KR102161280B1 - Method of isolating extracellular vesicles using semi-dry size-exclusion chromatography - Google Patents

Abstract

The present invention relates to a method for separating extracellular vesicles using size exclusion chromatography, and more particularly, the present invention provides size exclusion chromatography by preventing non-specific binding and loss of extracellular vesicles on a semi-dry size exclusion chromatography stationary phase. The present invention relates to a method for supplementing the disadvantages of the method for separating extracellular vesicles used and for rapidly and efficiently separating extracellular vesicles from various samples. The method for separating extracellular vesicles according to the present invention does not require expensive equipment such as an ultracentrifuge, and can efficiently separate the extracellular vesicles while preserving the shape and properties, as well as conventional semi-dry size exclusion chromatography. By greatly improving the loss of extracellular vesicles in the cell, it can be actively used to separate high-purity extracellular vesicles in combination with a conventional extracellular vesicle separation method. In addition, the method for separating extracellular vesicles of the present invention can be quickly utilized for clinical diagnosis by applying to a small amount of samples in pretreatment and post-treatment steps.

Description

Separation of extracellular vesicles using semi-dry size-exclusion chromatography {Method of isolating extracellular vesicles using semi-dry size-exclusion chromatography}

The present invention relates to a method of separating extracellular vesicles using size exclusion chromatography, and in more detail, the present invention prevents the extracellular vesicles from being adsorbed or non-specifically bound and lost on a semi-dry size exclusion chromatography stationary phase. The present invention relates to a method for quickly and efficiently separating the extracellular vesicles from various samples and to compensate for the shortcomings in the method for separating extracellular vesicles using semi-dry size exclusion chromatography.

Extracellular vesicles are biological nanoparticles secreted from various types of cells in vivo or in a test tube, exist in body fluids such as blood, urine, saliva, tears, etc., contain a lipid bilayer derived from cells, and range from 20 to 10,000 nm. It is a membrane-structured endoplasmic reticulum having various sizes.

Because the extracellular vesicle binds to other cells and tissues and acts as a transporter that transfers intracellular substances such as membrane components, proteins, and RNA, the proteins, lipids, amino acids, and proteins of the original cell (blast) that secreted the extracellular vesicles Since it contains RNA as it is, it is an important basis for knowing the physiological and pathological characteristics of the parent cell. In addition, as it is known that nucleic acids, growth hormones, proteins, etc. contained in the extracellular vesicles are protected by phospholipids in the form of cell membranes, they can perform more stable functions than soluble growth factors and cytokines. The importance is gradually increasing, and it is expected to be used for various purposes including diagnosis and treatment of diseases by analyzing substances contained in the extracellular vesicles.

The extracellular vesicles are as small as nanometers in size, and there are numerous substances in addition to the extracellular vesicles in cell culture fluids in body fluids. Therefore, for the analysis of extracellular vesicles, it is important to separate extracellular vesicles from samples such as in body fluids and cell culture fluids, and separation of extracellular vesicles is the most essential technology in all fields that utilize them.

Conventional techniques for separating extracellular vesicles include ultra-centrifugation, size exclusion chromatography, immunoaffinity isolation, microfluidics chip or polymer. Method, etc., of which ultracentrifugation was the most widely used. However, in the case of ultracentrifugation, the separation step is complicated, requiring a lot of labor and time, and requires expensive equipment, and the yield and purity are significantly low, so clinical results that require rapid results using only a small amount of samples are required. It is not suitable for use as a diagnostic and therapeutic method requiring large amounts of extracellular vesicles.

Accordingly, a size-exclusion chromatography purification method using the physical characteristic that extracellular vesicles have a relatively high molecular weight compared to various substances present in a biological sample including them is becoming increasingly common. On the other hand, the size exclusion chromatography method has the advantage of increasing the purity of the extracellular vesicles, but it has the disadvantage that the final purified material is greatly diluted due to the principle of size exclusion chromatography, and the development time to increase the purity of the extracellular vesicles Since this is relatively long, there is a problem that there are many difficulties in subsequent analysis or application.

