KR102059535B1 - Method for Isolation of Exosomes and Lipoproteins from Biological Sample - Google Patents

Abstract

The present invention relates to a method for separating exosomes and lipoproteins from a biological sample mixed with exosomes and lipoproteins. The fraction containing exosomes separated by the method of the present invention is obtained by greatly reducing the amount of lipoproteins. As confirmed that the purity of the exosomes increased, the method of the present invention can be usefully used to effectively separate exosomes and lipoproteins from a sample mixed with exosomes and lipoproteins.

Description

Method for isolation of exosomes and lipoproteins from biological samples {Method for Isolation of Exosomes and Lipoproteins from Biological Sample}

The present invention relates to a method for separating lipoproteins and exosomes from a biological sample mixed with lipoproteins and exosomes.

Extracellular vesicles are concepts that include exosomes, ectosomes, microvesicles, and apoptotic bodies that are released or secreted by cells. It is a bio-nanoparticle having a size of nm and produced from intracellular multi vesicular bodies (MVB).

These extracellular vesicles are relatively easy to separate from many different types of biofluids such as blood, cerebrospinal fluid, urine, amniotic fluid, breast milk, saliva, and semen, and depending on the cells from which they are derived, Derived), oncosome (from cancer cells), prostasome (from prostate cells), cardiosome (from cardiomyocytes), and the like. Cell vesicles contain various nucleotides or marker proteins depending on the cell or intracellular organelles from which they are derived. For example, oncosomes, which are extracellular vesicles derived from cancer cells, contain mRNAs of genes that induce the growth of cancer cells, and extracellular vesicles derived from antigen-presenting cells include major histocompatibility complexes. As such, extracellular vesicles contain high concentrations of cell-specific proteins and nucleotides, so they exist in about 0.01% of all protein bodies in normal biological fluids. Parcels have the advantage of being relatively easy to detect. In addition, although the types of proteins and nucleotides present in extracellular vesicles are very small, the exosome analysis is very useful for diagnosing specific diseases because the substances in extracellular vesicles can exhibit the unique characteristics of the cells from which they originate. It is useful, and research on this has been actively conducted recently.

In diagnosing or treating exosomes, it is important to obtain high-quality exosomes. Various methods have been studied regarding the isolation of exosomes, including Korean Patent Publication No. 10-2016-0115988. Examples include ultracentrifuges, density centrifuges, use of columns, PEG precipitation, chromatography, immuno-magnetic separation (IMS). And acoustic separation and acoustic purification. In the case of column chromatography, much attention has recently been given in that a relatively high purity exosome can be obtained in a short time. However, it has been reported that liposomal proteins are eluted with exosomes when exosomes are separated using column chromatography. Therefore, it is important to remove liposomal proteins to further increase the purity of exosomes. . In the past, column chromatography studies focused only on exosome elution sections and little in-depth analysis of lipoprotein elution was performed. However, lipoprotein isolation is essential for exosome research because lipoproteins with sizes and densities similar to exosomes are always present in cell culture or blood.

Under the above background, the present inventors have attempted to develop a method for separating exosomes and lipoproteins from biological samples, and can analyze exosomes and lipoproteins by analyzing particles in each section eluted using columns in cell culture and blood. The present invention has been completed by optimizing the conditions and identifying how to obtain pure exosomes using them.

Republic of Korea Patent Publication No. 10-2016-0115988

An object of the present invention is to provide a method for efficiently separating lipoproteins and exosomes from a biological sample mixed with lipoproteins and exosomes.

In order to achieve the above object, the present invention comprises a step of flowing a sample through a solid substrate having a pore size of 5 to 50 nm to perform size-exclusion chromatography (SEC) from the sample, Provided are methods for separating lipoproteins and exosomes.

The method for separating exosomes from the biological sample of the present invention can greatly increase the purity of the obtained exosomes by greatly reducing the amount of lipid protein relative to exosomes, which can be usefully used for separating exosomes and lipid proteins in a sample.

