JP7357346B2 - Exosome isolation method, exosome isolation kit, exosome removal method, separation solution acquisition method, and exosome isolation column - Google Patents

Description

The present invention relates to an exosome isolation method, an exosome isolation kit, and an exosome removal method.

Exosomes are extracellular vesicles that are secreted from cells and have a diameter of about 50 to 150 nm and are covered with a lipid bilayer membrane. Inside exosomes, information specific to the cells that secrete them, such as microRNAs (hereinafter referred to as "miRNAs"), is highly preserved, making them useful as new biomarkers for various diseases such as cancer and life phenomena. It is attracting attention as

Furthermore, in recent years, it has become clear that exosomes derived from mesenchymal stem cells have anti-inflammatory and complex immunoregulatory effects, and research is actively being conducted to utilize exosomes themselves as therapeutic agents. Therefore, techniques for isolating exosomes in a less damaged state (intact state) are becoming increasingly important. The global market size for exosome-based treatments and diagnostics is predicted to reach 180 million US dollars by 2023, and exosome research is considered one of the research fields with the highest growth expectations. .

As a method for isolating exosomes, ultracentrifugation using an ultracentrifuge is generally used. However, this ultracentrifugation method is difficult to cope with large-scale (large-scale purification) and multi-analyte use. Furthermore, problems such as exosomes being damaged by strong centrifugal force have also been pointed out. For future exosome research and the spread of exosome-based treatments, there is a need for the development of isolation methods that are more reproducible and can handle large quantities and multiple samples.

For example, Patent Document 1 discloses a method for isolating exosomes in an intact state with extremely little damage by using a peptide containing four or more lysines in close proximity to each other.

International Publication WO2019/039179 Publication

However, it is known that exosomes are not uniform and vary widely depending on the type and state of differentiated cells. For example, both CD9 and CD63 are known as markers of exosomes, but it is thought that the correlation between the expression levels of CD9 and CD63 in exosomes is not very high. That is, there are exosomes that express a relatively high amount of CD9 (CD9-positive exosomes) and exosomes that express a relatively high amount of CD63 (CD63-positive exosomes). It is known that CD63-positive exosomes are secreted in large quantities from tumorigenic cells (Reference: Ricklefs FL et al. J Extracell Vesicles. 2019;8(1);1588555). Therefore, especially in the field of cancer diagnosis, it is required to be able to efficiently isolate CD63-positive exosomes. Furthermore, in the future, for example, if exosomes themselves are to be used as therapeutic agents, methods that can isolate various types of exosomes will be needed. However, with the exosome isolation method described in Patent Document 1, the types of exosomes that can be isolated are unknown.

As a result of intensive studies by the present inventors, the method for isolating exosomes described in Patent Document 1 (method using a peptide containing consecutive lysines through alpha bonding, described later) shows that CD9-positive exosomes are more likely to be isolated than CD63-positive exosomes. It was found that it could be isolated preferentially (see Comparative Example 2 below). The purpose of the present invention is to provide a method for easily and efficiently isolating various types of exosomes, particularly CD63-positive exosomes, in an intact state (or in a near-intact state), an exosome isolation kit, and an exosome isolation kit. The objective is to provide a separate column. Another object of the present invention is to provide an exosome removal method that can easily remove various types of exosomes from a sample containing exosomes.

The present inventors have found that a peptide containing consecutive lysines through epsilon binding can bind to a variety of exosomes, and in particular can preferentially bind to CD63-positive exosomes. Furthermore, the present inventors have found that when using a carrier carrying a peptide containing consecutive lysines through epsilon binding, exosomes bound to the carrier can be dissociated from the carrier under mild conditions. Based on these findings, the present inventors have completed a method that can preferentially isolate CD63-positive exosomes among a variety of exosomes.

That is, one embodiment of the present invention has the following configuration.
[1] A method for isolating exosomes, which comprises isolating exosomes from a sample containing exosomes, the sample and a carrier carrying a peptide containing 20 to 40 consecutive lysines through epsilon bonds; a complex forming step in which the exosome and the peptide supported on the carrier form a complex by contacting with each other, and the complex obtained by the complex forming step and the metal A method for isolating exosomes, comprising a dissociation step of dissociating the exosomes from the complex by bringing them into contact with a dissociation buffer containing cations.
[2] The exosome isolation method according to [1], wherein the content of CD63-positive exosomes in the exosomes contained in the complex is 10% or more of the content of CD9-positive exosomes. .
[3] The method for isolating exosomes according to [1] or [2], wherein the carrier to which the peptide is bound is used by filling a column.
[4] An exosome isolation kit for carrying out the exosome isolation method according to any one of [1] to [3], comprising the peptide, the carrier, and the dissociation buffer. An exosome isolation kit.
[5] A method for removing exosomes from a sample containing exosomes, which comprises contacting the sample with a carrier carrying a peptide containing 20 to 40 consecutive lysines through epsilon bonding. a complex forming step in which the exosome and the peptide supported on the carrier form a complex by binding, and the carrier supports the complex formed by the complex forming step. and the sample to obtain a separation liquid from which at least a portion of the exosomes have been removed from the sample.
[6] An exosome isolation column for performing the exosome isolation method described in [3] or the exosome removal method described in [5], which is filled with the carrier to which the peptide is bound. An exosome isolation column characterized by:
[7] An exosome isolated by the exosome isolation method described in any one of [1] to [3].
[8] A separated liquid obtained by the exosome removal method described in [5].

According to one embodiment of the present invention, it is possible to easily isolate various types of exosomes in an intact state (or in a near-intact state).

FIG. 2 is a diagram showing the structures of ε-polylysine and α-polylysine. FIG. 3 is a diagram showing an outline of the experimental operation procedure of Example 1. 2 is a diagram showing a purification chart and purification results of Example 1. FIG. FIG. 3 is a diagram showing an outline of the experimental operation procedure of Example 2. 3 is a diagram illustrating an outline of the experimental operation procedure of Comparative Example 2. FIG. FIG. 3 is a diagram showing the results of marker analysis of exosomes isolated by the method according to Example 2 and Comparative Example 2. FIG. 2 is a diagram showing the results of comparison of exosomes isolated by the method according to Example 2 and Comparative Example 2.

An embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to each configuration described below, and various changes can be made within the scope of the claims. The present invention also includes embodiments and examples obtained by appropriately combining technical means disclosed in different embodiments and examples. Note that all academic literature and patent literature described in this specification are incorporated as references in this specification. In addition, unless otherwise specified in this specification, the numerical range "A to B" is intended to be "above A (including A and greater than A) and less than or equal to B (including B and less than B)."

As used herein, the term "peptide" is used interchangeably with "polypeptide" and refers to a compound in which two or more amino acids are linked by a peptide bond. Furthermore, the term "peptide bond" includes not only the bond between the amino group at the α-position and the carboxyl group in an amino acid, but also the bond between the amino group included in the side chain of the amino acid and the carboxyl group at the α-position. Here, a peptide bond in which the amino group included in the side chain of an amino acid is an amino group at the ε position of lysine is referred to as an "epsilon bond." In this specification, the one-letter or three-letter notation defined by IUPAC and IUB is used as appropriate for the notation of amino acids.