Semi-dry size exclusion chromatography is gradually being used as a technique to compensate for the disadvantages of such size exclusion chromatography. Semi-dry size exclusion chromatography is characterized in that the volume of the mobile phase present in the column is minimized by equilibrating the stationary phase in the column semi-dry by a scanning or rotary method before loading a sample. After loading a sample onto the equilibrated column, the extracellular vesicles are eluted, thereby greatly reducing the dilution rate of the separated extracellular vesicles, as well as enabling rapid separation of the sample. However, when purifying extracellular vesicles, there is a limitation in that the separation yield is deteriorated because they are easily adsorbed onto the semi-dry stationary bed. Therefore, there is an urgent need to develop an efficient separation and purification technology capable of completely maintaining the structure and function of the extracellular vesicles and complementing the weaknesses of the size exclusion chromatography technology.

An object of the present invention is to (a) equilibrate a stationary bed packed in a column in a semi-dry manner, (b) loading a mixture of a sample containing a blocking material and extracellular vesicles into the stationary bed, and (c) cells in the column It is to provide a method for separating extracellular vesicles comprising the step of eluting the external vesicles.

Another object of the present invention is (a) semi-dry equilibration of the stationary phase filled in the column using a solution containing a blocking material, (b) loading a sample containing extracellular vesicles into the column, and ( c) to provide a method for separating extracellular vesicles comprising the step of eluting the extracellular vesicles from the column.

The present invention is to solve the above-described problems, and provides a separation method capable of improving separation efficiency and improving the disadvantages of semi-dry size exclusion chromatography, which is a conventional method for separating extracellular vesicles.

In the method of separating extracellular vesicles of the present invention, in a method using a conventional semi-dry size exclusion chromatography, the extracellular vesicles in the sample are adsorbed to the stationary bed or are lost by non-specific binding by introducing a blocking material that non-specifically binds to the stationary phase. By preventing, it is possible to increase the separation efficiency of extracellular vesicles through semi-dry size exclusion chromatography.

The term "extracellular endoplasmic reticulum" of the present invention collectively refers to biological nanoparticles derived from cells of Archaea, Prokarya, or Eukarya, and extracellular vesicles (exosome), agrosomes (argosomes) , Dexosomes, ectosomes, exovesicles, oncosomes, prominosomes, prostasomes, tolerosomes, microparticles ( microparticles, microvesicles, nanovesicles, blebbing vesicles, budding vesicles, exosome-like vesicles, matrix vesicles , Membrane vesicle, shedding vesicle, membrane particle, shedding microvesicle, membrane bleb, epididimosome, promininosome promininosome), a texosome, or an archeosome, but is not limited thereto.

In the method for separating extracellular vesicles of the present invention, a blocking material smaller than the size of the extracellular vesicles is mixed with a sample containing the extracellular vesicles, and then loaded onto a size exclusion column equilibrated in a semi-dry manner, thereby adsorbing and adsorbing the The present invention relates to a method of effectively separating extracellular vesicles from a sample by interfering with non-specific binding substances.

One embodiment of the present invention includes the steps of (a) semi-drying the stationary bed packed in a column, (b) loading a mixture of a sample containing a blocking material and extracellular vesicles into the stationary bed, and (c) the column Eluting the extracellular vesicles in; It provides a method for separating extracellular vesicles comprising a.

The method for separating the extracellular vesicles according to the present invention is schematically shown in FIG. 1.

The method of separating extracellular vesicles according to the present invention includes a step [Step (a)] of semi-drying the stationary phase packed in the column after filling the stationary phase in a size exclusion chromatography column.