1 is a schematic diagram of a method for separating exosomes using a conventional column.
2 is a result of separation of exosomes using a conventional column:
A: exosomes observed in fraction # 8; And
B: Size distribution of exosomes observed in fractions # 7-9.
FIG. 3 shows particle Z-average size in each fraction (fractions # 8 to # 15) of cell culture, mouse serum and human serum identified through dynamic light scattering (DLS).
Figure 4 shows the results of detection of exosomes and lipoproteins of each fraction (fractions # 9 to # 15) of cell culture, mouse serum and human serum via Western blot:
ApoB: lipoprotein; And
CD63: exosome marker protein.
Figure 5 shows the results of detection of exosomes and lipoproteins according to column beads (2B column or 6B column) in each fraction of cell culture and human serum (fractions # 8 to # 15):
ApoB: lipoprotein; And
CD63: exosome marker protein.
6 shows the results of microscopic observation of the exosomes of fractions # 12 to # 14 where human serum was fractionated by 6B column (CL-6B):
A: exosomes captured using magnetic beads coated with an anti-CD63 antibody;
B: size of captured exosomes; And
C: Gold labeled exosomes.
Figure 7 shows the ratio of exosomes and lipoproteins according to the separation method:
ApoB: lipoprotein;
CD63: exosome marker protein;
2B (8-10): 2B column fractions # 8- # 10;
2B (12-14): 2B column fractions # 12- # 14;
6B (8-10): 6B column fractions # 8- # 10;
6B (12-14): 6B column fractions # 12- # 14;
ExoQuick: precipitate obtained by centrifugation after ExoQuick treatment; And
serum: Untreated serum.
Figure 8 shows the efficiency of removal of other proteins and exosome separation in human serum after fractionation after precipitation:
ApoB: lipoprotein;
CD63: exosome marker protein;
2B (12-14) -exo: a precipitate obtained by mixing 2B column fractions # 12- # 14 and Exoquick solution at 1: 1 (v / v);
6B (12-14) -exo: a precipitate obtained by mixing 6B column fractions # 12- # 14 and Exoquick solution at 1: 1 (v / v);
serum-exo: a precipitate obtained by mixing an unfractionated serum with an Exoquick solution at 1: 1 (v / v);
ExoQuick: a precipitate obtained by mixing unfractionated serum and Exoquick solution at 5: 1 (v / v); And
serum: Untreated serum.

Hereinafter, the present invention will be described in detail.

The present invention is to separate the lipoprotein and exosomes from a mixture of lipoproteins and exosomes, comprising performing size exclusion chromatography by flowing a sample through a solid substrate having a pore size of 5 to 50 nm. Provide a method.

The solid substrate may comprise a column packed with nonporous rigid beads or particles;

Columns packed with rigid reticulated foam;

A column packed with hard monolithic beds of sintered beads or non-bead sintered solid media with internal flow channels;

Columns packed with woven or non woven hard fibers;

Columns packed with hard yarn or optionally hollow monofilament fibers;

Spiral wound cartridges; or,

It may include, but is not limited to, a combination of at least three selected from the group consisting of beads, rigid reticulated foam, sintered beads, fibers, yarns, and monofilaments.

The substrate may be composed of agarose, sepharose, cellulose, controlled pore glass, silica gel, hydrophilic material including dextran or organic artificial polymer including polyacrylamide polystyrene, but is not limited thereto. Sepharose CL-6B containing beads composed of Sepharose was used.

The column may have a bead diameter of 20 to 180 μm, specifically 40 to 165 μm, but is not limited thereto.

The sample may be one or more selected from the group consisting of blood, cerebrospinal fluid, urine, amniotic fluid, breast milk, saliva, semen and cell culture, but is not limited thereto.

The separation method of the present invention may further include the step of precipitation after performing chromatography. The precipitation can be carried out by methods known in the art.

Exosomes separated by the separation method of the present invention may be included in the 40 to 50% section of the total eluate after the chromatography, but is not limited thereto, the 40 to 50% section by the separation method of the present invention The ratio of exosomes to lipoproteins in the eluate of

In a specific embodiment of the present invention, the inventors overlap the exosomes and lipoprotein elution section of the serum in the conventional method (Fig. 4), the separation of the exosomes and lipoprotein elution section from the serum when using the CL-6B column It was clearly seen in the observation using markers (FIG. 5) and microscopic observations (FIG. 6). The method showed the highest separation efficiency of exosomes compared to lipoproteins compared to the conventional 2B column method and the ExoQuick method. It was found to be high (Fig. 7), and also after the CL-6B column, when precipitated using Exoquick it was confirmed that the precipitation of lipoproteins can be reduced to increase the purity of exosomes (Fig. 8) .

Therefore, the separation method of exosomes and lipoproteins in the biological sample according to the present invention can be usefully used to increase the purity of the obtained exosomes by greatly reducing the amount of lipid protein relative to exosomes.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following Examples and Experimental Examples are only for illustrating the present invention, the present invention is not limited by the following Examples and Experimental Examples.