[1. Exosome isolation method]
A method for isolating exosomes according to an embodiment of the present invention (hereinafter referred to as "isolation method of the present invention") is a method for isolating exosomes from a sample containing exosomes, comprising:
By bringing the sample into contact with a carrier carrying a peptide containing 20 to 40 consecutive lysines through epsilon bonding, the exosome and the peptide carried on the carrier are bonded. a complex forming step of forming a complex, and
The method includes a dissociation step of bringing the complex obtained by the complex formation step into contact with a dissociation buffer containing a metal cation to dissociate the exosome from the complex.

Since the method for isolating exosomes according to one embodiment of the present invention has the above configuration, it achieves the following effects (1) to (3).
(1) Since there is no need to use an ultracentrifuge or the like, exosomes can be isolated easily and in a short time.
(2) Since it utilizes the fact that consecutive lysines through epsilon binding bind to the membranes of various exosomes, including exosomes with relatively high expression levels of CD63 (CD63-positive exosomes), it can capture a wide range of exosomes and can be released.
(3) Exosomes are dissociated from the complex under mild conditions using a dissociation buffer containing metal cations that does not (or has little) effect on the membrane structure of exosomes and the structure of proteins present in the membrane. There is. Therefore, exosomes can be isolated from the complex without damaging the exosomes (in other words, in an intact state or a near-intact state).

By isolating intact (or near-intact) exosomes, it is possible to perform, for example, (1) functional analysis of exosome physiological effects, and (2) biomarkers (exosome-derived proteins, nucleic acids such as miRNA). It is possible to use it for etc.

In addition, exosomes are expected to be applied to the treatment of a wide range of diseases, including cancer, Alzheimer's disease, myocardial infarction, cerebral infarction, and infectious diseases. Therefore, by isolating intact (or nearly intact) exosomes, the isolated exosomes themselves can be used as (3) therapeutic agents and (4) carriers for drug delivery systems. In particular, the use of exosomes as a therapeutic drug in (3) above includes (A) supplementary use in regenerative medicine, (B) use for the purpose of immune control, and (C) use as a vaccine. (Reference: Experimental Medicine, 2016, Vol. 34, p1390-1396: Next-generation medicine built by exosomes).

The above (A) to (C) will be explained by giving specific examples. Exosomes derived from mesenchymal stem/stromal cells (MSCs) are the most studied exosomes. MSC-derived exosomes are known to have cell division-promoting effects, anti-apoptotic effects, and anti-inflammatory effects. Thereby, it is possible to reduce secondary damage to tissues surrounding the damaged tissue, and it is possible to promote recovery of the damaged tissue. Therefore, it is expected to be applied to many diseases such as skin and liver regeneration and recovery from myocardial infarction ((A) auxiliary use in regenerative medicine). In addition, MSC-derived exosomes have complex immunoregulatory effects, and are expected to be applied to the treatment of autoimmune diseases and immune regulation after organ transplantation ((B) Use for immune regulation) ).

In addition, exosomes secreted from antigen-presenting cells, which are collected after reacting antigen-presenting cells with cancer antigen peptides, present antigenic peptides and have the effect of activating CD4 ⁺ and CD8 ⁺ T cells. be. Furthermore, it is also considered to use exosomes derived from cancer cells as vaccines by directly using them as antigens ((C) Use as vaccines).

In particular, it is known that CD63-positive exosomes, which can be efficiently isolated by the isolation method of the present invention, are secreted in large quantities from tumorous cells. The isolation method of the present invention can comprehensively isolate various types of exosomes, including exosomes with a relatively high expression level of CD9 (CD9-positive exosomes), CD63-positive exosomes, and the like. Therefore, according to the exosomes isolated by the isolation method of the present invention, more comprehensive and less omitted analysis can be realized in the above (1) functional analysis of exosome physiological effects and the like. Particularly in the field of cancer, effective use can be expected in (1) elucidation of cancer pathology through functional analysis of physiological effects of exosomes.

In addition, the exosomes isolated by the isolation method of the present invention can be expected to be effectively used as a cancer diagnostic tool by using them as the above-mentioned (2) biomarkers. Furthermore, the ability to isolate more diverse types of exosomes can be expected to expand the range of possible applications for use as a carrier for the above (3) therapeutic drugs and the above (4) drug delivery system.

Below, the materials used in the exosome isolation method according to one embodiment of the present invention will be explained first, and then each step of the exosome isolation method will be explained.

[1-1. material〕
(sample)
In the isolation method of the present invention, the "sample containing exosomes" (herein also simply referred to as sample) is not particularly limited in other compositions as long as it is a mixture containing exosomes. Examples include biological samples.

As used herein, "biological sample" means a specimen collected from the body of an organism. Biological samples include, for example, blood, plasma, serum, saliva, urine, lacrimal fluid, sweat, breast milk, amniotic fluid, cerebrospinal fluid (cerebrospinal fluid), bone marrow fluid, pleural fluid, ascites fluid, synovial fluid, aqueous humor, These include, but are not limited to, vitreous humor and the like. The selection of biological samples can be appropriately determined by those skilled in the art depending on the purpose. For example, from the viewpoint of ease of collection, blood, plasma, serum, saliva, urine, etc. are preferably used.

In one embodiment of the present invention, the origin of the biological sample is not particularly limited as long as it is derived from a biological species that possesses exosomes. The biological species from which the biological sample is derived is preferably, for example, a mammal. Examples of mammals include mice (Mus musculus), cows (Bos Taurus), humans (Homo sapiens), and the like.

(peptide)
The peptide used in the isolation method of the present invention (hereinafter appropriately referred to as the "peptide of the present invention") is a peptide containing 20 to 40 lysines (also referred to as "lysine") consecutively formed by epsilon bonds. Therefore, it can bind to exosomes in the sample. In particular, such a peptide can suitably bind to CD63-positive exosomes.

The inventors of the present invention have discovered that the peptide of the present invention can bind to various exosomes including CD63-positive exosomes because the peptide of the present invention contains 20 to 40 consecutive lysines through epsilon binding. Ta. In addition, the inventors of the present invention found that in the method for isolating exosomes described in Patent Document 1, CD9-positive exosomes are isolated preferentially over CD63-positive exosomes (see Comparative Example below). (see 2). That is, lysines connected by epsilon bonds have a higher binding affinity with CD63-positive exosomes than lysines connected by peptide bonds (also referred to as "alpha bonds") connected by α-position amino groups and carboxyl groups. This is a finding discovered through intensive research by the inventors of the present invention. Therefore, according to the isolation method of the present invention, CD63-positive exosomes can be easily isolated.

In the exosomes that bind to the peptide of the present invention, the content of CD63-positive exosomes is 10% or more of the content of CD9-positive exosomes, more preferably 15% or more of the content of CD9-positive exosomes, and more preferably The content of CD9-positive exosomes is 20% or more, more preferably the content of CD9-positive exosomes is 25% or more, more preferably 30% or more of the content of CD9-positive exosomes, and more preferably CD9-positive exosomes. 35% or more of the content of CD9-positive exosomes, more preferably 40% or more of the content of CD9-positive exosomes, more preferably 45% or more of the content of CD9-positive exosomes, and more preferably content of CD9-positive exosomes. The content is 50% or more of the amount, more preferably 55% or more of the content of CD9-positive exosomes, and more preferably 60% or more of the content of CD9-positive exosomes.

