JP2021067576A - Exosome isolation method, exosome isolation kit, and exosome removal method - Google Patents

Abstract

To allow for isolating various types of exosomes easily from an exosome-containing sample.SOLUTION: A method of isolating exosomes is provided, comprising: a complex forming step for contacting a sample with a peptide carried by a carrier to form a complex; and a dissociation step for contacting the complex with a dissociation buffer containing metal cations to dissociate exosomes from the complex.SELECTED DRAWING: Figure 1

Description

The present invention relates to methods for isolating exosomes, kits for isolating exosomes, and methods for removing exosomes.

Exosomes are extracellular vesicles secreted from cells having a diameter of about 50 to 150 nm and covered with a lipid bilayer membrane. Since information specific to the cell of the secretory source such as protein, mRNA, and microRNA (hereinafter referred to as "miRNA") is highly stored inside the exosome, various diseases such as cancer and biological phenomena occur. It is attracting attention as a new biomarker.

In recent years, it has become clear that mesenchymal stem cell-derived exosomes have anti-inflammatory and complex immunoregulatory effects, and studies using exosomes themselves as therapeutic agents are being actively conducted. Therefore, the importance of a technique for isolating exosomes in a state of less damage (intact state) is increasing. The global market for exosome treatment and diagnosis is projected to reach US $ 180 million in 2023, making exosome research one of the most promising areas of research. ..

As a method for isolating exosomes, an ultracentrifugal separation method using an ultracentrifuge is generally used. However, it is difficult for this ultracentrifugation method to cope with large scale (mass purification) and large sample size. Furthermore, problems such as damage to exosomes due to strong centrifugal force have been pointed out. For future exosome research and widespread use of exosome-based therapies, it is necessary to develop an isolation method that is more reproducible and can handle a large number of samples.

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

International Publication WO2019 / 039179

However, it is known that exosomes are not uniform and vary widely depending on the type and state of differentiated cells. For example, although both CD9 and CD63 are known as markers for exosomes, it is considered that the correlation between the expression level of CD9 and the expression level of CD63 in exosomes is not very high. That is, there are exosomes having a relatively high expression level of CD9 (CD9-positive exosomes) and exosomes having a relatively high expression level of CD63 (CD63-positive exosomes). It is known that a large amount of CD63-positive exosomes are secreted 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 that CD63-positive exosomes can be efficiently isolated. Further, in the future, for example, when exosomes themselves are used as therapeutic agents, a method capable of isolating various types of exosomes is required. However, in the method for isolating exosomes described in Patent Document 1, the types of exosomes that can be isolated are unknown.

As a result of diligent studies by the present inventors, in the method for isolating exosomes described in Patent Document 1 (method using peptides containing four or more lysines in close proximity to each other), CD9-positive exosomes are prioritized over CD63-positive exosomes. It was found that the lysine can be isolated (see Comparative Example 2 described later). An object of the present invention is to provide a method for isolating various types of exosomes, particularly CD63-positive exosomes, and an exosome isolation kit in a convenient and intact state (or in a state close to intact). Another object of the present invention is to provide a method for removing exosomes, which can easily remove various types of exosomes from a sample containing exosomes.

As a result of diligent studies to solve the above problems, the present inventors can bind a peptide containing arginine, which has a high affinity for phospholipids, to various exosomes, and in particular, preferentially binds to CD63-positive exosomes. I found out what I could do.

That is, one embodiment of the present invention has the following configuration.
[1] An exosome isolation method for isolating an exosome from a sample containing the exosome, wherein the sample, a peptide containing four or more arginines in close proximity to each other, and a carrier capable of supporting the peptide are contacted. A complex forming step of forming a complex formed by binding the exosome and the peptide supported on the carrier by contacting the sample with the peptide supported on the carrier. The exosome, which comprises contacting the complex obtained by the complex forming step with a dissociation buffer containing a metal cation to dissociate the exosome from the complex. Isolation method.
[2] The method for isolating an exosome according to [1], wherein the arginine contained in the peptide is continuous.
[3] The method for isolating an exosome according to [1] or [2], wherein the amount of arginine contained in the peptide is 8 or more.
[4] The single exosome according to any one of [1] to [3], wherein the content of the CD63-positive exosome is 10% or more of the content of the CD9-positive exosome. How to release.
[5] The method for isolating an exosome according to any one of [1] to [4], wherein the sample is derived from a living body and contains impurities other than the exosome.
[6] The method for isolating an exosome according to any one of [1] to [5], wherein the carrier is magnetic beads.
[7] An exosome isolation kit for performing the method for isolating an exosome according to any one of [1] to [6], which comprises the peptide, the carrier, and the dissociation buffer. An exosome isolation kit.
[8] A method for removing exosomes from a sample containing exosomes, wherein the sample, a peptide containing four or more arginines in close proximity to each other, and a carrier capable of supporting the peptide are brought into contact with each other. Alternatively, a complex forming step of contacting the sample with the peptide supported on the carrier to form a complex formed by binding the exosome and the peptide supported on the carrier, and Includes a separation step of separating the carrier carrying the complex obtained by the complex forming step and the sample to obtain a separation solution from which at least a part of the exosome has been removed from the sample. A method for removing an exosome, which comprises.

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

It is a figure which shows the outline of the experimental operation procedure of Example 1. FIG. It is a figure which shows the result of the amount of exosomes isolated by the method which concerns on Example 1. FIG. It is a figure which shows the result of the amount of exosomes isolated by the method which concerns on Example 1 and Comparative Example 2. It is a figure which shows the outline of the experimental operation procedure of Example 2. FIG. It is a figure which shows the outline of the experimental operation procedure of Example 3. FIG. It is a figure which shows the marker analysis result of the exosome isolated by the method which concerns on Example 3 and Comparative Example 2. It is a figure which shows the comparative result of the exosome isolated by the method which concerns on Example 3 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 the configurations described below, and various modifications can be made within the scope of the claims. The present invention also includes embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples, respectively, within the technical scope of the present invention. In addition, all the academic documents and patent documents described in the present specification are incorporated as references in the present specification. Further, unless otherwise specified in the present specification, "A to B" representing a numerical range is intended to be "A or more (including A and larger than A) and B or less (including B and smaller than B)".

In the present specification, the term "peptide" is used interchangeably with "polypeptide" and means a compound in which two or more amino acids are bound by a peptide bond. In the present specification, the notation of amino acids uses the one-letter notation or the three-letter notation defined by IUPAC and IUB as appropriate.

[1. Exosome isolation method]
The method for isolating exosomes according to an embodiment of the present invention (hereinafter, appropriately referred to as "method for isolating the present invention") is a method for isolating exosomes, which isolates exosomes from a sample containing exosomes.
By contacting the sample with a peptide containing four or more arginines in close proximity to each other and a carrier capable of supporting the peptide, or by contacting the sample with the peptide supported on the carrier. , A complex forming step of forming a complex formed by binding the exosome and the peptide supported on the carrier, and
It includes a dissociation step of contacting the complex obtained by the complex forming step with a dissociation buffer containing a metal cation to dissociate the exosome from the complex.

