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WO2025121435A1 - Procédé de séparation de vésicules extracellulaires et kit de séparation de vésicules extracellulaires - Google Patents

Procédé de séparation de vésicules extracellulaires et kit de séparation de vésicules extracellulaires Download PDF

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WO2025121435A1
WO2025121435A1 PCT/JP2024/043415 JP2024043415W WO2025121435A1 WO 2025121435 A1 WO2025121435 A1 WO 2025121435A1 JP 2024043415 W JP2024043415 W JP 2024043415W WO 2025121435 A1 WO2025121435 A1 WO 2025121435A1
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extracellular vesicles
type lectin
asgr1
cells
vesicles
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浩章 舘野
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National Institute of Advanced Industrial Science and Technology AIST
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor

Definitions

  • the present disclosure relates to a method for isolating extracellular vesicles and a kit for isolating extracellular vesicles.
  • Extracellular vesicles are spherical membrane vesicles composed of a lipid bilayer membrane of approximately 30 to 1000 nm, and are secreted extracellularly from all cells.
  • Extracellular vesicles contain bioinformation molecules such as DNA, RNA (mRNA, miRNA, ncRNA, etc.), proteins, and sugar chains within the vesicles, which act as signal transmitters to mediate various physiological functions.
  • Extracellular vesicles are being used to diagnose various diseases such as cancer by detecting the miRNA contained therein.
  • extracellular vesicles derived from mesenchymal stem cells are being used in therapeutic preparations because they have anti-inflammatory, immunoregulatory, and tissue repair effects.
  • JP 2021-012200 A International Publication No. 2016/088689
  • the present disclosure aims to provide a method for isolating extracellular vesicles and a kit for isolating extracellular vesicles.
  • the present disclosure includes the following aspects.
  • [1-1] Contacting extracellular vesicles in a sample with a C-type lectin; Separating the C-type lectin-bound extracellular vesicles from the C-type lectin;
  • a method for separating extracellular vesicles in a sample comprising: [1-2] Contacting extracellular vesicles in a sample with a C-type lectin in the presence of calcium ions; Separating C-type lectin-bound extracellular vesicles from the C-type lectin in the absence of calcium ions;
  • the separation method according to [1-1], [1-3] The isolation method according to [1-1] or [1-2], wherein the C-type lectin is ASGR1, DCSIGN, and/or CLEC1B.
  • the present disclosure provides a method for isolating extracellular vesicles and a kit for isolating extracellular vesicles.
  • FIG. 1 shows silver staining (A) of extracellular vesicles isolated by the TIM4 method in Example 1, and Western blots using anti-CD9 antibody (B), anti-CD63 antibody (C), and anti-81 antibody (D).
  • FIG. 1 shows the results of Western blotting using the lectin used, protein concentration, particle number, particle size of extracellular vesicles (mean value ⁇ standard error), anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody in the separation of extracellular vesicles using human C-type lectin in Example 2.
  • Example 3 silver staining (A) of extracellular vesicles separated using human C-type lectin, and Western blots using anti-CD9 antibody (B), anti-CD63 antibody (C), and anti-81 antibody (D) are shown.
  • Example 4 the protein concentration, particle number per ml, and particle size (mean value ⁇ standard error) of extracellular vesicles isolated from iPS cell culture supernatant using three types of C-type lectins (ASGR1, DCSIGN, CLEC1B), ultracentrifugation, and the TIM4 method are shown.
  • A Silver staining (A) of extracellular vesicles separated from iPS cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and TIM4 method, and Western blot using anti-CD9 antibody (B), anti-CD63 antibody (C), and anti-81 antibody (D) in Example 5.
  • the right figure shows the protein concentration, particle number, particle number per ml, and particle size (mean value ⁇ standard error) of extracellular vesicles obtained by each separation method.
  • A Silver staining (A) of extracellular vesicles separated from HEK293T cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and TIM4 method, and Western blot using anti-CD9 antibody (B), anti-CD63 antibody (C), and anti-81 antibody (D) in Example 6.
  • the right figure shows the protein concentration, particle number, particle number per ml, and particle size (mean value ⁇ standard error) of extracellular vesicles obtained by each separation method.
  • FIG. 1 shows the protein concentration, particle number, particle number per ml, and particle size (mean value ⁇ standard error) of extracellular vesicles obtained by each separation method.
  • FIG. 13 shows the results of miRNA expression analysis of extracellular vesicles isolated from HEK293T cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and TIM4 method in Example 7.
  • FIG. 13 shows the results of heat map analysis of the expression levels of miRNA in extracellular vesicles isolated from HEK293T cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and TIM4 method in Example 8.
  • FIG. 13 shows the results of glycan microarray analysis of ASGR1 in Example 9. 1 shows a list of glycans used in the glycan array in Example 9.
  • Example 1 is a graph showing the results of flow cytometry analysis of extracellular vesicles using ASGR1-immobilized beads in Example 10.
  • the vertical axis indicates the average fluorescence intensity.
  • Beads is a group in which beads on which ASGR1 is not immobilized are reacted with cell culture supernatant and reacted with an anti-CD63 antibody
  • Isotype is a group in which ASGR1-immobilized beads are reacted with cell culture supernatant and reacted with a control antibody
  • CD63 is a group in which ASGR1-immobilized beads are reacted with cell culture supernatant and reacted with an anti-CD63 antibody.
  • FIG. 12 shows the results of proteomic analysis of extracellular vesicles separated from HEK293T cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and TIM4 method (analysis of protein types in extracellular vesicles separated by each separation method) in Example 12.
  • FIG. 12 shows the results of proteomic analysis of extracellular vesicles separated from HEK293T cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and the TIM4 method in Example 12 (analysis of the proportion (%) of proteins associated with each cellular component in extracellular vesicles separated by each separation method).
  • FIG. 12 shows the results of proteomic analysis of extracellular vesicles separated from HEK293T cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and the TIM4 method in Example 12 (analysis of the proportion (%) of proteins associated with each cellular
  • Example 13 shows the results of proteomic analysis of extracellular vesicles separated from HEK293T cell culture supernatant by C-type lectin (ASGR1), ultracentrifugation (UC), and the TIM4 method in Example 12 (heat map analysis of the expression levels of exosome markers in extracellular vesicles separated by each separation method).
  • Graph showing the results of flow cytometry analysis of extracellular vesicles isolated from HEK293T cell culture supernatant using TIM4-immobilized beads (A) or ASGR1-immobilized beads (B) in Example 13, using buffer, control antibody (isotype), and exosome marker (CD9, CD63, or CD81) antibody.
