JP2023123105A - Method for purifying extracellular vesicle - Google Patents

Abstract

To provide a method capable of separating, concentrating and purifying an extracellular vesicle such as an exosome from a large amount of cell culture solution or the like, and acquiring an extracellular vesicle having more excellent effect of treating a disease, and to provide a composition containing an extracellular vesicle having excellent effect of treating a disease which is obtained by the method.SOLUTION: Provided herein is a method for separating an extracellular vesicle from a sample and purifying it, including: (A1) a process of treating a sample including an extracellular vesicle by using a hollow fiber membrane to concentrate a desired extracellular vesicle; (B1) a process of bringing concentrated liquid of the obtained extracellular vesicle into contact with a carrier carrying a peptide including continuous four or more lysines to form a complex in which the extracellular vesicle and the peptide are bonded; and (B2) a process of bringing the complex into contact with a dissociation buffer including a metal cation to dissociate the extracellular vesicle from the complex.SELECTED DRAWING: None

Description

The present invention relates to a method for purifying extracellular vesicles.

Exosomes are extracellular vesicles covered with a lipid bilayer membrane that are secreted by most cell types, generally with a particle size of 50–150 nm and several membrane-expressed marker proteins (CD9, CD63, CD81, etc.). Defined. These extracellular vesicles contain cell membrane proteins, intracellular proteins, nucleic acids such as DNA, mRNA, and miRNA, and are said to be involved in intercellular interactions. In such intercellular interactions, physiological and pathological functions have attracted attention, and attempts to diagnose diseases using exosomes as indicators and to use exosomes themselves for the treatment of diseases are being investigated.

On the other hand, mesenchymal stem cells (MSCs) are progenitor cells with pluripotency that were first isolated from bone marrow by Friedenstein (see Non-Patent Document 1). It has been clarified that mesenchymal stem cells exist in various tissues other than bone marrow, such as umbilical cord and fat, and administration of mesenchymal stem cells is expected as a new treatment method for various intractable diseases (See Patent Documents 1 and 2). According to recent research, it has also been found that part of the therapeutic effect of diseases by mesenchymal stem cells is due to extracellular vesicles released by mesenchymal stem cells. Disclosed are purified populations of type cell-derived vesicles (eg, exosomes and/or microvesicles), compositions, and methods of treatment using the vesicles.

Until now, ultracentrifugation and size exclusion chromatography using serum and cell culture supernatant as starting materials have been used to purify exosomes. However, all of these conventional purification methods are laboratory scale, and the recovery rate of exosomes is as low as 20 to 30%, which is inconvenient that they are not suitable for mass production for industrial use. Under these circumstances, there is a demand for the development of a new system that can separate, concentrate, and purify exosomes from large amounts of cell culture media and the like.

JP 2012-157263 A Japanese Patent Publication No. 2012-508733 Japanese Patent Publication No. 2019-501179

Pittenger F. M. et al. , Science, 284, pp. 143-147, 1999

In the circumstances described above, the present invention is capable of separating, concentrating, and purifying extracellular vesicles such as exosomes from a large amount of cell culture media, etc., and providing extracellular vesicles that are more effective in treating diseases. The purpose is to provide a method that can be obtained.

In order to solve the above problems, the present inventors conducted various studies and found that a large amount of cells can be obtained by a method combining a concentration step using a hollow fiber membrane and an affinity purification step using a specific peptide supported on a carrier. We found that extracellular vesicle populations containing high-purity exosomes can be easily separated, concentrated, and purified from the culture supernatant. It was also found that extracellular vesicles obtained by this method exhibit superior anti-inflammatory effects compared to extracellular vesicles obtained by conventional methods. That is, the gist of the present invention is as follows.

[1] A method for separating and purifying extracellular vesicles from a sample containing extracellular vesicles,
(A1) a step of treating a sample containing extracellular vesicles with a hollow fiber membrane to concentrate the desired extracellular vesicles;
(B1) The extracellular vesicle concentrate thus obtained is brought into contact with a carrier carrying a peptide containing four or more consecutive lysines to form a complex in which the extracellular vesicle and the peptide are bound. and (B2) contacting the complex with a dissociation buffer containing metal cations to dissociate the extracellular vesicles from the complex. Cell purification method.
[2] The method for purifying extracellular vesicles according to [1], wherein the extracellular vesicle-containing sample is cell culture supernatant, biological tissue, or its treated fluid.
[3] The method for purifying extracellular vesicles according to [1] or [2], further comprising (A0) removing contaminants before the (A1) step.
[4] Purification of extracellular vesicles according to any one of [1] to [3], further comprising a step of (A2) exchanging the solvent of the extracellular vesicle concentrate after step (A1). Method.
[5] Any one of [1] to [4], further comprising (A3) a step of further concentrating the extracellular vesicle concentrate after the solvent exchange after step (A1) or step (A2). A method for purifying extracellular vesicles as described.
[6] Using a hollow fiber membrane in step (A0), step (A2) or step (A3),
The method for purifying extracellular vesicles according to [5].
[7] The method for purifying extracellular vesicles according to any one of [1] to [6], wherein the hollow fiber membrane has a cut-off molecular weight of 100 kDa to 500 kDa.
[8] The method for purifying extracellular vesicles according to [6] or [7], wherein the hollow fiber membrane used in the step (A0) has a pore size of 0.2 μm to 0.8 μm.
[9] The extracellular small molecule according to any one of [1] to [8], wherein the material of the hollow fiber membrane is polyethersulfone (PES), modified polyethersulfone (mPES) or polyacrylonitrile (PAN). Cell purification method.
[10] The method for purifying extracellular vesicles according to any one of [1] to [9], wherein the hollow fiber membrane constitutes a hollow fiber membrane module.
[11] In step (B1), ε-amino group and carboxyl group are peptide-bonded between 4 or more consecutive lysine residues in the peptide containing 4 or more consecutive lysines, [1] to [ 10], the extracellular vesicle purification method according to any one of the above.
[12] of [1] to [11], wherein in step (B1), the carrier material is at least one selected from the group consisting of cellulose, agarose, polystyrene, polyacrylamide, polymethacrylic acid, and silica; The method for purifying extracellular vesicles according to any one of the above.

According to the present invention, extracellular vesicles such as exosomes can be easily separated, concentrated and purified from a large amount of cell culture media and the like, and the purity of the obtained exosomes is excellent. Furthermore, according to the present invention, it is possible to obtain extracellular vesicles that are highly effective in treating diseases, particularly extracellular vesicles that exhibit anti-inflammatory effects. Moreover, according to the present invention, there is the advantage that extracellular vesicles are less likely to be destroyed and aggregated because they are not damaged by centrifugation unlike conventional methods. Furthermore, the method of the present invention can easily scale up the system and automate each step.