As shown in Figure 1, the amino groups contained in the side chains of lysines (ε-polylysine) that are connected by epsilon bonds are stronger than the amino groups contained in the side chains of multiple lysines (α-polylysines) that are connected by alpha bonds. is also located close to the backbone of the polypeptide. In this way, even if the lysine is the same, the structure of the polypeptide differs greatly if the peptide bonding site is different. This is thought to cause a difference in binding to exosomes between lysines that are continuous by epsilon bonds and lysines that are continuous by α bonds.

The peptide of the present invention can bind to exosomes with a binding strength sufficient to isolate exosomes if it contains 20 to 40 consecutive lysines through epsilon binding. The peptide of the present invention preferably contains 20 or more lysines connected by epsilon bonds, more preferably 25 or more lysines connected by epsilon bonds, and 30 or more lysines connected by epsilon bonds. It is more preferable to contain 35 or more lysines, and more preferably to contain 35 or more lysines connected by epsilon bond. Further, it is preferable that the number of consecutive lysines contained in the peptide of the present invention through epsilon bonds is 40 or less. When the peptide of the present invention has the above structure, it has the advantage that it becomes easier to bind to exosomes.

The peptide of the present invention is not particularly limited in other configurations as long as it contains 20 to 40 consecutive lysines through epsilon bonds, and may be a peptide consisting only of lysine or may contain amino acids other than lysine. It may also be a peptide. Furthermore, the peptide of the present invention may be a complex peptide that further contains a structure other than the peptide, such as a sugar chain or an isoprenoid group. Amino acids contained in the peptides of the present invention may be modified. Furthermore, the amino acids contained in the peptide of the present invention may be of L type or D type.

The peptides of the invention may be readily made according to any technique known in the art, and may be expressed, for example, as natural peptides produced by microorganisms. Microorganisms that produce consecutive lysines through epsilon bonds are described, for example, in the reference (Shima, S. and Sakai H. (1977). Polylysine produced by Streptomyces. Agricultural and Biological Chemistry 41: 1807-1809). Furthermore, the peptide may be expressed by a transformant into which an expression vector for the peptide has been introduced, or may be chemically synthesized. That is, polynucleotides encoding the peptides of the invention are also within the scope of the invention. As a chemical synthesis method, a liquid phase method can be mentioned. Furthermore, commercially available ε-polylysine can be used as the peptide of the present invention.

Since the peptide of the present invention can consist of at least 20 lysines, the lower limit of the molecular weight of the peptide of the present invention is 2581.42 [=(146.19×20)−(18.02×19)]. I can say that. Although the upper limit of the molecular weight of the peptide of the present invention is not particularly limited, it can be said that about 300,000 is the upper limit from the viewpoint of operability such as solubility and viscosity.

The peptide of the present invention may be composed of a peptide having a single molecular weight, or may be composed of a mixture of peptides having different molecular weights.

(carrier)
The carrier used in the isolation method of the present invention (hereinafter appropriately referred to as "the carrier of the present invention") means a carrier that can support the peptide of the present invention. The carrier of the present invention can bind to the peptide of the present invention bound to exosomes to form a complex in which the exosomes, the peptide of the present invention, and the carrier of the present invention are bound in this order.

In the isolation method of the present invention, by using a carrier, it becomes possible to efficiently and easily isolate a complex containing exosomes.

The structure of the carrier is not particularly limited as long as it is a structure that can directly or indirectly support (retain) the peptide of the present invention. The carrier of the present invention is preferably a support that does not weaken the function of the peptide of the present invention bound to the carrier, such as glass, nylon membrane, semiconductor wafer, latex particles, cellulose particles, microbeads, silica beads, Examples include magnetic beads. The carrier of the present invention is preferably cellulose particles, which are suitable for column purification because they allow easy recovery of complexes containing exosomes and isolation of exosomes. Cellulose particles are widely used in the separation and purification of proteins, DNA, cells, etc., and are a carrier that can be fully understood by those skilled in the art.

The mode of binding the peptide and the carrier is not particularly limited, and may be a well-known mode. Further, the binding between the peptide and the carrier may be direct or indirect. Furthermore, the peptide and the carrier may be bonded via a linker made of a polypeptide.

The type of peptide that binds to the carrier may be one type or a combination of multiple types. When multiple types of peptides are used in combination, the combination is not particularly limited.

Note that the peptide of the present invention may be biotinylated and bound to streptavidin held on the carrier of the present invention.

(dissociation buffer)
In the isolation method of the present invention, exosomes are separated from the complex by bringing the complex into contact with a dissociation buffer containing metal cations (hereinafter referred to as "the dissociation buffer of the present invention") in the dissociation step described below. Dissociate and isolate exosomes. The dissociation buffer of the present invention does not contain protein denaturants and can dissociate exosomes from complexes under mild conditions, so exosomes can be isolated in an intact state (nearly intact state). be able to. The present inventors are the first to discover that the binding between the peptide of the present invention and exosomes can be dissociated using such a mild dissociation buffer, and those skilled in the art who are unaware of this new finding should Inventions cannot be easily conceived. Further, the effect that exosomes can be easily isolated in an intact state (nearly intact state) is a remarkable and advantageous effect of the present invention.

The metal cations contained in the dissociation buffer of the present invention are not particularly limited, and include, for example, monovalent metal cations such as sodium ions, potassium ions, lithium ions, silver ions, and copper (I) ions, and magnesium ions. , divalent metal cations such as calcium ions, zinc ions, nickel ions, barium ions, copper (II) ions, iron (II) ions, tin (II) ions, cobalt (II) ions, and lead (II) ions. ions, and trivalent metal cations such as aluminum ions and iron (III) ions. Among these metal cations, sodium ions, potassium ions, magnesium ions, zinc ions, and nickel ions are preferred as the metal cations contained in the dissociation buffer of the present invention, since they have high water solubility. Furthermore, since exosomes can be sufficiently dissociated from the complex, the dissociation buffer of the present invention preferably contains sodium ions, potassium ions, or magnesium ions among the above-mentioned metal cations, either alone or in combination. .

The purpose of the isolation method of the present invention is to easily isolate exosomes in an intact state (or in a near-intact state). Therefore, in the isolation method of the present invention, "isolating exosomes in an intact state (or in a near-intact state)" means to isolate exosomes in a sample without damaging them (almost without damaging them). It means separating and isolating substances other than exosomes (e.g. proteins, etc.) inside. In other words, in the isolation method of the present invention, "isolating exosomes in an intact state (or in a near-intact state)" means that the membrane structure (specifically, the lipid bilayer membrane structure) of the exosomes is maintained. Exosomes in the sample are separated from substances other than exosomes in the sample and isolated while the structure of the proteins present in the membrane is maintained (almost preserved) and the structure of the proteins present in the membrane is preserved (almost preserved). It means to do.