The term "column" of the present invention is a unit filled with a porous stationary phase used in size exclusion chromatography, and the "fixed phase" refers to particles having pores of various sizes for fractionating materials according to molecular weight. Depending on the size of the pores in the stationary phase, the resolution of separation varies according to the molecular size of molecules present in the sample. For example, when the pores in the stationary phase are large, relatively large molecules are efficiently separated, and relatively small molecules are eluted without separation. On the other hand, when the size of the pores in the stationary phase is small, the degree of separation of large molecules is low, but it may be effective in separating molecules above and below a certain size. Therefore, in general, a stationary phase having pores of a size that provides optimum separation efficiency is selected in consideration of the size of molecules to be separated and the size of contaminants in the sample. The most widely used stationary phases in size exclusion chromatography are Sepharose (GE Healthcare), Superose (GE Healthcare), Sephadex; Silica-based; Sigma), etc., and in one embodiment of the present invention, a Sephacryl S500 stationary bed having pores of a size capable of separating the extracellular vesicles, which are nanoparticles, from proteins of various sizes was used. It is not limited thereto. The semi-dry size exclusion chromatography development method of the present invention uses a rotary type, but is not limited thereto.

The term "equilibration" of the present invention is a process performed before loading a sample to be separated into a column. After filling the stationary phase in the column, loading a buffer solution and washing the stationary phase by rotation to move the column to a semi-dry state This is the step of minimizing the volume of the phase.

In the stationary phase equilibration step of the present invention, a buffer solution used in conventional size exclusion chromatography may be used.

Next, the method of separating extracellular vesicles of the present invention includes loading a mixture of a sample containing a blocking material and extracellular vesicles onto the equilibrated stationary phase [step (b)].

The term "sample" of the present invention includes a biological sample or cell culture medium, tissue sample, etc. including extracellular endoplasmic reticulum, and specifically, a mammalian cell culture medium, a bacterial cell culture medium, a yeast culture medium, a tissue extract, a cancer tissue , Serum, plasma, saliva, tears, sweat, urine, feces, cerebrospinal fluid (CSF), ascite, amniotic fluid, semen, milk, dust, fresh water, sea water, soil and fermentation One or more may be selected from the group consisting of food, but is not limited thereto.

The term "blocking material" of the present invention refers to a material that adsorbs or non-specifically binds to the stationary phase of size exclusion chromatography, and is a material capable of preventing the extracellular vesicles from binding to the stationary phase by competing with the extracellular vesicles. . Preferably, the blocking material may include a protein material having a size smaller than that of the extracellular vesicles to be separated. The blocking material that can be used in the method of separating extracellular vesicles of the present invention may be one material having the above properties, or may be a combination of two or more materials. The blocking material is a single material capable of adsorbing or non-specific bonding with a conventional fixed bed used in size exclusion chromatography, or a combination of various materials, and refers to a material capable of forming adsorption or complex non-specific bonding with the fixed bed. Preferably, the blocking material of the present invention may be a material having a size smaller than that of the extracellular vesicles. In one embodiment of the present invention, the blocking material is a single protein or protein complex such as fetal bovine serum (FBS), or fetal bovine albumin (BSA), but blocking as described above Any material that can play a role is not limited thereto.

The term "non-specific binding" of the present invention refers to non-covalent bonds between various types of molecules, specifically hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals forces (van der Waals force), and the like. Nonspecific binding between molecules may be due to one type of binding, or two or more types of binding may act simultaneously. In the present invention, the fixed phase of the size exclusion chromatography may bind or non-specifically bind to the extracellular vesicles or blocking materials by adsorption, and the extracellular vesicles and the blocking materials may competitively bind to the stationary phase and non-covalently.

Through the above step, the blocking material is adsorbed to the stationary phase or forms a non-specific bond, thereby inhibiting binding of the extracellular vesicles contained in the sample to the stationary phase, and consequently maximizing the separation efficiency of the extracellular vesicles.

