CN118685347A - A method for extracting and separating high-purity exosomes - Google Patents

Abstract

The invention discloses a method for extracting and separating high-purity exosomes, which comprises the following steps: sequentially centrifuging and filtering samples to obtain a supernatant without large-particle impurities; adsorbing exosomes in the supernatant by using an ion exchange chromatography packing, and eluting by using an eluent; and adding the obtained exosome solution into a centrifugal column filled with MixQ S filler, and centrifuging to obtain filtrate, namely the high-purity exosome solution. The invention innovatively introduces the centrifugal column filled with MixQ S filler, the effect of removing impurities can be achieved by simple centrifugal operation, and the obtained exosome has good integrity. The method is simple and easy to operate, can obtain the exosome with high purity, and has important significance for downstream application of the exosome.

Description

A method for extracting and separating high-purity exosomes

Technical Field

The present invention belongs to the technical field of exosome separation and extraction, and specifically relates to a method for extracting and separating high-purity exosomes.

Background Art

Exosomes are membrane-structured vesicles secreted by cells, with a diameter of 40 to 160 nm. They contain a variety of nucleic acids, proteins, lipids and other components, and play an important role in intercellular communication. Exosomes have many advantages, such as good tolerance and biocompatibility, low immunogenicity, the ability to cross natural barriers, and the ability to be engineered. Currently, they have been widely used as delivery carriers in the treatment of cardiovascular diseases, kidney damage, immune diseases, nervous system diseases, cancer and other diseases.

Methods for the separation and extraction of exosomes include ultracentrifugation, ultrafiltration and size exclusion chromatography, polymer precipitation, immunoaffinity chromatography, etc. At present, ultracentrifugation is the "gold standard" for the separation of exosomes, but this method usually requires a centrifugal force of more than 100,000g, and the separation operation is complicated and the cycle is long. Ultrafiltration and size exclusion chromatography are both methods for separation based on the size of exosomes. Ultrafiltration is to pass the sample through a membrane filter with different pore sizes and separate it according to the size and molecular weight of the particles. Size exclusion chromatography is also separated according to the size of the particles. Larger particles leave the chromatographic column first and are separated, while smaller particles leave later and are therefore collected. Polymer precipitation is to add polyethylene glycol to form a precipitate in the solution, and then centrifuge at 1500g at a low speed to separate the exosomes. This method is suitable for a large number of samples, but the purity of the resulting exosomes is low. Immunoaffinity chromatography is based on the antigen molecules unique to the surface of exosomes, which are separated by binding to antibodies. This method has strong specificity but low yield, and it is difficult to completely remove antibodies from the sample.

Recently, studies have shown that exosomes can be extracted through ion exchange chromatography fillers using the principle of charge. This method can use a simple centrifuge device to extract exosomes. However, the inventors found during their research that although exosomes in cell supernatant and urine can be extracted through ion exchange chromatography fillers, electron microscopy results show that there are a large number of impurities in the background of the obtained exosomes, which is obviously not conducive to the actual use of exosomes.

Summary of the invention

In view of the problem that a large amount of impurities exist in exosomes extracted by ion exchange fillers, the present invention provides a method for extracting and purifying exosomes, which is simple and effective.

The technical solution of the present invention is specifically as follows:

A method for extracting and separating high-purity exosomes, comprising the following steps:

S1. Centrifuge and filter the sample to obtain a supernatant without large particles;

S2, adsorbing the exosomes in the supernatant with ion exchange chromatography filler, and then eluting with eluent;

S3. Add the obtained exosome solution into a centrifugal column filled with MixQ700S filler, and the filtrate obtained by centrifugation is a high-purity exosome solution.

In the method of the present invention, the sample in step S1 is specifically a biological fluid such as cell culture fluid and urine. After the inventors identified and analyzed the exosomes extracted by ion exchange chromatography fillers, they found that the impurities in the background of the exosomes were mostly small crystalline particles under electron microscopy. Further analysis found that it contained salt particles and other non-exosomal proteins. Through a large number of experimental explorations, the inventors found that the impurities in the effective exosomes can be removed by simple low-speed centrifugation using a centrifugal column filled with MixQ700S fillers, and the background of the electron microscope image obtained by identification is clean. MixQ700S is a composite mode filler, in which the filler particle size is 70um, a core-shell structure microsphere, the shell layer is a neutral hydrophilic layer, which can prevent proteins above 700KD from entering the interior, and the core layer is an anion exchange and hydrophobic interaction functional layer, which is often used to separate and purify proteins.