  • FIG. 1 shows the results of flow cytometry analysis of extracellular vesicles isolated from 201B7 iPS cell culture supernatant using TIM4-immobilized beads (A) or ASGR1-immobilized beads (B) in Example 14, using buffer, control antibody (isotype), and exosome marker (CD9, CD63, or CD81) antibody.
  • Example 15 is a graph showing the results of ELISA analysis in Example 15, in which the culture supernatants of HEK293T cells (A), 201B7 iPS cells (B), adipose-derived mesenchymal stem cells (C), or bone marrow-derived mesenchymal stem cells (D) were reacted on an ASGR1-Fc-immobilized plate, followed by detection with a CD63 antibody.
  • the separation method according to this embodiment is a method for separating extracellular vesicles from a sample containing extracellular vesicles using a C-type lectin.
  • the separation method is a method for separating extracellular vesicles in a sample from other components in the sample.
  • extracellular vesicles include vesicles classified in various ways based on their origin and the size of small membrane vesicles. Specifically, in the present specification, extracellular vesicles include exosomes, microvesicles, apoptotic vesicles, adiposomes, and the like.
  • Exosomes are small membrane vesicles derived from late endosomes, composed of a lipid bilayer membrane, and having phosphatidylserine on the membrane surface. The diameter of the vesicles is usually about 50 nm to about 250 nm. Exosomes are known to contain proteins such as tetraspanins such as CD9, CD63, and CD81, Alix, TSG101, Lamp-1, and Flotillin (Thery et al., Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines, Journal of Extracellular Vesicles, 2018, Vol.
  • proteins such as tetraspanins such as CD9, CD63, and CD81, Alix, TSG101, Lamp-1, and Flotillin
  • Microvesicles are small membrane vesicles derived from plasma membranes, composed of a lipid bilayer membrane, and having phosphatidylserine on the membrane surface. Microvesicles are usually about 100 nm to about 1000 nm. Microvesicles are known to contain proteins such as integrins, selectins, and CD40 ligands.
  • Apoptotic vesicles are small membrane vesicles derived from apoptotic cells, composed of a lipid bilayer membrane and having phosphatidylserine on the membrane surface. Apoptotic vesicles are usually about 50 nm to about 500 nm in size. Apoptotic vesicles are known to contain histones. Adiposomes are small membrane vesicles derived from fat cells, composed of a lipid bilayer membrane, and having phosphatidylserine on the membrane surface. Adiposomes are usually about 100 nm to about 1000 nm in size. Adiposomes are known to contain MFG-E8 (milk fat globule-EGF factor 8).
  • both naturally occurring and artificially synthesized extracellular vesicles are subject to separation by the separation method according to this embodiment.
  • the extracellular vesicles contained in the sample may contain extracellular vesicles other than the target for separation, and the extracellular vesicles other than the target for separation may be naturally occurring or artificially synthesized extracellular vesicles.
  • the cells from which the extracellular vesicles are derived are not limited to the extent that they secrete the extracellular vesicles, and may be plant cells, animal cells, or microbial cells, and are not limited by the species of origin, developmental origin, tissue or organ of origin, cell function, etc.
  • the plant cells may be, for example, cells derived from either seed plants or non-seed plants, and may be cells derived from any organ such as stems, leaves, flowers, or roots.
  • the microbial cells may be, for example, cells of either prokaryotes or eukaryotes, and more specific non-limiting examples include Escherichia coli, Staphylococcus aureus, halophiles, acidophiles, thermophiles, yeast, fungi, bacteria, etc.
  • the animal cells may be, for example, cells derived from any of mammals, birds, reptiles, amphibians, fish, etc., and may be endoderm-derived, ectoderm-derived, mesoderm-derived, or stem cells, or may be derived from epithelial tissue, connective tissue, muscle tissue, or nerve tissue.
  • Non-limiting examples of cells include blood cells, muscle cells, skin cells, nerve cells, glandular cells, etc.
  • cells may be primary cells taken from an individual organism or tissue, or may be cultured cells. More specific, non-limiting examples of cells from which extracellular vesicles are derived include various stem cells such as mesenchymal stem cells (MSCs) (including adipose-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells), induced pluripotent stem cells (iPS cells), and embryonic stem cells (ES cells), cell lines (HEK293 cells, CHO cells, HeLa cells, MCF-7 cells, COS cells, PC12 cells, etc.), and primary cultured cells (skin fibroblasts, endothelial cells, muscle cells, hematopoietic cells, etc.).
  • the cells from which the extracellular vesicles are derived may be human-derived stem cells, cell lines established from human cells, or primary cultured cells derived from humans.
  • Naturally derived extracellular vesicles also include extracellular vesicles that have been isolated from plant cells, animal cells, microbial cells, etc., and have been treated with an enzyme that acts on glycans, resulting in a reduced or absent amount of glycans with low specificity for C-type lectins, or an increased proportion of glycans with high specificity for C-type lectins, compared to naturally occurring extracellular vesicles.
  • Non-limiting examples of enzyme treatments include sialidase and galactosidase.
  • Artificially synthesized extracellular vesicles can be obtained by culturing cultured plant cells, cultured animal cells, or cultured microbial cells that secrete extracellular vesicles in an appropriate medium under appropriate conditions and secreting the extracellular vesicles into the medium.
  • Artificially synthesized extracellular vesicles may be extracellular vesicles in which, compared to a case in which no genetic manipulation is performed, the amount of glycans with low specificity for C-type lectins in the secreted extracellular vesicles is reduced or absent, or the proportion of glycans with high specificity for C-type lectins is increased, by, for example, knocking out a specific gene related to glycan synthesis in cultured plant cells, cultured animal cells, or cultured microbial cells.
  • Non-limiting examples of genetic manipulation include knocking out core type 1 glycan synthesis enzyme (C1GALT1) and knocking out the molecular chaperone Cosmc for C1GALT1.
  • the extracellular vesicles contained in the sample may be the extracellular vesicles to be separated.
  • the extracellular vesicles to be separated have glycans that have binding ability to C-type lectin, and the other extracellular vesicles do not have glycans that have binding ability to C-type lectin. Therefore, the separation method according to this embodiment, which includes a step of contacting the extracellular vesicles in the sample with the C-type lectin and a step of separating the extracellular vesicles bound to the C-type lectin from the C-type lectin, can separate the extracellular vesicles that have binding ability to the C-type lectin.
