b, as shown in FIG. 3; alternately pushing syringes B and C up and down, starting the filtration process; permeate began to flow to the collection tube, while medium/large EV separated in the retentate; continue the filtration process until the syringe is completely empty and no more permeate flows; a new clean syringe (C) is inserted in position 5.

In the embodiment of the present invention, the washing residual small granule cell exosomes preferably comprises: disconnecting sample injector B while closing the fluid valve at position 3; PBS syringe B was reconnected, the fluid valve at position 3 was activated and the filter was washed with PBS until the syringe was completely emptied to remove the remaining smaller particles (figure 4). This step was repeated once.

In an embodiment of the invention, the enrichment of the small EV preferably comprises filtering the permeate containing the small EV, soluble proteins and other substances using HBM-LS TFF-Easy to further concentrate the small molecule particles, the concentrated sample will be further purified using size exclusion chromatography columns for downstream applications and analysis.

The present invention preferably uses TFF-Easy for exosome purification and concentration, and TFF-Easy is preferably constructed as shown in FIG. 5, the same as the outside of the device shown in FIG. 2, except that the pore size of the filter is different, and TFF-Easy is a filter containing polysulfone hollow fibers (20nm pores) that can concentrate and remove small proteins and molecules in the dilution matrix (cell conditioned medium, urine, etc.) before EV purification. Water and small molecules (<20nm) pass through the hollow fiber pores, while EV is concentrated in the retentate; EV can be easily recovered from the cartridge using a syringe. TFF-Easy was supplied aseptically. Beta irradiation may be used to sterilize once the filter is used. TFF-Easy was not autoclaved. The apparatus was stored at room temperature.

When the concentration is performed in the embodiment of the invention, the concentration preferably comprises filtration and sample injection, tangential flow filtration and recovery of concentrated EV.

The filter sample injection of the invention preferably comprises: removing the screw from the sample injection nozzle (position 1 in fig. 5); a syringe filter is inserted in the nozzle. The device is compatible with a variety of filter types. The size of the filter holes can be selected by the user as desired. To retain all EV subtypes, it is recommended to use a filter pore size of 0.22 μm; remove the sample with syringe a and insert into the top of the filter; inserting a clean empty syringe into the tangential flow filtration nozzle (position 2 in FIG. 5); rotating the valve to the position indicated by the arrow 3 in fig. 6; the sample is injected into the filter by pushing the plunger of syringe a; the sample passed through the filter and filled syringe B.

The tangential flow filtration of the present invention preferably comprises: rotating the valve 3 to the position shown in figure 7; opening the permeation nozzle, and detaching the screw; inserting a new clean syringe (C) into position 5; 2 permeate collection tubes were placed below the nozzle at position 4, as shown in fig. 7; alternately pushing syringes B and C up and down to begin the concentration process; continuing the concentration process until the desired volume is obtained; the permeate started to flow to the collection tube, while the retentate in the syringe contained the concentrated EV.

The recovery of the concentrated EV according to the present invention preferably includes: to collect concentrate EV, the concentrate product is pushed into syringe B; the fluid valve 6 is rotated to the position shown in figure 8 and the syringe C is then disconnected; filling syringe C with air; rotating the fluid valve 6 to the position shown in figure 9; insert syringe C and inject air into the apparatus, then pull up the plunger of syringe B to collect all remaining volume of concentrated sample; rotating the valve 3 to the position shown by the arrow on the right side of figure 9 and disconnecting the syringe B; the concentrated samples were collected in clean test tubes.

The invention, after said concentration, performs a Size exclusion chromatography (Size exclusion chromatography SEC) purification, said SEC purification preferably comprising the use of SEC columns packed with porous gel particles. The SEC size exclusion chromatographic column preferably comprises EXO-spin series products of Cell guide Systems company, qEVsingle, qEVoriginal, qEV1, qEV2, qEV10, qEV100 and the like of the iZON company and matched exosome Automatic extraction equipment, namely an Izon Automatic extraction Collector; or other similar products, the EXO-spin family of products are exemplified in the examples, but they are not to be construed as limiting the scope of the invention.

The SEC according to the invention is preferably a method for separating particles in solution according to particle size, the chromatographic column being packed with stable porous gel particles. When a solution containing exosomes is added into an SEC chromatographic column, small molecules can pass through the pores of gel particles, but larger molecules cannot pass through the pores, so that the small particles in the same column have a longer moving path than large particles and take longer time to flow through, and the physical separation of particles with different sizes is realized. Thus, the eluents of different stages will contain particles of different sizes, first eluting larger exosome particles, then smaller protein and lipid particles. The conventional SEC-based exosome purification needs a precipitation step, but in the technical scheme of the invention, the original concentration of an exosome sample is high based on the exosome production process of a Fiber Cell low vesicle culture system, and meanwhile, the extremely low vesicle pollution in the system is ensured, and the concentrated sample is directly purified by using an SEC chromatographic column, so that the influence of a precipitator on a final exosome sample is avoided.

