CN115449505A - Purification method for separating exosome and exosome kit - Google Patents

Abstract

The invention discloses a purification method for separating exosomes and an exosome kit, comprising: centrifuging a liquid to be extracted containing exosomes to remove impurities to obtain an initial extraction liquid of exosomes; The initial extract is concentrated by ultrafiltration using a hollow fiber column or an ultrafiltration membrane bag to obtain a concentrated solution; the concentrated solution is subjected to composite chromatography to obtain a flow-through liquid; the flow-through liquid is obtained using a hollow fiber column or an ultrafiltration membrane After the membrane bag is concentrated and replaced, the purified exosome solution is obtained. The purification method for separating exosomes according to the embodiment of the present invention can meet the requirements of large-scale exosome preparation, and can also obtain high-purity exosomes at low cost and high efficiency.

Description

A purification method and exosome kit for separating exosomes

technical field

The invention relates to the technical field of biochemistry, in particular to a purification method for isolating exosomes and an exosome kit.

Background technique

Exosomes are extracellular vesicles with a phospholipid bilayer structure secreted by a class of cells, with a size of 30-150nm. They can be secreted by almost all types of cells and widely exist in serum, plasma, urine, cerebrospinal fluid and semen, etc. in various body fluids. Exosomes carry nucleic acids and proteins, etc., and play an important role in signal transduction in vivo. At present, exosomes are mainly used as therapeutic drugs in the following two aspects. One is as a drug delivery carrier, and the delivered drugs are mainly drug molecules (doxorubicin, paclitaxel, etc.), nucleic acids (miRNA, siRNA, etc.), proteins, etc.; One is that exosomes are directly used as therapeutic drugs, mainly MSC exosomes, and MSC-exo has been reported to treat various diseases. However, how to isolate a large number of exosomes with biological functions is one of the problems that restrict the later clinical application.

Contents of the invention

The present invention is proposed in response to the above problems. The present invention provides a purification method for isolating exosomes, which is used to solve the constraints of exosome separation, to meet the large-scale demand for exosomes, and to achieve low-cost and high-efficiency Obtain high-purity exosomes.

The present invention also provides a purification method for separating exosomes, which is characterized in that, comprising:

Centrifuge the liquid to be extracted containing exosomes to remove impurities, such as cell debris, etc., to obtain the initial extraction liquid of exosomes;

performing composite chromatography on the initial extract or the treated initial extract to obtain a flow-through;

At least one of the primary extract and the flow-through liquid is concentrated by ultrafiltration using a hollow fiber column or an ultrafiltration membrane bag to obtain a purified exosome solution.

The method according to the embodiment of the present invention can not only meet the extraction of a large amount of exosomes and meet the clinical needs, but also can extract a small amount of exosomes to meet the needs of scientific research, without the need for multiple chromatography, and the operation steps are simple, High-salt solution is not required, so there is no effect on the structure and activity of exosomes, and high-purity and high-activity exosomes can be obtained with a single chromatography.

According to an embodiment of the present invention, when centrifuging the liquid to be extracted, the range of centrifugal acceleration used is between 2000g-10000g. This centrifugal acceleration facilitates the separation of impurities such as cell fragments.

According to an embodiment of the present invention, the fluid to be extracted includes body fluid and/or culture fluid.

According to an embodiment of the present invention, the body fluid includes one or more mixtures of serum, plasma, semen, ascites, amniotic fluid, cerebrospinal fluid, urine, milk and breast milk.

According to an embodiment of the present invention, the culture medium includes one or both of cell culture supernatant and bacterial culture supernatant.

According to an embodiment of the present invention, performing composite chromatography on the treated primary extraction liquid, the obtained flow-through liquid includes: performing ultrafiltration concentration on the primary extraction liquid using a hollow fiber column or an ultrafiltration membrane bag to obtain a concentrated liquid; carry out composite chromatography on the concentrated solution, and obtain the flow-through liquid.