  • the extracellular vesicles to be separated are at least CD9-positive, CD63-positive, and/or CD81-positive extracellular vesicles. In one embodiment, the extracellular vesicles to be separated are at least CD9-positive, CD63-positive, and CD81-positive extracellular vesicles. At least CD9-positive, CD63-positive, and/or CD81-positive extracellular vesicles express glycans on their surface, as described below in "C-type lectin," and are therefore easily separated from a sample containing extracellular vesicles by the separation method using C-type lectins according to this embodiment.
  • extracellular vesicles are separated from a sample containing extracellular vesicles using C-type lectin.
  • the sample is not limited to the extent that it contains extracellular vesicles, and may be a biological sample or an experimental sample collected from a living body.
  • Biological samples include, for example, body fluid samples, solid samples, and semi-solid samples in which extracellular vesicles are present.
  • body fluids include body fluids in a broad sense, such as blood (plasma, serum, whole blood), digestive fluids such as gastric juice, bile, pancreatic juice, and intestinal juice, tissue fluids such as interstitial fluid, intercellular fluid, and interstitial fluid, body cavity fluid, serous cavity fluid, pleural fluid, ascites, pericardial fluid, cerebrospinal fluid, synovial fluid, aqueous humor, lymph, amniotic fluid, saliva, sputum, urine, semen, sweat, tears, nasal mucus, vaginal fluid, and milk.
  • solid samples include, for example, tissue pieces and feces collected from individual organisms. Semi-solid samples include vomit, etc.
  • biological samples include plant or fruit extracts, cultured cell culture fluids, and microbial culture fluids.
  • Culture fluids include culture fluids containing cells and microorganisms, and their supernatants.
  • the microbial culture medium may be, for example, a yeast culture medium, and more specifically, may be mash produced in the sake production process, or a sake product containing at least a portion of the components of mash or containing extracellular vesicles derived from mash.
  • the species from which the biological sample is derived is not limited as long as it has extracellular vesicles, and may be an animal, a plant, or a microbial cell.
  • the species is the same as the species of the cells from which the extracellular vesicles are derived, as described in the section "Extracellular vesicles.”
  • the sample When the sample is a biological sample, it may be stored after collection from the subject until the separation method is performed according to methods well known to those skilled in the art.
  • Non-limiting examples of the experimental sample include culture supernatant containing extracellular vesicles, physiological saline containing extracellular vesicles, buffer containing extracellular vesicles, etc.
  • the extracellular vesicles may be artificially synthesized extracellular vesicles as described in the "Extracellular vesicles" section above.
  • C-type lectins Lectin is a general term for proteins or glycoproteins that exist in plants, animals, microorganisms, etc. and have a specific binding activity mainly to glycans.
  • C-type lectins are one of the major structurally defined lectin families, and in a broad sense include lectins that bind to glycans and ligands other than glycans, regardless of whether they are calcium-dependent or not.
  • the C-type lectin is preferably a lectin having calcium-dependent sugar chain binding activity, and more preferably a C-type lectin receptor (transmembrane receptor), in consideration of the binding affinity to the extracellular vesicles to be separated.
  • the C-type lectin receptor is not limited by its structure, and may be any C-type lectin receptor classified into, for example, group 2, group 4, group 5, group 6, group 14, etc.
  • the C-type lectin receptor is not limited by other functions within the scope of having binding affinity to extracellular vesicles, and is not limited by the level of binding affinity to extracellular vesicles.
  • the binding ability between a C-type lectin receptor and an extracellular vesicle means the binding ability between a C-type lectin receptor and a glycan expressed on the surface of an extracellular vesicle.
  • the glycan is not limited to the extent that it is expressed on the surface of the extracellular vesicle in a form capable of binding to a C-type lectin, nor is it limited by its expression level.
  • the glycan is a glycan having ⁇ GalNAc or ⁇ GalNAc at the non-reducing end.
  • glycans having ⁇ GalNAc or ⁇ GalNAc at the non-reducing end include A-type glycans (GalNAc ⁇ 1-3Gal ⁇ 1-4GlcNAc ⁇ 1-), Core6 (GlcNAc ⁇ 1-6GalNAc ⁇ 1-), di-GalNAc ⁇ (GalNAc ⁇ 1-3GalNAc ⁇ 1-), LDN (GalNAc ⁇ 1-4GlcNAc ⁇ 1-), Forssman (GalNAc ⁇ 1-3GalNAc ⁇ 1-3Gal ⁇ 1-4Gal ⁇ 1-4Glc ⁇ 1-), Forssman disaccharide (GalNAc ⁇ 1-3GalNAc ⁇ 1-), ⁇ GalNAc (GalNAc ⁇ 1-), Tn (GalNAc ⁇ 1-), A-di (GalNAc ⁇ 1-3Gal ⁇ 1-), GA2 (A-type gly
  • the glycan is ⁇ Gal (Gal ⁇ 1-).
  • the sugar chain may exist as a glycoprotein, and examples of such glycoproteins include BSM (bovine submaximal mucin), asialo-BSM, and the like.
  • the sugar chain may be LeX (Gal ⁇ 1-4(Fuc ⁇ 1-3)GlcNAc), LeA (Gal ⁇ 1-3(Fuc ⁇ 1-4)GlcNAc), a high mannose type sugar chain, an O-type sugar chain (di-sialyl T (Sia ⁇ 2-3Gal ⁇ 1-3(Sia ⁇ 2-6)GalNAc), etc.), etc.
  • the C-type lectin receptor is A-type glycan (GalNAc ⁇ 1-3Gal ⁇ 1-4GlcNAc ⁇ 1-), Core6 (GlcNAc ⁇ 1-6GalNAc ⁇ 1-), Tn (GalNAc ⁇ 1-), di-GalNAc ⁇ (GalNAc ⁇ 1-3GalNAc ⁇ 1-), LDN (GalNAc ⁇ 1-4GlcNAc ⁇ 1-), Forssman (GalNAc ⁇ 1-3GalNAc ⁇ 1-3Gal ⁇ 1-4Gal ⁇ 1-4Glc ⁇ 1-), ⁇ GalNAc (GalNAc ⁇ 1-), Tn (GalNAc ⁇ 1-, A-di (GalNAc ⁇ 1-3Gal ⁇ 1-), BSM (sialylated Tn), ⁇ Gal (Gal ⁇ 1-), BSM (Bovine It is preferable that the C-type lectin receptor is a C-type lectin
  • C-type lectin receptors were ASGR1 (Asialoglycoprotein receptor 1 (UniProt No. P07306), DCSIGN (CD209 antigen (UniProt No. Q9NNX6), or CLEC1B (C-type lectin domain family 1 member B (UniProt No. Q9NNX6)). No. Q9P126), and more preferably ASGR1.