Preferably, in the above method, the centrifugation condition in step S1 is 1500-3000g for 10-20min, and the filtration is to filter the centrifugal supernatant with a 0.22μm pore size filter membrane. The method of the present invention first removes large particle impurities in the sample by centrifugation, and then removes non-exosome large vesicles and most of the miscellaneous proteins by filtration; after these pre-treatments, the sample can obtain a supernatant without large particle impurities, which is conducive to subsequent adsorption and elution.

Preferably, in the above method, step S2 specifically comprises: incubating the ion exchange chromatography filler and the supernatant in a binding buffer, centrifuging and transferring the precipitate to a purification column, washing with a washing solution, and then eluting with an eluent.

Preferably, in the above method, the ion exchange chromatography filler in step S2 is a weak anion exchange chromatography filler, wherein the weak anion exchanger comprises diethylaminoethyl.

More preferably, in step S2 of the above method, the binding buffer is 200-500 mM Tris-HCl buffer (pH 7.0-8.0), the washing solution is 50-200 mM Tris, 10-150 mM NaCl (pH 7.2-8.0), and the elution solution is 50-100 mM HEPES, 100-500 mM NaCl.

Preferably, in the above method, the centrifugation condition of step S3 is: 300-1000g centrifugation for 30-90s.

Compared with the prior art, the present invention has the following beneficial effects:

In order to solve the problem of impurities in the process of extracting and separating exosomes by adsorption of ion exchange chromatography fillers, the present invention innovatively introduces a centrifugal column with self-filled MixQ700S filler, and the effect of completely removing impurities can be achieved through a simple centrifugation operation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a TEM electron microscope image of the exosomes extracted and separated in Example 1.

Figure 2 is a TEM electron microscope image of the exosomes extracted and separated in Comparative Example 1.

DETAILED DESCRIPTION

The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The term "comprises" and any variation thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

If no specific techniques or conditions are specified in the following examples, all of them are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

Example 1

In this example, HEK293T cell culture fluid is used as an example, and the extraction and identification of exosomes are performed according to the method of the present invention, which specifically includes the following steps:

(1) Take 10 ml of cell culture medium and centrifuge it at 4°C and 1500 g for 10 min to remove suspended cells and cell debris. Pass the resulting supernatant through a 0.22 μm pore size filter to remove larger microvesicles. Pipette 5 ml of the filtrate into a 10 ml centrifuge tube.

(2) Use a pipette to add 500 μl of binding buffer (200 mM Tris-HCl buffer, pH 8.0) into the centrifuge tube containing the filtrate in step (1), close the lid, and invert to mix.

(3) Pipette 300 μl of DEAE resin solution into the centrifuge tube obtained in step (2), cover the lid tightly, mix by continuous inversion at room temperature, incubate for 15 min, centrifuge at 1500 g for 2 min, gently remove the centrifuge tube from the centrifuge, take 500 μl of supernatant with a pipette (do not discard), carefully pour out the remaining supernatant, gently blow up the resin with the liquid in the pipette (500 μl), transfer all of it to the purification column (which has been placed in the collection tube), let it stand for 2 min, centrifuge at 2000 g for 2 min at room temperature, pipette 500 μl of washing solution (pH 8.0, 50 mM Tris, 10 mM NaCl) and add it to the purification column, let it stand for 5 min, centrifuge at 2000 g for 2 min at room temperature, and discard the filtrate and collection tube.

(4) Transfer the purification column to a 1.5 ml centrifuge tube with low protein adsorption, add 200 μl of elution buffer (pH 7.2, 50 mM HEPES, 100 mM NaCl), incubate for 5 min, centrifuge at 350 g for 2 min at room temperature, transfer the filtrate back to the purification column, incubate for 5 min, and centrifuge at 2500 g for 3 min at room temperature. The liquid obtained in the centrifuge tube is the concentrated exosome solution.

(5) Add the concentrated exosome solution to a centrifugal column pre-filled with MixQ700S filler and centrifuge at 300 g for 90 seconds. The filtrate is the exosome solution with impurities removed.