Since the method for isolating exosomes according to the embodiment of the present invention has the above-mentioned constitution, the effects (1) to (3) described later are exhibited.
(1) Since it is not necessary to use an ultracentrifuge or the like, exosomes can be isolated in a short time and easily.
(2) Since a peptide containing four or more arginines binds to the membranes of various exosomes including exosomes (CD63-positive exosomes) having a relatively high expression level of CD63, it widely captures exosomes. , Can be isolated.
(3) Instead of a dissociation buffer containing a surfactant or the like that affects the membrane structure, a dissociation buffer containing a metal cation that does not (or has little) affects the membrane structure of the exosome and the structure of the protein present in the membrane. The exosomes are dissociated from the complex under the mild conditions used. Therefore, exosomes can be isolated from the complex without damaging the exosomes (in other words, in an intact or near-intact state).

By isolating exosomes in an intact state (or a state close to intact), for example, (1) functional analysis of the physiological action of exosomes, and (2) biomarkers (proteins derived from exosomes, nucleic acids such as miRNA). It can be used for such purposes.

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

Specific examples of the above (A) to (C) will be described. Exosomes derived from mesenchymal stem / stromal cells (MSCs) are the most studied exosomes. MSC-derived exosomes are known to have cell division promoting, anti-apoptotic, and anti-inflammatory effects. This makes it possible to reduce secondary damage to the tissue surrounding the damaged tissue and 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 for regenerative medicine). In addition, MSC-derived exosomes have a complex immunomodulatory effect, and are expected to be applied to the treatment of autoimmune diseases and immunoregulation after organ transplantation ((B) Utilization for the purpose of immunoregulation. ).

In addition, the antigen peptide is presented to the exosomes secreted from the antigen-presenting cells, which are collected after reacting the antigen-presenting cells with the cancer antigen peptide, and have an action of activating CD4 positive and CD8 positive T cells. is there. It is also considered that the exosome derived from cancer cells is directly used as an antigen to use the exosome as a vaccine ((C) use as a vaccine).

In particular, it is known that a large amount of CD63-positive exosomes that can be efficiently isolated by the isolation method of the present invention are secreted from tumorigenic cells. The isolation method of the present invention can comprehensively isolate various types of exosomes, including exosomes having 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, it is possible to realize a more comprehensive and less leaky analysis of the functional analysis of (1) the physiological action of the exosomes. In particular, in the field of cancer, it can be expected to be effectively used for elucidation of cancer pathological conditions by functional analysis such as (1) physiological action of exosomes.

Further, the exosome isolated by the isolation method of the present invention can be expected to be effectively used as a cancer diagnostic tool by using it as the biomarker (2) described above. Furthermore, with regard to the use of the (3) therapeutic agent and the (4) drug delivery system as a carrier, it is expected that the range of application will be expanded by isolating a wider variety of exosomes.

Hereinafter, the material used for the method for isolating exosomes according to the embodiment of the present invention will be described first, and then each step of the method for isolating exosomes will be described.

[1-1. material〕
(sample)
In the isolation method of the present invention, the "sample containing exosomes" (also simply referred to as "sample" in the present specification) is not particularly limited as long as it is a mixture containing exosomes, and other configurations are not particularly limited, for example. Biological samples containing exosomes can be mentioned.

As used herein, the term "biological sample" means a sample taken from the body of an organism. Biological samples include, for example, blood, plasma, serum, saliva, urine, tears, sweat, breast milk, amniotic fluid, cerebrospinal fluid (cerebrospinal fluid), bone marrow fluid, pleural effusion, ascites, joint fluid, aqueous humor, It includes, but is not limited to, plasma fluid and the like. The selection of biological samples can be appropriately set by those skilled in the art, depending on the intended purpose. For example, from the viewpoint of ease of collection, blood, plasma, serum, saliva, urine and the like are preferably used.

In one embodiment of the invention, the origin of the biological sample is not particularly limited as long as it is derived from an exosome-carrying species. As the species from which the biological sample is derived, for example, mammals are preferable. 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 "peptide of the present invention") is a peptide containing four or more arginines in close proximity to each other and can bind to exosomes in a sample. .. In particular, such peptides can suitably bind to CD63-positive exosomes.

In the description of the peptides of the present invention, "close to each other" means that the arginines constituting the peptides of the present invention are adjacent to each other (in other words, continuous) or between the arginines of the present invention. It means that it contains ~ 5 (more preferably 1-3, more preferably 1) amino acids other than arginine.

The inventors of the present invention have found that the peptides of the present invention can bind to various exosomes including CD63-positive exosomes by containing four or more arginines in close proximity to each other. Further, the inventors of the present invention have found that in the method for isolating exosomes described in Patent Document 1 described above, CD9-positive exosomes are preferentially isolated over CD63-positive exosomes (comparative examples described later). 2). That is, peptides containing four or more arginines in close proximity to each other have a higher binding affinity to CD63-positive exosomes than peptides containing four or more lysines in close proximity to each other. This is a finding found by diligent 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.

The exosome that binds to the peptide of the present invention has a CD63-positive exosome content of 10% or more of the CD9-positive exosome content, more preferably 15% or more of the CD9-positive exosome content, 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. Is 35% or more, 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 the content of CD9-positive exosomes. The amount is 50% or more, 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.

The peptide of the present invention can bind to an exosome with a binding strength sufficient to isolate an exosome if it contains four or more arginines in close proximity to each other, but five or more arginines in close proximity to each other can be bound to the exosome. It is preferably contained, more preferably 6 or more arginines in close proximity to each other, further preferably 7 or more arginines in close proximity to each other, and particularly preferably 8 or more arginines in close proximity to each other. When the peptide of the present invention has the above-mentioned constitution, the peptide has an advantage that it is more easily bound to an exosome.

The peptide of the present invention is not particularly limited in other configurations as long as it contains four or more arginines in close proximity to each other, and may be a peptide consisting only of arginine or a peptide containing amino acids other than arginine. Good. Furthermore, the peptide of the present invention may be a complex peptide further containing a structure other than the peptide such as a sugar chain or an isoprenoid group. The amino acids contained in the peptides of the present invention may be modified. Further, the amino acid contained in the peptide of the present invention may be L-type or D-type.

The peptide of the present invention can be easily prepared according to any method known in the art, and may be expressed, for example, by a transformant into which an expression vector of the peptide has been introduced, or may be chemically synthesized. That is, the nucleotides encoding the peptides of the invention are also within the scope of the invention. Examples of the chemical synthesis method include a solid phase method and a liquid phase method. In the solid phase method, for example, various commercially available peptide synthesizers (Model MultiPep RS (Intavis AG), etc.) can be used. In addition, commercially available polyarginine can be used as the peptide of the present invention. Commercially available polyarginine is not particularly limited, and examples thereof include poly-L-arginine (molecular weight 5000-15000, manufactured by Sigma-Aldrich).

Since the peptide of the present invention can consist of at least four arginines, it can be said that the lower limit of the molecular weight of the peptide of the present invention is 642.78 [= (174.21 × 4) − (18.02 × 3)]. .. The upper limit of the molecular weight of the peptide of the present invention is not particularly limited, but it can be said that the upper limit is about 300,000 from the viewpoint of operability such as solubility and viscosity.

The peptides of the present invention may consist of peptides having a single molecular weight or 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 "carrier of the present invention") means a carrier capable of carrying the peptide of the present invention. The carrier of the present invention can bind to the peptide of the present invention bound to an exosome to form a complex bound in the order of exosome-the peptide of the present invention-the carrier of the present invention.

In the isolation method of the present invention, the complex containing exosomes can be efficiently and easily isolated by using a carrier.