The dissociation buffer may contain substances other than metal cations as long as they do not affect the membrane structure of exosomes and the structure of proteins present in the membrane.

The concentration of metal cations in the dissociation buffer is not particularly limited as long as it can dissociate exosomes from the complex and does not affect the membrane structure of exosomes and the structure of proteins present in the membrane. In other words, the optimal cation concentration may vary depending on the binding strength between the peptide of the present invention and exosomes used, the type (valence) of the metal cation, etc., so the optimal concentration should be determined after considering the appropriate concentration. do it. Note that the concentration of metal cations in the dissociation buffer of the present invention is not particularly limited, but for example, it is preferably 0.01M to 5M, more preferably 0.05M to 2M, and It is more preferably from .1M to 1M, even more preferably from 0.2M to 0.7M, particularly preferably from 0.2 to 0.4M.

Further, the pH of the dissociation buffer can be set as appropriate. The pH of the dissociation buffer is preferably 5 to 10, more preferably 6 to 9, even more preferably 7 to 8, and particularly preferably 7.3 to 7.5. It is preferable that the pH of the dissociation buffer is within the above range because it does not affect the membrane structure of exosomes and the structure of proteins present in the membrane.

[1-2. Process]
(Complex formation process)
The complex formation step included in the isolation method of the present invention (hereinafter referred to as the "complex formation step of the present invention") is a process in which a sample containing exosomes and the peptide of the present invention supported on the carrier of the present invention are A complex formed by binding the exosome and the peptide supported on the carrier (a complex in which the exosome - the peptide of the present invention - the carrier of the present invention are bound in this order; hereinafter referred to as "the present invention" as appropriate) This is a complex forming step in which a compound of the invention is formed. The method of contacting the sample with the peptide of the present invention supported on the carrier of the present invention is such that the exosomes in the sample and the peptide of the present invention supported on the carrier of the present invention bind to each other, thereby forming the complex of the present invention. Other methods are not limited as long as they are carried out under conditions that allow the formation of.

For example, in contacting a sample containing exosomes with the peptide of the present invention supported on the carrier of the present invention, for example, a columnar or cylindrical container (column) is filled with the carrier of the present invention on which the peptide of the present invention is supported. A method using a column (herein referred to as a "peptide column") prepared by the following method is mentioned. By passing a solution containing a sample through the peptide column, it is possible to bring the sample into contact with the peptide of the present invention supported on the carrier of the present invention, thereby obtaining the complex of the present invention. In this case, a peptide column may be prepared by using a commercially available epsilon polypeptide carrier, etc. in which the peptide of the present invention is preliminarily supported on the carrier of the present invention, and filling the column with the epsilon polypeptide carrier, etc. .

The contact time between the peptide column and the sample is preferably sufficient for the exosomes in the sample and the peptide of the present invention supported on the carrier of the present invention to form the complex of the present invention. The above-mentioned time can be set as appropriate by adjusting the flow rate of the solution containing the sample, the time from the addition of the solution to the start of passage, etc.

A conventionally known physical method can be used for passing the sample solution through the peptide column. Examples include methods (1) to (3) below.
(1) A method in which the sample solution is added onto a peptide column placed vertically, and the sample solution is passed through the peptide column by gravity.
(2) Add the sample solution to one side of the peptide column and apply pressure from the direction in which the sample solution was added, or by suctioning from the direction opposite to the direction in which the sample solution was added. A method of passing sample solution through.
(3) A method of adding a sample solution to one side of a peptide column, loading the peptide column into a centrifuge tube, and centrifuging the centrifuge tube, thereby passing the sample solution through the peptide column. Note that the method (3) above is a method using a so-called conventionally known spin column. That is, the column filled with the carrier of the present invention may be a spin column.

In addition, the complex formation step involves contacting the peptide of the present invention, the carrier of the present invention, and the sample without using a column, and then forming a complex in which the exosomes, the peptide of the present invention, and the carrier of the present invention are bound in this order. It may be formed. In this case, magnetic beads may be used as the carrier of the present invention.

(Washing process)
Preferably, the isolation method of the present invention includes a washing step.

The washing step is a step in which the complex of the present invention, such as a column, is washed an appropriate number of times using an appropriate washing solution. As long as the washing step is carried out under conditions that do not dissociate the complex, the type of washing liquid used, the number of washings, etc. are not particularly limited.

Examples of the washing liquid used in the washing step include physiological saline and phosphate buffered saline (PBS).

In the complex forming step of the present invention, when a peptide column or a carrier column is used, an appropriate amount of the washing solution may be passed through the column after the complex of the present invention is formed.

(Dissociation step)
The dissociation step included in the isolation method of the present invention (hereinafter referred to as the "dissociation step of the present invention") is a method for combining the complex of the present invention obtained by the complex formation step and the present invention containing a metal cation. This is a step of dissociating exosomes from the complex of the present invention by contacting the complex with a dissociation buffer of the present invention.

The method for bringing the complex of the present invention into contact with the dissociation buffer of the present invention is not limited to any other method as long as it is carried out under conditions that dissociate exosomes from the complex of the present invention. For example, it is possible to bring the complex of the present invention into contact with the dissociation buffer of the present invention by passing the dissociation buffer of the present invention through a column on which the complex of the present invention has been formed in the complex formation step. be. Physical methods for passing the dissociation buffer of the present invention through the column include methods (1) to (3) described in the section (complex formation step). When the column and the dissociation buffer of the present invention are brought into contact, the exosomes are dissociated from the complex and migrate to the dissociation buffer. Therefore, exosomes can be isolated by isolating the dissociation buffer after passing through the column.

The contact time between the carrier and the dissociation buffer in the column is preferably a sufficient time for exosomes to dissociate from the complex of the present invention. The above-mentioned time can be set as appropriate by adjusting the flow rate of the dissociation buffer, the time from the addition of the dissociation buffer to the start of passage, and the like.

Alternatively, when a column is not used, the dissociation buffer of the present invention may be added to a container containing the complex of the present invention and mixed. By contacting the complex of the present invention with the dissociation buffer of the present invention, exosomes are dissociated from the complex of the present invention and transferred to the dissociation buffer. Then, by separating the dissociation buffer and the carrier of the present invention and isolating the dissociation buffer, exosomes can be isolated.

In the dissociation step of the present invention, the peptide of the present invention may remain bound to the carrier of the present invention or may be dissociated from the carrier of the present invention. Further, after the above separation, the peptide of the present invention may be contained in the isolated dissociation buffer. From the viewpoint of isolating exosomes with higher purity, it is preferable that the peptide of the present invention remains bound to the carrier of the present invention in the dissociation step of the present invention, and after the above separation, the peptide of the present invention is added to the isolated dissociation buffer. Preferably, the peptide of the invention is not included.

Note that exosomes isolated by the isolation method of the present invention are also included within the technical scope of the present invention.