According to an embodiment of the present invention, the initial extract is concentrated by ultrafiltration using a hollow fiber column or an ultrafiltration membrane bag to obtain a concentrated solution, which includes: vacuum filtering the initial extract; The initial extract is concentrated by ultrafiltration using a hollow fiber column or an ultrafiltration membrane bag to obtain a concentrate.

According to an embodiment of the present invention, the molecular cutoff of the hollow fiber column or the ultrafiltration membrane package is 100kd-700kd.

According to an embodiment of the present invention, the concentrated solution is subjected to composite chromatography, and the obtained flow-through liquid includes: performing composite chromatography on the concentrated solution through anion exchange, hydrophobicity, and molecular sieve binding, and the obtained flow-through liquid liquid.

According to an embodiment of the present invention, the particle size of the filler for composite chromatography is 20-200 μm, and the ligand is octylamine.

The present invention also provides an exosome kit, including exosomes, which are prepared by the purification method for isolating exosomes described in the above examples.

The technical solution of the present invention has at least one of the following effects:

According to the purification method for separating exosomes according to the embodiment of the present invention, this method can not only meet the extraction of a large amount of exosomes and meet the clinical needs, but also can extract a small amount of exosomes to meet the needs of scientific research without the need for multiple Secondary chromatography has simple operation steps and does not require high salt solution, so it has no effect on the structure and activity of exosomes, and high-purity and high-activity exosomes can be obtained with a single chromatography.

Description of drawings

Fig. 1 is the flowchart of the purification method of the isolated exosome of the embodiment of the present invention;

Figure 2 is the size and distribution diagram of 293 exosomes corresponding to Example 1 of the present invention;

Figure 3 is an electron micrograph of 293 exosome composite chromatography corresponding to Example 1 of the present invention;

Figure 4 is the size and distribution diagram of 293 exosomes corresponding to Comparative Example 1;

Fig. 5 is the electron micrograph of the 293 exosomes corresponding to Comparative Example 1 after ultracentrifugation;

Figure 6 is a comparison chart of the detection of 293 exosome marker proteins corresponding to Example 1 and Comparative Example 1;

Figure 7 is the size and distribution of serum exosomes corresponding to Example 2 of the present invention;

8 is an electron micrograph of the composite chromatography of serum exosomes corresponding to Example 2 of the present invention;

Figure 9 is the size and distribution of serum exosomes corresponding to Comparative Example 2;

10 is an electron micrograph of the serum exosomes corresponding to Comparative Example 2 after ultracentrifugation;

Figure 11 is a comparison chart of the detection of serum exosome marker proteins corresponding to Example 2 and Comparative Example 2;

Figure 12 is the size and distribution of MSC exosomes corresponding to Example 3 of the present invention;

Figure 13 is an electron micrograph of MSC exosome composite chromatography corresponding to Example 3 of the present invention;

Figure 14 is the size and distribution of MSC exosomes corresponding to Comparative Example 3;

Figure 15 is an electron micrograph of MSC exosomes corresponding to Comparative Example 3 after ultracentrifugation;

Fig. 16 is a comparison chart of MSC exosome marker protein detection corresponding to Example 3 and Comparative Example 3.

detailed description

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are some, not all, embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention belong to the protection scope of the present invention.

Firstly, the technical problem of the present invention will be described below.

Exosome isolation and purification methods include ultracentrifugation, immunoaffinity chromatography, ultrafiltration centrifugation, gel filtration chromatography, polymer precipitation and other methods. Among them, ultracentrifugation is the most commonly used exosome separation method. This method can obtain relatively pure exosomes, but this method is time-consuming and labor-intensive, the number of processed samples is small, and the separation efficiency is relatively low. Immunoaffinity chromatography has higher yield and purity than ultracentrifugation, but it is not suitable for large samples, and eluting exosomes from magnetic beads may damage the structure of exosomes. The ultrafiltration centrifugation method is simple and efficient and does not affect the biological activity of exosomes, but the separation efficiency is low and it is not suitable for the separation of large samples. Gel filtration chromatography is also called size exclusion chromatography or molecular sieve. The exosomes obtained from the separation are complete in shape, but its production capacity is severely restricted by its low flow rate and low sample loading. The polymer precipitation method is simple and fast, and the recovery rate of exosomes is relatively high, but there are still a large amount of impurity proteins, so the purity of the isolated exosomes is low.