The carrier is not particularly limited in other configurations as long as it is a structure capable of directly or indirectly supporting (retaining) 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 that binds to the carrier. For example, glass, nylon membrane, semiconductor wafer, latex particles, cellulose particles, microbeads, silica beads, etc. Examples include magnetic beads. As the carrier of the present invention, magnetic beads are particularly preferable because the complex containing exosomes can be easily recovered and the exosomes can be easily isolated. Magnetic beads are widely used in the separation and purification of proteins, DNA, cells and the like, and are carriers that can be fully understood by those skilled in the art.

(Method of binding the peptide of the present invention to the carrier of the present invention)
As described above, the peptide of the present invention can be supported by the carrier of the present invention by binding the peptide of the present invention to the carrier of the present invention.

In one embodiment of the present invention, the method for binding the peptide to the carrier is not particularly limited, and a well-known method can be used as appropriate. Further, the binding between the peptide and the carrier may be direct or indirect. For example, using the peptide of the present invention to which biotin is bound and the carrier of the present invention to which streptavidin is bound, an indirect bond between the peptide and the carrier is formed through the binding between biotin and streptavidin. It is possible. Since streptavidin forms a tetramer, four "peptide-bound biotins" can be bound to one "carrier-bound streptavidin." That is, when the carrier of the present invention has one streptavidin, four peptides of the present invention are bound to one carrier of the present invention. Therefore, the carrier of the present invention can bind to one or more exosomes via four peptides of the present invention. Therefore, the binding force between the carrier of the present invention and exosomes becomes extremely high, or the carrier of the present invention can bind to a plurality of exosomes. Therefore, by using the above-mentioned binding method, exosomes can be isolated in extremely high yield.

The method of binding biotin to the peptide, in other words, the method of producing a biotin-bound peptide (hereinafter, also appropriately referred to as “biotin-labeled peptide”) is not particularly limited. For example, biotin and the peptide may be directly bound or indirectly bound. From the viewpoint of maintaining the structure of the peptide and the function based on the peptide as normal as possible, it is preferable to indirectly bind biotin to the peptide. When indirectly binding biotin to a peptide, for example, any linker may be linked to the peptide of the present invention, and then biotin may be bound to the linker linked to the peptide. From the viewpoint of maintaining the structure of the peptide and the function based on the peptide as normally as possible, it is preferable that the peptide has a linker at its amino-terminal (N-terminal) and / or carboxy-terminal (C-terminal) for binding to biotin. ..

The linker may be a linker composed of a polypeptide (also referred to as a "peptide linker" in the present specification), or a well-known cross-linking agent or spacer arm capable of linking biotin and a peptide. You may.

In the case of a peptide linker, the length of the linker and the types of amino acids constituting the linker can be appropriately set by those skilled in the art. The length of the peptide linker is not particularly limited, and is usually 1 to 20, preferably 1 to 10 (for example, 10, 9, 8, 7, 6, 5, 5). A linker consisting of 4, 3, 2, 1) amino acids is used. The type of amino acid used in the peptide linker is also not particularly limited, but for example, glycine (G), serine (S), threonine (T) and the like can be used. In particular, peptide linkers such as GGS, GGGS (SEQ ID NO: 1), GGGGS (SEQ ID NO: 2), and peptide linkers in which these are repeated a plurality of times (for example, GGGSGGGS (SEQ ID NO: 3), GGGSGGGGSGGGGS (SEQ ID NO: 4), etc.) ) Is preferably used.

Well-known cross-linking agents capable of linking biotin and peptides include, but are not limited to, N-succinimidylamide, N-maleinimide, isothiocyanate, bromoacetamide and the like.

The spacer arm is not particularly limited as long as it is well known, but for example, a spacer arm containing a carbon chain is used, and more preferably, a hexanonate having a carbon chain having 6 carbon atoms is used.

The method for producing streptavidin bound to the carrier is not particularly limited. For example, the carrier and streptavidin may be directly bound or indirectly bound. As appropriate, streptavidin bound to the carrier may be prepared according to a known method. Magnetic beads to which streptavidin is bound are commercially available, for example, Dynabeads (registered trademark, registered trademark,
Thermo Fisher Scientific Co., Ltd.) is known.

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

(Dissociation buffer)
In the isolation method of the present invention, in the dissociation step described later, the exosome is removed from the complex by contacting the complex with a dissociation buffer containing a metal cation (hereinafter, appropriately referred to as “dissociation buffer of the present invention”). Dissociate and isolate the exosome. Since the dissociation buffer of the present invention can dissociate exosomes from the complex under mild (mild) conditions without containing a surfactant and / or a protein denaturing agent that affects the membrane structure, the exosomes can be dissociated. Can be isolated in an intact state (a state close to intact). The present inventors have found for the first time that such a mild dissociation buffer can dissociate the binding between the peptide of the present invention and an exosome.

The metal cation contained in the dissociation buffer of the present invention is not particularly limited, and for example, monovalent metal cations such as sodium ion, potassium ion, lithium ion, silver ion, and copper (I) ion, and magnesium ion. , Calcium ion, Zinc ion, Nickel ion, Barium ion, Copper (II) ion, Iron (II) ion, Tin (II) ion, Cobalt (II) ion, Lead (II) ion and other divalent metal cations Examples thereof include ions, aluminum ions, trivalent metal cations such as iron (III) ions, and the like. Among these metal cations, the metal cations contained in the dissociation buffer of the present invention are preferably sodium ion, potassium ion, magnesium ion, zinc ion, and nickel ion because of their high water solubility. Furthermore, since exosomes can be sufficiently dissociated from the complex, the dissociation buffer of the present invention preferably contains sodium ion, potassium ion, or magnesium ion among the above-mentioned metal cations alone or in combination. ..

An object of the isolation method of the present invention is to isolate exosomes in a simple and intact state (or in a state close to intact). Therefore, in the isolation method of the present invention, "isolating an exosome in an intact state (or in a state close to an intact state)" means that the exosome in the sample is sampled without damaging the exosome (almost without damaging it). It means to separate and isolate from substances other than exosomes (for example, proteins). In other words, in the isolation method of the present invention, "isolating an exosome in an intact state (or in a state close to an intact state)" means that the membrane structure of the exosome (specifically, the lipid bilayer structure) was maintained. Separate and isolate exosomes in the sample from substances other than exosomes in the sample in a state (almost preserved state) and in a state where the structure of the protein present in the membrane is preserved (almost preserved state). Means that.

The dissociation buffer may contain a substance other than the metal cation as long as it does not affect the membrane structure of the exosome and the structure of the protein present on the membrane.

The concentration of metal cations in the dissociation buffer is not particularly limited as long as it is possible to dissociate the exosome from the complex and does not affect the membrane structure of the exosome and the structure of the protein present on the membrane. That is, since the optimum cation concentration may change depending on the binding strength between the peptide of the present invention used and the exosome, the type (valence) of the metal cation, etc., the optimum concentration should be examined and determined as appropriate. do it. The concentration of metal cations in the dissociation buffer of the present invention is not particularly limited, but is preferably, for example, 0.01 to 5M, more preferably 0.05 to 2M, and 0. It is more preferably 1 to 1 M, further preferably 0.2 to 0.7 M, and particularly preferably 0.2 to 0.4 M.

Further, the pH of the dissociation buffer can be appropriately set. The pH of the dissociation buffer is preferably 5 to 10, more preferably 6 to 9, further preferably 7 to 8, and particularly preferably 7.3 to 7.5. When the pH of the dissociation buffer is in the above range, it does not affect the membrane structure of the exosome and the structure of the protein present on the membrane, which is preferable.