FIG. 1 shows electron micrographs of extracellular vesicles purified by various methods.

The present invention will be described in detail below.

<Method for purifying extracellular vesicles>
The present invention provides a method for separating and purifying extracellular vesicles from a sample containing extracellular vesicles,
(A1) a step of treating a sample containing extracellular vesicles with a hollow fiber membrane to concentrate the desired extracellular vesicles; contacting a carrier carrying a peptide containing 4 or more lysines to form a complex in which the extracellular vesicle and the peptide are bound; and (B2) comprising the complex and a metal cation. It is characterized by including a step of contacting with a dissociation buffer to dissociate the extracellular vesicles from the complex. Before the step (A1), (A0) removing contaminants, and after the step (A1), (A2) exchanging the solvent of the extracellular vesicle concentrate, and/or (A3) It may further include a step of further concentrating the extracellular vesicle concentrate after the solvent exchange. According to the method of the present invention, extracellular vesicles such as exosomes can be easily separated, concentrated, and purified from a large amount of cell culture medium by the step of using a hollow fiber membrane, and four or more consecutive lysines can be separated. The step ((B1) step and (B2) step) using a carrier supported peptide containing, it is possible to further improve the purity of exosomes. Here, "concentration" in the case of "concentrating the desired extracellular vesicles" means that the ratio of extracellular vesicles of a desired size is increased from a sample containing extracellular vesicles using a hollow fiber membrane. Collecting the liquid which does not pass through the hollow fiber membranes and collecting the liquid which passes through the hollow fiber membranes are also included. It also includes simply reducing the amount of solvent to increase the concentration of extracellular vesicles of the desired size.

(extracellular vesicle)
Extracellular vesicles are a general term for nanometer-sized vesicles released from cells, and are classified into exosomes, microvesicles, apoptotic bodies, and the like. Extracellular vesicles in the present invention contain at least exosomes, preferably high-purity exosomes.

(exosome)
Exosomes are extracellular vesicles with a diameter of about 50 nm to 150 nm, which are derived from cells, and are present in large amounts in all body fluids such as blood, saliva, and urine. Exosomes contain cell membrane-derived lipids and proteins on their surfaces, and contain nucleic acids and proteins such as mRNA and miRNA inside, and contain information derived from the released cells. Therefore, the information contained in exosomes can also be used as a biomarker, such as a diagnostic marker. In addition, it is known that these biological information molecules composed of nucleic acids and proteins function in cells that have taken up exosomes, and it has been revealed that intercellular communication occurs through the exchange of nucleic acids and proteins via exosomes. . Therefore, it is believed that exosomes can be used not only for diagnosis but also for prevention and treatment of diseases.

The term diameter or particle size when specifying the size of extracellular vesicles or exosomes, as used herein, refers to the largest dimension of an extracellular vesicle, as extracellular vesicles are not necessarily spherical. and The diameter or particle size may be measured using conventional techniques for measuring nanoparticle size, such as microscopy techniques (eg, transmission electron microscopy) or light scattering techniques. It can also be measured using Nanoparticle Tracking Analysis (NTA), which is based on both light scattering and Brownian motion analysis. In the present invention, the diameter or particle size of extracellular vesicles is determined using NanoSight (Malvern Panalytical, NanoSight L10) for extracellular vesicles with a particle size of 50 nm or more, and for extracellular vesicles with a particle size of less than 50 nm. , measured using a transmission electron microscope. In addition, for counting the number of exosomes, an exosome counting system (ExoCounter, JVC Kenwood) or the like can be used that employs a surface antigen-specific sandwich detection method with antibodies of discs and nanobeads.

Molecular weight is sometimes used in specifying the size of extracellular vesicles and exosomes, where the term molecular weight refers to the sum of the atomic weights of all atoms in the molecule. Therefore, the molecular weight of extracellular vesicles or exosomes is the total atomic weight of all atoms contained in the extracellular vesicles or exosomes. The molecular weight of extracellular vesicles and exosomes can be determined using membranes with specific cutoffs. As is well known to those skilled in the art, when a composition containing exosomes passes through a 300 kDa cut-off membrane, extracellular vesicles and exosomes present in the resulting residue have a molecular weight of 300 kDa or greater. On the other hand, the molecular weight of exosomes present in the recovered liquid is less than 300 kDa.

(Sample containing extracellular vesicles)
In the method of the present invention, the "sample containing extracellular vesicles" is not particularly limited as long as it is a mixture containing extracellular vesicles. Examples include collected specimens and the like. Specifically, culture supernatant of various cells, blood, plasma, serum, saliva, urine, tears, sweat, breast milk, amniotic fluid, cerebrospinal fluid (cerebrospinal fluid), bone marrow fluid, pleural fluid, ascites, synovial fluid, eye Examples include, but are not limited to, aqueous humor, vitreous humor, and the like. A sample containing extracellular vesicles can be appropriately selected depending on the purpose. Plasma, serum, saliva, urine and the like are preferred, and culture supernatants of various cells are more preferred.

The cells in the culture supernatant of the above various cells are not particularly limited, and may be animal cells, plant cells, fungi, or bacteria. Algae may be used, but cells that can be cultured under in vitro conditions are preferred, and mammalian cells that can be cultured under in vitro conditions are more preferred. In addition, as long as the cells can be cultured under in vitro conditions, they may be cells that form tissues excised from mammals or cells that have been established in vitro.

Mammal-derived cells that can be cultured under in vitro conditions include, for example, mesenchymal stem cells, other stem cells (embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, skin stem cells, etc.), tissue progenitor cells, tissue cells (bone cells, osteoblasts, adipocytes, chondrocytes, skin cells, nerve cells, muscle cells, blood cells, fibroblasts, liver cells, etc.); Among these, mesenchymal stem cells are preferable from the viewpoint that the obtained extracellular vesicles have various physiological activities and have a therapeutic effect on inflammatory diseases and the like. Mesenchymal stem cells include, for example, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells, depending on the origin of the mesenchymal stem cells. More preferred are adipose-derived mesenchymal stem cells. In the present invention, extracellular vesicle-derived cells may be autologous, allogeneic, or heterologous, but are preferably allogeneic.

The method for obtaining the culture supernatant of the various cells described above is not particularly limited, but can be performed, for example, as follows.