Based on the above technical problems, the present invention provides a purification method for separating exosomes. This method can not only meet the extraction of a large number of exosomes and meet the clinical needs, but also can extract a small amount of exosomes to meet the needs of scientific research. It does not require multiple chromatography, the operation steps are simple, and high salt solution is not required, so there is no effect on the structure and activity of exosomes, and high-purity and high-activity exosomes can be obtained with a single chromatography.

The purification method for isolating exosomes according to the embodiment of the present invention will be specifically described below. As shown in Fig. 1, the method includes S110-S140.

S110, centrifuging the to-be-extracted solution containing exosomes to remove impurities to obtain an initial exosome-extracted solution. Wherein, the impurities may be cell fragments and the like.

In an embodiment of the present invention, the liquid to be extracted may be one or more mixed body fluids of serum, plasma, semen, ascites, amniotic fluid, cerebrospinal fluid, urine, milk and breast milk. It may also be one or both of cell culture supernatant and bacterial culture supernatant.

In one embodiment of the present invention, the centrifugal acceleration used in the centrifugation process of the liquid to be extracted can be between 2000g-10000g, for example, it can be 2000g, 3000g, 4000g, 5000g, 6000g, 7000g, 8000g, 9000g or 10000g.

S120, the primary extract is concentrated by ultrafiltration using a hollow fiber column or an ultrafiltration membrane bag to obtain a concentrate.

In an embodiment of the present application, before performing ultrafiltration and concentration on the initial extract, vacuum filtration may also be performed on the initial extract. For example, the supernatant of the primary extract obtained after centrifugation is filtered in a disposable vacuum filtration flask to remove vesicles larger than a set size.

In an embodiment of the present invention, the hollow fiber column or the ultrafiltration membrane package has a molecular cutoff of 100kd-700kd, for example, 100kd, 200kd, 300kd, 400kd, 500kd, 600kd or 700kd.

S130, subjecting the concentrated solution to composite chromatography to obtain a flow-through solution.

In one embodiment of the present application, the composite chromatography further includes:

S131, preparation of buffer. It can further include eluent configuration, balance solution configuration and column preservation solution.

Wherein, the eluent can be obtained after vacuum filtration of NaOH solution. The equilibrium solution can be obtained by filtering the phosphate buffer solution through a vacuum filter bottle. The column preservation solution can be obtained by filtering 20% ethanol through a vacuum filter bottle.

S132, get on the plane.

Purify using a protein purifier. After starting up and cleaning, connect the protein purifier to the chromatographic column, wash with double distilled water until the conductance, UV and pH values are stable, use phosphate buffered saline to balance until the conductance, UV and pH values are stable, and then reset the UV280 to zero. A certain amount of ultrafiltration-concentrated sample, such as 50ml, is loaded using the AKTA sample loading pump at a sample loading speed of 2.5ml/min. The sample is collected according to the value of UV280, and the obtained flow-through is collected.

S133, column cleaning. Use phosphate buffered saline solution to elute to equilibrium, then wash the chromatography column with NaOH solution. After cleaning the chromatography column with at least 10 column volumes, clean the system pump and loading pump with ddH20 until the pH value drops to 7.0, and replace the 20% ethanol solution. Clean the system pump and sample pump.

In the embodiment of the present invention, the particle size of the filler involved in the composite chromatography is 20-200 μm, and the ligand is octylamine. The filler not only has the function of a molecular sieve, but also is an anion exchange and a multimodal filler combined with hydrophobicity.

In S140, after the flow-through solution is concentrated and replaced by a hollow fiber column or an ultrafiltration membrane bag, a purified exosome solution is obtained.