[1-2. Process]
(Complex formation step)
In the complex formation step (hereinafter, appropriately referred to as “complex formation step of the present invention”) included in the isolation method of the present invention, a sample containing an exosome, a peptide of the present invention, and a carrier of the present invention are brought into contact with each other. A complex (exosome-) formed by binding the exosome and the peptide carried on the carrier by contacting the sample with the peptide of the present invention carried on the carrier of the present invention. This is a complex forming step of forming a complex in which the peptide of the present invention and the carrier of the present invention are bound in this order; hereinafter, appropriately referred to as “complex of the present invention”). That is, in the complex formation step of the present invention, after the peptide of the present invention is brought into contact with the carrier of the present invention and the sample in a state where the peptide of the present invention is not supported on the carrier of the present invention, the exosome-the present invention A complex may be formed in which the peptide-the carrier of the present invention is bound in this order, or the peptide of the present invention is brought into contact with the sample in a state where the peptide of the present invention is already carried on the carrier of the present invention to form the complex of the present invention. It means that a complex may be formed. However, it can be said that the latter aspect is more preferable from the viewpoint of forming the complex of the present invention more efficiently.

As a method of contacting the sample containing the exosome with the peptide of the present invention and the carrier of the present invention, the exosome in the sample, the peptide of the present invention and the carrier of the present invention are bound to each other to form a complex of the present invention. Other methods are not limited as long as they are carried out under possible conditions. For example, a method of adding the peptide of the present invention and the carrier of the present invention to a sample and mixing them can be mentioned. The order of addition and mixing when contacting the sample, the peptide of the present invention, and the carrier of the present invention is not particularly limited. For example, (1) the peptide of the present invention and the carrier of the present invention may be added to the sample at the same time and mixed, or (2) the peptide of the present invention is added to the sample and mixed, and then the carrier of the present invention is added. The carrier of the present invention may be added to the sample and mixed, and then the peptide of the present invention may be added and mixed. However, it can be said that the procedure (2) above is preferable because the peptide of the present invention binds to an exosome and the peptide of the present invention binds to the carrier of the present invention. Contact of the sample with the peptide and the carrier according to the method described above can form a complex of the present invention containing an exosome, the peptide and the carrier.

Further, as a method of contacting the sample with the peptide of the present invention carried on the carrier of the present invention, the exosome in the sample and the peptide of the present invention carried on the carrier of the present invention are bound to each other, and the present invention Other methods are not limited as long as they are carried out under conditions that allow the formation of a complex. For example, a method of adding the peptide of the present invention supported on the carrier of the present invention to a sample and mixing the samples can be mentioned.

The contact time between the sample and the peptide of the present invention and the carrier of the present invention, or the contact time between the sample and the peptide of the present invention carried on the carrier of the present invention is sufficient to form the complex of the present invention. The time is not limited, and it can be determined after considering the optimum conditions as appropriate.

Further, in the contact between the sample containing the exosome and the peptide of the present invention and the carrier of the present invention, for example, the carrier of the present invention or the carrier of the present invention in which the carrier of the present invention is supported in a columnar or cylindrical container (column). It is also possible to use a column containing a carrier (referred to as a "carrier column" in the present specification) prepared by filling the above. As a method for obtaining the complex of the present invention by passing a solution containing a sample and a solution containing a peptide through a carrier column, the exosome in the sample, the peptide of the present invention, and the carrier of the present invention are bound to each other, respectively. Other methods are not limited as long as they are carried out under conditions that can form the complex of the invention.

The contact time between the carrier column and the peptide and / or sample or the sample containing the exosome-peptide complex is sufficient for the exosome, peptide and carrier in the sample to form the complex of the present invention. Is preferable. During the above time, the flow rate of the sample solution (a sample containing an exosome and / or a solution containing the peptide of the present invention, or a solution containing an exosome-peptide complex) to the carrier column, or the sample solution is added. It can be set appropriately by adjusting the time from the start of the liquid flow to the start of the liquid passage.

As a physical method for passing the sample solution through the carrier column, a conventionally known method can be used. For example, the following methods (1) to (3) can be mentioned.
(1) A method in which the sample solution is added onto a carrier column placed in the vertical direction, and the sample solution is passed through the carrier column by gravity.
(2) The sample solution is added to one of the carrier columns, and the pressure is applied from the direction in which the sample solution is added, or the sample solution is sucked in the direction opposite to the direction in which the sample solution is added. A method of passing a sample solution.
(3) A method in which a sample solution is added to one of the carrier columns, the carrier column is loaded into a centrifuge tube, and the centrifuge tube is centrifuged to allow the sample solution to pass through the carrier column. The method (3) is a method using a so-called conventionally known spin column.

(Dissociation process)
The dissociation step included in the isolation method of the present invention (hereinafter, appropriately referred to as “dissociation step of the present invention”) is the present invention containing the complex of the present invention obtained by the complex forming step and a metal cation. This is a step of dissociating the exosome from the complex of the present invention by contacting with the dissociation buffer of the present invention.

The method for contacting the complex of the present invention 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, a method of adding the dissociation buffer of the present invention to a container containing the complex of the present invention and mixing the mixture can be mentioned. The contact time between the complex of the present invention and the dissociation buffer of the present invention is not particularly limited as long as it is a time sufficient for the exosomes to dissociate from the complex. Contact between the complex of the present invention and the dissociation buffer of the present invention dissociates the exosome from the complex of the present invention and moves it to the dissociation buffer. Then, the exosome can be isolated by separating the dissociation buffer and the carrier of the present invention and isolating the dissociation buffer.

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. In addition, the peptide of the present invention may be contained in the isolated dissociation buffer after the separation. From the viewpoint of isolating the exosome 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 separation, the present invention is placed in the isolated dissociation buffer. It is preferable that the peptide of the invention is not contained.

When a column is used in the complex forming step, the complex of the present invention is passed by passing the dissociation buffer of the present invention through the column in which the complex of the present invention is formed in the complex forming step. Can be brought into contact with the dissociation buffer of the present invention. Examples of the physical method for passing the dissociation buffer of the present invention through the column include the methods (1) to (3) described in the section (complex forming step). When the column is brought into contact with the dissociation buffer of the present invention, the exosome is dissociated from the complex and transferred to the dissociation buffer. Therefore, exosomes can be isolated by isolating the dissociation buffer after column passage.

The isolation method of the present invention may include steps other than the above-mentioned complex formation step and dissociation step. Other steps include, for example, a cleaning step and a recovery step.

(Recovery process)
The recovery step is a step of recovering the complex of the present invention obtained by the complex forming step, which is provided between the complex forming step and the dissociation step. As the recovery step, a conventionally known method can be appropriately adopted depending on the complex forming step, the carrier and the like.

The carrier is [1-1. In the case of a structure as described in the section (Carrier) of [Material], the carrier containing the complex of the present invention can be recovered by centrifugation. For example, it is possible to recover the carrier containing the complex of the present invention by centrifuging under the condition of 500 to 4000 × g.