In one embodiment of the present invention, it was confirmed that the separation yield of extracellular vesicles was improved when a mixture of a blocking material and a sample was loaded, and in particular, when bovine serum (FBS) or bovine serum albumin (BSA) was included as a blocking material, the yield It was found that this is maximized (Fig. 3). In addition, the yield of isolated extracellular vesicles increased in proportion to the concentration of the blocking substance (Fig. 4), and the blocking effect of the bovine serum albumin was not affected by the concentration of commonly used salt (NaCl) (Fig. 5 ).

Next, the method of separating extracellular vesicles of the present invention includes the step of eluting the extracellular vesicles from the column [step (c)].

When a sample containing extracellular vesicles is loaded onto a semi-dry size exclusion chromatography column and the mobile phase is developed by rotation, large molecules such as extracellular vesicles are selectively eluted out of the sample, and the remaining small sized substances stay inside the column, resulting in a larger size. Large extracellular vesicles can be isolated from the sample. In general, extracellular vesicles have a molecular size of 1,000 kDa or more and belong to particles that are larger than other impurities, so they are preferentially eluted together with the mobile phase in the development of a rotary mobile phase. The molecular weight of the eluted material is different depending on the size and pore size of the porous stationary phase, the length of the column, the flow rate of the mobile phase, etc., and the size of the eluted molecule is constant under the same conditions. In an embodiment of the present invention, after adding HBS buffer solution (20 mM HEPES, 150 mM NaCl, pH 7.4) to a column (8 x 20 mm) filled with Sephacryl S500, washing with a rotary (300 x g, 5 minutes) In the semi-dry stationary phase equilibrated, 2 mg of the sample extracellular vesicles and various concentrations of blocking material were mixed and loaded, and the column was rotated at 700 xg for 5 minutes, and the eluate was analyzed.

Another embodiment of the present invention is the step of (a) equilibrating a stationary phase filled in a column in a semi-dry manner using a solution containing a blocking material, (b) loading a sample containing extracellular vesicles into the column And (c) provides a method for separating extracellular vesicles comprising the step of eluting the extracellular vesicles from the column.

In the above embodiment, after filling the stationary phase in a size exclusion chromatography column, the step of equilibrating the stationary phase filled in the column using a solution containing a blocking material in a semi-dry manner [Step (a)] is characterized in that it comprises.

It was confirmed that the stationary phase can be first blocked by using a buffer solution containing a blocking material in the equilibrium stage of the stationary bed, and thus, separation efficiency can be improved even if a separate blocking material is not mixed with the sample (Fig. 6). .

In addition, in the separation method of the present invention, a blocking material may be mixed and used in both the blocking step [(a) step] and the sample loading step [(b) step]. In this case, it was confirmed through the following examples that the stationary phase can be blocked in two steps, so that the efficiency of separating the extracellular vesicles can be maximized (Fig. 7).

In the separation method of the present invention, the type and concentration of the blocking material and the mixing ratio of the equilibration buffer solution or the sample may be modified by those skilled in the art by optimizing according to the properties of the sample to be separated or the extracellular vesicles and the purpose of separation. have.

The method for separating extracellular vesicles according to the present invention does not require expensive equipment such as an ultracentrifuge, and since the sample is not exposed to extreme environments during the separation process, it is efficiently separated while preserving the shape and properties of the extracellular vesicles. There is an advantage that you can do it. In addition, the method of the present invention greatly improves the loss of extracellular vesicles in the conventional semi-dry size exclusion chromatography, so that it can be actively used to separate high-purity extracellular vesicles by combining with the conventional extracellular vesicle separation method. In addition, separation efficiency can be maximized by applying it to a step before or after performing the conventional method. In addition, the method of separating the extracellular vesicles of the present invention can effectively separate the extracellular vesicles, and can be quickly utilized for clinical diagnosis by applying to a small amount of samples in the pretreatment and post-treatment steps.