Example  1. Existing column  Under conditions Exosomes  Detachable confirmation

The column prepared for the chromatography may have a variety of volumes, but in the present invention, the filling beads were filled in a representative diameter of 10 mm and a height of 50 mm. In order to confirm the degree of separation of exosomes by conventional column chromatography, the cell culture medium was prepared using a sepharose CL-2B column (Sigma-Aldrich Co.) having a diameter of 60 to 200 μm and a pore size of 75 nm. Size Exclusion Chromatography (SEC) was performed as follows.

Specifically, prior to loading the sample, the fill beads were washed with 300 ml of PBS. After washing, the prepared sample was loaded as soon as the mobile phase buffer was exhausted in addition to the packed column beads. The volume of the sample loaded on the column was made to not exceed 5% of the total volume of the charged beads. In this experiment, 0.3 to 0.5 ml of sample was loaded. The pressure of the mobile phase was flowed at 2-4 Pa. The solution which received 0.5 ml of eluate which flowed out from the time of sample loading was made into 1 fraction. The samples were all eluted within 25-30 sections on the basis of 280 nm absorbance, and the separated sample fractions were identified by Transmission Electron Microscopy (TEM).

As a result, the exosomes (indicated by the black arrows) were observed in the TEM analysis of fractions # 7 to # 9 as shown in FIG. 2 (FIG. 2A), and the size distribution of the observed exosomes averaged 48.5 ± 13.1. nm appeared (FIG. 2B).

Example 2 Comparison of the Tendency of Exosome and Lipoprotein Elution in Cell Culture and Serum

In order to confirm the separation of exosomes from serum in addition to the cell culture medium, cell culture medium, mouse serum and human serum were fractionated as in Example 1, and Z-average size (DLS) was used for each fraction using dynamic light scattering (DLS). Z-Average size) was measured. The serum was centrifuged for 30 min at 10000 rcf, 4 ° C for 30 min, and the obtained supernatant was used. Western blots were performed for exosome marker proteins and lipoproteins using fractions as samples. As the primary antibody, an antibody against CD63, an exosome marker protein (sc-15363 manufactured by Santa Cruz), and an ApoB-100 antibody (sc-25542, manufactured by Santa Cruz), a lipoprotein antibody, were used. Specifically, the antibody against CD63 was diluted 1: 500 (0.1% (v / v)) in a solution in which 5% (w / v) skim milk was dissolved in TBST, and an antibody against ApoB-100 was used. Diluted to 1: 1000 (0.1% (v / v)) and used.

As a result, the difference in the average size of the particles in the DLS results in fractions # 9 and 10 was remarkable, and the particle size in the serum was found to be smaller to 35-53% of the cell culture (Fig. 3). . Exosomes present in the blood were found to be smaller than exosomes in cell culture.

When the cell culture was fractionated, it was confirmed that the exosomes and lipoprotein particles were eluted in the fractions # 9 and 10, but when the serum was fractionated, the exosomes were eluted later than the lipoproteins (FIG. 4). Compared with the DLS size, lipid proteins were mainly detected at z-means 30-150 nm and exosomes at 20-65 nm.

Example  3. column On the bead  According Exosomes Lipoprotein  Verify optimization of separation

The cell culture and human serum were fractionated as in Example 1 by replacing with a sepharose CL-6B column (Sigma-Aldrich Co.) having a pore size of about 20 nm with a bead size of 40-165 μm and Western blot as in Example 2. Z-average size was measured for each fraction by using dynamic light scattering (DLS).

As a result, as shown in FIG. 5A, the serum is eluted with exosomes and lipoproteins when fractionated into a 2B column, but the fraction of exosomes and lipoproteins from serum is increased as the lipoproteins are eluted when fractionated with 6B column. As the separation of the elution section became clear, lipoproteins were first separated from fractions # 9 to # 11, and exosomes were separated from fractions # 12 to # 14. Since the exosomes present in the blood are small in size, fractionation with a sepharose CL-6B column with a 20 nm pore size resulted in elution of lipoproteins and exosomes. However, in the cell culture solution, the fraction of lipoprotein and exosomes was also eluted when fractionated by 6B column.

Example  4. By column Fractionated  In section Exosomes  Check for existence

In order to determine whether exosomes are actually present in the fractions of fractions # 12 to # 14, which fractionated human serum in the column 6B in Example 3, the # 9 fraction was used as a control to capture anti-CD63 magnetic beads and CD63-immuno-gold labeling treatment was performed and confirmed by TEM.

Specifically, fractions of # 12 to # 14 were fixed with 2% PFA and loaded onto a TEM grid and washed with 50 mM PBS. Blocked with 3% BSA and reacted with antibody against CD63 (Santa Cruz, sc-15363), washed with PBS and PBS / 0.5% BSA, and reacted with Protein A-gold (10 nm). After completion of the reaction, washed again with PBS, fixed with 1% glutaraldehyde (glutaraldehyde), washed with tertiary distilled water and the phase contrast with 2% phosphotungstic acid (pH 7-8) solution was performed. . Observation was made with a 200 kV electron transmission microscope.