[2. How to remove exosomes]
A method for removing exosomes according to an embodiment of the present invention (hereinafter referred to as "removal method of the present invention" as appropriate) is a method for removing exosomes from a sample containing exosomes, comprising:
By bringing the sample into contact with a carrier carrying a peptide containing 20 to 40 consecutive lysines through epsilon bonding, the exosome and the peptide carried on the carrier are bonded. a complex forming step of forming a complex, and
The method includes a separation step of separating the sample from the carrier supporting the complex formed in the complex formation step to obtain a separated liquid from which at least a portion of the exosomes have been removed from the sample. .

Since the method for removing exosomes according to one embodiment of the present invention has the above configuration, it achieves the following effects (1) and (2).
(1) Since there is no need to use an ultracentrifuge or the like, exosomes can be removed easily and in a short time.
(2) Since it utilizes the fact that consecutive lysines bind to the membranes of various exosomes, including CD63-positive exosomes, a wide range of exosomes can be captured and removed from the sample.

Due to the effects of (1) and (2) above, the removal method of the present invention makes it possible to obtain a separated liquid from which various types of exosomes, including CD63-positive exosomes, are efficiently removed from a sample. Therefore, a separated liquid with extremely few remaining exosomes can be easily obtained by a simple method.

Such an exosome removal method is useful, for example, for producing serum from which exosomes have been removed. When culturing cultured cells, fetal bovine serum (FBS) is generally used. Usually, FBS contains bovine-derived exosomes, so if you want to collect exosomes secreted from human cells, it is necessary to use FBS from which bovine-derived exosomes have been removed in advance. In order to produce such FBS, a method is generally used in which exosomes are removed by ultracentrifugation at 100,000 xg for 16 hours or more at 4°C. However, this ultracentrifugation method requires a long time to remove exosomes, and it is difficult to completely remove exosomes from FBS (for example, if ultracentrifugation time is 16 hours, approximately 20% of exosomes are There are problems such as (remaining). On the other hand, according to the removal method of the present invention, it is only necessary to pass FBS containing bovine-derived exosomes through a column, so FBS from which bovine-derived exosomes have been removed can be easily produced.

[2-1. material〕
The removal method according to one embodiment of the present invention can be carried out using the above-described peptide of the present invention and carrier of the present invention. Furthermore, the "sample containing exosomes" in the removal method of the present invention is the same as the sample used in the isolation method of the present invention. Therefore, regarding materials [1-1. Materials] can be used.

[2-2. Process]
The removal method of the present invention includes a complex formation step and a separation step. The complex forming step is the same as the complex forming step included in the isolation method of the present invention described above, so it is similar to [1-2. The explanation of (complex formation step) in step] can be referred to.

(separation process)
The separation step included in the removal method of the present invention (hereinafter appropriately referred to as "separation step of the present invention") is as follows:
The carrier of the present invention carrying the complex of the present invention formed in the complex formation step is separated from the sample after contact with the peptide of the present invention and the carrier of the present invention, and at least one of the exosomes is extracted from the sample. This is a separation step in which a separated liquid is obtained from which part has been removed.

As a method for separating the complex of the present invention and a sample, when a peptide column or a carrier column is used in the complex formation step of the present invention, the separated liquid can be collected as a flow-through, and the complex of the present invention can be separated from the sample. Stay within the column. Therefore, it is easy to separate the complex of the present invention and the sample.

Furthermore, when a column is not used, the complex of the present invention can be separated from the sample by, for example, centrifugation. For example, the complex of the present invention can be separated from a sample by centrifugation under conditions of 500 g to 4000 g.

Furthermore, when the carrier contained in the complex of the present invention is a magnetic bead, the complex can be easily separated from the sample by applying magnetic force from the outside using a magnetic material such as a conventionally known magnetic stand. I can do it.

The removal method of the present invention may include steps other than the above-described complex forming step and separation step. Other steps include, for example, a dissociation step.

(Dissociation step)
The method of the dissociation step is the same as the dissociation step included in the isolation method of the present invention described above, and therefore [1-2. The explanation of (dissociation step) in [Process] can be referred to. The dissociation step allows the carrier of the present invention to be regenerated to the state before contact with the sample containing exosomes. Further, if the peptide of the present invention remains bound to the carrier of the present invention after the dissociation step, the peptide of the present invention can also be reused in a state bound to the carrier of the present invention.

In the dissociation step, the exosomes can be dissociated from the complex of the present invention under mild conditions, so that the carrier of the present invention and the peptide of the present invention are less susceptible to damage such as denaturation. Therefore, the carrier of the present invention and the peptide of the present invention can be used in the removal method of the present invention multiple times by undergoing a dissociation step.

Note that the separated liquid obtained by the removal method of the present invention is also included within the technical scope of the present invention.

[3. Exosome isolation kit]
The exosome isolation kit according to one embodiment of the present invention (hereinafter referred to as the "kit of the present invention" as appropriate) is a kit for carrying out the above-described isolation method of the present invention or removal method of the present invention, and includes: It includes the carrier of the present invention on which the peptide of the present invention described above is supported, and the dissociation buffer of the present invention. Therefore, regarding the explanation of the configuration of the kit, please refer to [1. Exosome isolation method] can be referred to.

Furthermore, the kit of the present invention may include a column that holds the carrier of the present invention carrying the peptide of the present invention. Further, the column may be filled in advance with the carrier of the present invention carrying the peptide of the present invention. The sample solution, etc. may be passed through the column by gravity, or the sample solution, etc. may be passed through the column by suction from one side of the column. Further, the column may be a conventionally known spin column.

In addition to the above configuration, the kit of the present invention may also include a container (for example, a bottle, plate, tube, dish, etc.) containing a specific material. Kits of the invention may include containers containing diluents, solvents, wash solutions, or other reagents. The term "comprising" used in the description of the kit of the present invention may mean that the kit is contained in any of the individual containers constituting the kit.

The kit of the present invention may also include instructions for carrying out the isolation method of the present invention or the removal method of the present invention.

[4. Exosome isolation column]
The exosome isolation column according to one embodiment of the present invention (hereinafter referred to as the "column of the present invention") is a column for carrying out the above-described isolation method of the present invention or removal method of the present invention, and includes: This is a column packed with the carrier of the present invention supporting the peptide of the present invention described above. The sample solution, etc. may be passed through the column by gravity, or the sample solution, etc. may be passed through the column by suction from one side of the column. Further, the column includes a conventionally known spin column.

The column of the present invention comprises [1. Exosome isolation method] and [2. Exosome removal method]. Therefore, for a description of the column of the present invention, see [1. Exosome isolation method] and [2. [Exosome Removal Method]] can be referred to.