In the present invention, the cell culture method is not particularly limited as long as it is suitable for each cell, and conventionally known methods for those skilled in the art are used. It is carried out under 7% CO ₂ environment, 5%-21% O ₂ environment. Also, the timing and method of passage of cells are not particularly limited as long as they are suitable for each cell, and can be carried out in the same manner as in the past while observing the condition of the cells.

As the medium used for cell culture, those conventionally known to those skilled in the art can be selected and used for each cell type, and are not particularly limited. From the viewpoint of using the extracellular vesicles derived from the culture supernatant for humans, etc., a serum-free medium that does not contain animal serum is preferable. When the extracellular vesicles derived from the culture supernatant are used for purposes other than administration to humans or the like, the medium may contain animal-derived serum.

The serum-free medium is not particularly limited as long as it does not contain animal serum. A known basal medium having a composition containing additives other than animal serum can be used. The composition of the basal medium can be appropriately selected according to the type of cells to be cultured. For example, Minimum Essential Medium (MEM) such as Eagle's Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium α (MEM-α), Mesenchymal Stem Cell Basal Medium (MSCBM), Ham's F-12 and F -10 medium, DMEM/F12 medium, Williams medium E, RPMI-1640 medium, MCDB medium, 199 medium, Fisher medium, Iscove's modified Dulbecco's medium (IMDM), McCoy's modified medium and the like.

Other additives added to the basal medium include amino acids, inorganic salts, vitamins and other additives such as carbon sources and antibiotics. Concentrations of these additives to be used are not particularly limited, and they can be used at commonly used concentrations.

(Hollow fiber membrane, hollow fiber membrane module)
A hollow fiber membrane is one form of use of a permeable membrane, and refers to a membrane body formed into a thin straw-like tubule. In the hollow fiber membrane module, a bundle of hollow fiber membranes in which a large number of the above hollow fiber membranes are bundled is stored in a sealed state in a housing container made of a synthetic resin such as vinyl chloride or a metal such as aluminum. In general, the diameter (inner diameter) of each hollow fiber membrane is about 100 μm to several mm, but it is preferably 100 μm to 500 μm, more preferably 100 μm to 300 μm, in order to efficiently circulate the liquid. is more preferred. In addition, the length of each hollow fiber membrane can be adjusted according to the application. The length, so-called effective length, is preferably 120 mm to 450 mm, more preferably 100 mm to 300 mm, even more preferably 150 mm to 250 mm. The thickness of the hollow fiber membrane is generally about 10 μm to 100 μm, preferably 20 μm to 60 μm, more preferably 20 μm to 50 μm, even more preferably 30 μm to 50 μm. In the present invention, from the viewpoint of purification efficiency, it is preferable to use a hollow fiber membrane module (the hollow fiber membrane constitutes the hollow fiber membrane module).

The membrane area of the hollow fiber membranes in the hollow fiber membrane module used in the method of the present invention is a converted value in the middle part of the thickness, that is, the average value of the outer surface area and the inner surface area, considering the diameter difference between the inside and outside of the hollow fiber membrane. can be Since the membrane area of the hollow fiber membrane and the processing speed are in a proportional relationship, using a hollow fiber membrane module with a large membrane area increases the processing speed, so the time required for processing can be shortened. It can reduce the damage taken by cells. On the other hand, since there is a proportional relationship between the membrane area of the hollow fiber module and the dead volume (remaining amount in the flow path) during purification, the use of a hollow fiber module with a large membrane area increases the loss accordingly. Therefore, it is preferable to select a hollow fiber membrane module having an appropriate membrane area according to the purpose. The membrane area of the hollow fiber membrane in the hollow fiber membrane module used in the method of the present invention is appropriately selected from the range of about 1 cm ² to 3,000 cm ² according to the purpose. For example, when the purpose is to concentrate to a small volume, it is preferable to use a module with a small membrane area of about 20 cm ² . In addition, when the purpose is concentration, filtration, removal of contaminants, etc. of the cell culture supernatant, for example, the membrane area is about 115 cm ² , 1,000 cm ² , or 1,600 cm ² depending on the amount of treatment. Preference is given to using hollow fiber membrane modules.

The pore size of the hollow fiber membrane (including the hollow fiber membrane provided in the hollow fiber membrane module) used in the method of the present invention can be selected according to the purpose. When removing substances, select a pore size that does not allow permeation of only the contaminants. Such a pore size is 0.05 μm to 2 μm, preferably 0.1 μm to 1 μm, more preferably 0.2 μm to 0.8 μm, and 0.2 μm to 0.5 μm. is more preferred, and 0.22 μm is particularly preferred. When intentionally leaving microvesicles, it is preferable to use vesicles with a pore size of 0.05 μm to 1 μm. In addition, when removing unnecessary small size particles and debris from the culture supernatant etc. after removing contaminants larger than extracellular vesicles, leaving the necessary extracellular vesicles, the cutoff molecular weight It is preferred to use a cut-off membrane with a λ of 100 kDa to 1,000 kDa, more preferably a cut-off membrane of 120 kDa to 800 kDa, even more preferably a cut-off membrane of 150 kDa to 500 kDa, and a cut-off membrane of 300 kDa. is particularly preferred. In this case, by continuously diluting and concentrating the culture supernatant after removing contaminants, unwanted substances of small size are removed, and at the same time, the solvent is exchanged to obtain extracellular small cells of the required size. You can get cells. Furthermore, when further concentrating the extracellular vesicles obtained in the above step, a hollow fiber membrane having a size that allows medium and solvent to permeate may be used, and a cut-off membrane with a cut-off molecular weight of 10 kDa to 500 kDa. , more preferably a 50 kDa to 300 kDa cutoff membrane, even more preferably a 100 kDa to 200 kDa cutoff membrane, and particularly preferably a 100 kDa cutoff membrane.

The material of the hollow fiber membrane provided in the hollow fiber membrane module used in the method of the present invention is not particularly limited as long as it is a material that can be used for the purpose of appropriately separating cell-derived extracellular vesicles. Sulfone (PES), modified polyethersulfone (mPES), polyacrylonitrile (PAN) and the like. Among these, polyethersulfone (PES) and modified polyethersulfone (mPES) are preferable, and modified polyethersulfone (mPES) is more preferable, from the viewpoint of high hydrophilicity and low protein adsorption.