  • the separation method according to the present embodiment makes it easier to separate CD9-positive, CD63-positive, and/or CD81-positive extracellular vesicles.
  • the C-type lectin receptor is ASGR1, it makes it easier to separate CD9-positive, CD63-positive, and/or CD81-positive extracellular vesicles with higher purity.
  • the use of C-type lectin receptors has the advantage that it is easy to separate types of extracellular vesicles different from those that can be obtained by conventional methods (e.g., the TIM4 method, ultracentrifugation, etc.).
  • the use of C-type lectin receptors makes it easy to separate extracellular vesicles that encapsulate and/or express genes (e.g., miRNA) and/or proteins different from those obtained by conventional methods.
  • the C-type lectin receptor is preferably ASGR1, DCSIGN, and/or CLEC1B, and more preferably ASGR1. The ability to separate different types of extracellular vesicles allows for their analysis and use for disease diagnosis and/or treatment.
  • the C-type lectin is not limited by its preparation method, and may be a naturally derived lectin or an artificially synthesized lectin.
  • a naturally derived lectin it may be one isolated from biological tissues or cells of plants, animals, microorganisms, etc. according to a method well known to those skilled in the art.
  • an artificially synthesized lectin it can be prepared by expressing a recombinant lectin in mammalian cells, plant cells, yeast, or E. coli by a method well known to those skilled in the art. It can also be prepared in an in vitro translation system.
  • the C-type lectin may be a commercially available lectin provided by reagent companies such as Acro Biosystems, R&D systems, Sigma-Aldrich, Takara Bio, and Fujifilm Wako Pure Chemical Industries.
  • C-type lectins also include proteins or peptides other than full-length proteins, so long as they maintain their ability to bind to the target glycan, and may be, for example, structures in which a portion of the protein is deleted, structures in which an arbitrary peptide is added to a full-length protein, extracellular domains, proteins or peptides containing an extracellular domain, sugar-binding domains, proteins or peptides containing a sugar-binding domain, etc.
  • the C-type lectin may be immobilized on any carrier suitable for the separation method according to the present embodiment.
  • the carrier may be appropriately selected from carriers known to those skilled in the art, and may be in the form of a microplate, a tube, a disk-shaped piece, particles (beads), or the like. When the carrier is a particle or the like, the carrier may be packed in a purification or separation column.
  • the carrier may be a porous body having through-holes, such as a sponge monolith. The size of the through-holes is not limited, and may be about 1 nm to about 100 ⁇ m.
  • the material of the carrier can also be appropriately selected from substances well known to those skilled in the art, and may be polystyrene, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylamide, polyglycidyl methacrylate, polypropylene, polyolefin, polyimide, polyurethane, polyester, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber, agarose, dextran, glass, silicon oxide, diatomaceous earth, porous glass, ground glass, alumina, silica gel, metal oxides, iron, cobalt, nickel, magnetite, chromite, etc.
  • the size of the particles can be appropriately selected by a person skilled in the art depending on the application, and may be, for example, about 10 nm to about 100 ⁇ m, about 100 nm to about 10 ⁇ m, or about 1 ⁇ m to about 5 ⁇ m.
  • the method for immobilizing the C-type lectin on the carrier can be appropriately selected from methods known to those skilled in the art, and may be, for example, affinity binding, chemical binding, or physical adsorption.
  • the Fc region of an antibody may be added to the lectin, and the lectin may be bound to beads to which protein G has been added by utilizing the binding force between the Fc region and protein G, biotinylated lectin may be immobilized on streptavidin-immobilized beads, or lectin may be immobilized on beads activated with N-hydroxysuccinimide (NHS) or epoxy groups by amine coupling.
  • NHS N-hydroxysuccinimide
  • the separation method is a method for separating extracellular vesicles in a sample, comprising the steps of contacting extracellular vesicles in the sample with a C-type lectin, and separating the extracellular vesicles bound to the C-type lectin from the C-type lectin.
  • the sample containing extracellular vesicles may be a biological sample collected from a living organism or an experimental sample. Regardless of the type of sample, the sample may be pretreated prior to the separation method according to this embodiment. Pretreatment may involve, for example, concentrating the extracellular vesicles by removing undesired substances in the sample. More specifically, substances in the sample can be selected based on their size or characteristics and undesired substances can be removed by ultrafiltration, tangential flow filtration, ultracentrifugation, ion exchange separation, or the like.
  • the contact between the extracellular vesicles and the C-type lectin in the sample can be achieved by, for example, mixing the C-type lectin with a sample containing the extracellular vesicles.
  • a sample containing the extracellular vesicles For example, when the C-type lectin is immobilized on a carrier such as a microplate, a tube, or a disk-shaped piece, the mixture may be achieved by dropping a liquid sample containing the extracellular vesicles onto the side on which the C-type lectin is exposed.
  • the C-type lectin immobilized on a particulate carrier such as beads and a sample containing the extracellular vesicles may be mixed in one solution.
  • the non-immobilized C-type lectin and a sample containing the extracellular vesicles may be mixed in one solution.
  • the mixed C-type lectins and at least a portion of the extracellular vesicles bind to each other due to their binding properties to form a complex.
  • the solution used in the step of contacting the extracellular vesicles in the sample with the C-type lectin is not limited as long as it can dissolve or suspend the extracellular vesicles and the C-type lectin in a stable state and does not prevent the contact and/or binding between the C-type lectin and the extracellular vesicles, and examples of the solution include water or a buffer solution having a pH of about 7.0 to about 8.0, preferably about 7.2 to about 7.6 (e.g., Tris buffer, Hepes buffer, etc.).
  • the step of contacting extracellular vesicles in a sample with C-type lectin is preferably carried out in the presence of calcium ions.
  • the C-type lectin according to this embodiment is preferably a lectin having calcium-dependent glycan binding activity, and in this case, the presence of calcium ions improves the binding ability to extracellular vesicles.
  • a buffer that does not bind to calcium to form a precipitate may be used.
  • the calcium ion may be derived from any source, such as calcium chloride, calcium hydroxide, calcium bicarbonate, calcium iodide, calcium bromide, or calcium acetate, and is preferably derived from calcium chloride, calcium bicarbonate, or calcium iodide, and more preferably from calcium chloride or calcium bicarbonate.
  • the calcium ion concentration can be adjusted by adding one or more of these ionic compounds before or at the time of contacting the extracellular vesicles in the sample with the C-type lectin, and the final concentration is about 0.5 to about 100 mM, preferably about 0.5 to about 10 mM, more preferably about 0.5 to about 5.0 mM, and even more preferably about 0.5 to about 2.0 mM. When it is about 0.5 to about 2.0 mM, it may be about 1.0 mM.