When the carrier is magnetic beads, the carrier can be easily recovered by applying a magnetic force from the outside using a magnetic material such as a conventionally known magnetic stand. When the carrier is a magnetic bead and is recovered using a magnetic material, it has the following advantages (1) and (2) as compared with the centrifugation method: (1) The carrier which is a magnetic bead has the following advantages. Since it is attracted to the magnetic material, it is possible to remove the supernatant almost completely, and the purity of the obtained exosome is high; (2) When the supernatant is removed, the magnetic beads are removed together. The risk is reduced and the loss of the obtained exosome can be prevented (the isolation rate of the exosome can be improved).

When a carrier column is used in the complex forming step of the present invention, the carrier containing the complex of the present invention stays in the column. Therefore, the above-mentioned recovery step is not required.

(Washing process)
The isolation method of the present invention preferably further includes a washing step before the dissociation step.

The washing step is a step of washing the recovered complex of the present invention or the complex of the present invention in the column with an appropriate cleaning solution an appropriate number of times. The type of cleaning liquid used, the number of cleanings, and the like are not particularly limited as long as the cleaning step is performed under conditions where the complex does not dissociate.

Examples of the cleaning solution used in the cleaning step include physiological saline or Phosphate buffered saline (PBS).

When one embodiment of the present invention includes a recovery step, the cleaning step includes the following methods. (1) By mixing the recovered carrier containing the complex of the present invention with the cleaning solution, the carrier is resuspended in the cleaning solution. (2) Again, the carrier containing the complex of the present invention is recovered (that is, the carrier containing the complex of the present invention and the washing liquid are separated).

When a carrier column is used in the complex forming step of the present invention, an appropriate amount of the washing liquid may be passed through the column after the complex of the present invention is formed.

[2. How to remove exosomes]
The method for removing exosomes according to an embodiment of the present invention (hereinafter, appropriately referred to as "the method for removing exosomes of the present invention") is a method for removing exosomes from a sample containing exosomes.
By contacting the sample with a peptide containing four or more arginines in close proximity to each other and a carrier capable of supporting the peptide, or by contacting the sample with the peptide supported on the carrier. , A complex forming step of forming a complex formed by binding the exosome and the peptide supported on the carrier, and
It includes a separation step of separating the carrier carrying the complex obtained by the complex formation step and the sample to obtain a separation solution from which at least a part of the exosome has been removed from the sample. ..

Since the method for removing exosomes according to the embodiment of the present invention has the above-mentioned constitution, the following effects (1) and (2) are exhibited.
(1) Since it is not necessary to use an ultracentrifuge or the like, exosomes can be removed quickly and easily.
(2) Since arginine utilizes the fact that it binds to the membranes of various exosomes including CD63-positive exosomes, exosomes can be widely captured and isolated.

Due to the effects of (1) and (2) above, a separation solution in which various types of exosomes including CD63-positive exosomes are efficiently removed can be obtained from a sample by the removal method of the present invention. Therefore, a separation solution having extremely little residual exosome can be easily obtained by a simple method.

Such a method for removing exosomes is useful, for example, for producing serum from which exosomes have been removed. When culturing cultured cells, fetal bovine serum (FBS) is generally used. Normally, FBS contains bovine-derived exosomes, so if it is desired to recover exosomes secreted from human cells, it is necessary to use an FBS from which bovine-derived exosomes have been removed in advance. In order to prepare such an FBS, a method of removing exosomes by ultracentrifugation at 100,000 × g for 16 hours or more at 4 ° C. is generally used. However, it is difficult to completely remove exosomes from FBS, which requires a long time to remove exosomes by this ultracentrifugation method (for example, when the ultracentrifugation time is 16 hours, about 20% of exosomes are in FBS. There are problems such as (remaining). On the other hand, according to the removal method of the present invention, exosomes can be easily removed from an FBS containing bovine-derived exosomes using a carrier, so that an FBS from which bovine-derived exosomes have been removed can be easily produced.

[3. Exosome isolation kit]
The exosome isolation kit according to one embodiment of the present invention (hereinafter, appropriately referred to as “kit of the present invention”) is a kit for carrying out the above-mentioned isolation method of the present invention or the removal method of the present invention. Includes the peptides of the invention described above, the carriers of the invention, and the dissociation buffers of the invention. Therefore, for the explanation of the configuration of the kit, see [1. Method of isolating exosomes] can be incorporated.

The peptide of the present invention and the carrier of the present invention included in the kit of the present invention may be included in the kit of the present invention in a state where the peptide of the present invention is pre-supported on the carrier of the present invention, and each of them is different. It may be included as a body.

Further, the kit of the present invention may include magnetic beads, magnetic beads on which streptavidin is immobilized, a magnetic substance such as a magnetic stand, biotin and the like.

In addition to the above configuration, the kit of the present invention may include a container (for example, a bottle, a plate, a tube, a dish, a column, etc.) containing a specific material. The kit of the present invention may include a container containing a diluent, solvent, cleaning solution or other reagent. The term "equipped" as used in the description of the kit of the present invention may be intended to be contained within any of the individual containers that make up 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.

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 from culture supernatants using polyarginine-immobilized magnetic beads>
〔Materials and methods〕
(Sample preparation)
Samples were prepared by the following method using the breast cancer cell line MCF-7 (JCRB cell bank) known as a prolific strain of exosomes. DMEM medium (Dulbecco's modified Eagle medium, Thermo Fisher Scientific) was used as the medium.
(1) In a dish, MCF-7 cells were seeded in 10 mL of DMEM medium (1.0 × 10 ⁵ cells / mL) and cultured at _{37 ° C. under the condition of 5% CO 2.} DMEM medium is pre-prepared with 10% (v / v) deactivated fetal bovine serum (HyClone FBS, GE Healthcare), 100units / mL penicillin (Nacalai Tesque), and 0.1 mg / mL streptomycin (Meiji Seika Pharma). Was added.
(2) The cells were cultured until they became 50 to 80% of the culture area of the dish.
(3) 3 mL of PBS (Nacalai Tesque) was slowly added to spread the whole dish, and then PBS was removed.
(4) The washing step of (3) was performed twice to remove exosomes derived from FBS from the dish.
(5) 10 mL of Advanced DMEM medium (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, and the cells in the culture supernatant peeled off from the dish were removed.
(7) The culture supernatant was centrifuged at 10000 × g for 30 minutes to remove debris in the culture supernatant.
(8) The culture supernatant was filtered through a 0.22 μm filter to prepare a sample (hereinafter referred to as “supernatant sample”). The supernatant sample was stored at −80 ° C. as needed.

(Preparation of magnetic beads carrying arginine)
Peptides containing four or more arginines in close proximity to each other were bound (supported) to magnetic beads (carriers) to prepare magnetic beads in which the peptides were supported.

As the carrier, magnetic beads on which streptavidin was immobilized (attached to the ExoIntact Exosome Purification Reagent Kit, HMT Biomedical Co., Ltd.) (also referred to as “streptavidin magnetic beads” in the present specification) were used.

The peptide includes a linker peptide consisting of GGGSGGGGS (SEQ ID NO: 3) or GGGSGGGSGGGGS (SEQ ID NO: 4) and a sequence consisting of 4, 8, or 16 consecutive arginine residues (hereinafter, "polyarginine"). "Peptide") was used. The amino acids used in this example are all L-type amino acids. The polyarginine peptide further has biotin bound to the N-terminus of the polyarginine peptide in order to bind to magnetic beads (carriers) having streptavidin. The polyarginine peptide to which the biotin is bound is hereinafter referred to as "biotin-labeled polyarginine peptide". The biotin-labeled polyarginine peptide used in this example was prepared by contract synthesis by Eurofins Genomics.