In the present example, when EXO-spin is used for the SEC purification, Exo-spin is preferably included ^TM EX04 column pretreatment and exosome purification steps (fig. 20).

Exo-spin according to the invention ^TM -EX04 chromatography column pretreatment, preferably comprising: incubating the column at room temperature for 15min to equilibrate the column temperature; taking down the bottom outlet plug, taking down the screw cap at the upper end, and placing the Exo-spin chromatographic column in or above the waste liquid collecting tube; removing the preservative buffer from the top of the column using a pipettor and immediately proceeding to the next step to prevent the bed from drying; adding 10mL PBS balance column buffer solution at the top end of the column, allowing the PBS buffer solution to flow through the column matrix under the action of gravity without centrifugation, allowing the buffer solution to enter a waste liquid collecting pipe after passing, and sucking and discarding; the above steps were repeated, and pretreatment was performed 2 times with column PBS buffer.

The exosome purification of the present invention preferably comprises: after the column equilibrated, carefully inject 1mL of TFF-concentrated exosome-concentrated sample into the top of the column; placing the chromatographic column in a waste liquid tube, and discharging the purified sample under the action of gravity; injecting 2mL of PBS into the top of the chromatographic column, allowing the PBS to drain into a waste liquid tube under the action of gravity and discarding; putting the chromatographic column into a new 50mL collecting pipe, adding 3-5 mL PBS again, discharging the PBS under the action of gravity, and collecting the eluent containing the exosome; the exosome purity can be improved to the maximum extent by eluting with 3mL of PBS, and the exosome yield can be improved to the maximum extent by eluting with 5mL of PBS; after purification of the first 1mL sample load, the column was washed with PBS to remove free protein from the column, and then more samples could be purified on the same column. For samples greater than 1mL, iterative loading allows loading of sample volumes up to 5 mL; the purified sample can be subjected to vesicle concentration test, exosome identification and downstream application.

The high-throughput purification method disclosed by the invention is simple in flow operation, can realize the purification of exosomes without a high-speed centrifugation step, and simultaneously ensures the low pollution of products.

Before the clinical exosome reagent is prepared, the invention preferably further comprises the step of replacing the solvent of the prepared exosome with physiological saline. In the present example, the exosomes quantified after purification were stored in PBS containing 0.2% BSA, and the buffer was replaced again by the method in TFF-Easy, and the buffer was replaced with physiological saline, and thus the exosomes were used in clinical experiments.

The following examples are provided to illustrate the purification method of high-throughput exosomes provided by the present invention in detail, but they should not be construed as limiting the scope of the present invention.

Examples

Selecting umbilical cord mesenchymal stem cells from the same parent source P5 generation in 2D T-175 Cell culture flask and 3D Fiber Cell system, respectively, using Rooster Nourich ^TM -MSC-XF mesenchymal stem cell expansion medium for cell expansion and Rooster Collection ^TM EV Pro exosome enrichment medium for exosome enrichment, cell supernatants from each of the 2D and 3D culture conditions were harvested as purified raw samples for fine purification of exosomes.

Wherein the 2D culture conditions are in Rooster Nourich ^TM When the cells cultured in the MSC-XF mesenchymal stem cell amplification culture medium reach 85% confluency, the cells are washed 4 times by using D-PBS and replaced by new Rooster Collect ^TM EV Pro medium for 48 hours,the supernatant was collected. 3D culture selection is changed into Rooster Collect ^TM Cell supernatants at day 6 of harvest after EV Pro medium.

The supernatants were used before purification by centrifugation at 16000 Xg for 1h at 4 ℃.

(I) 2D and 3D environmental harvest exosomes can realize high-power enrichment

The exosome particle size Analysis method is Nanoparticle Tracking Analysis (NTA), and the principle is to track and analyze brownian motion of each particle and calculate the hydrodynamic diameter and concentration of the nanoparticles by combining a stockers-Einstein equation. NTA technology has been recognized by the field of exosome research as one of the means of exosome characterization. Compared with other characterization modes, the NTA technology has simpler sample processing, can better ensure the original state of the exosome and has higher detection speed.

The concentration data of exosomes before and after purification of 2D and 3D environments were tested using NTA, respectively, and the results showed that the 2D harvest exosomes concentration before purification was 1.1E +9 particles/mL and the concentration after purification was 2.0E +10 particles/mL, achieving about 18-fold purification and concentration (fig. 10); 3D harvest exosomes were purified at a pre-4.2E +10 particles/mL concentration and at a post-purification concentration of 5.9E +11 particles/mL, achieving about 14-fold purification and concentration (FIG. 11).