(Peptide containing 4 or more consecutive lysines)
The “peptide containing 4 or more consecutive lysines (hereinafter also referred to as the unit “peptide of the present invention”)” used in the method of the present invention is a peptide containing 4 or more consecutively bound lysines, It can preferably bind to exosomes in a sample, particularly CD63-positive exosomes. Other configurations of the peptide of the present invention are not particularly limited as long as it contains four or more consecutive lysines. may In addition, the peptide of the present invention may be a composite peptide further comprising a non-peptide structure such as a sugar chain or an isoprenoid group. The amino acids contained in the peptides of the present invention may be modified, and the amino acids may be in L-form or D-form. Furthermore, the peptide of the present invention may be one in which the amino group at the α-position and the carboxyl group in the amino acid are peptide-bonded, and the amino group contained in the side chain of the amino acid and the carboxyl group at the α-position are peptide-bonded. It may be a peptide bond between the amino group at the α-position in the amino acid and the carboxyl group contained in the side chain of the amino acid, and the amino group contained in the side chain of the amino acid and the side of the amino acid A peptide bond with the carboxyl group contained in the chain may also be used.

Peptides of the present invention, containing four or more consecutive lysines, can bind to exosomes with a binding strength sufficient to isolate exosomes. Peptides of the present invention contain 4 or more consecutive lysines, and from the viewpoint of improving binding to exosomes, preferably contain 10 or more consecutive lysines, and 20 or more consecutive lysines. More preferably, it contains lysine, more preferably contains 25 or more consecutive lysines, even more preferably contains 30 or more consecutive lysines, and contains 35 or more consecutive lysines. is particularly preferred. Also, the peptide of the present invention preferably contains 40 or less consecutive lysines.

Contiguous 4 or more lysines contained in the peptide of the present invention, from the viewpoint of improving the binding property to exosomes, the L-form is preferred. In addition, from the viewpoint of improving the binding property to exosomes, the consecutive 4 or more lysines contained in the peptide of the present invention are those in which the lysine residues are peptide-bonded with the ε-amino group and the carboxyl group. preferable.

The peptides of the invention can be produced by methods known in the art. For example, it may be expressed by naturally occurring microorganisms, or may be expressed by transformants. Furthermore, the peptide of the present invention may be chemically synthesized by a liquid phase method or the like. A commercially available polylysine can also be used as the peptide of the present invention.

(Carrier)
The carrier used in the method of the present invention (hereinafter also simply referred to as "the carrier of the present invention") is a carrier carrying the peptide of the present invention. By carrying the peptide of the present invention, the carrier of the present invention can specifically bind exosomes. On the carrier of the present invention, exosomes and the peptide of the present invention form a complex.

Other constitutions of the carrier of the present invention are not particularly limited as long as it is a structure capable of directly or indirectly supporting 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. Examples include cellulose, agarose, polystyrene, polyacrylamide, polymethacrylic acid, silica, glass, nylon membrane, Examples include semiconductor wafers, latex, microbeads, and magnetic beads. Among these, the carriers of the present invention are cellulose, agarose, polystyrene, polyacrylamide, and polymethacryl, which are suitable for column purification in that they allow easy recovery of complexes containing exosomes and isolation of exosomes. Acid and silica are preferred, and cellulose is more preferred.

The mode of binding between the peptide of the present invention and the carrier is not particularly limited, and the binding between the peptide and the carrier may be direct or indirect. Also, the binding between the peptide of the present invention and a carrier may be via a linker comprising a polypeptide.

In addition, in the method of the present invention, a commercially available carrier supporting the peptide of the present invention can also be used. Examples of such commercial products include Exopure (ExoPUA (registered trademark), manufactured by Silicon Bio).

(dissociation buffer)
In the method of the present invention, a dissociation buffer containing metal cations (hereinafter simply referred to as Also referred to as "the dissociation buffer of the present invention") is used. The dissociation buffer of the present invention does not contain a protein denaturant or the like and can dissociate exosomes from the complex under mild conditions, so exosomes can be isolated in a nearly intact state.

The metal cation contained in the dissociation buffer of the present invention is not particularly limited, and examples include monovalent metal cations such as sodium ion, potassium ion, lithium ion, silver ion, and copper (I) ion; 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 trivalent metal cations such as aluminum ions and iron (III) ions; As the metal cations contained in the dissociation buffer of the present invention, sodium ions, potassium ions, magnesium ions, zinc ions, and nickel ions are preferable among these metal cations because of their high water solubility. Considering that extracellular vesicles (exosomes) can be sufficiently dissociated, it is more preferable to contain sodium ions, potassium ions, or magnesium ions alone or in combination.

The concentration of metal cations in the dissociation buffer of the present invention is capable of dissociating extracellular vesicles (exosomes) from the complex, and proteins present in the membrane structure and membrane of extracellular vesicles (exosomes) is not particularly limited as long as it does not affect the structure of In other words, the optimal concentration of cations can vary depending on the binding strength between the peptide of the present invention and extracellular vesicles (exosomes) used, and the type (valence) of the metal cations. can be determined after considering The concentration of metal cations in the dissociation buffer of the present invention is not particularly limited. .2 to 0.6M is more preferred.

The pH of the dissociation buffer of the present invention is preferably 5.0 to 9.0, more preferably 5.5 to 8.5, even more preferably 6.0 to 8.0. If the pH of the dissociation buffer is within the above range, it is preferable because it does not affect the membrane structure of extracellular vesicles (exosomes) and the structure of proteins present in the membrane.

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

(Regarding each step of the extracellular vesicle purification method of the present invention)
The method for purifying extracellular vesicles of the present invention is divided into steps and specifically explained, but the present invention is not limited to the methods shown below.

The extracellular vesicle purification method of the present invention comprises
(A0) a step of removing contaminants,
(A1) a step of treating a sample containing extracellular vesicles with a hollow fiber membrane to concentrate the desired extracellular vesicles;
(A2) a step of exchanging the solvent of the extracellular vesicle concentrate obtained;
(A3) a step of further concentrating the extracellular vesicle concentrate after solvent exchange;
(B1) The extracellular vesicle concentrate thus obtained is brought into contact with a carrier carrying a peptide containing four or more consecutive lysines to form a complex in which the extracellular vesicle and the peptide are bound. and (B2) contacting the complex with a dissociation buffer containing metal cations to dissociate the extracellular vesicles from the complex.

