1. A method of purifying exosomes, comprising the steps of:

(1) centrifuging 9000-11000 g of cell culture solution rich in exosome;

(2) centrifuging the supernatant obtained in the step (1) and an iopamidol solution together to obtain an exosome crude extract, wherein during centrifugation, iopamidol is positioned at the lower part of the supernatant obtained in the step (1), and the centrifugal force is 100000-110000 g; the concentration of the iopamidol solution is 40-42% (w/v); the volume ratio of the supernatant obtained in the step (1) to iopamidol is 500-600: 1;

(3) diluting the exosome crude extract by 4-6 times with a buffer solution, and centrifuging the exosome crude extract and the exosome pure together, wherein the exosome pure is positioned at the lower part of the exosome crude extract, and the centrifugal force is 100000-110000 g; the volume ratio of the exosome crude extract to the exosome pure is 2: 1-1.2; the exocrine purity is 25-30% (w/v) iopamidol solution;

(4) and (4) respectively collecting products after centrifugal separation in the step (3).

2. A method for purifying exosome according to claim 1, wherein in the step (3), the buffer solution is PBS buffer.

3. The method for purifying exosomes according to claim 1, wherein in the step (1), the centrifugation time is 25-35 min.

4. The method for purifying exosomes according to claim 1, wherein in the step (2), the centrifugation time is 65-75 min.

5. The method for purifying exosomes according to claim 1, wherein in the step (3), the centrifugation time is 65-75 min.

6. A continuous density gradient media for use in a method of purifying exosomes, the method of preparing the continuous density gradient media comprising the steps of: the exosome-purified was centrifuged for 70min at 100000g of centrifugal force.

7. A method for preparing a continuous density gradient medium for use in a method for purifying exosomes, the method comprising the steps of: the exosome-purified was centrifuged for 70min at 100000g of centrifugal force.

Background

A certain medium is used for forming a continuous or discontinuous density gradient in a centrifugal tube, a cell suspension or homogenate is placed at the top of the medium, and the cells or different components in the cells are layered under the action of gravity or a centrifugal force field to achieve the purpose of separation. The density gradient media commonly used are primarily sucrose and cesium chloride. Exosomes are nanoscale lipid vesicles formed by endosomal invagination to form multivesicular bodies and then release to the outside of cells through plasma membrane fusion, have a size of 30-150 nm, and a density range of 1.12-1.17 g/mL, and are lighter than DNA, RNA, and most proteins. Traditional differential ultracentrifugation is one of the most common methods for exosome separation and purification, but when exosome is centrifuged to the bottom of a centrifuge tube, the integrity of exosome cannot be ensured due to the action of centrifugal force, and the separation purity is limited. In some studies, the addition of buffer pads (Optiprep or Exojuice) or density gradient media (sucrose or cesium chloride) to ultracentrifuge tubes has been used to increase the purity or ensure the integrity of exosomes. The use of sucrose density gradient and cesium chloride density gradient centrifugation to achieve density gradient equilibrium is time consuming and difficult to remove, and sucrose solutions and cesium chloride solutions tend to form high osmotic pressures which are detrimental to the maintenance of exosome integrity. Therefore, it would be desirable to develop a new density gradient media that is easy to remove in safety considerations and that allows for the acquisition of high purity exosomes.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a purification method of exosomes.

In order to achieve the purpose, the invention adopts the technical scheme that: a method of purifying exosomes, the method comprising the steps of:

(1) centrifuging 9000-11000 g of cell culture solution rich in exosome;

(2) centrifuging the supernatant obtained in the step (1) and an iopamidol solution together to obtain an exosome crude extract, wherein during centrifugation, iopamidol is positioned at the lower part of the supernatant obtained in the step (1), and the centrifugal force is 100000-110000 g; the concentration of the iopamidol solution is 40-42% (w/v); the volume ratio of the supernatant obtained in the step (1) to iopamidol is 500-600: 1;

(3) diluting the exosome crude extract by 4-6 times with a buffer solution, and centrifuging the exosome crude extract and the exosome pure together, wherein the exosome pure is positioned at the lower part of the exosome crude extract, and the centrifugal force is 100000-110000 g; the volume ratio of the exosome crude extract to the exosome pure is 2: 1-1.2; the exocrine purity is 25-30% (w/v) iopamidol solution;

(4) and (4) respectively collecting products after centrifugal separation in the step (3).

The method can quickly form a continuous density gradient by ultracentrifugation of iopamidol with a certain concentration, can be used for separating high-purity exosomes, and is favorable for improving the purity of the obtained exosomes.

Preferably, in the step (3), the buffer solution is PBS buffer.

Preferably, in the step (1), the centrifugation time is 25-35 min.