(II) the purified exosomes have obvious exosome structures through electron microscope tests

Exosomes are many different nanoscale vesicles released by cells, which can be distinguished according to their morphology and size distribution. Since particles smaller than 300nm are not visible in optical methods, electron microscopy is required to characterize exosome product morphology. The results show that exosomes with obvious ring structure characteristics are obtained before and after purification under 2D/3D culture conditions (FIG. 12).

(III) the exosomes after purification have the expression of specific proteins through Western Blot test

And detecting the expression conditions of CD63, CD81, TSG101 and beta-actin in the sample by Western blot. The results indicate that under 2D/3D culture conditions, the fluid harvest had expression of the exosome marker protein before and at each stage of purification (FIG. 13).

And (IV) before and after purification, the composition of the exosome subgroup has no significant change after the exosomes are tested by NanoView.

As a biological medicine development, the method needs to analyze the exosome subgroup before and after purification so as to ensure that the exosome subgroup components and the ratio of the initial sample and the purified sample are in a stable state in the exosome purification process by the purification process.

The subgroup composition of exosomes before and after 2D/3D environmental purification was tested using a NanoView full-automatic exosome fluorescence detection analysis system. The results are as follows:

(1) the composition of the 2D/3D exosome subpopulation displayed in the CD63/CD81/CD9 capture channel did not change significantly before and after purification, demonstrating that the initial sample did not affect the composition of the exosome subpopulation by this purification process (fig. 14).

Similarly, by analyzing the fluorescence of the exosome surface markers captured in different channels and the change of the particle size distribution of the exosome surface markers, the particle size of a specific subgroup of 2D/3D exosomes is shown not to be significantly changed before and after purification, and the fact that the particle size distribution of the exosome subgroup is not influenced by an initial sample subjected to the purification process is proved (fig. 15 and fig. 16);

then, by visualizing the fluorescence of exosomes captured in different channels, the fluorescence intensity ratio of a specific subgroup of 2D exosomes is not significantly changed before and after purification, the fluorescence intensity ratio of the specific subgroup of 3D exosomes is significantly different before and after purification, the fluorescence intensity ratio of the specific subgroup of 3D exosomes expressed by the combination of CD63/CD81/CD9 in each capture channel is proved to have small influence on the composition of the subgroup of exosomes obtained under the 2D condition and has certain influence on the composition of the subgroup of exosomes obtained under the 3D condition, the reason may be that the concentration of exosomes obtained under the 3D condition is higher, the random capture detection result of a high-concentration vesicle sample can reflect the composition of a final product, and the result that the particle size of the specific subgroup of 2D/3D exosomes is not significantly changed before and after purification proves that the purification process does not cause the stability of main components of exosomes before and after 2D/3D environmental purification, the subpopulation of the high concentration sample detects the subpopulation composition of the complex multi-marker co-expression that is more reflective of exosomes. (FIG. 17, FIG. 18).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for purifying high-purity exosomes is characterized by comprising the following steps: sequentially performing TFF tangential flow purification and size exclusion chromatography purification on the supernatant of the cultured exosome secreting cells;

said supernatant comprises a mixture of a Rooster Noursh-based supernatant ^TM -MSC-XF mesenchymal stem cell expansion medium and Rooster Collection ^TM Cell supernatants collected from EV exosome-rich medium under 2D culture conditions or 3D bioreactor culture system.

2. The purification method of claim 1, further comprising centrifuging the supernatant at 3000g for 1h at 4 ℃ to remove impurities before purifying the supernatant.

3. The purification process of claim 1, wherein the TFF tangential flow purification apparatus comprises a hollow fiber module.

4. The purification method according to claim 4, wherein the hollow fibers in the hollow fiber module have a pore size of 200 nm.

5. The purification method of claim 1, further comprising concentrating the exosomes prior to performing the size exclusion chromatography purification.

6. The purification process according to claim 5, wherein the concentrating comprises using a filter element comprising polysulfone hollow fibers having a pore size of 20 nm.

7. The purification method according to claim 1, wherein the size exclusion chromatography purification comprises using a SEC column packed with porous gel particles.

8. The purification method of claim 7, wherein the size exclusion chromatography purification uses an eluent that is PBS.

9. Use of exosomes prepared by the purification method according to any one of claims 1 to 8 in the preparation of clinical exosome reagents.

10. The use of claim 9, further comprising replacing the solvent of the prepared exosomes with physiological saline prior to preparing the clinical exosome agent.