(A0) Step of removing contaminants In this step, a suction filtration unit or a hollow Filtration is performed using a fiber membrane module. If necessary, before filtration, the cell culture supernatant may be centrifuged under conditions such as 2,000×g for 20 minutes to precipitate cells and large debris, and the supernatant may be used. A membrane filter with a pore size of 0.22 μm is set in the suction filtration unit, and a conventional method can be used. Further, when a hollow fiber membrane module is used, the pore size of the hollow fiber membrane is 0.05 μm to 2 μm, preferably 0.1 μm to 1 μm, more preferably 0.2 μm to 0.8 μm. It is preferably 0.2 μm to 0.5 μm, particularly preferably 0.22 μm. When microvesicles are to be left, it is preferable to use vesicles with a pore size of 0.05 μm to 1 μm. In this step, the amount of sample that can be processed can be adjusted by changing the size of the filtration device or the hollow fiber membrane module.

Specific suction filtration units used in this step include, for example, filter systems such as Corning 431097 and ThermoFisher 566-0020. Examples of the hollow fiber membrane module include C02P20U05N for laboratory scale and S02P20U05N (both manufactured by Repligen) for large-scale processing.

(A1) A step of treating a sample containing extracellular vesicles with a hollow fiber membrane to concentrate the desired extracellular vesicles. In this step, the sample containing extracellular vesicles after removing contaminants. From (cell culture supernatant, etc.), leaving the necessary extracellular vesicles, unnecessary small-sized particles and debris are removed. Specifically, a sample (cell culture supernatant, etc.) from which contaminants have been removed is introduced into a feed bottle or the like, and a pump is operated to load the hollow fiber membrane module. Thereafter, a total of about 50 to 150 times the volume of the sample is permeated with PBS or the like to concentrate extracellular vesicles of a desired size while leaving them in the hollow fiber membrane module to obtain the desired concentrate of extracellular vesicles. can be obtained and washed. The filtrate is pumped to a waste liquid tank or the like. In this step, the size of extracellular vesicles to be collected can be adjusted by appropriately selecting the hollow fiber membranes provided in the hollow fiber membrane module. In the purification method of the present invention, the hollow fiber membrane used in this step is preferably a cut-off membrane of 100 kDa to 1,000 kDa, more preferably a cut-off membrane of 120 kDa to 800 kDa, more preferably 150 kDa to 500 kDa. is more preferred, and a 300 kDa cut-off membrane is particularly preferred. By selecting such a cut-off membrane, extracellular small particles such as exosomes with a particle size of 50 nm or more (50 nm or more and 220 nm or less, preferably 50 nm or more and 150 nm or less) can be extracted from samples such as culture supernatants of mesenchymal stem cells and the like. In addition to vesicles, it is possible to obtain an extracellular vesicle population having an anti-inflammatory effect that contains extracellular vesicles with a particle size of less than 50 nm (1 nm or more and less than 50 nm, preferably 10 nm or more and less than 50 nm). Also in this step, the amount of culture supernatant that can be treated can be adjusted by changing the size of the hollow fiber membrane module.

Specific hollow fiber membrane modules used in this step include commercially available products such as C02E30005N for laboratory scale and C04N30005N (both manufactured by Repligen) for large-scale processing.

(A2) A step of exchanging the solvent of the obtained extracellular vesicle concentrate In this step, the concentrate obtained in the above step is diluted with a target solution, an ultrafiltration device, a hollow fiber membrane , the concentration in the hollow fiber membrane module or the like is performed once or repeatedly to exchange the solvent of the concentrate obtained in the above step. A cut-off membrane having a pore size that allows medium and solvent to permeate is set in the ultrafiltration device. It is preferably a cut-off membrane with a cut-off molecular weight of 10 kDa to 500 kDa, more preferably a cut-off membrane of 50 kDa to 300 kDa, even more preferably a cut-off membrane of 100 kDa to 200 kDa, and a cut-off membrane of 100 kDa. is particularly preferred. In addition, when a hollow fiber membrane or a hollow fiber membrane module is used in this step, the hollow fiber membrane to be used may be of a size that allows the culture medium and solvent to permeate, as in the above. It is preferably a 10 kDa to 500 kDa cutoff membrane, more preferably a 50 kDa to 300 kDa cutoff membrane, even more preferably a 100 kDa to 200 kDa cutoff membrane, and preferably a 100 kDa cutoff membrane. Especially preferred. By selecting such a cut-off membrane, it is possible to replace the obtained extracellular vesicles with an appropriate solvent without losing them.

Specific hollow fiber membrane modules used in this step include commercially available products such as C02E10005N (manufactured by Repligen).

(A3) Step of further concentrating the extracellular vesicle concentrate after solvent exchange In this step, the extracellular vesicle concentrate after solvent exchange obtained in the above step is subjected to an ultrafiltration device, hollow fibers It is further concentrated using a membrane, hollow fiber membrane module, or the like. A cut-off membrane having a pore size that allows medium and solvent to permeate is set in the ultrafiltration device. It is preferably a cut-off membrane with a cut-off molecular weight of 10 kDa to 500 kDa, more preferably a cut-off membrane of 50 kDa to 300 kDa, even more preferably a cut-off membrane of 100 kDa to 200 kDa, and a cut-off membrane of 100 kDa. is particularly preferred. In addition, when a hollow fiber membrane module is used in this step, the hollow fiber membrane may be of a size that allows medium and solvent to permeate, and has a cut-off molecular weight cutoff of 10 kDa to 500 kDa. It is preferably a membrane, more preferably a 50 kDa to 300 kDa cutoff membrane, even more preferably a 100 kDa to 200 kDa cutoff membrane, particularly preferably a 100 kDa cutoff membrane. By selecting such a cut-off membrane, it is possible to remove unnecessary substances and concentrate the extracellular vesicles to an appropriate concentration without losing the obtained extracellular vesicles.

Specific hollow fiber membrane modules used in this step include commercially available products such as C02E10005N (manufactured by Repligen).

The concentrate of extracellular vesicles purified and concentrated as described above may be collected in the feed bottle and further concentrated using a small-scale hollow fiber membrane module as necessary. In that case, instead of using a pump, manual concentration using a syringe, an ultrafiltration device, or the like may be performed.

The obtained concentrate of extracellular vesicles may be filtered through a 0.22 μm filter in a clean bench for the purpose of sterilization and removal of aggregates generated in the purification step.

According to the purification method of the present invention, it is possible to process up to about 5 mL to 50 L of cell culture supernatant, so extracellular vesicles can be prepared in large quantities as needed for industrial use. It should be noted that each of the above steps can be controlled and automated by software that controls a series of operations.