EXAMPLES Hereinafter, one embodiment of the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

<1. Isolation and removal of exosomes using columns>
〔material and method〕
(Sample preparation)
Using the breast cancer cell line MCF-7 (JCRB Cell Bank), which is known to be a prolific exosome producer, samples were prepared by the following method. The medium used was DMEM medium (Dulbecco's modified Eagle medium, manufactured by Thermo Fisher Scientific).
(1) MCF-7 cells were seeded in 10 mL of DMEM medium (1.0×10 ⁵ cells/mL) in a dish, and cultured at 37° C. and 5% CO ₂ . The DMEM medium was preliminarily supplemented with 10% (v/v) inactivated fetal bovine serum (HyClone FBS; manufactured by GE Healthcare), 100 units/mL penicillin (manufactured by Nacalai Tesque), and 0.1 mg/mL streptomycin (Meiji Seika Pharma). ) was added.
(2) The cells were cultured until they accounted for 50% to 80% of the culture area of the dish.
(3) After slowly adding 3 mL of PBS (manufactured by Nacalai Tesque) and distributing it throughout the dish, the PBS was removed.
(4) The washing step in (3) was performed twice to remove FBS-derived exosomes from the dish.
(5) 10 mL of Advanced DMEM medium (manufactured by Thermo Fisher Scientific) was added and cultured. After culturing for 24 hours, the culture supernatant containing exosomes was collected.
(6) The culture supernatant was centrifuged at 2000×g for 10 minutes to remove cells detached from the dish in the culture supernatant.
(7) The culture supernatant was centrifuged at 10,000×g for 30 minutes to remove debris in the culture supernatant.
(8) The culture supernatant was filtered with a 0.22 μm filter to prepare a sample (hereinafter referred to as “pre-purification sample”). Samples before purification were stored at -80°C if necessary.

(column purification)
As Example 1 of the present invention, a column (ε-polylysine column) packed with a carrier bound to ε-polylysine (a peptide containing multiple consecutive lysines through epsilon bonds) was used to collect exosomes from a sample before purification. Isolation and removal were performed. As shown in Figure 2, the ε-polylysine column is made of 20 to 40 lysine residues prepared by the method described in the reference (Kobunshi Ronshu, 64, 12, 821-829 (2007)). - Porous spherical cellulose particles with immobilized polylysine were used. This ε-polylysine column was connected to a liquid chromatography system (AKTA purifier, manufactured by GE Healthcare) for purification. In the purification, BW Buffer (attached to ExoIntact Exosome Purification Reagent Kit, manufactured by HMT Biomedical Co., Ltd.) was used as Buffer X, and 1M NaCl solution was used as Buffer Y.

The column purification method will be described below with reference to FIG.
(1) The ε-polylysine column was equilibrated by feeding the solution at a flow rate of 0.2 mL/min and using 100% Buffer X.
(2) Add 1/10 volume (e.g. 0.2 mL for 2 mL sample) of Binding Buffer (supplied with ExoIntact Exosome purification reagent kit, manufactured by HMT Biomedical Co., Ltd.) to the above-mentioned pre-purification sample. , the entire amount was injected. In addition, all the fractions from after the injection to elution were collected in 1 mL portions.
(3) The pre-purification sample to which Binding Buffer was added was passed through an ε-polylysine column and collected as a flow-through fraction, and then the solution was sent as it was to wash the ε-polylysine column.
(4) Exosomes bound to the ε-polylysine column were eluted using a NaCl concentration gradient (Buffer Y, 0-100%, 50 min). As shown in FIG. 3, fractions A1 to A7 are flow-through fractions, fractions A8 to A11 are wash fractions, and fractions A12 and after are eluted fractions.

(Quantification and purity evaluation of exosomes)
The concentration of exosomes contained in each fraction obtained by the above column purification was measured using a CD9/CD63 ELISA kit (manufactured by Cosmo Bio). Measurement samples were prepared by diluting the pre-purification sample or each obtained fraction 20 times with assay buffer. Furthermore, a CD9/CD63 fusion protein was used as a standard protein. The measurement method was performed according to the protocol of the kit. The specific measurement method is shown below.
(1) Add 100 μL of diluted measurement sample or 100 μL of serially diluted sample of CD9/CD63 fusion protein (12.5 to 100 pg/mL) to an anti-CD9 antibody immobilized 96-well plate, and leave it for 2 hours. The reaction was allowed to take place.
(2) After washing three times with 300 μL of washing buffer (1×), 100 μL of HRP-labeled anti-CD63 antibody solution was added and allowed to react for 2 hours.
(3) After washing three times with 300 μL of washing buffer (1×), 100 μL of substrate solution was added to allow color reaction to occur for 10 minutes, and 50 μL of stop solution was added to stop the color reaction.
(4) Absorbance of A450 was measured using a plate reader (ARVO MX 1420, manufactured by PerkinElmer).

A calibration curve was created from the measurement results of serially diluted samples of CD9/CD63 fusion protein, which is a standard protein, and the concentration of exosomes contained in the measurement sample was converted to the concentration of CD9/CD63 fusion protein.

In addition, in order to evaluate purity, the protein concentration contained in the measurement sample was measured using a Micro BCA Protein assay kit (manufactured by Thermo Fisher Scientific). The total amount of exosomes (pg)/total amount of protein (μg) in each measurement sample was determined, and the purity of the exosomes was evaluated.

(Exosome isolation and purity evaluation using ultracentrifugation)
As Comparative Example 1, exosomes were isolated by ultracentrifugation. The specific method of ultracentrifugation is as follows.
(1) Using the above-mentioned pre-purification sample, ultracentrifugation was performed at 100,000 xg, 70 min, and 4°C.
(2) The supernatant was removed, suspended in 0.5 mL of PBS (manufactured by Nacalai Tesque), and ultracentrifuged again under the same conditions.
(3) The supernatant was removed and suspended in 0.05 mL of PBS (manufactured by Nacalai Tesque) to obtain an ultracentrifuged sample.

Similar to the method described above (Exosome Quantification and Purity Assessment), the exosome concentration of the ultracentrifuged samples was measured using a CD9/CD63 ELISA kit. In addition, protein concentration was measured using Micro BCA Protein assay kit (manufactured by Thermo Fisher Scientific). Then, the purity of exosomes in the ultracentrifuged sample was evaluated as total exosome amount (pg)/total protein amount (μg).

[Result: Isolation of exosomes]
As shown in the table in FIG. 3, in Example 1, no exosomes were detected in the flow-through fraction (A3). On the other hand, exosomes were detected in the elution fraction using the 1M NaCl concentration gradient. In particular, many exosomes were detected in the 0.2 to 0.4 M NaCl elution fractions (A15 and B15). Furthermore, the protein concentration was measured in the fraction in which exosomes were detected, and the purity (exosome amount (pg)/protein amount (μg)) was evaluated. As a result, it was found that the purity of the eluted fraction (A15) was approximately 31 times higher and the purity of the eluted fraction (B15) was approximately 56 times higher compared to the sample before purification (Figure 3). . Therefore, according to the isolation method of the present invention, it was shown that exosomes can be isolated with high purity.

Next, a comparison was made between Example 1 using the isolation method of the present invention and Comparative Example 1 using an exosome isolation method using an ultracentrifuge, which is known as the most standard method. Specifically, the purity of exosomes was compared between the elution fraction containing exosomes isolated according to Example 1 and the ultracentrifugation sample containing exosomes isolated according to Comparative Example 1. The results are shown in Table 1 below. The elution fraction of Example 1 had up to about 26 times higher exosome purity than the ultracentrifuged sample of Comparative Example 1. Therefore, it was shown that according to the isolation method of the present invention, exosomes can be isolated with extremely high purity (14.6 to 26 times higher purity) than the conventional isolation method using an ultracentrifuge.