1 is a schematic diagram of a method for separating extracellular vesicles according to the present invention.
2 is a result of isolation of sample extracellular vesicles derived from colon cancer cell line (SW480).
3 is a result of separation of extracellular vesicles through semi-dry size exclusion chromatography in a sample in which various blocking substances are mixed.
4 is a result of separation of extracellular vesicles through semi-dry size exclusion chromatography in samples in which various concentrations of bovine serum albumin are mixed.
5 is a result of separation of extracellular vesicles through semi-dry size exclusion chromatography in a sample in which 10% bovine serum albumin and salts of various concentrations are mixed.
6 is a result of separation of extracellular vesicles through semi-dry size exclusion chromatography in which the stationary phase is blocked with various concentrations of bovine serum albumin.
7 is a result of separation of extracellular vesicles through semi-dry size exclusion chromatography in which a stationary phase is blocked in a sample containing a blocking material.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1. Purification and analysis of specimen extracellular vesicles

The colon cancer cell line SW480 culture was centrifuged at 500 xg for 10 minutes and at 2,000 xg for 20 minutes to remove the precipitate. In order to first purify and precipitate the extracellular vesicles present in the supernatant, the extracellular vesicles were added with a polyethylene glycol solution (8.4% Polyethylene Glycol 6000, 250 mM NaCl, 20 mM HEPES, pH 7.4) for 16 hours. After refrigerated storage, the precipitated extracellular vesicles were harvested by centrifugation at 12,000 xg for 30 minutes, and the precipitate was dissolved in HEPES buffer (HBS, HEPES-buffered saline, 20 mM HEPES, 150 mM NaCl, pH 7.4).

In order to secondarily purify the extracellular vesicles using density and buoyancy, the sample was subjected to 30%-20%-5% Optiprep buoyancy density gradient ultracentrifugation at 200,000 xg for 2 hours. After ultracentrifugation, extracellular vesicles and supernatant density (1.08 ~ 1.12 g/ml) bands were harvested. The harvested band was loaded onto a column (10 x 100 mm) packed with Sephacryl S500, and the extracellular vesicles were finally purified through molecular size exclusion chromatography.

HPLC analysis and Western blotting were performed to analyze the purity of the sample extracellular vesicles finally isolated according to the method for purifying extracellular vesicles (Figs. 2(a) and 2(b)). In HPLC analysis, after loading the sample extracellular vesicles purified according to the above method on a TSKgel6000 column (7.5 x 600 mm) for HPLC, flowing at 0.5 ml/min using a HEPES buffer solution as a fluidized bed, and tracking the absorbance at 280 nm. The purity of the purified sample was verified. In Western blot, purification results were analyzed using extracellular vesicle marker proteins (Alix, CD63, CD9) and non-extracellular vesicle proteins (calnexin, Histone H2B). In addition, the size of the separated extracellular vesicles was confirmed by dynamic light scattering (DLS) (FIG. 2 (c)), and its shape was confirmed by a transmission electron microscope (FIG. 2(d)).

As a result, it was confirmed that the sample extracellular vesicles isolated from the colon cancer cell line had very high purity, and the average size was a nanoparticle having an intact double membrane of about 160 nm in diameter.

Example 2. Efficiency of separation of extracellular vesicles according to the type of blocking material

Separation efficiencies were compared using a mixture of extracellular vesicle samples and various blocking substances in order to search for factors capable of blocking non-specific binding of extracellular vesicles in the stationary phase of semi-dry size exclusion chromatography according to the method of the present invention.

The colon cancer-derived sample of extracellular vesicles 2 μg in the same amount of various low-molecular substances (Trehalose, Glycerol, DMSO), nanoparticles removed 10% (v / v) Fetal bovine serum, or 10% (v /v) After mixing with fetal bovine albumin, each was loaded onto a size exclusion chromatography column equilibrated in a semi-dry manner. Rotary size exclusion chromatography was performed while the column was centrifuged at 700 xg for 5 minutes (Fig. 3(a)).

In order to confirm the yield of the extracellular vesicles in the eluate, a Western blot was performed on the CD9 protein, which is a marker of the extracellular vesicles (Fig. 3(b)), and further nanoparticle analysis (NPA, Nanoparticle Analysis) was performed. Thus, the dissolution rate of the extracellular vesicles was confirmed (Fig. 3(c)).