  • the step of contacting the extracellular vesicles in the sample with the C-type lectin can be carried out by incubating at about 10° C. or lower, preferably at about 1 to about 6° C., more preferably at about 4° C., for about 0.5 to about 24 hours, preferably about 3 to about 20 hours, more preferably about 6 to 12 hours.
  • a carrier such as beads that specifically bind to the C-type lectin may be introduced into the complex of the C-type lectin and the extracellular vesicles to form a complex of the C-type lectin, the carrier, and the extracellular vesicles.
  • the extracellular vesicles bound to the C-type lectin can be separated from the C-type lectin by incubating the complex of the C-type lectin and the extracellular vesicles in an elution solution. This causes at least a portion of the extracellular vesicles bound to the C-type lectin to separate from the C-type lectin.
  • the solution used in this step is not limited as long as it can dissolve or suspend the extracellular vesicles and C-type lectin in a stable state and does not interfere with the separation of the C-type lectin from the extracellular vesicles, and examples of the solution include water or a buffer solution having a pH of about 7.0 to about 8.0, preferably about 7.2 to about 7.6 (e.g., Tris buffer, Hepes buffer, etc.).
  • the elution solution is preferably free of calcium ions, because the C-type lectin according to this embodiment is preferably a lectin having calcium-dependent glycan-binding activity, and the absence of calcium ions makes it easier for the extracellular vesicles to be separated from the C-type lectin.
  • the eluate may be an eluate containing a glycan recognized by a C-type lectin. This is because the use of such an eluate tends to improve the efficiency of separation of extracellular vesicles from C-type lectins.
  • Such a sugar chain may be any sugar chain that is recognized by the C-type lectin used, but when the C-type lectin is ASGR1, DCSIGN, or CLEC1B, for example, the sugar chain is preferably a sugar chain having ⁇ GalNAc or ⁇ GalNAc, ⁇ Man, ⁇ Fuc, or di-sialyl T (Sia ⁇ 2-3Gal ⁇ 1-3(Sia ⁇ 2-6)GalNAc) at the non-reducing end. Examples of ⁇ GalNAc and ⁇ GalNAc are as described above in "C-type lectins.”
  • the separation method according to this embodiment may include a step of washing the complex and removing undesired substances before the step of separating the extracellular vesicles bound to the C-type lectin from the C-type lectin in the elution solution.
  • the washing is preferably performed about once to about three times, and may be performed twice.
  • the washing solution is not limited to a range that can dissolve or suspend the extracellular vesicles and the C-type lectin in a stable state and does not interfere with the separation of the C-type lectin and the extracellular vesicles, and examples thereof include water or a buffer solution having a pH of about 7.0 to about 8.0, preferably about 7.2 to about 7.6 (e.g., Tris buffer, Hepes buffer, etc.).
  • a buffer solution having a pH of about 7.0 to about 8.0, preferably about 7.2 to about 7.6 (e.g., Tris buffer, Hepes buffer, etc.).
  • a carrier such as a microplate, a tube, or a disk-shaped piece
  • the surface on the carrier on which the complex of the C-type lectin and the extracellular vesicles is present can be washed by dropping a washing solution.
  • the complex of the bead-immobilized C-type lectin and extracellular vesicles can be washed by adding a washing solution, agglutinating or precipitating the complex using the beads, and then removing the supernatant.
  • the beads are magnetic beads
  • the complex containing the beads can be agglutinated or precipitated by magnetic force.
  • the separation method of this embodiment makes it possible to separate extracellular vesicles that have binding ability to C-type lectins.
  • ASGR1, DCSIGN, or CLEC1B is used as a C-type lectin
  • a population of extracellular vesicles containing at least CD9-positive, CD63-positive, and/or CD81-positive extracellular vesicles can be separated.
  • ASGR1, DCSIGN, or CLEC1B is used as a C-type lectin
  • a population of extracellular vesicles containing at least CD9-positive, CD63-positive, and CD81-positive extracellular vesicles can be separated.
  • the extracellular vesicles separated by the separation method of this embodiment may have an average particle size of about 50 nm to about 250 nm, about 100 nm to about 250 nm, about 150 nm to about 250 nm, or about 200 nm to about 250 nm.
  • the extracellular vesicles to be separated can be isolated from the sample by obtaining only the solution containing the free extracellular vesicles.
  • the separation method according to this embodiment can be used in a method for purifying extracellular vesicles by further isolating the extracellular vesicles separated from other components in a sample by the separation method as described above.
  • the separated extracellular vesicles can be used in a method for detecting extracellular vesicles by labeling them with a label (e.g., a specific antibody) that binds to a marker specific to the extracellular vesicles.
  • the population of extracellular vesicles separated by the separation method comprises at least CD9-positive, CD63-positive, and/or CD81-positive extracellular vesicles at a higher density compared to the sample containing extracellular vesicles prior to being subjected to the separation method. In one embodiment, the population of extracellular vesicles separated by the separation method comprises at least CD9-positive, CD63-positive, and CD81-positive extracellular vesicles at a higher density than the sample containing the extracellular vesicles prior to being subjected to the separation method.
  • the separation method according to the present embodiment can enrich for at least CD9-positive, CD63-positive, and/or CD81-positive extracellular vesicles, or at least CD9-positive, CD63-positive, and CD81-positive extracellular vesicles.
  • a diagnostic method includes the separation method according to this embodiment, and preferably includes a step of analyzing information molecules in the separated extracellular vesicles.
  • target symptoms or diseases include, but are not limited to, cancer, neurodegenerative diseases, psychiatric diseases, chronic diseases (lifestyle-related diseases), and the like.
  • the kit according to this embodiment is a kit for separating extracellular vesicles from a sample containing extracellular vesicles, the kit including a C-type lectin.
  • the kit is a kit for separating extracellular vesicles in a sample from other components in the sample.
  • the kit according to this embodiment can be used to carry out any of the separation methods described in the first embodiment.
  • the kit may contain, in addition to the C-type lectin, one or more reagents necessary for each step in the separation method described in the first embodiment, i.e., the "step of contacting extracellular vesicles in a sample with C-type lectin” and/or the "step of separating extracellular vesicles bound to C-type lectin from C-type lectin.”
  • the reagents are, for example, a buffer solution used in these steps, an ionic compound containing calcium ions, a sugar chain used in the elution solution, and/or a washing solution.