(B1) The extracellular vesicle concentrate thus obtained is brought into contact with a carrier carrying a peptide containing four or more consecutive lysines to form a complex in which the extracellular vesicle and the peptide are bound. Forming step (complex forming step)
In this step, the concentrate obtained in a series of steps A (sample containing desired extracellular vesicles (exosomes, etc.)) and the present invention containing 4 or more consecutive lysines supported on the carrier of the present invention By contacting with the peptide, the step of forming a complex in which the extracellular vesicles (exosomes, etc.) and the peptide are bound. The method of contacting the extracellular vesicles (exosomes, etc.) with the peptide of the present invention is not particularly limited, but for example, a columnar or cylindrical column with a carrier carrying the peptide of the present invention. A preferred method is a method using a column prepared by packing. By passing the concentrate obtained in step A through the column, the extracellular vesicles (exosomes, etc.) and the peptide of the present invention supported on the carrier of the present invention are brought into contact to obtain a complex. Is possible. In this step, a commercially available ε-polypeptide carrier or the like containing 4 or more lysines, in which the peptide of the present invention is previously supported on the carrier of the present invention, is used. The columns may be made by packing the columns.

As a specific method for passing the concentrate obtained in step A through the column, a method conventionally known to those skilled in the art can be used. For example, the column packed with the carrier supporting the peptide of the present invention is placed in a phosphate buffer, Tris buffer, Bis-Tris buffer, or After substitution with a binding buffer such as HEPES buffer (pH 6.0 to 8.0), etc., the concentrated solution obtained in step A (extracellular vesicles (exosomes, etc.) was added to the sample, gravity or centrifugal force or by pressurizing from the top or sucking from the bottom.In addition, the concentrated solution obtained in step A (sample containing extracellular vesicles (exosomes, etc.) contains a binding buffer May be mixed.

The contact time between the column and the sample is a time sufficient for extracellular vesicles (exosomes, etc.) in the sample and the peptide of the present invention supported on the carrier of the present invention to form a complex, and the sample It can be set as appropriate by adjusting the flow rate of the solution containing. Since the liquid flow rate varies depending on the viscosity of the sample, the optimum flow rate changes depending on the viscosity of the sample.

In this step, without using a column, the carrier to which the peptide of the present invention is bound and the sample are mixed and brought into contact, and extracellular vesicles (such as exosomes) - the peptide of the present invention - the carrier of the present invention in this order. Bound complexes may be formed.

A washing step is preferably included after this step and before step (B2), which will be described later. In the washing step, substances that do not bind to the peptide of the present invention and do not form a complex are removed by washing with a washing solution. The washing step is carried out under conditions in which the complex does not dissociate, and the type of washing solution used and the number of times of washing are not particularly limited. It is preferable to wash until the A260 (absorption at 260 nm) and A280 (absorption at 280 nm) are sufficiently lowered while checking with a monitor or the like. Examples of the washing liquid include buffers such as phosphate buffer (PBS).

(B2) A step of contacting the complex with a dissociation buffer containing metal cations to dissociate the extracellular vesicles from the complex. This step includes the complex and a dissociation buffer containing metal cations. (Dissociation buffer of the present invention described above) is brought into contact to dissociate extracellular vesicles such as exosomes from the complex.

As a method for contacting the complex with the dissociation buffer of the present invention, for example, the dissociation buffer of the present invention is passed through the column in which the complex of the present invention was formed in the complex formation step. A complex of the invention can be contacted with a dissociation buffer of the invention. As a specific method for passing the dissociation buffer of the present invention through the column, the dissociation buffer is added and passed through the column by gravity or centrifugal force, or by pressurizing from the top or sucking from the bottom. method. When the column is brought into contact with the dissociation buffer of the present invention, extracellular vesicles (exosomes, etc.) are dissociated from the complex and migrate into the dissociation buffer.

The contact time between the complex and the dissociation buffer of the present invention is a sufficient time for extracellular vesicles (such as exosomes) to dissociate from the complex, and the flow rate of the solution containing the sample is adjusted. It can be appropriately set by, for example.

In order to efficiently dissociate and recover extracellular vesicles (exosomes, etc.) from the above complex, gradually from a buffer solution that does not contain any dissociation buffer (dissociation buffer 0%) with the metal cation concentration as described above The content of the dissociation buffer may be gradually increased to 100% of the dissociation buffer, elution may be performed with a linear gradient of 0% to 100%, and fractions may be collected for each specific amount. After that, measure the content of extracellular vesicles (exosomes, etc.) in each fraction, collect only the fraction containing extracellular vesicles (exosomes, etc.), and extract the desired extracellular vesicle-containing Get the composition.

In this step, 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. From the standpoint of isolation, the peptide of the present invention preferably remains bound to the carrier of the present invention in this step.

When extracellular vesicles are purified using the mesenchymal stem cell culture supernatant as a starting material by the purification method of the present invention, the finally obtained extracellular vesicles (such as exosomes) have an average diameter of 50 nm or more and 220 nm or less. is preferably 50 nm or more and 200 nm or less, more preferably 50 nm or more and 150 nm or less, and even more preferably 120 nm or more and 150 nm or less.

When extracellular vesicles are purified using the mesenchymal stem cell culture supernatant as a starting material by the purification method of the present invention, the finally obtained extracellular vesicles have a diameter of 50 nm or more and 220 nm or less (preferably 50 nm or more and 150 nm). Below), extracellular vesicles such as exosomes account for 70% or more of the total number of particles, preferably 80% or more, and more preferably 90% or more.

When extracellular vesicles are purified using mesenchymal stem cell culture supernatant as a starting material by the purification method of the present invention, exosome markers CD9 and CD63 are expressed in the finally obtained extracellular vesicles. Exosome purity can be confirmed by measuring the percentage of vesicles. Extracellular vesicles obtained by the purification method of the present invention, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more are exosomes expressing CD9 and CD63. In addition, the total number of particles in the purified product containing extracellular vesicles (NTA method) and the number of exosome marker-positive particles (exocounter) are measured, respectively, and the exosome marker-positive particle rate relative to the total number of particles is calculated. Furthermore, since the extracellular vesicles obtained by the purification method of the present invention are less contaminated with impurities, the positive particle rate before purification is converted to 1, and the positive particle rate after purification is calculated. It is preferably 2.5 or more, more preferably 3 or more, and even more preferably 5.0 or more. Although the upper limit of this numerical value is preferably as high as possible, it is usually 20 or less, preferably 15 or less, and more preferably 10 or less. When the positive particle rate before purification is converted to 1, the positive particle rate after purification shows a higher value as impurity particles other than extracellular vesicles are removed, but the impurity particles are not removed. value close to 1.