In one embodiment of the present invention, the molecular cutoff of the hollow fiber column or the ultrafiltration membrane package is 100kd-700kd.

In some embodiments of the present invention, the above step 110-step 140 is just an exemplary illustration, and in some embodiments, the compound chromatography in step 130 can be directly performed first, and then the concentration in step 140 can be performed without going through the concentration process in step 120. In the actual preparation process, it can be reasonably selected according to the initial volume of the sample. For example, if the initial volume (such as serum) is relatively small, compound chromatography can be performed directly without concentration, and the concentration of exosomes can be further increased by concentration after the chromatography is completed. Conversely, it is also possible to only perform steps 110-130 without performing the final step 140 . That is, when the initial volume is particularly large, it can be concentrated first and then combined with chromatography. When a larger number of exosomes is required, secondary concentration can be used, because the volume will increase during the chromatographic purification process, thereby increasing the number of exosomes.

The purification method for separating exosomes according to the embodiment of the present invention can not only meet the extraction of a large amount of exosomes and meet the clinical needs, but also can extract a small amount of exosomes to meet the needs of scientific research without the need for multiple layers. Analysis, the operation steps are simple, no high-salt solution is required, so there is no effect on the structure and activity of exosomes, and high-purity and high-activity exosomes can be obtained with a single chromatography. Compared with the existing exosome purification methods, the extraction of different volumes of exosome solutions can be achieved by switching chromatographic columns of different specifications (loading capacity), for example, the volume range can reach between 2ml-600L.

In addition, the present invention also provides an exosome kit, including exosomes, wherein the exosomes are prepared by the purification method for isolating exosomes in the above-mentioned embodiment. For details, reference may be made to the preparation process of the above examples, and for the packaging process, reference may be made to the packaging method in the prior art.

In order to enable technical researchers in the field to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific examples.

In the embodiment of the present invention, the purified exosome solution can be obtained after concentration, composite chromatography and/or concentration of the liquid to be extracted containing exosomes. Solutions with exosomes include serum, plasma, semen, ascites, amniotic fluid, cerebrospinal fluid, urine, milk, and milk, as well as culture supernatants of cells or bacteria.

The packing used for the composite chromatography is the product of Capto core 400/700 of cityva company. The particle size is 20-200μm, and the ligand is octylamine. The filler is equilibrated with 5-20 column volumes of buffer, and then loaded according to the ratio of sample to filler volume ratio of 1:1-10:1. The residence time of the sample on the chromatography column should not be less than 1.5min. It can also be understood as the collected exosome purification solution. Proteins and other impurities smaller than exosomes will be adsorbed to the chromatographic column, while exosomes flow out directly, so as to achieve the purpose of separating exosomes from impurities.

Materials and components involved in the present invention that are not specifically described can be commercially available or prepared according to methods known to those skilled in the art, which are well known in the art and will not be repeated here.

In the following examples, the sources of samples are 293 cell supernatant, serum fluid and MSC cell supernatant respectively. The method used is the purification method for isolating exosomes in the embodiment of the present invention, which is compared with the existing common ultracentrifugation purification method.

Example 1

Isolation and purification of exosomes in the supernatant of HEK-293 suspension cells.

1. Take 1L of the supernatant of HEK-293 suspended cells, remove the cell pellet, and centrifuge at 10000g for 30min to further remove cell debris;

2. Filter the centrifuged supernatant with a 0.22 μm disposable vacuum filter bottle to remove vesicles larger than 0.22 μm;

3. Select a hollow fiber column with a cut-off size of 700KD, concentrate the filtered cell supernatant 20 times with a hollow fiber column, control the transmembrane pressure at 4psi, and concentrate to 50ml.

4. Composite chromatography

4.1 Preparation of buffer

Eluent: 1.0M NaOH solution, obtained by filtering with a 0.2 μm vacuum filter bottle after preparation.

Equilibrium solution: 0.01M phosphate buffer solution, pH 7.4, obtained by filtering with a 0.2 μm vacuum filter bottle after preparation.