  • the C-type lectin may be immobilized on a carrier as described in the first embodiment, but when it is not immobilized, the kit may include a carrier for immobilizing the C-type lectin and one or more reagents for immobilizing the C-type lectin on the carrier.
  • the kit can be used as a kit for purifying extracellular vesicles by further isolating the extracellular vesicles separated from other components in the sample by the separation method.
  • the kit can be used as a kit for detecting extracellular vesicles by labeling the separated extracellular vesicles with a label (e.g., a specific antibody) that binds to a marker specific to the extracellular vesicles.
  • the kit may include a means for labeling the marker specific to the extracellular vesicles.
  • a means may be an antibody that specifically binds to a marker specifically expressed in the extracellular vesicles.
  • the antibody is preferably an anti-CD9 antibody, an anti-CD63 antibody, and/or an anti-CD81 antibody.
  • the kit includes a C-type lectin and an antibody that specifically binds to the extracellular vesicles.
  • An antibody that specifically binds to a marker specifically expressed in extracellular vesicles may be modified as necessary for its detection. The modification may be appropriately selected from techniques known to those skilled in the art, and may be, for example, biotin, a fluorescent substance, an enzyme, etc.
  • the kit for detecting extracellular vesicles may be, for example, a kit for EIA (Enzyme Immunoassay) including chemiluminescent enzyme immunoassay (CLEIA), fluorescent immunoassay, ELISA (Enzyme-Linked Immunosorbent Assay), RIA (Radioimmunoassay), Western blotting, latex agglutination, immunochromatography, sandwich, flow cytometry, etc.
  • EIA Enzyme Immunoassay
  • CLIA chemiluminescent enzyme immunoassay
  • ELISA Enzyme-Linked Immunosorbent Assay
  • RIA Radioimmunoassay
  • Western blotting latex agglutination
  • immunochromatography sandwich
  • flow cytometry etc.
  • the kit according to this embodiment and various kits using the same contain the C-type lectin described above, and further contain one or more of the reagents described above, a means for labeling a marker specific to extracellular vesicles, positive/negative controls, and/or instructions for use of the kit.
  • the kit for separating extracellular vesicles in a sample according to this embodiment can be used as kits for diagnosing symptoms or diseases of a subject based on analysis of bioinformation molecules contained in the extracellular vesicles, using the extracellular vesicles purified by the kit.
  • the kit for detecting extracellular vesicles can be used to quantify extracellular vesicles in a sample, and based on the results, the symptoms and diseases of the subject can be diagnosed. That is, the kit for detecting extracellular vesicles according to this embodiment can be used as a kit for diagnosing the symptoms and diseases of the subject based on the detection or quantification results.
  • target symptoms or diseases include, but are not limited to, cancer, neurodegenerative diseases, psychiatric diseases, chronic diseases (lifestyle-related diseases), and the like.
  • the antibodies against CD9, CD63, and CD81 used were Exosome CD9 Antibody (Ts9) (Invitrogen, 10626D), Anti CD63 Human (Mouse) Unlabeled, 8A12 (Cosmo Bio Co., Ltd., SHI-EXO-MO2), and Anti CD81, Human (Mouse) Unlabeled, 12C4 (Cosmo Bio Co., Ltd.: SHI-EXO-MO3). Unless otherwise specified, each analysis was performed according to the method described in the previous Examples.
  • extracellular vesicles were isolated using the MagCaptureTM Exosome Isolation Kit PS (TIM4 method, Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. The obtained extracellular vesicles were subjected to protein quantification using a micro BCA assay (manufactured by PIERCE), and particle size and particle number were measured using a Nanosite (manufactured by Japan Quantum Design Co., Ltd.).
  • the extracellular vesicles separated by the rLSLN-immobilized beads were negative for CD9, CD63, and CD81, and CD9-positive, CD63-positive, or CD81-positive extracellular vesicles could not be separated.
  • Example 2 Separation of extracellular vesicles using human C-type lectin
  • the base sequence encoding the extracellular domain of human C-type lectin (Table 1, SEQ ID NOs: 1 to 23) was ligated to the XhoI/EcoRV restriction enzyme site of pSecTag/FRT/V5-His TOPO vector (ThermoFisher).
  • An expression vector was prepared by ligating the human IgG1 Fc region to the AgeI/PmeI restriction enzyme site.
  • the prepared expression vector was transfected into HEK293T cells using Lipofectamine LTX Reagent with PLUS Reagent (ThermoFisher), cultured in a CO2 incubator, and the culture supernatant was collected.
  • Human C-type lectin-Fc fusion protein was purified from the collected culture supernatant using rProtein A Sepharose Fast Flow (GE Healthcare Japan). The degree of purification of the purified protein was confirmed by electrophoresis and Western blotting, and the protein amount was quantified using the Pierce (registered trademark) BCA Protein Assay Kit (manufactured by ThermoFisher). A total of 23 types of human C-type lectin-Fc fusion proteins were produced. TIM4 and the other 23 types of human C-type lectins used in this example are as shown in Figure 2.
  • C-type lectin-immobilized beads were prepared by binding 12 ⁇ g of C-type lectin-Fc fusion protein to 50 ⁇ l of Dynabeads ProteinG (Veritas) at 4 ° C for 30 minutes. 50 ml of culture supernatant of 201B7 iPS cells cultured in mTeSR PLUS (Veritas) was collected and concentrated to 1 ml with 100 kDa cut Amicon Ultra (Merck). CaCl2 was added to a final concentration of 1 mM, mixed with the C-type lectin-immobilized beads, and incubated overnight at 4 ° C while mixing.
  • extracellular vesicles were isolated using the MagCapture(TM) Exosome Isolation Kit PS (TIM4 method, Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions.
  • the obtained extracellular vesicles were subjected to protein quantification using a micro BCA assay (PIERCE), and particle size and particle number were measured using a Nanosite (Japan Quantum Design). Furthermore, 36 ⁇ l of each sample was electrophoresed, transferred to a PVDF membrane, and Western blotted with antibodies against CD9, CD63, and CD81.
  • CLEC1B was found to be reactive to anti-CD9 antibody and anti-CD63 antibody, and had strong reactivity to anti-CD81 antibody. With respect to lectins other than those mentioned above, no reactivity was detected with anti-CD9 antibody, anti-CD63 antibody, or anti-CD81 antibody.
  • Example 3 Expression of markers in extracellular vesicles separated using human C-type lectins
  • the extracellular vesicles prepared in Example 2 were electrophoresed at 0.15 ⁇ g/well and Western blotted with antibodies against CD9, CD63, and CD81.