As a result, blocking substances having complex properties, including proteins, such as bovine serum or bovine serum albumin, effectively interfere with the binding of the stationary phase to the extracellular vesicles in semi-dry size exclusion chromatography, thereby significantly improving the yield of extracellular vesicles. I could see that.

Example 3. Efficiency of separation of extracellular vesicles according to the concentration of blocking substances

In order to confirm the separation efficiency of extracellular vesicles according to the concentration of bovine serum albumin, a sample in which various concentrations of bovine serum albumin were mixed was loaded onto the stationary phase of the semi-dry size exclusion chromatography. Specifically, 2 μg of purified colon cancer-derived sample extracellular vesicles and 2 μg of bovine serum albumin (0%, 5%, 10%) of various concentrations were mixed, and then loaded onto a size exclusion chromatography column equilibrated in a semi-dry manner. , Centrifugation was performed at 700 xg for 5 minutes to perform rotary size exclusion chromatography (Fig. 4(a)).

In order to confirm the yield of the extracellular vesicles in the eluate, Western blotting was performed on the CD9 protein, which is a marker of the extracellular vesicles, to confirm the dissolution rate of the extracellular vesicles (FIG. 4(b)). As a result, the separation efficiency of extracellular vesicles was significantly increased when 5% or 10% BSA was included compared to a sample not containing bovine serum albumin (0% BSA). In addition, it was found that the yield of extracellular vesicles was further improved by effectively interfering with the binding of the stationary phase and extracellular vesicles in dry size exclusion chromatography in proportion to the concentration of bovine serum albumin.

In addition, it was confirmed whether the concentration of salt (NaCl) added for the purpose of inhibiting hydrogen bonding and ionic bonding in size exclusion chromatography affects the blocking effect of bovine serum albumin. In the same method as described above, the extracellular vesicles were separated by mixing various concentrations (0.15M, 0.5M, 1.0M) of salt in the colon cancer-derived extracellular vesicle sample and 10% BSA mixture. As a result of confirming the extracellular vesicles in the eluate by Western blot and nanoparticle analysis, it was found that the blocking effect by bovine serum albumin was not affected by the concentration of salt (FIG. 5).

Example 4. After equilibration of the stationary phase using a blocking material, the separation efficiency of extracellular vesicles was confirmed

In another embodiment of the present invention, the column was first blocked before loading the sample containing the extracellular vesicles. Specifically, put the stationary phase in the prepared size exclusion chromatography column, and equilibrate the stationary phase of the size exclusion chromatography column in a semi-dry manner through centrifugation, and then a HBS buffer solution without bovine serum albumin (20 mM HEPES, 150 mM NaCl, pH 7 .4) After loading 700 μl, 700 μl of a buffer solution containing 1% bovine serum albumin, or 700 μl of a buffer solution containing 2% bovine serum albumin, the column was blocked while centrifuging. An additional buffer solution was added to the blocked column and centrifugation was repeated six times to wash excess albumin. After loading a sample of 2 μg of colon cancer-derived extracellular vesicles on the final washed column, the extracellular vesicles were eluted through centrifugation (FIG. 6(a)).

As a result of comparing the yield of extracellular vesicles of each column through Western blot (Fig. 6(b)), when the stationary phase of dry size exclusion chromatography using bovine serum albumin is first blocked, the extracellular vesicle yield is significantly improved. Became. From this, it was found that even without mixing the blocking material in the sample, the separation efficiency of the extracellular vesicles can be increased by effectively inhibiting the non-specific binding of the extracellular vesicles and the stationary phase.