Column preservation solution: 20% ethanol, obtained by filtering with a 0.2 μm vacuum filter bottle after preparation.

4.2 Computer

After the ATKA explorer 100/AVANT 150 system is started and cleaned, it is connected to a capto core 700 chromatography column (10ml composite chromatography column is selected), washed with double distilled water until the conductivity, UV and pH values are stable, and the pH is 7.4. 0.01M phosphate buffered saline solution is balanced to After the conductance, UV and pH values are stable, reset the UV280 to zero, and load 50ml of the ultrafiltered and concentrated sample using the AKTA sample loading pump at a sample loading speed of 2.5ml/min. Sample collection starts after 400mAU, stops when it drops to 400mAU, and the collected exosome purification solution is 55ml.

4.3 Column cleaning

pH 7.4 0.01M phosphate buffered saline solution to elute to equilibrium, 1.0M NaOH solution to wash the chromatography column, at least 10 column volumes, after the chromatography column is cleaned, ddH20 cleans the system pump and sample pump until the pH value drops to 7.0 , Replace the 20% ethanol solution to clean the system pump and sample pump.

The size and distribution of the obtained 293 exosomes were detected, and the results are shown in Figure 2. Figure 2 shows the particle size assay for exosome size and distribution. The particle size test results show the particle size of the extracted exosomes.

The morphology of the obtained 293 exosomes was detected, and the results are shown in Figure 3. Referring to the composite tomographic electron microscope image of 293 exosomes shown in Figure 3, complete exosomes can be seen.

Comparative Example 1 Separation and purification of exosomes from HEK-293 cell supernatant.

The exosomes in the supernatant of HEK-293 cells were separated and purified by the ultracentrifugation method. The specific steps are as follows: 200ml of the collected cell supernatant was centrifuged at 2000g for 15min to remove dead cells; the supernatant was centrifuged at 10000g for 30min to remove Impurities such as cell debris; take the supernatant and centrifuge at 120,000g for 70 minutes. It can be seen that the bottom layer of the tube is precipitated, and some samples will have color. The main component of this precipitate is exosomes; the precipitate is washed with PBS solution, blown and resuspended, and centrifuged again at 120,000g After 70 minutes, carefully discard the supernatant to remove some adsorbed impurities. The precipitate is exosomes, and the precipitate is resuspended in 100 μL PBS. Obtained exosomes were observed under an electron microscope. For details, please refer to the electron microscope image of 293 exosomes after ultra-separation shown in FIG. 3 .

The size and distribution of the obtained 293 exosomes were detected, and the results are shown in Figure 4. Figure 4 shows the particle size assay for exosome size and distribution. The particle size test results show the particle size of the extracted exosomes.

The morphology of the obtained 293 exosomes was detected, and the results are shown in Figure 5. Refer to the electron microscope image of 293 exosomes shown in Figure 5 after ultracentrifugation.

For the detection of 293 exosome marker proteins in Example 1 and Comparative Example 1, refer to the comparison chart of 293 exosome marker protein detection shown in FIG. 6 . It can be seen from Figure 6 that both Comparative Example 1 and Example 1 can detect the exosome marker protein TSG101, with an average molecular weight of 46KDa; detect the exosome marker protein CD81, with an average molecular weight of 22KDa; detect the exosome marker Protein CD9, the average molecular weight is between 23-27KDa.

Example 2 Separation and purification of exosomes in human serum

1. Take 2mL of the isolated human serum, add 3ml of PBS to dilute to 5ml, and use a 0.22μm disposable needle filter to filter out vesicles larger than 0.22μm.

2. Composite chromatography

2.1 Preparation of buffer eluent: 1.0M NaOH solution, after preparation, filter with a 0.2 μm vacuum filter bottle.

Equilibrium solution: 0.01M phosphate buffer solution, pH 7.4, after the preparation is completed, filter with a 0.2 μm vacuum filter bottle.

Column preservation solution: 20% ethanol, after the preparation is completed, use a 0.2 μm vacuum filter bottle to filter.