[Result: Exosome removal]
Furthermore, as shown in the table in FIG. 3, in Example 1, no exosomes were detected in the flow-through fraction (A3). This indicates that according to the removal method of the present invention, exosomes can be removed from the sample with high efficiency to below the detection limit.

<2. Isolation of exosomes using ε-polylysine and α-polylysine>
〔material and method〕
(Exosome isolation and marker analysis using ε-polylysine)
As Example 2 of the present invention, exosomes were isolated using ε-polylysine. FIG. 4 shows an overview of the isolation method of Example 2 of the present invention. The ε-polylysine immobilized carrier has <1. The porous spherical cellulose particles on which ε-polylysine was immobilized were used. In Example 2, centrifugation was performed at 300×g for 2 minutes.

The specific method is shown below.
(1) 0.02 mL of Binding Buffer (attached to ExoIntact Exosome purification reagent kit, manufactured by HMT Biomedical Co., Ltd.) was added to 0.2 mL of the above-mentioned pre-purification sample.
(2) ε-polylysine immobilized carrier (0.2 mL) was added to a 1.5 mL tube and centrifuged to precipitate the ε-polylysine immobilized carrier.
(3) The supernatant was removed, 0.2 mL of BW Buffer (attached to ExoIntact Exosome purification reagent kit, manufactured by HMT Biomedical Co., Ltd.) was added and mixed with the carrier, and the carrier was washed.
(4) After carrying out the washing operation in (3) twice, the ε-polylysine-immobilized carrier was suspended in 0.2 mL of BW Buffer to obtain a suspension of the ε-polylysine-immobilized carrier.
(5) 100 μL of the suspension of the obtained ε-polylysine immobilized carrier and 220 μL of the pre-purification sample to which Binding Buffer was added were mixed and reacted for 30 minutes.
(6) This reaction solution was centrifuged and the supernatant was removed. Washing was performed by adding and mixing 0.2 mL of BW Buffer.
(7) After performing the washing operation in (6) three times, the ε-polylysine immobilized carrier was suspended in 0.2 mL of BW Buffer.
(8) The obtained suspension was divided into one for detection of CD9-positive exosomes and one for detection of CD63-positive exosomes, and 100 μL each was dispensed into 1.5 mL tubes. As a negative control, two 1.5 mL tubes were prepared in which only the ε-polylysine immobilized carrier washed with 50 μL of BW Buffer was dispensed.
(9) The 1.5 mL tube was centrifuged to remove the supernatant, 100 μL of blocking solution was added, and the mixture was reacted for 20 min. The blocking solution was a solution in which 2% BSA (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and 0.025% Tween 20 (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) were added to TBS (pH 7.4, manufactured by Nacalai Tesque). be.
(10) Furthermore, 100 μL of the primary antibody solution was added, and the mixture was reacted for 30 min. As the primary antibody solution, anti-CD9 antibody (Anti-CD9 Human (Mouse) Unlabeled 12A12, manufactured by Cosmo Bio) or anti-CD63 antibody (Anti-CD63, Monoclonal Antibody (3-13), Fuji Film Wako Pure Chemical Industries, Ltd.) (manufactured by Co., Ltd.) diluted 1000 times with a blocking solution, respectively.
(11) After adding the primary antibody solution and reacting for 30 minutes, remove the supernatant by centrifugation and add 0.1 mL of TBS-T (a solution of TBS (pH 7.4) with 0.025% Tween 20 added). Washed twice with
(12) Each 1.5 mL tube was centrifuged to remove the supernatant, 100 μL of secondary antibody solution was added, and the mixture was mixed and reacted for 30 min. As the secondary antibody solution, Anti-Mouse-HRP (Goat Anti-Mouse IgG H&L (HRP), ab97023, manufactured by Abcam) diluted 50,000 times with a blocking solution was used.
(13) After adding the secondary antibody solution and reacting for 30 minutes, the 1.5 mL tube was centrifuged to remove the supernatant and washed three times with 0.1 mL of TBS-T.
(14) The 1.5 mL tube was centrifuged to remove the supernatant and suspended in 0.05 mL of TBS (pH 7.4).
(15) 90 μL of luminescent substrate (ELISA-Star peroxidase chemiluminescent substrate, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added to 10 μL of the obtained suspension, and after reaction for 5 min, the luminescence value was measured using a plate reader.

The amount of CD9-positive exosomes or CD63-positive exosomes bound to the ε-polylysine immobilized carrier was quantified by subtracting the luminescence value of the negative control from the obtained luminescence value.

(Exosome isolation and marker analysis using α-polylysine)
As Comparative Example 2, exosomes were isolated using α-polylysine (a peptide containing multiple consecutive lysines through alpha bonds). FIG. 5 shows an outline of the isolation method of Comparative Example 2. In Comparative Example 2, α-polylysine containing eight lysine residues and magnetic beads were used. Exosomes were collected using ExoIntact Exosome purification reagent kit (manufactured by HMT Biomedical Co., Ltd.). In addition, preparation of magnetic beads on which α-polylysine is immobilized (hereinafter referred to as "α-polylysine-immobilized magnetic beads"), mixing of α-polylysine-bound magnetic beads and a pre-purification sample to which Binding Buffer has been added, And washing was performed according to the protocol of the same kit. A magnetic stand (manufactured by Thermo Fisher Scientific) was used to separate the magnetic beads and the solution.

The specific method is shown below.
(1) 0.02 mL of Binding Buffer was added to the pre-purification sample.
(2) Exosomes were bound to the α-polylysine-immobilized magnetic beads by adding 8 μL of α-polylysine-immobilized magnetic beads to 220 μL of the pre-purification sample to which Binding Buffer had been added and mixing for 30 minutes. For α-polylysine-immobilized magnetic beads, mix and wash 20 μL of Biotin-labeled EX peptide (biotin-labeled α-polylysine) and 20 μL of Streptavidin Magnetic Beads (1 μm, 10 mg/mL), and then add 20 μL of BW Buffer. It was prepared by suspending it in
(3) After the reaction, the cells were washed three times with 0.2 mL of BW Buffer and resuspended in 200 μL of BW Buffer.
(4) The obtained suspension was divided into one for detection of CD9-positive exosomes and one for detection of CD63-positive exosomes, and 100 μL each was dispensed into 1.5 mL tubes. Furthermore, as a negative control, two 1.5 mL tubes were prepared in which 4 μL of α-polylysine-immobilized magnetic beads alone were dispensed.
(5) After leaving the 1.5 mL tube on a magnetic stand for 1 minute, the supernatant was removed, 100 μL of blocking solution was added, and the mixture was mixed and reacted for 20 minutes. The same blocking solution as in Example 2 was used.
(6) Furthermore, 100 μL of the primary antibody solution was added, and the mixture was reacted for 30 min. Note that the same primary antibody solution and secondary antibody solution as in Example 2 were used.
(7) After reacting with the primary antibody solution for 30 minutes, the 1.5 mL tube was left standing on a magnetic stand for 1 minute, the supernatant was removed, and the tube was washed twice with 0.1 mL of TBS-T.
(8) The 1.5 mL tube was placed on a magnetic stand for 1 minute, the supernatant was removed, 100 μL of the secondary antibody solution was added, and the mixture was mixed and reacted for 30 minutes.
(9) After reacting with the secondary antibody solution for 30 minutes, the 1.5 mL tube was left standing on a magnetic stand for 1 minute, the supernatant was removed, and the tube was washed three times with 0.1 mL of TBS-T.
(10) The 1.5 mL tube was placed on a magnetic stand for 1 minute, the supernatant was removed, and the tube was suspended in 0.04 mL of TBS (pH 7.4).
(11) 90 μL of the same luminescent substrate as in Example 2 was added to 10 μL of the obtained suspension, and after reaction for 5 minutes, the luminescence value was measured using a plate reader.