Preferably, in the step (2), the centrifugation time is 65-75 min.

Preferably, in the step (3), the centrifugation time is 65-75 min.

The invention also relates to a continuous density gradient medium for the purification method of exosomes, and the preparation method of the continuous density gradient medium comprises the following steps: the exosome-purified was centrifuged for 70min at 100000g of centrifugal force.

The continuous density gradient medium has gradually and continuously improved density from the upper layer to the bottom layer, can be used for separating high-purity exosomes, and can improve the purity of the obtained exosomes.

The invention also relates to a preparation method of the continuous density gradient medium for the exosome purification method, which comprises the following steps: the exosome-purified was centrifuged for 70min at 100000g of centrifugal force.

The continuous density gradient medium obtained by the preparation method has the advantages that the density is gradually and continuously improved from the upper layer to the bottom layer, the continuous density gradient medium can be used for separating high-purity exosomes, and the purity of the obtained exosomes can be improved.

The invention has the beneficial effects that: the purification method can quickly form a continuous density gradient by ultracentrifugation of iopamidol with a certain concentration, can be used for separating high-purity exosomes, and is favorable for improving the purity of the obtained exosomes.

Drawings

FIG. 1 is a graph showing the results of density distribution of a continuous density gradient medium according to an embodiment of the present invention.

FIG. 2 is a flow chart of a method for purifying exosomes according to an embodiment of the present invention.

FIG. 3 is a graph representing the purification results of exosomes according to the example of the present invention.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

Example 1

A method of purifying exosomes as an embodiment of the present invention, the method comprising the steps of:

(1) centrifuging 50mL of cell culture solution rich in exosome for 30min at 4 ℃ and 10000 g;

(2) transferring the supernatant obtained in the step (1) to an ultracentrifuge tube (BECKMAN, Cat #: 326823), wherein 100 mu L of 40% (w/v) iopamidol solution is added into the bottom of the ultracentrifuge tube in advance; centrifuging 100000g for 70min to obtain an exosome crude extract, wherein the volume ratio of the supernatant obtained in the step (1) to the iopamidol is 500:1 during centrifugation;

(3) diluting 1mL of the crude exosome extract to 5mL by using a PBS buffer solution, uniformly mixing, transferring into a new ultracentrifuge tube (BECKMAN, Cat #: 326819), adding 500uL of exosome pure (frozen and unfrozen) into the bottom of the ultracentrifuge tube in advance, and centrifuging for 70min at 100000 g; the exocrine pure is 25% (w/v) iopamidol solution;

(4) the bottom layer was collected, and each layer had 100. mu.L of liquid, 5 layers from the bottom up in the order of #5, #4, #3, #2, #1, and stratified samples were collected.

The preparation method of the cell culture solution rich in exosomes comprises the following steps: HEK293T cells were obtained from the institute of cell research, national academy of sciences, using DMEM medium (Gibco) containing 10% exosome-free fetal bovine serum (Excell Bio) and 1% penicillin/streptomycin (Gibco) at 37 ℃ with 5% CO₂Culturing in a constant temperature incubator. Collecting cell culture fluid rich in exosome when the cell fusion degree reaches 90%。

Example 2

A method of purifying exosomes as an embodiment of the present invention, the method comprising the steps of:

(1) 50mL of exosome-rich cell culture medium was centrifuged at 9000g for 25min at 4 ℃;

(2) transferring the supernatant obtained in the step (1) to an ultracentrifuge tube (BECKMAN, Cat #: 326823), wherein 100 mu L of 42% (w/v) iopamidol solution is added into the bottom of the ultracentrifuge tube in advance; 110000g of the mixed solution is centrifuged for 65min to obtain a crude extract of the exosome, and the volume ratio of the supernatant obtained in the step (1) to the iopamidol is 550:1 during centrifugation;

(3) diluting 1mL of the crude exosome extract to 5mL by using a PBS buffer solution, uniformly mixing, transferring into a new ultracentrifuge tube (BECKMAN, Cat #: 326819), adding 500uL of exosome pure (frozen and unfrozen) into the bottom of the ultracentrifuge tube in advance, and centrifuging for 65min at 100000 g; the exocrine pure is 28% (w/v) iopamidol solution;

(4) the bottom layer was collected, and each layer had 100. mu.L of liquid, 5 layers from the bottom up in the order of #5, #4, #3, #2, #1, and stratified samples were collected.