  • the population of extracellular vesicles separated using beads immobilized with three types of C-type lectins contained CD9-positive, CD63-positive, and/or CD81-positive extracellular vesicles.
  • Example 4 Comparison of extracellular vesicles separated by different separation methods Furthermore, separation of extracellular vesicles was examined using beads immobilized with three types of C-type lectins (ASGR1, DCSIGN, CLEC1B) and compared with extracellular vesicles separated by ultracentrifugation and the TIM4 method.
  • C-type lectin-immobilized beads were prepared in the same manner as in Example 2, and extracellular vesicles were separated using the C-type lectin-immobilized beads and the TIM4 method.
  • UC ultracentrifugation method
  • 20 ml of the iPS cell culture supernatant was filtered through a 0.22 ⁇ m filter, placed in an ultracentrifuge tube, and ultracentrifuged at 100,000 ⁇ g for 70 minutes at 4° C. in an ultracentrifuge (Beckman Optima MAX-XP). After removing the supernatant, a small amount of PBS was added to the ultracentrifuge tube, and the mixture was stirred with a vortex to recover an eluate containing extracellular vesicles.
  • Example 5 Separation of extracellular vesicles derived from iPS cells by ASGR1 Among the three types of C-type lectins (ASGR1, DCSIGN, CLEC1B), ASGR1 was further examined.
  • a solution (100 ⁇ l) containing extracellular vesicles separated from the culture supernatant (20 ml) of 201B7 iPS cells using ASGR1-immobilized beads was electrophoresed with the same particle number (4.5 ⁇ 10 7 / well) of extracellular vesicles separated by ultracentrifugation and TIM4 method, and Western blotted with antibodies against CD9, CD63, and CD81. The results are shown in Figure 5.
  • the extracellular vesicles separated by ASGR1-immobilized beads had a particle diameter of about 220 nm, and the reactivity of antibodies against CD9, CD63, and CD81 was the highest compared to the extracellular vesicles separated by the TIM4 method and ultracentrifugation method. Therefore, it was shown that when ASGR1 was used, a larger number of intact extracellular vesicles positive for markers (CD9, CD63, CD81) and having a particle diameter (average value) of about 220 nm were separated from the extracellular vesicles with the same particle number compared to the ultracentrifugation method and the TIM4 method. This shows that when ASGR1 is used, CD9-positive, CD63-positive, and/or CD81-positive extracellular vesicles can be separated with a higher purity compared to the ultracentrifugation method and the TIM4 method.
  • Example 6 Separation of extracellular vesicles derived from HEK293T cells by ASGR1 HEK293T cells were cultured in RPMI1640, and 20 ml of the resulting culture supernatant was collected, and extracellular vesicles (100 ⁇ l as a solution) were separated using ASGR1-immobilized beads, the TIM4 method, and ultracentrifugation. As a result, as with the ultracentrifugation and TIM4 methods, extracellular vesicles with a particle diameter of about 200 nm could be separated using ASGR1-immobilized beads.
  • the same number of particles (4.5 ⁇ 10 7 /well) were electrophoresed and western blotted with antibodies against CD9, CD63, and CD81.
  • the results are shown in Figure 6.
  • the extracellular vesicles separated by ASGR1-immobilized beads had a particle diameter (average value) of about 200 nm, and the reactivity of antibodies against CD9, CD63, and CD81 was the highest compared to the extracellular vesicles separated by the TIM4 method and ultracentrifugation method.
  • UC ultracentrifugation
  • TIM4 the TIM4 method
  • ASGR1 3D-Gene DNA chip
  • Example 8 Heat map analysis of miRNA amounts in extracellular vesicles isolated by different isolation methods The amounts of miRNA in extracellular vesicles purified by three methods, ultracentrifugation (UC), TIM4, and ASGR1, were analyzed using Heatmapper (http://www.heatmapper.ca/) and shown in a heat map. The results are shown in Figure 8. It was found that extracellular vesicles purified using ASGR1 contained more miRNAs than extracellular vesicles purified using other methods.
  • Example 9 Analysis of glycan affinity of ASGR1 by glycan microarray ASGR1 was mixed with Cy3-conjugated AffiniPure Goat Anti-Human IgG and Fc ⁇ Fragment Specific antibody (Jackson ImmunoResearch) and analyzed by glycan microarray (Tateno et al. Glycobiology, Vol. 18, Issue 10, Pages 789-798, 2008). As shown in FIG.
  • ASGR1 was found to exhibit high reactivity to ⁇ Gal, Asialo-BSM (bovine submaximal mucin), A-di (GalNAc ⁇ 1-3Gal ⁇ 1-PAA), Core6 (GlcNAc ⁇ 1-6GalNAc ⁇ 1-), Tn (GalNAc ⁇ 1-), di-GalNAc ⁇ (GalNAc ⁇ 1-3GalNAc ⁇ 1-), BSM (bovine submaximal mucin), LDN (GalNAc ⁇ 1-4GlcNAc ⁇ 1-), Forssman disaccharide (GalNAc ⁇ 1-3GalNAc ⁇ 1-), and ⁇ GalNAc (GalNAc ⁇ 1-), and to exhibit high affinity for glycans having ⁇ GalNAc at the non-reducing end.
  • a list of the glycans used in the glycan array is shown in FIG.
  • Example 10 Flow cytometry analysis of extracellular vesicles using ASGR1-immobilized beads 24 ⁇ g of the ASGR1-Fc fusion protein prepared in Example 2 was mixed with 50 ⁇ l of Dynabeads Protein G (Veritas), and allowed to bind at 4° C. for 30 minutes to prepare ASGR1-immobilized beads. 10 ml of culture supernatant of 201B7 iPS cells cultured in mTeSR PLUS (Veritas) was collected and concentrated to 1 ml with 100 kDa cut Amicon Ultra (Merck). CaCl2 was added to a final concentration of 1 mM, mixed with ASGR1-immobilized beads, and incubated overnight at 4 ° C. with mixing.
  • Dynabeads Protein G Veritas
  • Example 11 Separation of extracellular vesicles derived from HEK293T cells using ASGR1
  • ASGR1-immobilized beads were separated from the culture supernatant of HEK293T cells using ASGR1-immobilized beads, the TIM4 method, and ultracentrifugation.
  • the results of observation of the separated extracellular vesicles under a transmission electron microscope are shown in FIG. 12.
  • the extracellular vesicles separated by ASGR1-immobilized beads were vesicles having a lipid bilayer membrane and had a particle diameter of approximately 200 to 300 nm.