Example 5. Checking the separation efficiency of extracellular vesicles during equilibration of the stationary phase and sample loading using a blocking material

In another embodiment of the present invention, after blocking a size exclusion chromatography column, a mixture of a sample and a blocking material was loaded to confirm the efficiency of separation of extracellular vesicles. Specifically, the fixed phase was put on the prepared size exclusion chromatography column, and the fixed phase of the size fold chromatography column was equilibrated in a semi-dry manner through centrifugation. Next, 700 μl of a buffer solution or a buffer solution to which 700 μl of 2% bovine serum albumin was added was loaded, and the column was blocked while centrifuging. The blocked column was further loaded with a buffer solution and centrifuged 6 times to remove excess albumin. An extracellular vesicle sample and a 10% bovine serum albumin mixture sample, or an extracellular vesicle sample to which bovine serum albumin was not added, respectively, were loaded onto the final washed column, and then the extracellular vesicles were eluted through centrifugation (Fig. 7 ( a)), the yield of the final purified extracellular vesicles was compared by Western blot (Fig. 7(b)). As a result, when the stationary phase of dry size exclusion chromatography was first blocked using bovine serum albumin, and then an extracellular vesicle sample containing 10% albumin was loaded and eluted, the extracellular vesicle separation efficiency was found to be the best. From this, by blocking the column before loading the sample, and loading the sample with an additional blocking material, non-specific binding between the extracellular vesicles and the stationary phase of semi-dry size exclusion chromatography can be most effectively inhibited, and the elution rate of the extracellular vesicles is reduced. It was found that it can be significantly improved.

Claims (15)

(a) equilibrating the stationary phase charged in the column in a semi-dry manner using a solution containing no blocking material;
(b) loading a mixture of a sample containing a blocking substance and an extracellular vesicle into the stationary bed; And
(c) eluting extracellular vesicles from the column;
Extracellular vesicle separation method comprising a.
The method of claim 1,
The blocking material is a material that competes with the extracellular vesicles at the adsorption site or non-specific binding site of the stationary bed.
The method of claim 1,
The method of separating extracellular vesicles, wherein the blocking material includes a protein material having a size smaller than that of the extracellular vesicles to be separated.
The method of claim 1,
The blocking material is cow serum (Fetal bovine serum), or bovine serum albumin (Fetal bovine albumin) extracellular endoplasmic reticulum separation method.
The method of claim 1,
The sample is mammalian cell culture medium, bacterial cell culture medium, yeast culture medium, tissue extract, cancer tissue, serum, plasma, saliva, tears, sweat, urine, feces, cerebrospinal fluid (CSF), ascite , Amniotic fluid, semen, milk (milk), dust, fresh water, seawater, soil and fermented food, characterized in that at least one selected from the group consisting of extracellular vesicle separation method.
The method of claim 1,
Further comprising a pretreatment step of the sample before loading the sample, or
Further comprising a post-treatment step of the eluate after the elution step, or
Or extracellular vesicle separation method, characterized in that it further comprises both pre-treatment and post-treatment steps.
The method of claim 6,
The pretreatment steps of the sample include centrifugation, ultracentrifugation, filtration, ultrafiltration, sonication, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity chromatography, polymer-based precipitation, and organic solvent precipitation. The method of separating extracellular vesicles using one or more methods selected from the group consisting of.
delete delete (a) equilibrating the stationary phase filled in the column in a semi-dry manner using a solution containing a blocking material;
(b) loading a sample containing a blocking material and an extracellular vesicle into the column; And
(c) eluting extracellular vesicles from the column;
Extracellular vesicle separation method comprising a.
The method of claim 10,
The blocking material is a material that competes with the extracellular vesicles at the adsorption site or non-specific binding site of the stationary bed.
The method of claim 10,
The method of separating extracellular vesicles, wherein the blocking material includes a protein material having a size smaller than that of the extracellular vesicles to be separated.
The method of claim 10,
The blocking material is cow serum (Fetal bovine serum), or bovine serum albumin (Fetal bovine albumin) extracellular endoplasmic reticulum separation method.
The method of claim 10,
The method of separating extracellular vesicles further comprising the step of further equilibrating the equilibrated stationary phase after step (a) with a solution containing no blocking material.
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