2.2 on the computer

After the ATKA explorer 100/AVANT 150 system is started and cleaned, connect the capto core 700 chromatography column (4.7ml composite chromatography column is selected), wash with double distilled water until the conductivity, UV and pH values are stable, pH 7.4 0.01M phosphate buffered saline solution After equilibrating until the conductance, UV and pH values are stable, reset UV280 to zero, and load 5ml of the diluted serum using the AKTA sample loading pump at a sample loading speed of 1ml/min. Collect samples according to the value of UV280, and reach 400mAU at UV280 Then start to collect samples, and stop collecting samples when it drops to 400mAU, and the collected purified exosomes are 7ml.

2.3 Column cleaning

pH 7.4 0.01M phosphate buffered saline solution to elute to equilibrium, 1.0M NaOH solution to wash the chromatography column, at least 10 column volumes after the chromatography column is cleaned, ddH20 to clean the system pump and sample pump until the pH value drops to 7.0, Replace the 20% ethanol solution to clean the system pump and sample pump.

The size and distribution of the serum exosomes obtained in Example 1 were detected, and the results are shown in Figure 7. Figure 7 shows the particle size assay for exosome size and distribution. The particle size test results show the particle size of the extracted exosomes.

The morphology of exosomes in serum exosomes obtained in Example 1 was detected, and the results are shown in FIG. 8 . Referring to the composite tomographic electron micrograph of serum exosomes shown in Figure 8, complete exosomes can be seen.

Comparative Example 2 Separation and Purification of Exosomes from Human Serum

Exosomes in human serum were separated and purified by ultracentrifugation, and the specific steps were as follows:

Centrifuge 5ml of the separated human serum at 2,000×g for 15 minutes to remove dead cells; take the supernatant and centrifuge at 10,000g for 30 minutes to remove impurities such as cell debris; take the supernatant and centrifuge at 120,000g for 70 minutes, and you can see that the bottom of the tube is precipitated. Some samples will have color, and the main component of the precipitate is exosomes; rinse the precipitate with PBS solution, pipette and resuspend, centrifuge again at 120,000g for 70 minutes, carefully discard the supernatant, remove some adsorbed impurities, and the precipitate is exosomes Resuspend the pellet in 100 μL PBS.

The size and distribution of the serum exosomes obtained in Comparative Example 2 were detected, and the results are shown in FIG. 9 . Figure 9 shows the particle size assay for exosome size and distribution. The particle size test results showed the particle size distribution of the extracted exosomes.

The morphology of the serum exosomes obtained in Example 2 was detected, and the results are shown in FIG. 10 . Refer to the electron micrograph of serum exosomes after ultracentrifugation shown in FIG. 10 .

For the detection of serum exosome marker proteins in Example 2 and Comparative Example 2, refer to the comparison chart of serum exosome marker protein detection shown in FIG. 11 . It can be seen from Figure 11 that both Comparative Example 2 and Example 2 can detect the exosome marker protein TSG101, with an average molecular weight of 46KDa; detect the exosome marker protein CD81, with an average molecular weight of 22KDa; detect the exosome marker Protein CD9, the average molecular weight is between 23-27KDa.

Example 3 Separation and purification of human mesenchymal stem cell supernatant exosomes

1. Take 2L of hUC-MSC cell supernatant, remove the cell pellet, and centrifuge at 10000g for 30min to further remove cell debris.

2. Filter the centrifuged supernatant with a 0.22 μm disposable vacuum filter bottle to remove vesicles larger than 0.22 μm.

3. Concentration: Select a hollow fiber column with a cut-off size of 700KD, concentrate the filtered cell supernatant 20 times with a hollow fiber column, control the transmembrane pressure at 4psi, and concentrate to 100ml

4. Composite chromatography

4.1 Preparation of buffer eluent: 1.0M NaOH solution, obtained by filtering with a 0.2 μm vacuum filter bottle after preparation. Equilibrium solution: 0.01M phosphate buffer solution, pH 7.4, obtained by filtering with a 0.2 μm vacuum filter bottle after preparation.