The exosome solution obtained in step (5) was subjected to electron microscopy, and the results are shown in Figure 1: the electron microscopy photo shows a clean background, no obvious impurities, and a large number of exosomes with complete morphology.

Comparative Example 1

The samples used in this example are the same as those in Example 1, and exosomes are extracted by the following steps:

(1) Take 10 ml of cell culture medium and centrifuge it at 4°C and 1500 g for 10 min to remove suspended cells and cell debris. Pass the resulting supernatant through a 0.22 μm pore size filter to remove larger microvesicles. Pipette 5 ml of the filtrate into a 10 ml centrifuge tube.

(2) Pipette 500 μl of binding buffer (200 mM Tris-HCl buffer, pH 8.0) into the centrifuge tube containing the filtrate in step (1), close the lid, and invert to mix.

(3) Pipette 300 μl of DEAE resin into the centrifuge tube obtained in step (2), cover the lid tightly, mix by continuous inversion at room temperature, incubate for 15 min, centrifuge at 1500 g for 2 min, gently remove the centrifuge tube from the centrifuge, use a pipette to take 500 μl of supernatant (do not discard), carefully pour out the remaining supernatant, use the liquid in the pipette (500 μl) to gently blow up the resin, transfer all of it into the purification column (which has been placed in the collection tube), let it stand for 2 min, centrifuge at 2000 g for 2 min at room temperature, pipette 500 μl of washing solution (pH 8.0, 50 mM Tris, 10 mM NaCl) and add it to the purification column, let it stand for 5 min, centrifuge at 2000 g for 2 min at room temperature, and discard the filtrate and collection tube.

(4) Transfer the purification column to a 1.5 ml centrifuge tube with low protein adsorption, add 200 μl of elution buffer (pH 7.2, 50 mM HEPES, 100 mM NaCl), incubate for 5 min, centrifuge at 350 g for 2 min at room temperature, transfer the eluate back to the purification column, incubate for 5 min, and centrifuge at 2500 g for 3 min at room temperature. The liquid obtained in the centrifuge tube is the exosome solution.

The exosome solution obtained in step (4) was subjected to electron microscopy, and the results are shown in FIG2 : The electron microscopy photograph of the exosomes obtained in this example showed an unclean background and many small crystalline particles.

In summary, the present invention can overcome the defects of the existing method by introducing a centrifugal column with self-filled MixQ700S filler, obtain high-purity exosomes, and ensure the good integrity of the exosomes. It can be seen that the method of the present invention can obtain high-quality exosomes, which is of great significance to the downstream applications of exosomes.

It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims (10)

1. A method for extracting and separating high-purity exosomes, which is characterized by comprising the following steps:

s1, sequentially centrifuging and filtering a sample to obtain a supernatant without large-particle impurities;

s2, adsorbing exosomes in the supernatant by using an ion exchange chromatography packing, and eluting by using an eluent;

And S3, adding the obtained exosome solution into a centrifugal column filled with MixQ S filler, and centrifuging to obtain filtrate, namely the high-purity exosome solution.

2. The method of claim 1, wherein the step S1 sample comprises a cell culture fluid, urine.

3. The method according to claim 1, wherein the centrifugation of step S1 is: centrifuging at 1500-3000g for 10-20min.

4. The method of claim 1, wherein step S1 filtration employs a 0.22 μm pore size filter.

5. The method according to claim 1, wherein step S2 is specifically: after incubation of the ion exchange chromatography packing, cell supernatant in binding buffer, centrifugation and transfer of the precipitate to a purification column, washing with washing liquid and eluting with eluting liquid.

6. The method according to claim 1 or 5, wherein the ion exchange chromatography packing of step S2 is a weak anion exchange chromatography packing, wherein the weak anion exchanger comprises diethylaminoethyl.

7. The method of claim 6, wherein the binding buffer is Tris-HCl at a pH of 7.0-8.0, 200-500 mM.

8. The method according to claim 6, wherein the washing solution is pH 7.2-8.0, 50-200mM Tris, 10-150mM NaCl.

9. The method of claim 6, wherein the eluent is pH 7.2-7.5, 50-100mM HEPES, 100-500mM NaCl.

10. The method according to claim 1, wherein the centrifugation of step S3 is: centrifuging for 30-90s at 300-1000 g.