The names of the biotin-labeled polyarginine peptides used in this example and the amino acid sequences of the polyarginine peptides contained in the respective biotin-labeled polyarginine peptides are as follows.
1. 1. BIOTIN-R4; GGGSGGGSGGGSRRRRR (SEQ ID NO: 5) The N-terminal of the peptide consisting of the amino acid sequence is modified with biotin.
2. BIOTIN-R8; GGGSGGGSGGGSRRRRRRRRR (SEQ ID NO: 6) The N-terminal of the peptide consisting of the amino acid sequence is modified with biotin.
3. 3. BIOTIN-R16; GGGSGGGSRRRRRRRRRRRRRRRRRRR (SEQ ID NO: 7) The N-terminal of the peptide consisting of the amino acid sequence is modified with biotin.

Using the biotin-streptavidin interaction, specifically, magnetic beads carrying a polyarginine peptide were produced by the following method.
(1) Add 20 μL (0.2 mg) of streptavidin magnetic beads to a 1.5 mL microtube (also simply referred to as a “tube”), set the tube on a magnet stand, let stand for 1 minute, and then stand on top. Qing was removed.
(2) The tube was removed from the magnet stand, 0.2 mL of BW Buffer (attached to the ExoIntact Exosome Purification Reagent Kit, HMT Biomedical Co., Ltd.) was added and mixed. The tube was set on a magnet stand and allowed to stand for 1 minute to remove the supernatant. The operation (cleaning) of (2) was performed twice in total.
(3) 200 μL of BW Buffer was added to the tube, and the streptavidin magnetic beads were resuspended in the BW Buffer to obtain washed streptavidin magnetic beads (180 μL).
(4) 20 μL of biotin-labeled polyarginine peptide (Biotin-R4, Biotin-R8, or Biotin-R16; 100 μM) was added to each tube.
(5) The tube was mixed at room temperature for 20 minutes, and the biotin-labeled polyarginine peptide was brought into contact with the streptavidin magnetic beads to bind (ie, support) the biotin-labeled polyarginine peptide to the streptavidin magnetic beads. ..
(6) The tube was set on a magnet stand and allowed to stand for 1 minute to remove the supernatant.
(7) The same operation (washing) as in (2) above was performed a total of three times.
(8) 50 μL of BW Buffer was added to the tube, and the streptavidin magnetic beads were resuspended in the BW Buffer to obtain streptavidin magnetic beads (0.2 mg / 50 μL) carrying a biotin-labeled polyarginine peptide.

In the present specification, streptavidin magnetic beads carrying a biotin-labeled polyarginine peptide are referred to as polyarginine-immobilized magnetic beads. Further, in the present specification, in particular, streptavidin magnetic beads carrying Biotin-R4, streptavidin magnetic beads carrying Biotin-R8, and streptavidin magnetic beads carrying Biotin-R16 are used, respectively. They are referred to as "R4 immobilized magnetic beads", "R8 immobilized magnetic beads", and "R16 immobilized magnetic beads".

(Preparation of magnetic beads carrying lysine)
As Comparative Example 1, a peptide containing eight lysines was bound (supported) to a magnetic bead (carrier) to prepare a magnetic bead in which the peptide was supported.

As the carrier, streptavidin magnetic beads were used as in the case of the polyarginine-immobilized magnetic beads.

As the peptide, a peptide containing a linker peptide consisting of GGGSGGGSGGGGS (SEQ ID NO: 4) and a sequence consisting of eight consecutive lysine residues (hereinafter referred to as "polylysine peptide") was used. The amino acids used in Comparative Example 1 are all L-type amino acids. In order to bind the polylysine peptide to magnetic beads (carrier) having streptavidin, biotin is bound to the N-terminal of the polylysine peptide. The polylysine peptide to which the biotin is bound is hereinafter referred to as "biotin-labeled polylysine peptide". The biotin-labeled polylysine peptide used in Comparative Example 1 was prepared by contract synthesis by Eurofin Genomics.

The name of the biotin-labeled polylysine peptide used in Comparative Example 1 and the amino acid sequence of the polylysine peptide contained in the biotin-labeled polylysine peptide are as follows.
4. Biotin-K8; The N-terminal of a peptide consisting of the amino acid sequence shown by GGGSGGGGSGGGSKKKKKKKKK (SEQ ID NO: 8) modified with biotin.

The magnetic beads carrying the polylysine peptide were prepared by performing the same operations as in the above-mentioned methods (1) to (8) of the above-mentioned methods (preparation of magnetic beads carrying arginine).

In the present specification, streptavidin magnetic beads carrying a biotin-labeled polylysine peptide are referred to as polylysine-immobilized magnetic beads. Further, in the present specification, in particular, streptavidin magnetic beads carrying Biotin-K8 are referred to as "K8-immobilized magnetic beads".

(Isolation and removal of exosomes from culture supernatants using polyarginine-immobilized magnetic beads)
As Example 1 of the present invention, exosomes were isolated and removed from the supernatant sample using polyarginine-immobilized magnetic beads. In addition, polylysine-immobilized magnetic beads were used as Comparative Example 1 to isolate and remove exosomes from the supernatant sample.

An isolation method using polyarginine-immobilized magnetic beads will be described below with reference to FIG.
(1) 50 μL of Binding Buffer (attached to ExoIntact Exosome Purification Reagent Kit, HMT Biomedical Co., Ltd.) was added to 0.5 mL of the supernatant sample.
(2) 50 μL of R4 immobilized magnetic beads, R8 immobilized magnetic beads, or R16 immobilized magnetic beads was added to a tube containing 550 μL of the supernatant sample to which Binding Buffer was added.
(3) The exosomes in the supernatant sample were brought into contact with the polyarginine-immobilized magnetic beads by mixing the tubes at room temperature for 60 minutes, and the exosomes were bound to the polyarginine peptide. That is, in the step (3), a complex containing an exosome, a polyarginine peptide, and streptavidin magnetic beads (carrier) was formed.
(4) The tube was set on a magnet stand and allowed to stand for 1 minute to remove the supernatant.
(5) The complex was washed by performing the following operations: (5-1) The tube was removed from the magnet stand, 1.0 mL of BW Buffer was added to the tube and mixed; (5-2) Tube It was set on a magnet stand and allowed to stand for 1 minute to remove the supernatant.
(6) The operation (washing) of (5) above was performed twice more (three times in total), and polyarginine-immobilized magnetic beads (0.2 mg) containing the exosomes in the supernatant sample as a complex were collected.
(7) As a dissociation buffer, 50 μL of NaCl solution [10 mM Tris-HCl (pH 7.5), 0.5 M NaCl] was added and mixed.
(8) The mixture was allowed to stand for 10 minutes to dissociate exosomes from the complex, and the dissociation buffer (50 μL) was collected and used as an elution sample.

Using K8-immobilized magnetic beads (50 μL) as Comparative Example 1 and streptavidin magnetic beads (0.2 mg / 50 μL) in which a peptide is not supported as a negative control, the same operations as in (1) to (7) above are performed. This was performed to obtain an elution sample of exosomes.