When extracellular vesicles are purified using mesenchymal stem cell culture supernatant as a starting material by the purification method of the present invention, the finally obtained extracellular vesicles induce inflammatory macrophages into anti-inflammatory macrophages. It has an action, that is, an anti-inflammatory action, but this action is not seen in extracellular vesicles such as exosomes isolated and purified by conventionally known purification methods. Therefore, the extracellular vesicles obtained by the purification method of the present invention can be used in anticipation of preventive and/or therapeutic effects on inflammatory diseases.

<Extracellular vesicle purification system>
The scope of the present invention also includes an extracellular vesicle purification system including all of the reagents, devices, instruments, and control software for realizing the extracellular vesicle purification method of the present invention. For specific reagents, devices, instruments, control software, and details of the purification process, the description in the section on the method for purifying extracellular vesicles can be applied.

EXAMPLES The present invention will be described in more detail based on examples below, but the present invention is not limited to these examples.

[Purification of extracellular vesicles]
1. Preparation of mesenchymal stem cell culture supernatant Human adipose-derived mesenchymal stem cells (AD-MSC, Lonza) were cultured in a serum-free medium for mesenchymal stem cells (manufactured by Rohto Pharmaceutical Co., Ltd.) for 2 to 5 days, and the medium was collected. did. The collected medium was centrifuged at 400×g to obtain a mesenchymal stem cell culture supernatant.

2. Purification of extracellular vesicles (1) Example 1:
A composition containing extracellular vesicles was obtained from the mesenchymal stem cell culture supernatant obtained above using a hollow fiber membrane.

Specifically, first, the mesenchymal stem cell culture supernatant is filtered using a 0.22 μm filter or a 0.2 μm hollow fiber membrane module for removing contaminants (C02P20U05N, manufactured by Repligen) to remove contaminants. did. The material of the hollow fiber membranes of the hollow fiber membrane module is modified polyethersulfone (mPES).

The culture supernatant from which contaminants were removed was introduced into the feed bottle. Using the main pump, the culture supernatant was loaded from the feed bottle into a hollow fiber membrane module for purification (C02E30005N, manufactured by Repligen) and concentrated. Subsequently, the following liquid A was added to the concentrate for dilution, and then concentrated again using a hollow fiber membrane module. The material of the hollow fiber membranes of the hollow fiber membrane module is modified polyethersulfone (mPES). By this step, contaminants with a molecular weight of about 300 kDa or less were removed, and a concentrated solution of extracellular vesicles in which the solvent was replaced with solution A was obtained. As liquid A, a phosphate buffer (pH 7.0) containing 100 mM sodium chloride was used.

Next, the extracellular vesicle concentrate (70 to 90 mL) obtained in the above step was purified and concentrated using an exosome purification column for liquid chromatography (ExoPUA column, manufactured by Silicon Bio Co., Ltd.). . The ExoPUA column is a column packed with a cellulose carrier on which peptides (peptides containing four or more lysines in close proximity to each other) that bind to the lipid bilayer membrane of exosomes are immobilized. After equilibrating the ExoPUA column with liquid A (20 mL) using a tube pump, the extracellular vesicle concentrate (70 to 90 mL) was sent to the ExoPUA column. Next, liquid A (20 mL) was fed to wash impurities other than extracellular vesicles in the column. After that, liquid B (20 mL) was fed to elute the extracellular vesicles in the column and collected in a fractionating tube to obtain the extracellular vesicle-containing composition of Example 1. As liquid B, a phosphate buffer (pH 7.0) containing 600 mM sodium chloride was used.

(2) Comparative Example 1: Ultracentrifuge (UC)
The mesenchymal stem cell culture supernatant obtained above was centrifuged at 2,000×g for 20 minutes, and then filtered by suction using a suction filtration filter (Corning, 431096) to remove contaminants. The filtered culture supernatant was ultracentrifuged in the following steps. That is, 20 mL of the filtered culture supernatant was filled in an ultracentrifuge tube (Koki Holdings, S309156A) and centrifuged at 120,000 x g, 70 min, 4°C using an ultracentrifuge (Koki Holdings, CS100FNX). ultracentrifuged. The supernatant was removed from the ultracentrifugation tube, 20 mL of the filtered culture supernatant was newly filled, and the same operation was repeated to obtain a pellet prepared from a total of 80 mL of the culture supernatant. Further, 20 mL of D-PBS(-) was filled, followed by ultracentrifugation in the same manner, and the supernatant was removed. A pellet formed at the bottom of the ultracentrifugation tube was dissolved with 0.5 mL of D-PBS(-) to obtain an extracellular vesicle-containing composition of Comparative Example 1.

(3) Comparative Example 2: Polymer Sedimentation Method (Total Exosome Isolation Reagent: TEIR)
Using "Total Exosome Isolation Reagent (from cell culture media)" manufactured by Thermo Fisher SCIENTIFIC, purification was performed according to the attached protocol. 80 mL of the culture supernatant obtained above was centrifuged at 2,000×g for 20 minutes, and then suction filtered using a suction filtration filter (Corning, 431096) to remove contaminants. A 50 mL centrifugation tube (TPP, 91050) was filled with the filtered culture supernatant and Total Exosome Isolation Reagent at a ratio of 2:1, thoroughly mixed, and allowed to stand overnight at 4°C. The next day, it was centrifuged at 10,000×g, 60 min, 4° C. (KUBOTA, 6200). The supernatant was removed from the centrifuge tube, the inside of the container was washed with 1 mL of D-PBS(-), and the solution components were removed again. After the solution on the wall surface was removed by flashing, the pellet formed at the bottom was dissolved with 0.5 mL of D-PBS(−) to obtain an extracellular vesicle-containing composition of Comparative Example 2.

(4) Comparative Example 3: Size exclusion chromatography method (SEC)
Purification was performed using iZON's "qEV35" according to the attached protocol. 80 mL of the mesenchymal stem cell culture supernatant obtained above was centrifuged at 2,000×g for 20 minutes, followed by suction filtration using a suction filtration filter (Corning, 431096) to remove contaminants. Amicon Ultra-15 was filled with the filtered culture supernatant, and centrifugation was repeated at 5,000×g, 20 min, 4° C. until the volume of the supernatant reached 0.15 mL. The qEV35 column was fixed on a stand, the upper and lower caps were removed, and the buffer was discharged. A total of 3.5 mL of D-PBS(-) was added to the column and drained. A column was filled with 0.15 mL of the concentrated culture supernatant, and then filled and discharged with D-PBS(−) so that the total volume was 1 mL. 0.2 mL of D-PBS(−) was added/discharged and the discharged solutions were collected, and fractions 1 to 3 thereof were used as the extracellular vesicle-containing composition of Comparative Example 3.