The amount of CD9-positive exosomes or CD63-positive exosomes bound to the α-polylysine-immobilized magnetic beads was quantified by subtracting the luminescence value of the negative control from the obtained luminescence value.

〔result〕
As shown in FIG. 6, in Example 2 of the present invention using an ε-polylysine immobilized carrier, both CD9-positive exosomes and CD63-positive exosomes were detected compared to Comparative Example 2 using α-polylysine-immobilized magnetic beads. It was found that it was possible to combine them well.

Furthermore, as shown in Figure 7, as a result of comparing the value (CD63/CD9) obtained by dividing the amount of CD63-positive exosomes bound to the carrier by the amount of CD9-positive exosomes bound to the carrier, it was found that for the ε-polylysine immobilized carrier, The CD63/CD9 value increased approximately 6.7 times compared to α-polylysine-immobilized magnetic beads. In other words, ε-polylysine-immobilized carriers bind well to both CD9-positive exosomes and CD63-positive exosomes, whereas α-polylysine-immobilized magnetic beads bind to CD63-positive exosomes better than binding to CD9-positive exosomes. It was suggested that the bond was very weak.

From the above, it has been shown that when ε-polylysine is used to isolate exosomes, various types of exosomes including CD63-positive exosomes can be isolated more preferably than when α-polylysine is used.

<3. Isolation of exosomes using spin column>
〔material and method〕
As Example 3 of the present invention, exosomes were isolated using a spin column. A microbio spin chromatography column (manufactured by Bio-Rad) was used as the spin column. In Example 3, centrifugation was performed at 300×g for 1 min.
(1) A spin column was set in a 2 mL tube, 0.5 mL of ε-polylysine carrier was added, and the storage solution was removed by centrifugation. The flow-through was discarded, and the ε-polylysine carrier was equilibrated by adding 0.6 mL of BW Buffer (attached to ExoIntact Exosome purification reagent kit, manufactured by HMT Biomedical) and centrifuging.
(2) 0.1 mL of Binding Buffer (attached to ExoIntact Exosome purification reagent kit, manufactured by HMT Biomedical) was added to 1.0 mL of the above-mentioned pre-purification sample.
(3) Exosomes were bound to the ε-polylysine carrier by adding 0.55 mL of the sample obtained in (2) to the ε-polylysine carrier and mixing for 10 seconds.
(4) The spin column was centrifuged and the flow-through was removed. For all the remaining samples obtained in (2), the operation (3) was performed for each 0.55 mL to bind the exosomes to the ε-polylysine carrier.
(5) The ε-polylysine carrier was washed by adding 0.6 mL of BW Buffer to the ε-polylysine carrier, centrifuging it, and removing the flow-through. This washing operation was performed three times in total.
(6) Set the spin column after washing into a new 2.0 mL tube, and add 0.2 mL of Elution Buffer (included with ExoIntact Exosome purification reagent kit, manufactured by HMT Biomedical) to the ε-polylysine carrier for 10 seconds. By mixing, exosomes were eluted into Elution Buffer.
(7) The spin column was centrifuged and the flow-through (exosome elution fraction) was collected. This elution step was performed again, and the collected exosome elution fractions were combined.

〔result〕
The purity of exosomes was compared between the exosome elution fraction purified by a spin column and the ultracentrifugation sample containing exosomes isolated according to Comparative Example 1 described above. The results are shown in Table 2 below.

The exosome elution fraction isolated by the spin column had approximately 3.7 times higher exosome purity than the ultracentrifugation sample of Comparative Example 1. Therefore, it was shown that the isolation method of the present invention using a spin column can isolate exosomes with higher purity than the conventional isolation method using an ultracentrifuge.

According to the present invention, various types of exosomes including CD63-positive exosomes can be easily isolated or removed from a sample in an intact state (nearly intact state). Therefore, according to the present invention, it becomes possible to easily and precisely analyze substances contained inside various types of exosomes. Therefore, the present invention is useful in various technical fields, particularly in the pharmaceutical and medical fields.

Claims (7)

A method for isolating exosomes, the method comprising: isolating exosomes from a sample containing exosomes,
By bringing the sample into contact with a carrier carrying a peptide containing 20 to 40 consecutive lysines through epsilon bonding, the exosome and the peptide carried on the carrier are bonded. a complex forming step of forming a complex, and
Isolation of exosomes, comprising a dissociation step of dissociating the exosomes from the complex by contacting the complex obtained by the complex formation step with a dissociation buffer containing a metal cation. Method. The method for isolating exosomes according to claim 1, wherein the content of CD63-positive exosomes in the exosomes contained in the complex is 10% or more of the content of CD9-positive exosomes. The method for isolating exosomes according to claim 1 or 2, characterized in that the carrier to which the peptide is bound is used by filling a column. An exosome isolation kit for carrying out the exosome isolation method according to any one of claims 1 to 3, comprising:
An exosome isolation kit comprising the carrier carrying the peptide and the dissociation buffer. A method for removing exosomes, the method comprising: removing exosomes from a sample containing exosomes,
By bringing the sample into contact with a carrier carrying a peptide containing 20 to 40 consecutive lysines through epsilon bonding, the exosome and the peptide carried on the carrier are bonded. a complex forming step of forming a complex, and
a separation step of separating the carrier carrying the complex formed in the complex formation step from the sample to obtain a separated liquid from which at least a portion of the exosomes have been removed from the sample; Characteristic exosome removal method. A method for obtaining a separated liquid, the method comprising: obtaining a separated liquid from which at least a portion of the exosomes have been removed from a sample containing exosomes,
By bringing the sample into contact with a carrier carrying a peptide containing 20 to 40 consecutive lysines through epsilon bonding, the exosome and the peptide carried on the carrier are bonded. a complex forming step of forming a complex, and
a separation step of separating the carrier carrying the complex formed in the complex formation step and the sample to obtain the separated liquid from which at least a portion of the exosomes have been removed from the sample; A method for obtaining a separated liquid, characterized by: An exosome isolation column for carrying out the method for isolating exosomes according to claim 3 , the method for removing exosomes according to claim 5 , or the method for obtaining a separated liquid according to claim 6 ,
An exosome isolation column, characterized in that it is filled with the carrier carrying the peptide.

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