(Quantification of exosomes)
The exosome concentration of the eluted sample obtained by the above-mentioned polyarginine-immobilized magnetic beads was measured using a CD9 / CD63ELISA kit (Cosmo Bio Co., Ltd.). As a measurement sample, a supernatant sample or each eluted sample was prepared by diluting it 20-fold with assay buffer. In addition, 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) To an anti-CD9 antibody-immobilized 96-well plate, a 100 μL diluted measurement sample or a 100 μL serially diluted sample of the CD9 / CD63 fusion protein (12.5 to 100 pg / mL) was added and allowed to stand for 2 hours. It was reacted at the table.
(2) After washing 3 times with 300 μL of washing buffer (1 ×), 100 μL of HRP-labeled anti-CD63 antibody solution was added and the reaction was allowed to stand for 2 hours.
(3) After washing three times with 300 μL of washing buffer (1 ×), 100 μL of the substrate solution was added to cause a color reaction for 10 minutes, and 50 μL of a stop solution was added to stop the color development reaction.
(4) The absorbance of A450 was measured with a plate reader (ARVO MX 1420, PerkinElmer).

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

In addition, the exosome concentration of the negative control and the exosome concentration of Comparative Example 1 using the K8-immobilized magnetic beads were also measured using the CD9 / CD63ELISA kit in the same manner.

[Result: Isolation of exosomes from culture supernatant]
In FIG. 2, Example 1 using R4-immobilized magnetic beads, R8-immobilized magnetic beads, or R16-immobilized magnetic beads and only streptavidin magnetic beads (denoted as “magnetic beads only” in FIG. 2) are used. The measurement result of the exosome concentration with the negative control that was present is shown. As shown in FIG. 2, since no exosomes were detected by the negative control, it was found that almost no exosomes were bound to the streptavidin magnetic beads themselves. On the other hand, in Example 1 using R4 immobilized magnetic beads, R8 immobilized magnetic beads, or R16 immobilized magnetic beads, exosomes were detected in the eluted sample.

Therefore, it was shown that the isolation method of the present invention using a peptide containing four or more arginines (polyarginine peptide) can isolate exosomes by specifically binding exosomes to the polyarginine peptide. .. Furthermore, when the exosome concentrations of Example 1 using the R4 immobilized magnetic beads, the R8 immobilized magnetic beads, or the R16 immobilized magnetic beads were compared, it was shown that the exosome concentration of the R8 immobilized magnetic beads was the highest. .. Therefore, it was shown that the isolation method of the present invention using a peptide containing 8 or more arginines can isolate exosomes extremely efficiently.

Next, FIG. 3 shows the measurement results of the exosome concentration between Example 1 using R8-immobilized magnetic beads and Comparative Example 1 using K8-immobilized magnetic beads. It was found that the exosome concentration was improved about 2.4 times in Example 1 using the R8-immobilized magnetic beads as compared with Comparative Example 1 using the K8-immobilized magnetic beads. Therefore, it was shown that the isolation method of the present invention using polyarginine-immobilized magnetic beads can isolate exosomes more efficiently than the isolation method using polylysine-immobilized magnetic beads having the same chain length.

[Result: Removal of exosomes from culture supernatant]
As shown in FIGS. 2 and 3, it was shown that the exosomes were bound to the polyarginine-immobilized magnetic beads and eluted in the eluted sample of Example 1. Therefore, it can be said that the supernatant after mixing the polyarginine-immobilized magnetic beads and the exosomes and allowing them to stand on a magnet stand is a separation solution in which at least a part of the exosomes is removed from the sample containing the exosomes. From this, it was shown that the removal method of the present invention can remove exosomes from a sample with high efficiency.

<2. Isolation of exosomes from serum using polyarginine-immobilized magnetic beads>
〔Materials and methods〕
(Sample preparation)
Using Human Serum (BSL) as serum, the sample was prepared by the following method. MicroSpin S-400 HR Columns (GE Healthcare) was used as the spin column.
(1) The spin column was set in a 1.5 mL tube, the resin of the spin column was vortexed, and then the mixture was allowed to stand for 1 minute.
(2) A spin column was attached to the attached collection tube, and the mixture was centrifuged at 735 × g for 1 min to remove the preservative solution.
(3) The spin column was attached to a new 1.5 mL tube, and 0.1 mL of serum was slowly added to the spin column.
(4) Centrifugation of 735 × g for 2 minutes was performed to remove debris in serum.
(5) 0.4 mL of BW Buffer was added to 0.1 mL of the supernatant to prepare a 0.5 mL sample (hereinafter referred to as “serum sample”).

(Isolation and quantification of exosomes from serum using polyarginine-immobilized magnetic beads)
As Example 2 of the present invention, exosomes were isolated from serum using polyarginine-immobilized magnetic beads. FIG. 4 shows an outline of the isolation method according to Example 2 of the present invention. The polyarginine-immobilized magnetic beads are <1. Isolation and removal of exosomes from culture supernatants using polyarginine-immobilized magnetic beads> The R8-immobilized magnetic beads used in> were used.

The specific method is shown below.
(1) 50 μL of R8-immobilized magnetic beads were added to 0.5 mL of serum sample.
(2) The exosomes in the serum sample were brought into contact with the R8-immobilized magnetic beads by mixing the tubes at room temperature for 60 minutes, and the exosomes were bound to the polyarginine peptide. That is, in the step (2), a complex containing an exosome, a polyarginine peptide, and streptavidin magnetic beads (carrier) was formed.
(3) The tube was set on a magnet stand and allowed to stand for 1 minute to remove the supernatant.
(4) The complex was washed by performing the following operations: (4-1) The tube was removed from the magnet stand, 1.0 mL of BW Buffer was added to the tube and mixed; (4-2) Tube was added. It was set on a magnet stand and allowed to stand for 1 minute to remove the supernatant.
(5) The operation (washing) of (4) above was performed twice more (three times in total), and R8-immobilized magnetic beads (0.2 mg) containing the exosomes in the serum sample as a complex were collected.
(6) As a dissociation buffer, 50 μL of NaCl solution [10 mM Tris-HCl (pH 7.5), 0.5 M NaCl] was added and mixed.
(7) The mixture was allowed to stand for 10 minutes to dissociate exosomes from the complex, and the dissociation buffer (50 μL) was collected and used as an elution sample.

The obtained eluted sample is <1. The exosome concentration was measured using the CD9 / CD63ELISA kit in the same manner as in the quantification method performed in> Isolation and removal of exosomes from the culture supernatant using polyarginine-immobilized magnetic beads>.

〔result〕
In Example 2 of the present invention using R8-immobilized magnetic beads, it was found that 132 pg / mL exosomes could be recovered from serum. Therefore, it was shown that the isolation method of the present invention using polyarginine-immobilized magnetic beads can recover exosomes not only from the culture supernatant but also from serum containing a large amount of impurities other than exosomes. That is, in the isolation method of the present invention, exosomes can be efficiently isolated from a sample derived from a living body.

<3. Isolation of various exosomes with polyarginine-immobilized magnetic beads>
〔Materials and methods〕
(Isolation of exosomes and marker analysis using polyarginine-immobilized magnetic beads)
As Example 3 of the present invention, exosomes were isolated using polyarginine-immobilized magnetic beads. FIG. 5 shows an outline of the isolation method according to Example 3 of the present invention. Samples for isolating exosomes are <1. Isolation and removal of exosomes from culture supernatants using polyarginine-immobilized magnetic beads> The supernatant sample used in was used. The polyarginine-immobilized magnetic beads are <1. Isolation and removal of exosomes from culture supernatants using polyarginine-immobilized magnetic beads> The R8-immobilized magnetic beads used in> were used. As Comparative Example 2, <1. Isolation and removal of exosomes from culture supernatants using polyarginine-immobilized magnetic beads> The K8-immobilized magnetic beads used in> were used.