3. Characterization of extracellular vesicle-containing composition (1) Protein concentration and exosome concentration and purity In addition to extracellular vesicles, the culture supernatant contains abundant proteins necessary for culture and proteins secreted by cells. . These proteins correspond to impurities in the extracellular vesicles that are finally purified, and there is concern that they may affect the quality of the extracellular vesicles and, in turn, their safety and efficacy. In this experiment, the protein concentration and exosome concentration (CD9/CD63 ELISA kit) of the extracellular vesicle-containing composition of Example 1 and Comparative Examples 1 to 3 were measured, and the ratio of exosome concentration to protein concentration (ppm) was measured respectively. The value was used as the purity of each composition. That is, the larger the amount of impurities brought in, the lower the purity, and the more impurities are removed, the higher the purity. The results are shown in Table 1 below.

As shown in Table 1, the purity of the culture supernatant before purification was 0.054, whereas the purity after purification was 20, 12, 2.0 in Example 1 and Comparative Examples 1 to 3, respectively. 4 and 8.3. Thus, it was confirmed that extracellular vesicles were purified by any method, and it was confirmed that the extracellular vesicles purified by Example 1 had the highest purity.

(2) Total number of particles and exosome marker (CD63/CD9) positive particle number and positive particle rate Among the particles in the culture supernatant, in addition to extracellular vesicles, fragments of dead cells, viruses, aggregates of proteins The possibility of inclusion of particles such as lumps and precipitated salts cannot be denied. These particles correspond to impurities in the extracellular vesicles that are finally purified, and there are concerns that they may affect the quality of the extracellular vesicles and, in turn, the safety and efficacy of the extracellular vesicles. Therefore, the total number of particles (NTA method) and the number of exosome marker-positive particles (exocounter) of the extracellular vesicle-containing composition of Example 1 and Comparative Examples 1 to 3 were measured, respectively, exosome markers for the total number of particles A positive particle rate was calculated. Furthermore, the positive particle rate before purification was converted to 1, and changes in the positive particle rate after purification are shown in Table 2 below. That is, the more impurity particles other than extracellular vesicles are removed, the higher the value is, but the value is close to 1 when the impurity particles are not removed.

As a result, as shown in Table 2, the fluctuations in the exosome marker-positive particle rate after purification were 5.6, 0.9, 0.9 and 0.8 in Example 1 and Comparative Examples 1 to 3, respectively. there were. In Example 1, the positive particle rate was higher than in Comparative Examples 1 to 3, and it was confirmed that impurity particles were removed. On the other hand, in Comparative Examples 1 to 3, it was confirmed that the particles were non-selectively recovered without removing the impurity particles. Since the extracellular vesicles of Example 1 have a high positive particle rate, they also have an excellent anti-inflammatory effect.

(3) Electron Microscopic Observation The extracellular vesicles of Example 1 and Comparative Examples 1 to 3 were observed by a transmission electron microscope (FIG. 1). As a result, it was found that the exosomes purified by the present invention have a well-maintained spherical lipid bilayer membrane structure and remarkably low contaminants in the surroundings.

According to the present invention, extracellular vesicles such as exosomes can be easily separated, concentrated and purified from a large amount of cell culture media and the like, and the purity of the obtained exosomes is excellent. Furthermore, according to the present invention, it is possible to obtain extracellular vesicles that are highly effective in treating diseases, particularly extracellular vesicles that exhibit anti-inflammatory effects. Moreover, according to the present invention, there is the advantage that extracellular vesicles are less likely to be destroyed and aggregated because they are not damaged by centrifugation unlike conventional methods. Furthermore, the method of the present invention can easily scale up the system and automate each step.

Claims (12)

A method for separating and purifying extracellular vesicles from a sample containing extracellular vesicles,
(A1) a step of treating a sample containing extracellular vesicles with a hollow fiber membrane to concentrate the desired extracellular vesicles;
(B1) The extracellular vesicle concentrate thus obtained is brought into contact with a carrier carrying a peptide containing four or more consecutive lysines to form a complex in which the extracellular vesicle and the peptide are bound. and (B2) contacting the complex with a dissociation buffer containing metal cations to dissociate the extracellular vesicles from the complex. Cell purification method. 2. The method for purifying extracellular vesicles according to claim 1, wherein the sample containing extracellular vesicles is cell culture supernatant, biological tissue or its treated fluid. 3. The method for purifying extracellular vesicles according to claim 1, further comprising (A0) removing contaminants before the (A1) step. 4. The method for purifying extracellular vesicles according to any one of claims 1 to 3, further comprising the step of (A2) exchanging the solvent of the extracellular vesicle concentrate after the step (A1). 5. The cell according to any one of claims 1 to 4, further comprising (A3) a step of further concentrating the extracellular vesicle concentrate after the solvent exchange after the step (A1) or (A2). Method for purification of outer vesicles. (A0) step, (A2) step or (A3) step, using a hollow fiber membrane,
The method for purifying extracellular vesicles according to claim 4. The method for purifying extracellular vesicles according to any one of claims 1 to 6, wherein the hollow fiber membrane has a cut-off molecular weight of 100 kDa to 500 kDa. 8. The method for purifying extracellular vesicles according to claim 6 or 7, wherein the hollow fiber membrane used in step (A0) has a pore size of 0.2 μm to 0.8 μm. Purification of extracellular vesicles according to any one of claims 1 to 8, wherein the material of the hollow fiber membrane is polyethersulfone (PES), modified polyethersulfone (mPES) or polyacrylonitrile (PAN). Method. The method for purifying extracellular vesicles according to any one of claims 1 to 9, wherein the hollow fiber membrane constitutes a hollow fiber membrane module. 11. Any one of claims 1 to 10, wherein in the step (B1), the ε-amino group and the carboxyl group are peptide-bonded between the 4 or more consecutive lysine residues in the peptide containing 4 or more consecutive lysines. 2. The method for purifying extracellular vesicles according to item 1. 12. The method according to any one of claims 1 to 11, wherein in step (B1), the carrier material is at least one selected from the group consisting of cellulose, agarose, polystyrene, polyacrylamide, polymethacrylic acid, and silica. A method for purifying extracellular vesicles as described.