Column preservation solution: 20% ethanol, obtained by filtering with a 0.2 μm vacuum filter bottle after preparation.

4.2 Computer

After the ATKA explorer 100/AVANT 150 system is started and cleaned, it is connected to a capto core 700 chromatography column (10ml composite chromatography column is selected), washed with double distilled water until the conductivity, UV and pH values are stable, and the pH 7.4 is balanced with 0.01M phosphate buffered saline solution After the conductance, UV and pH values are stable, reset the UV280 to zero, and load 100ml of the ultrafiltered and concentrated sample using the AKTA sample loading pump at a sample loading speed of 2.5ml/min, and collect the sample according to the UV280 value. After reaching 400mAU, the sample collection was started, and when it dropped to 400mAU, the sample collection was stopped, and the collected purified exosomes were 105ml.

4.3 Column cleaning

pH 7.4 0.01M phosphate buffered saline solution to elute to equilibrium, 1.0M NaOH solution to wash the chromatography column, at least 10 column volumes, after the chromatography column is cleaned, ddH20 cleans the system pump and sample pump until the pH value drops to 7.0 , Replace the 20% ethanol solution to clean the system pump and sample pump.

5. Concentration: Select a hollow fiber column with a cut-off size of 700KD, and further concentrate the purified exosome solution with a hollow fiber column to a volume of 5ml.

The size and distribution of MSC exosomes obtained in Example 3 were detected, and the results are shown in Figure 12. Figure 12 shows the particle size assay for exosome size and distribution. The particle size test results show the particle size of the extracted exosomes.

The morphology of MSC exosomes obtained in Example 3 was detected, and the results are shown in FIG. 13 . Referring to the MSC exosome composite tomographic electron microscope image shown in Figure 13, complete exosomes can be seen.

Comparative Example 3 Separation and Purification of Exosomes from the Supernatant of hUC-MSC Cells

The exosomes in the supernatant of hUC-MSC cells were separated and purified by ultracentrifugation, and the specific steps were as follows: 200ml of the collected cell supernatant was centrifuged at 2000g for 15min to remove dead cells; the supernatant was centrifuged at 10000g for 30min to remove Impurities such as cell debris; take the supernatant and centrifuge at 120,000g for 70 minutes. It can be seen that the bottom layer of the tube is precipitated, and some samples will have color. The main component of this precipitate is exosomes; the precipitate is washed with PBS solution, blown and resuspended, and centrifuged again at 120,000g After 70 minutes, carefully discard the supernatant to remove some adsorbed impurities. The precipitate is exosomes, and the precipitate is resuspended in 100 μL PBS.

The size and distribution of MSC exosomes obtained in Example 3 were detected, and the results are shown in Figure 14. Figure 14 shows the particle size assay for exosome size and distribution. The particle size test results show the particle size distribution of the extracted exosomes.

The morphology of MSC exosomes obtained in Example 3 was detected, and the results are shown in FIG. 15 . Refer to the electron micrograph of serum exosomes after ultracentrifugation shown in FIG. 15 .

For the detection of MSC exosome marker proteins in Example 3 and Comparative Example 3, refer to the comparison chart of MSC exosome marker protein detection shown in FIG. 16 . It can be seen from Figure 16 that both Comparative Example 3 and Example 3 can detect the exosome marker protein TSG101, with an average molecular weight of 46KDa; detect the exosome marker protein CD81, with an average molecular weight of 22KDa; detect the exosome marker Protein CD9, the average molecular weight is between 23-27KDa.