Example 3

A method of purifying exosomes as an embodiment of the present invention, the method comprising the steps of:

(1) centrifuging 50mL of cell culture fluid rich in exosomes for 35min at 4 ℃ and 11000 g;

(2) transferring the supernatant obtained in the step (1) to an ultracentrifuge tube (BECKMAN, Cat #: 326823), wherein 100 mu L of 40% (w/v) iopamidol solution is added into the bottom of the ultracentrifuge tube in advance; centrifuging at 90000g for 75min to obtain crude exosome extract, wherein the volume ratio of the supernatant obtained in the step (1) to iopamidol is 600:1 during centrifugation;

(3) diluting 1mL of the crude exosome extract to 5mL by using a PBS buffer solution, uniformly mixing, transferring into a new ultracentrifuge tube (BECKMAN, Cat #: 326819), adding 600uL of exosome pure (frozen and unfrozen) into the bottom of the ultracentrifuge tube in advance, and centrifuging for 75min at 110000 g; the exocrine pure is 30% (w/v) iopamidol solution;

(4) the bottom layer was collected, and each layer had 100. mu.L of liquid, 5 layers from the bottom up in the order of #5, #4, #3, #2, #1, and stratified samples were collected.

Effect test

1. Preparation of continuous density gradient medium: 5mL of the exo-pure solution is added into an ultracentrifuge tube (BECKMAN, Cat #: 326819) and placed in a refrigerator at the temperature of 20 ℃ below zero for 1h or a refrigerator at the temperature of 80 ℃ below zero for 30 min. And taking out the centrifuge tube, and performing ultracentrifugation after the exogenetic pure solution is dissolved, wherein the centrifugation condition is 100000g and 70 min. After the centrifugation, the layers were separated from top to bottom, and the density was measured by sucking the liquid every 200. mu.L. The density calculation formula is mass/volume. Each 100. mu.L of liquid was weighed by a precision electronic balance. 25 consecutive detection point data are obtained. The centrifugation conditions were also changed and the centrifugation was carried out overnight after freezing and thawing, and 25 consecutive monitoring points were obtained in the same manner. The results are shown in FIG. 1. A total of 25 layers were taken (FIG. 1A), and the density data was plotted from top to bottom to see the density profile (FIG. 1B).

As can be seen from FIG. 1, the density of the continuous density gradient medium increases gradually and continuously from the upper layer to the bottom layer.

And (3) analyzing the characteristics of the exosome: a25% (w/v) solution of iopamidol was found to have a density of 1.142g/mL by mass to volume ratio and an osmolality of 316. + -.3 mOsm/kg by Micro-Osmometer Model 210. Similar to the osmotic pressure of PBS. The osmotic pressure of PBS was 310. + -.2 mOsm/kg.

2. And (3) carrying out particle size analysis, electron microscope detection and WB detection on the collected layered sample to detect the exosome marker molecules.

(1) And (3) identifying the protein immunoblotting: taking 20 mu L of an exosome sample, adding 5x protein loading buffer, uniformly mixing, heating at 95 ℃ for 10min, cooling on ice, separating by using 10% SDS-PAGE protein gel, transferring the protein on the gel to a PVDF membrane, sealing by using 5% skimmed milk for 1h, incubating a TSG101 antibody for 1h, incubating a horseradish peroxidase-labeled secondary antibody for 1h, and finally, using an ECL chemiluminescence substrate to react to detect a fluorescence signal in a Tanon gel imaging instrument.

(2) Exosome nano-particle size analysis: taking 1 mu L of exosome sample, diluting the exosome sample by 1000 times with PBS, slowly injecting the diluted liquid into a sample groove of a Nanosight instrument by using a 1mL disposable injector, and counting the number and size distribution of exosome particles in the sample by using Nanosight detection and analysis software.

(3) And (3) identifying the form of the exosome by a transmission electron microscope: 10 μ L of the exosome sample was taken, fixed with 10 μ L of 2.5% glutaraldehyde, counterstained with 1% uranyl acetate, dropped onto a copper mesh, and pictures were taken under a Hitachi H-7650 transmission electron microscope.

Samples collected from different layers are labeled from top to bottom as EP1, EP2, EP3, EP4 and EP5, and partial extraction of exosome proteins is carried out for detecting an exosome marker molecule TSG101, and exosome signals are found in a third layer (EP3) and a fourth layer (EP4) (FIG. 3A) and are strongest in the fourth layer. Meanwhile, electron microscope detection is carried out on the third layer sample and the fourth layer sample, exosomes with vesicle-like structures in the fourth layer can be observed (fig. 3C), particle size analysis results show that a peak value exists at a position of 130nm, the distribution range is below 200nm (fig. 3E), and the exosome purity is high. The above effect experiment is the effect obtained by the verification of the embodiment 1, and the effects of the embodiments 2 to 3 are similar to those of the embodiment 1, and therefore, the description thereof is omitted.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.