As a result, it was confirmed that the exosomes (indicated by the black arrows) were captured in the magnetic beads (FIG. 6A). As in Example 2, the # 9 fraction of the DLS was 72.9 ± 28.1 nm, but the # 12 fraction to the # 14 fraction. Was found to be 32.7 ± 7.02 (FIG. 6B), which means that in Example 2, the exosome fraction was found in the # 12 fraction to the # 14 fraction (FIG. 4), with the result that the fraction size corresponding to this interval was 20-65 nm. It corresponds to (FIG. 3). It was also confirmed that the exosomes were fractionated through CD63-immuno-gold labeling (FIG. 6C).

Example  5. According to the separation method Exosomes Lipoprotein  Confirmation of Separation Efficiency

According to the separation method, how the relative amounts of exosomes and lipoproteins differ, perform Western blot as in Example 2, and then quantify the blotting portion of the sample by using the Bio-Rad ChemiDoc Image Software through density measurement It was.

Human serum samples disclosed in Example 2 were fractionated. The column chromatography method used CL-2B and CL-6B as the method described in Example 1, was compared using a commercialized product ExoQuick (System Biosciences, Inc.). The separation method using Exoquick is as follows. The serum samples were centrifuged at 10,000 rcf for 30 minutes to remove microvesicles and cell debris, and then the obtained supernatant and Exoquick solution (Cat # .4478360) were mixed at 5: 1 and refilled. It was suspended and stored at 4 ° C. for 30 minutes. It was then centrifuged again at 10,000 rcf for 10 minutes and the pellet formed was resuspended in PBS to be used as the sample.

As a result, in the density centrifugation method using ExoQuick as shown in Figure 7A it was confirmed that the lipoprotein is separated with a significant amount with the exosomes. In the fraction using 2B column, the amount of lipoprotein was the most in the # 8- # 10 fraction, and the lipoprotein and the exosome were in the # 12- # 14 fraction. In the fraction using 6B column, lipoproteins were higher from fractions # 8 to # 10, and lipoproteins were not detected and exosomes were detected in fractions # 12- # 14. Therefore, when comparing the ratio of lipoprotein and exosomes in the fraction, not just the detection amount, as shown in FIG. 7B, the method of exosome separation efficiency is higher than that of lipoproteins using the CL-6B column. It was found that the method of liposomal protein separation efficiency is higher than that of exosomes was obtained by using the column CL-2B fraction # 8- # 10 fractions.

Example  6. In case of precipitating serum after fractionation Exosomes Lipoprotein  Confirmation of Separation Efficiency

In the case of further precipitated using ExoQuick to the fraction of high separation efficiency (fraction # 12- # 14) of the exosomes identified in Example 5 The exosome separation efficiency was compared.

Specifically, the human serum samples were fractionated with CL-2B and CL-6B columns, and the # 12- # 14 fractions and the Exoquick solution (Cat #. 4478360) for which the exosomes were detected were referred to Example 5 above. Precipitated by mixing at 1: 1 (v / v), and precipitated by mixing 1: 1 (v / v) of the serum with no fraction and Exoquick solution for comparison (Serum-exo in FIG. 8). Unprepared serum and Exoquick solution was prepared by mixing 5: 1 (v / v) to precipitate (Exoquick in Figure 8). The separation of each sample was confirmed by SDS-PAGE, and the amount of exosomes and lipoproteins was compared by Western blot.

As a result, soluble proteins such as albumin (albumin: ˜65k Da) were found to be mostly removed through conventional fractionation techniques (FIG. 8A). Exosomes were 2B (12-14) -exo precipitated using CL-2B column, 6B (12-14) -exo precipitated using CL-6B column, serum-exo and Exoquick precipitated without fractionation. Was detected in (Fig. 8B), the amount of exosomes compared to lipoproteins was found to be the highest separation efficiency of exosomes and lipoproteins when precipitated using a CL-6B column (Fig. 8C).

Claims (7)

A method of separating lipoproteins and exosomes from serum, comprising performing size exclusion chromatography by flowing serum through Sepharose CL-6B having a pore size of 5 to 50 nm.
delete delete delete delete The method of claim 1, further comprising the step of precipitation after performing the chromatography.
The method of claim 1, wherein the separated exosomes are included in the 40-50% section of the total eluate after the chromatography.

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