Below in conjunction with chart, the experimental results of above-mentioned three comparative examples and three embodiments are summarized, specifically as shown in table 1 below:

Table 1

As shown in Table 1, it can be seen from Example 1 of the present invention and Comparative Example 1 that the method of the embodiment of the present invention can process a relatively large volume (1 L) of exosomes to be extracted in a short period of time, and does not require many repeated operations. In Comparative Example 1, the ultracentrifugation method was used to process 200ml at a time, which was time-consuming and laborious, and large-scale exosome extraction could not be achieved. Moreover, the purification efficiency of Example 1 is more than 20 times that of Comparative Example 1, indicating that the purification efficiency of the composite chromatography adopted in the present invention is much higher than that of Chaoli. From Example 2 of the present invention and Comparative Example 2, it can be seen that the Example of the present application can also process small volume (2ml) of exosomes to be extracted. Compared with Comparative Document 2, the composite chromatography method of the present invention, Even if less liquid to be extracted is processed, a large amount of exosomes can be obtained, and the purification efficiency is more than 100 times that of Comparative Example 2, which also shows that the purification efficiency of the composite chromatography of the embodiment of the present invention is much higher for ultra-centrifugal purification efficiency. From Example 3 and Comparative Example 3, it can be seen that the purification efficiency of Example 3 is about 60 times that of Comparative Example 3, and it can also be shown that the purification method for separating exosomes in the embodiment of the present invention is far superior to the existing ultra-centrifugal Purification method.

Compared with the ultracentrifugation method, the exosome purification method according to the embodiment of the present invention is more time-saving, efficient and convenient to operate, not only can meet the large-scale exosome demand, but also can extract a small amount of exosome, Obtaining exosomes has the advantages of low cost, high purity and good activity, and simple operation.

While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that it will include all embodiments falling within the scope of the appended claims.

Claims (10)

1. A method of purifying an isolated exosome, comprising:

centrifuging the extract to be extracted containing the exosomes, and removing impurities to obtain primary extract of the exosomes;

carrying out composite chromatography on the primary extract or the treated primary extract to obtain a percolation solution;

and carrying out ultrafiltration concentration on at least one of the primary extract and the flow-through liquid by adopting a hollow fiber column or an ultrafiltration membrane pack to obtain a purified exosome solution.

2. A method for the purification of an isolated exosome according to claim 1, characterised in that the centrifugation acceleration used when the solution to be extracted is centrifuged is in the range 2000-10000 g.

3. A purification method for isolated exosomes according to claim 1 or 2, wherein the liquid to be extracted comprises a body fluid and/or a culture liquid comprising one or both of a culture supernatant of cells, a culture supernatant of bacteria.

4. A purification method for isolating exosomes according to claim 3, wherein the body fluid comprises one or a mixture of serum, plasma, semen, ascites, amniotic fluid, cerebrospinal fluid, urine, milk and milk.

5. The method for purifying an isolated exosome according to claim 1, wherein the flow-through solution obtained by performing composite chromatography on the treated primary extraction solution comprises:

carrying out ultrafiltration concentration on the primary extract by adopting a hollow fiber column or an ultrafiltration membrane pack to obtain a concentrated solution;

and carrying out composite chromatography on the concentrated solution to obtain the percolation solution.

6. A method for purifying an exosome according to claim 5, wherein the primary extract is subjected to ultrafiltration concentration by using a hollow fiber column or an ultrafiltration membrane package to obtain a concentrated solution, and the method comprises the following steps:

carrying out vacuum filtration on the primary extraction solution;

and carrying out ultrafiltration concentration on the initial extract after vacuum filtration by adopting a hollow fiber column or an ultrafiltration membrane pack to obtain a concentrated solution.

7. A purification method for isolated exosomes according to claim 1, wherein the molecular cut-off of said hollow fiber column or ultrafiltration membrane is 100kd-700kd.

8. A method for purifying an isolated exosome according to claim 1, wherein the flow-through solution obtained by subjecting the primary extract or the treated primary extract to complex chromatography comprises:

and (3) carrying out anion exchange, hydrophobic and molecular sieve combination on the primary extract or the treated primary extract for composite chromatography to obtain a flow-through solution.

9. A method for the purification of an isolated exosome according to claim 1, wherein the particle size of the filler of the complex chromatography is 20-200 μm, and the ligand is octylamine.

10. An exosome kit comprising exosomes prepared by the purification method for isolating exosomes described in the above example.

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