  • Example 12 Proteomic analysis of extracellular vesicles separated by different separation methods
  • extracellular vesicles were separated from the culture supernatant of HEK293T cells using ASGR1-immobilized beads, the TIM4 method, and ultracentrifugation.
  • a volume equivalent to 1.5 ⁇ g of total protein was taken from each suspension of extracellular vesicles, subjected to acetone precipitation, and the precipitate was dissolved in solubilization buffer.
  • the lysate was subjected to trypsin hydrolysis to obtain a peptide mixture.
  • the MS/MS data obtained by liquid chromatography tandem mass spectrometry (LC-MS/MS) of the peptide mixture was collated with a human amino acid sequence database. Both information on peptide identification and peptide detection intensity were integrated, and the peptide detection intensity was integrated for each protein (protein metric value) (FIG. 13).
  • the data obtained as described above was analyzed using FunRich software (http://www.funrich.org/) to analyze the proportion of proteins associated with each cellular component (FIG. 14).
  • exosome-associated proteins were detected in 49.80% of extracellular vesicles separated with ASGR1-immobilized beads, 47.98% by the TIM4 method, and 47.34% by the ultracentrifugation method.
  • the results of this example demonstrated that exosomes can be isolated more selectively (with higher purity) by using ASGR1 for separation than by conventional methods.
  • extracellular vesicles isolated using ASGR1-immobilized beads contained the greatest amount of exosome markers compared to those isolated using the TIM4 method and ultracentrifugation method.
  • Example 13 Flow cytometry analysis of extracellular vesicles derived from HEK293T cells using an exosome marker antibody The culture supernatant obtained by culturing HEK293T cells was incubated overnight at 4°C with ASGR1-immobilized beads or TIM4-immobilized beads (MagCapture (trademark) Exosome Isolation Kit PS, Fujifilm Wako Pure Chemical Industries, Ltd.).
  • the beads were then suspended in wash buffer (Tris-HCl; NaCl; pH 7.4) supplemented with 1 mM CaCl 2 and incubated with 10 ⁇ g/ml of each of a labeled isotype control antibody (clone number: A-1, MC2B-TF2, Cosmo Bio Co., Ltd.) or an exosome marker (CD9, CD63, or CD81) antibody (clone number: 12A12, SHI-EXO-M01, -M02, and -M03 (manufacturer: CAC), Cosmo Bio Co., Ltd.).
  • Flow cytometry data was analyzed using CytoFLEX (Beckman Coulter, Inc.) and is shown in FIG.
  • the detection patterns of exosomes positive for exosome markers CD9, CD63, and CD81 were different between the ASGR1-immobilized bead group and the TIM4-immobilized bead group.
  • the results of this Example demonstrated that the expression patterns of each exosome marker in exosomes obtained from HEK293T cells differed between exosomes isolated using ASGR1-immobilized beads and those isolated using the TIM4 method, i.e., different exosome groups were isolated.
  • Example 14 Flow cytometry analysis of iPS cell-derived extracellular vesicles using exosome marker antibody Culture supernatant of 201B7 iPS cells (human iPS cell line) was incubated with ASGR1-immobilized beads or TIM4-immobilized beads (MagCaptureTM Exosome Isolation Kit PS, Fujifilm Wako Pure Chemical Industries, Ltd.) overnight at 4°C.
  • the beads were then suspended in wash buffer (Tris-HCl; NaCl; pH 7.4) supplemented with 1 mM CaCl 2 and incubated with 10 ⁇ g/ml of each of a labeled isotype control antibody (clone number: A-1, MC2B-TF2, Cosmo Bio Co., Ltd.) or an exosome marker (CD9, CD63, or CD81) antibody (clone number: 12A12, SHI-EXO-M01, -M02, and -M03 (manufacturer: CAC), Cosmo Bio Co., Ltd.).
  • Flow cytometry data was analyzed using CytoFLEX (Beckman Coulter, Inc.) and is shown in FIG.
  • the detection patterns of exosomes positive for exosome markers CD9, CD63, and CD81 were different between the ASGR1-immobilized bead group and the TIM4-immobilized bead group.
  • the results of this example demonstrated that the expression patterns of each exosome marker in exosomes obtained from iPS cells differed between exosomes isolated using ASGR1-immobilized beads and those isolated using the TIM4 method, i.e., different groups of exosomes were isolated.
  • Example 15 ELISA analysis of extracellular vesicles derived from different cells using CD63 antibody HEK293T cells were cultured in RPMI1640, iPS cells in mTeSR1 (STEMCELL Technologies), and adipose-derived mesenchymal stem cells (ADSC, Lot No. 2118, Life Technologies) and bone marrow-derived mesenchymal stem cells (Yub622, RIKEN CELL BANK) in MesenPRO RS (trademark) Medium (catalog number: 12746-012, GIBCO) (supplemented with 2 mM L-glutamine and 1% penicillin-streptomycin).
  • the culture supernatant was diluted in three stages (2-fold (0.5), 4-fold (0.25), 8-fold (0.125)) as shown on the horizontal axis of Figures 18A to 18D.
  • the culture supernatant was washed away, and peroxidase-labeled anti-CD63 antibody (SHI-EXO-M02 (Cosmo Bio Co., Ltd.) labeled with peroxidase (LK11, Dojindo Laboratories)) (0.5 ⁇ g/ml, 100 ⁇ l) was added and reacted at room temperature for 2 hours.
  • TMB solution manufactured by Fujifilm Wako Pure Chemical Industries
  • CD63 an exosome marker
  • the method for isolating extracellular vesicles and the kit for isolating extracellular vesicles according to the present disclosure can separate extracellular vesicles from a sample containing extracellular vesicles, and therefore can be suitably used in diagnosis, treatment, and the like using extracellular vesicles, and has industrial applicability.

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Abstract

La présente invention aborde le problème consistant à fournir : un procédé de séparation de vésicules extracellulaires; et un kit de séparation de vésicules extracellulaires.　L'invention concerne un procédé de séparation pour séparer des vésicules extracellulaires dans un échantillon, le procédé de séparation comprenant : la mise en contact de vésicules extracellulaires dans un échantillon avec une lectine de type C; et la séparation des vésicules extracellulaires de produits liés des vésicules extracellulaires et de la lectine de type C. L'invention concerne également un kit de séparation de vésicules extracellulaires d'un échantillon contenant les vésicules extracellulaires, le kit comprenant une lectine de type C.
PCT/JP2024/043415 2023-12-08 2024-12-09 Procédé de séparation de vésicules extracellulaires et kit de séparation de vésicules extracellulaires Pending WO2025121435A1 (fr)

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