The specific method is shown below.
(1) 0.02 mL of Binding Buffer was added to 0.2 mL of the supernatant sample.
(2) 8 μL of R8-immobilized magnetic beads or K8-immobilized magnetic beads was added to a tube containing 220 μL of the supernatant sample to which Binding Buffer was added.
(3) By mixing the tubes at room temperature for 30 minutes, the exosomes in the supernatant sample were brought into contact with the R8-immobilized magnetic beads or K8-immobilized magnetic beads, and the exosomes were bound to the polyarginine peptide or polylysine peptide. .. That is, in the step (2), a complex containing an exosome and a polyarginine peptide or a polylysine peptide and a streptavidin magnetic bead (carrier) was formed.
(4) The tube was set on a magnet stand and allowed to stand for 1 minute to remove the supernatant.
(5) The complex was washed by performing the following operations: (5-1) The tube was removed from the magnet stand, 0.2 mL of BW Buffer was added to the tube and mixed; (5-2) Tube was added. It was set on a magnet stand and allowed to stand for 1 minute to remove the supernatant.
(6) The operation (washing) of (5) above was performed twice more (three times in total) and resuspended in 200 μL of BW Buffer.
(7) The obtained suspension was divided into a suspension for detecting CD9-positive exosomes and a suspension for detecting CD63-positive exosomes, and 100 μL of each was dispensed into a 1.5 mL tube. As a negative control, two 1.5 mL tubes were prepared by dispensing only 4 μL of R8-immobilized magnetic beads or K8-immobilized magnetic beads.
(8) After allowing the 1.5 mL tube to stand on the magnet stand for 1 minute, the supernatant was removed, 100 μL of the blocking solution was added, and the mixture reaction was carried out for 20 minutes. For the blocking solution of R8-immobilized magnetic beads, add 2% Block Ace (KAC) and 0.025% Tween 20 (Fuji Film Wako Pure Chemical Industries, Ltd.) to TBS (pH 7.4, Nacalai Tesque). The added solution was used. For the blocking solution of K8-immobilized magnetic beads, 2% BSA (Fuji Film Wako Pure Chemical Industries, Ltd.) and 0.025% Tween 20 (Fuji Film Wako Pure Chemical Industries, Ltd.) were added to TBS (pH 7.4, Nacalai Tesque). Was used.
(9) Further, 100 μL of the primary antibody solution was added, and the mixture reaction was carried out for 30 minutes. As the primary antibody solution, anti-CD9 antibody (Anti-CD9 Human (Mouse) Unlabeled 12A12, Cosmo Bio Co., Ltd.) or anti-CD63 antibody (Anti-CD63, Monoclonal Antibody (3-13), Fuji Film Wako Pure Chemical Industries, Ltd.) ) Was diluted 1000-fold with a blocking solution.
(10) After adding the primary antibody solution and reacting for 30 minutes, the mixture was allowed to stand on a magnet stand for 1 minute, the supernatant was removed, and 0.025% Tween 20 was added to 0.1 mL of TBS-T (TBS (pH 7.4)). Was washed twice with the solution to which the above was added.
(11) Each 1.5 mL tube was allowed to stand on a magnet stand for 1 minute, the supernatant was removed, 100 μL of a secondary antibody solution was added, and a mixture reaction was carried out for 30 minutes. As the secondary antibody solution, Anti-Mouse-HRP (Goat Anti-Mouse IgG H & L (HRP), ab97023, Abcam) diluted 50,000 times with a blocking solution was used.
(12) After adding the secondary antibody solution and reacting for 30 minutes, the 1.5 mL tube was allowed to stand on the magnet stand for 1 minute, the supernatant was removed, and the mixture was washed 3 times with 0.1 mL TBS-T.
(13) The 1.5 mL tube was allowed to stand on the magnet stand for 1 minute, the supernatant was removed, and the tube was suspended in 0.04 mL of TBS (pH 7.4).
(14) 90 μL of a luminescent substrate (ELISA-Star peroxidase chemiluminescent substrate, Fuji Film Wako Pure Chemical Industries, Ltd.) was added to 10 μL of the obtained suspension, and after a 5-minute reaction, the luminescence value was measured with a plate reader.

The amount of CD9-positive exosomes or CD63-positive exosomes bound to R8-immobilized magnetic beads or K8-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 3 of the present invention using R8-immobilized magnetic beads, both CD9-positive exosomes and CD63-positive exosomes were better than in Comparative Example 2 using K8-immobilized magnetic beads. It was found that it can be isolated.

Further, FIG. 7 shows a comparison of values (CD63 / CD9) obtained by dividing the amount of CD63-positive exosomes bound to the magnetic beads by the amount of CD9-positive exosomes bound to the magnetic beads. As shown in FIG. 7, the value of CD63 / CD9 in the R8-immobilized magnetic beads was about 10 times higher than that in the K8-immobilized magnetic beads. In other words, it was suggested that the K8-immobilized magnetic beads had very weak binding to CD63-positive exosomes as compared to binding to CD9-positive exosomes. On the other hand, it was suggested that the R8-immobilized magnetic beads had good binding to CD9-positive exosomes and CD63-positive exosomes.

From the above, it was shown that when polyarginine is used to isolate exosomes, various types of exosomes including CD63-positive exosomes can be more preferably isolated as compared with the case where polylysine is used.

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 (close to intact state). Therefore, according to the present invention, it becomes possible to easily and in detail analyze substances contained inside various types of exosomes. Therefore, the present invention is useful in various technical fields, especially in the pharmaceutical and medical fields.

Claims (8)

An exosome isolation method for isolating exosomes from a sample containing exosomes.
By contacting the sample with a peptide containing four or more arginines in close proximity to each other and a carrier capable of supporting the peptide, or by contacting the sample with the peptide supported on the carrier. , A complex forming step of forming a complex formed by binding the exosome and the peptide supported on the carrier, and
Isolation of exosomes, which comprises a dissociation step of contacting the complex obtained by the complex forming step with a dissociation buffer containing a metal cation to dissociate the exosome from the complex. Method. The method for isolating an exosome according to claim 1, wherein the arginine contained in the peptide is continuous. The method for isolating an exosome according to claim 1 or 2, wherein the amount of arginine contained in the peptide is 8 or more. The method for isolating an exosome according to any one of claims 1 to 3, wherein the content of the CD63-positive exosome is 10% or more of the content of the CD9-positive exosome. The method for isolating an exosome according to any one of claims 1 to 4, wherein the sample is derived from a living body and contains impurities other than the exosome. The method for isolating an exosome according to any one of claims 1 to 5, wherein the carrier is magnetic beads. An exosome isolation kit for performing the method for isolating an exosome according to any one of claims 1 to 6.
An exosome isolation kit comprising said peptide, said carrier and said dissociation buffer. A method for removing exosomes from a sample containing exosomes, wherein the sample, a peptide containing four or more arginines in close proximity to each other, and a carrier capable of supporting the peptide are brought into contact with each other, or A complex forming step of contacting the sample with the peptide supported on the carrier to form a complex formed by binding the exosome and the peptide supported on the carrier, and the composite. It is characterized by comprising a separation step of separating the carrier carrying the complex obtained by the body forming step and the sample to obtain a separation solution from which at least a part of the exosome has been removed from the sample. How to remove exosomes.

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